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Foods and Diets in Health

Part 1: Basic food science


Part 2: Diet and disease
Part 3: Foods, diets and disease

A compilation of lecture series delivered for


Food Science at College of Human Health, FSU, Tallahassee

Innovations And Solutions Inc.

3945 West Pensacola Street, Tallahassee, Florida 32304


Web-based Selected Lecture Series

Foods and Diets in Health


Part 3: Foods, diets and disease
________________________________________________

Course Number: Human Health HU 4657 and BCE 4003c and 4004c
Florida State University and Florida A&M University
Elective Course at: Maharana Pratap A&T University, Udaipur, Rajasthan, and Uttar
Pradesh Technical University, Lucknow, Uttar Pradesh, India

Part 1: Basic food science


Part 2: Diet and disease
Part 3: Foods, diets and disease

Editors: Rakesh Sharma,Ph.D, M.Sc-Ph.D(IIT-D)


Bharati D Shrinivas, Ph.D(Nagpur)

Sponsoring Institution: Innovations and Solutions Inc. USA(R)


Address: Center of Nanoscience and Biotechnology,
Innovations and Solutions Inc.
901 West Jefferson Street, Tallahassee A26, Florida 32304
Copyright© 2009. The material here is a compilation from web-based information and
research articles solely for sharing and educational purposes. Prohibited the material to
use for any profit making business without prior permission from sole editors or original
authors by email.

Cited text and materials is from web sources: Sribd.com, Novapublishers.com, nsti.org.

Cited material sources:


NSTI 2007,2008 and 2009 Conference CDs
Free distributed material from scribd.com

Contact Email:
rksz2009@yahoo.com
bharuds312@gmail.com
Table of contents

Part 1: Basic food science


Lecture 1: Food science
Lecture 2: Principles of food and health
Lecture 3: Food components and diets
Lecture 4: Classification of foods
Lecture 5: Chemistry of foods I
Lecture 6: Chemistry of foods II
Lecture 9: Metabolism of carbohydrates in food
Lecture 10: Metabolism of lipids in food
Lecture 11: Metabolism of proteins in food
Lecture 12: Metabolism of vitamins and minerals in food
Lecture 13: Metabolism of nucleic acids in food
Lecture 14: Physiological relationship of food metabolism in the body
Lecture 15: Applied techniques: Analysis of foods
Lecture 16: Applied techniques: Evaluation of foods and nutrition
Lecture 17: Applied techniques: Nutrition assessment and dietetics

Part 2: Diet and disease


Lecture 1: Principles of diet in health and disease
Lecture 2: Dietary groups
Lecture 3: Dietary surveys in community
Lecture 4: Nutritional diseases and disorders
Lecture 5: Therapeutic diets and nutraceuticals
Lecture 6: Analytical methods of nutritional analysis and diet preparation
Lecture 7: Clinical dietetics in hospital practice
Lecture 9: Public health nutrition
Lecture 10: Ditetics as profession

Part 3: Foods, diets and disease


Lecture 1: A survey of foods and diets in disease
Lecture 2: Dietary fibers and gut motility
Lecture 3: Prification, structure, health benefits: Feruloyl Arabinoxylans in fibers
Lecture 4: Rhinacanthus nasutus: antimutagen properties
Lecture 5: Dietary fibers in cultured cells
Lecture 6: Bamboo shoots as dietary fibers
Lecture 7: Tropical and temperate fruits
Lecture 8: Relationship of physical activity and dietary fiber consumption
Lecture 9: Cardiovascular prevention by soluble fiber
Lecture 10: Processing techniques and their effect on fruit phytochemicals
Lecture 11: Grafting of biofibers
Lecture 12: Bioactive foods and nutraceutical supplementation criteria in
cardiovascular protection
Lecture 13: Comparison of cholesterol lowering diets: Apple, Casein

Appendix
Paper 1: Cholesterol 7a-Hydroxylase Activity Is Increased by Dietary Modification
with Psyllium Hydrocolloid, Pectin, Cholesterol and Cholestyramine in Rats.
Few words on 3D imaging
Paper 2: Cholesterol 7a-Hydroxylase Activities From Human and Rat Liver Are
Modulated In Vitro Posttranslationally by Phosphorylation/Dephosphorylation
Foods, Diets and Disease
Editors: Rakesh Sharma, Bharati D Shrinivas © 2009 Innovations And Solutions, Inc.
___________________________________________________________________________

Lecture 2

DIETARY FIBERS AND GUT MOTILITY

Rakesh Sharma, Bharati D Shrinivas

ABSTRACT
The relationship between dietary fibers and motility of the digestive tract is essential for the
accomplishment of their functions, but is somewhat complex, as there may be reciprocal influences.
In fact, it is well known that fibers can modify gastrointestinal motility, but it is not as much known that
gastrointestinal motility may modify the functions of fibers and reverse their beneficial effects.
The effects of motility on fiber functions obviously is not irrelevant with respect to the fiber
characteristics (e.g. viscosity, water holding capacity, fermentability etc.) and vice-versa. Consequently
the kind of fibers and the kind of gut motility should be taken into account when considering their
functional interactions.
Fibers may influence both the motor activity of the stomach, by delaying in most cases gastric emptying,
and that of the small intestine, where the most frequent fiber effect is an acceleration of transit, whereas
in the colon the transit may be variously modified.
However, when the motor activity is impaired, the presence of fibers in the gut lumen may became
deleterious. In fact, if there is an inefficient gastric motility with gastric stasis, the accumulation of fibers
in the gastric cavity induces a further worsening of gastric motility and in some cases may lead to an
abnormal fermentation of fibers and formation of bezoars. A delayed intestinal transit may favour the
small intestinal bacterial overgrowth, which is responsible of an out of place fiber fermentation in the
small intestine with many patho-physiological problems, and, in addition, if there is a chronic pseudo-
obstruction, may provoke an episode of functional obstruction. The altered transit in the colon due to
constipation or diarrhoea may produce differences in fiber fermentation, that in its turn may modify the
colonic transit with an abnormal production of gases. In addition, when there is a condition of severely
altered colonic transit the addition of a large quantity of fibers to diet may further worsen the motor
activity leading to an impaction.
For these reasons the addition of fibers to a diet, that usually is beneficial for the gut transit, should be
done with caution in patients with the above mentioned alterations of gut motility.
2 Rakesh Sharma, Bharti D Shrinivas

INTRODUCTION
Dietary fibers carry out many physiological functions in the gastrointestinal tract aimed to health
preservation through their fermentation by colonic bacteria and the maintenance of a normal
escretory function.
The addition of dietary fibers to a diet is found to increase the frequency of bowel movements and
this fact suggests that fibers influence gut motor activity and transit. However the effect of fibers on
gastrointestinal motility depends both on the segment of the gut considered and on the kind of
fibers. In fact the various segments of the gastrointestinal canal have different patterns of motor
activity and each kind of fiber has a different behaviour in the intestinal lumen that depends on its
intrinsic characteristics.
To investigate the relationships between dietary fibers and gut motility it is necessary first of all to
define the characteristics of the gastroduodenal, small intestinal and colonic motor activities and
afterwards those of dietary fibers.

CHARACTERISTICS OF GUT MOTILITY DURING FUNCTIONAL PERIODS


Gastrointestinal motility carries out various functions during both the interdigestive and digestive
periods through the performance of different motor patterns that will be briefly summarized
hereafter before discussing the characteristics and the effects of fibers.

Interdigestive Period

The interdigestive period is characterized by a peculiar motor pattern called “interdigestive migrating
motor complex” (IMMC) [1.2] characterized by a phase of peristaltic hyperactivity (Figure 1) called
phase III or activity front, lasting a few minutes and propagating from the stomach through the
duodenum, jejunum and ileum down to the ileo-cecal valve every about 90 minutes. The phase III is
preceded by a period of irregular motor activity, called phase II, with pressure waves that
progressively increase in frequency and amplitude and is followed more or less abruptly by a phase of
motor silence, called phase I, that takes up about one third or one quarter of the entire cycle.
The principal function (Table 1) of the IMMC is clearing the gastrointestinal lumen from the not
absorbed leftovers of the meal, bacteria and foreign stuffs with the aid of the cyclic output of
gastric, duodenal, pancreatic, biliary and IgA secretions (Figure 2), that takes place just before the
phase III of peristaltic activity.
In this manner it performs a “washing” and a “disinfection” of the gastrointestinal tract from the
stomach down to the ileo-caecal valve, so avoiding the gastric stasis of indigestible fibers and
preventing the bacterial overgrowth in the stomach and intestine that may give rise to abnormal
fermentations. For this reason it is also called the “intestinal housekeeper” [3].
Dietary Fibers and Gut Motility 3

Table 1. Functions of the Interdigestive Migrating Motor Complex (Immc) and Secretory Cyclic
Activities of the Gastrointestinal Tract

1. to eliminate from the gastrointestinal lumen the not digested and not
absorbed substances that remain after the completion of digestive and
absorptive activity.
2. to perform a cyclic washing, cleaning and “disinfection” of the
gastrointestinal lumen with the gastric, duodenal, biliary pancreatic and
immuno-globulinic A secretions that increase just before the appearance of
the activity front of IMMC, that sweeps them along the gastrointestinal
canal.
3. to avoid the stasis and reflux of secretions from the duodenum to the
stomach and from the cecum to the ileum.

Figure 1. Prolonged gastrointestinal manometric recording during fasting. Trace 1 is from the gastric corpus, traces
2 and 3 are from the antrum and traces 4-6 from the duodenum.

Note in the top tracing two periods of gastroduodenal intense motor activity at about 90 min interval starting in the
gastric corpus and propagating through the antrum in the duodenum. In the bottom panel one can see that this
motor activity is represented by strong peristaltic contractions that reach the maximal frequency of 3/min in the
stomach and 12/min in the duodenum. This cyclic motor activity is called phase III or activity front of the
interdigestive migrating motor complex (IMMC), is preceded by a period of increasing motor activity (phase II)
and is followed by a period of motor silence (phase I). (Modified from Bortolotti M. La dispepsia. Piccin Nuova
Libraria, Padova, 1996)
4 Rakesh Sharma, Bharti D Shrinivas

Digestive Period
When a meal is ingested, the IMMC cycling is immediately interrupted al all levels of the
gastrointestinal tract (Figure 3) and is replaced by a motor activity similar to that of phase II with phasic
waves of variable amplitude. The duration of interruption is proportional to the caloric content of the
meal, being about 4-5 hours for a meal of 500 Kcal, and is longer for lipids with respect to proteins and
carbohydrates. Indigestible fibers do not stop the IMMC cycling [4,5]. The reappearance of IMMC takes
place first in the small intestine and afterwards in the stomach. The ingested meal is received and
processed by the stomach until it is ready for emptying in the intestine. Gastric emptying is controlled by
various gastric and intestinal neuro-hormonal mechanisms that are activated by the physico-chemical
characteristics of the meal.

Figure 2. Secretory cyclic activities that takes place in the upper gut in correspondence with the phase III of IMMC.

Note: The gastric, duodenal, bilio-pancreatic and IgA secretions that are pushed
forward by the peristaltic contractions of the IMMC activity front and carry out a
“washing” and “disinfection” of the gastrointestinal lumen during the interdigestive
period.

Liquids are emptied more rapidly than solids, while meals containing proteins, lipids and carbohydrates
are emptied more slowly than inert ingesta and the emptying rate depends on the caloric density, being
slower as the latter is higher [6].
Solid meals requires a mechanical treatment by the gastric motor activity aimed to reduce the volume of
the meal particles to a diameter <1-2 mm.
Dietary Fibers and Gut Motility 5

Figure 3.

Note: The intake of a meal stops the occurrence of IMMC phases at all levels of the
entire gastrointestinal tract with appearance of an irregular motor activity that
continues until the processes of digestion and absorption are carried out. At this
point the cycling activity reappears, before in the intestine and after in the stomach,
to clean up the not digested and not absorbed leftovers of the meal.

The pylorus works as a filter blocking the particles with a diameter >1-2 mm, so that the antral motor
activity may grind and triturate them to a suitable diameter [7]. However, if the viscosity of the liquid
phase is high, the pylorus lazes the capacity of discriminating the particles with higher diameter that,
consequently are abnormally emptied [8,9] and may induce maldigestion.

The solid particles, which have a specific weight inferior or superior to that of water, are emptied with
more difficulty, as well as the hard particles with respect to those which are compressible.

The emptying of non digestible solids is very important, because they include dietary fibers. The latter
ones, that are not grinded and reduced to a diameter <1-2 mm, remain in the stomach until the other
foods adequately triturated are emptied and the digestive and absorptive processes are accomplished. At
this point the activity front of the IMMC reappears and cleans up the gastric lumen from the residual
solid stuff by means of peristaltic contractions with a great propulsive capacity that propels the particles
of the gastric content into the intestine even when their diameter is >2 mm. Hence the gastric emptying
6 Rakesh Sharma, Bharti D Shrinivas
of fibers requires this kind of motor activity and for this reason is emptied with a more or less substantial
delay with respect to the other more digestible components of a meal.

Table 2. Functions of the Digestive Motor Activity of the Gastrointestinal Tract

1. to receive and store the meal in the gastric fundus with receptive
relaxation and reservoir function (reception and storage of the meal),
2. to deliver progressively the meal with gastric secretions from the
fundus to the antrum with adaptive contractions (intragastric distribution of
the meal),
3. to triturate solids in particles of 1-2 mm with strong antral
contractions against the closed pylorus (mixing and grinding of the meal).
4. to selectively and progressively deliver the properly treated gastric
chyme into the duodenum (emptying of the meal).
5. to mix the nutrients with the digestive secretions and to distribute the
chyme on the maximal possible mucosal surface of the intestine for a rapid
and complete absorption (mixing and absorption).

The digestive motor activity of the small intestine is represented by segmenting waves the majority of
which (80%) are not propulsive. In fact they have the task of mixing the nutrients with the digestive
secretions and distributing the chyme on the maximal possible mucosal surface of the intestine for a
rapid and complete absorption.
Once the digestion and absorption of the meal is accomplished, about 5 hours after a normal meal, the
not absorbed residues, including dietary fibers, are swept away together bacteria, insoluble material, etc
by the peristaltic activity of the IMMC phase III.

The motor activity of the ileo-colonic junction has the function of regulating the passage of intestinal
contents into the colon [10], preventing at the same time the reflux of colonic content back into the
ileum, to avoid the small intestinal bacterial overgrowth (SIBO). The terminal ileum shows a reflex
clearing activity in connection with some products of bacterial fermentation refluxed from the caecum,
as, for example, the short chain fatty acids. The ileocolonic transit is rapid post prandially and slow and
erratic during fasting [11]. The ileo-caecal sphincter does not discriminate between solids and liquids
[12], while distal small bowel selectively retains dietary fibers [13].

In this manner the ileo-caecal junction has the task of functionally separating the small intestinal
environment from that of the colon. If this functional separation is altered, the colonic bacteria growth
more or less proximally in the intestinal lumen, fermenting the not absorbed nutrients, as well as dietary
fibers, and cause the small intestinal bacterial overgrowth.
Dietary Fibers and Gut Motility 7
The motor activity of the colon gives rise to a colonic transit far more slow than that of the other
portions of gut, representing about 90% of the total transit time.

The characteristic of the colonic motor activity can be easily observed with the technique of prolonged
manometric monitoring in physiologic and pathophysiologic conditions [14,15].

During the interdigestive period it does not show a motor activity similar to that of the
gastrointestinal migrating motor complex, even if it presents periods of inactivity especially during the
nocturnal rest [16] alternated with periods of activity [17]. The latter ones are characterized by stationary
and propagated contractions, including “mass movements” that usually precede defecation.
After meal ingestion a “gastrocolic reflex” [18] takes place with segmenting and migrating
contractions that give rise to propulsion as well as retropulsion of the colonic content and may end in a
mass movement. The activation of this reflex is initially due to a stimulation of mechanical receptors of
the gastric wall, while the subsequent phase depends essentially on lipidic content of the meal, that acts
through neuro-hormonal mechanisms [19].
The functions of the colonic motility are aimed (Table 3) to delay the transit of the chyme arriving
from the ileum, to allow water, salts and short chain fatty acids absorption with segmental non
propagated contractions, to store the feces allowing fermentation and to propel the colonic content into
the rectum with peristaltic contractions (mass movements) for defecation.

Table 3. Functions of Colonic Motility

1. to delay the transit of the chyme arriving from the ileum, to allow
water, salts and short chain fatty acids absorption with segmental non
propagated contractions (mixing and absorption),
2. to store the feces allowing the fermentation by colonic bacteria
(storage),
3. to propel the colonic content into the rectum with peristaltic
contractions (mass movements) for defecation (propulsion and defecation).

The colonic transit conditions both water absorption and bacterial fermentation. So one of the
consequences of slow transit is a greater production of gas, that, if there is a presence of only
saccharolytic bacteria, may be hydrogen, or, if there is the presence of methanogenic bacteria, also
methane, as happens in the 30-50% of the European population [20].
The time interval between the arrival of the intestinal content in the cecum and the expulsion of feces
from the rectum is that allowed for water absorption and bacterial fermentation of the not absorbed
nutrients and dietary fibers. More the transit is rapid, less time is allowed for water absorption and
bacterial fermentation, while slower the transit is, more time is available for water absorption and
bacterial fermentation.
The not fermented dietary fibers are propelled along the colon and represent the bulk of feces together
with the bacterial mass and other not digested residues.
8 Rakesh Sharma, Bharti D Shrinivas

CHARACTERISTICS OF FIBERS THAT INFLUENCE GUT MOTILITY


Dietary fibers may modify gut motor activity and transit in different manners that depend on the tract of
the gut considered and on the intrinsic characteristics of the fibers, such as viscosity, solubility, water
holding capacity, fecal bulking activity and fermentability (Table 4).

Table 4. Characteristics of fibers that may Influence Gut Motility

1. Viscosity: capacity of a fiber dispersed or dissolved in water to


gelatinize and form a dense and viscid solution.
2. Solubility. capacity of dissolve in water without leaving particles in
suspension.
3. Water holding capacity (WHC): capacity of swelling and retain water
in its matrix
4. Fecal bulking index (FBI): index that measures the ability of swelling
and retain water.
5. Fermentability: capacity of being destroyed by intestinal bacteria
forming other chemical compounds.

Viscosity and Solubility

Viscosity is the capacity of a fiber dispersed or dissolved in water to gelatinize and form a dense viscid
solution, whereas solubility is the capacity of a fiber of dissolving in water, without leaving particles in
suspension.
The fibers that give viscous solutions are in general soluble (glucomannan, guar gum, psyllium, etc.)
(Table 5), while insoluble fibers give dispersions in water that are less viscous.
The viscosity of a fiber solution depends on various factors including pH. In fact fibers as pectin and
alginate tend to gelatinize, when exposed to a pH less than 3, consequently, when the gastric pH is
higher than 3, a condition that occurs during the intake of a meal or during a treatment with antisecretory
drugs, the gelification of some fiber may be more difficult.
In addition the viscosity may change in a non linear fashion, when the “shear rate” of the fluid varies, as
happen in the gastrointestinal canal, in consequence of the diameter of the lumen and the motility
patterns [21]. An interesting experiment was conducted by Dikeman et al [22] to determine the
viscosities of both soluble and insoluble dietary fibers during gastric and small intestinal digestive
simulation and multiple shear rates, obtaining different values of viscosity for the same fiber depending
on the gastrointestinal tract and digestive period.
Table 5. Solubility and Viscosity of the Most Common Dietary Fibers

Fiber Solubility Viscosity


Pectin yes yes
Psyllium yes yes
Dietary Fibers and Gut Motility 9
Glucomannan yes yes
Guar gum yes yes
Linseed yes yes
Guar gum partially hydrolized yes no
Maltodestrin resistant partially hydrolized yes no
Inuline yes no
Cellulose no no
Bran no no

Water Holding Capacity and Fecal Bulking Index

Water holding capacity (WHC) of a fiber is the capacity of swelling and retain water in its matrix. This
contributes to increase the volume of intestinal content. With regard to the colonic content the point of
reference is the fecal bulking index (FBI), that measures the real effect of a fiber on fecal swelling and
differs strongly among the various fibers (Table 6). WHC influences remarkably not only the fecal mass,
but also the fermentability of the fiber and the absorption of nutrients in the intestine

Fermentability

Fermentability is the capacity of being destroyed by intestinal bacteria with formation of other chemical
compounds. Some of these products, such as short chain fatty acids and gases may influence gut motility.
There are significant differences in fermentability among the various fibers (Table 7 ), that depend not only
on the chemical composition, but also on the water holding capacity that favors the penetration of bacteria
inside the matrix of the fiber.

Table 6. Fecal Bulking Index (FBI) of Some Fibers

Fiber FBI
Pectin 3.6
Oat bran 13.1
Guar gum 16.1
Linseed grounded 33.1
Ispaghula (90%
441
fiber)

Table 7. Degree of Fermentability of Some Fibers

Low or partial High


Cellulose Pectins
Hemicellulose Gums
Lignin Inulin
Resistant stark Oligosaccharides
10 Rakesh Sharma, Bharti D Shrinivas

EFFECT OF FIBERS ON GUT MOTILITY


The influence of fibers on motility may be reciprocal. In fact, while on one hand, it is well accepted that
the fibers may modify gut motility, on the other hand, is not as much known that the gut motility may
interfere with the beneficial effects of fibers.
There are motility alterations that may involve either tract of the alimentary canal and may be influenced
by the characteristics of the fibers. Consequently, when the dietary fibers are administered to a subject or
a patient, the kind of fiber and the kind of gut motility should be taken into account, keeping care to their
functional interactions.

Stomach

The characteristic of the digestive motility of the stomach, that does not empty the particles of the meal
with a diameter >1-2 mm, produces a retention in the gastric lumen of the dietary fibers that cannot be
reduced to a diameter <1-2 mm by the antral motor activity during gastric digestion. For this reason this
kind of fibers affects gastric motility, delaying gastric emptying. Different preparations of the same fiber
may influence gastric motility in different manners. In fact coarse bran delays gastric emptying, while
fine bran does not [23]. This fact probably may account for different results obtained in various studies
with the same kind of fibers.
The fibers that form viscous solutions delay gastric emptying, while the fibers that are insoluble have
less effect on gastric emptying, provided that are reduced to a diameter <1-2 mm, otherwise they are
emptied at the end of the digestive period.
As the viscosity of a solution of fibers depends on pH, when the pH of the gastric juice is less than 3,
they tend to gelatinize, as happens with pectin and alginate, but when the gastric pH is higher than 3, as
during the intake of a meal or during a treatment with antisecretory drugs, the gelification of some fibers
may occur with more difficulty.
In addition the viscosity may change in a non linear fashion when the “shear rate” of the fluid varies, as
happen in the gastrointestinal canal, in consequence of the diameter of the lumen and the motility
patterns during interdigestive and digestive periods [21).

Clinical Corollary: Stomach

The restraining effect the fibers on gastric emptying may be beneficial, as, when taken in
correspondence of a meal, they induces early satiety [24,25] and, consequently, may help to lose weight.
Another beneficial effect of fibers that delay gastric emptying may be the prevention of dumping
syndrome in patients who underwent gastric resection [26] and the improvement of the glucose
tolerance in non insulin-dependent diabetes, because they slow down the glucose absorption [27,28].
However the fiber effect that delays gastric emptying may become dangerous in particular conditions
connected with gastric dysmotility. In fact, in patients with delayed gastric emptying due to impairment
of neuromuscular functions, especially if this impairment is so severe as to induce gastroparesis (Figure
Dietary Fibers and Gut Motility 11
4), the absence of the interdigestive cyclic motor activity that cleans the gastric lumen at the end of the
digestive period, may cause the retention of dietary fibers in the gastric lumen. This fact worsen
dyspeptic symptoms and, in particular conditions of anacidity, favors an abnormal fermentation with gas
production, that determines gastric distension and bloating.
Another consequence of the retention of fibers is the formation of bezoars and, in particular, of
pharmacobezoars comprised of medications in a background of pathologic gastric motility or excessive
use of anticholinergics [29].

Figure 4. A) Radiogram performed about 6 hours after ingestion of barium in a patient with both gastroparesis and
intestinal pseudo-obstruction. Note that the barium is still present in the stomach and small intestine.
12 Rakesh Sharma, Bharti D Shrinivas
B) Gastrointestinal manometric recording in a patient with both gastroparesis and intestinal pseudo-obstruction.
The recording sites (1-6) are the same of Figure1, whereas the trace n. 7 is from the jejunum.
Note that during the entire recording time lasting about 120 min there is a complete absence of motor activity in the
stomach (traces 1-3) and small intestine (traces 4-7), with only sporadic and low amplitude contractions in the
intestine.

Intestine

The fibers that produce viscous solution, such as guar gum, delay intestinal transit [30], whereas other
fibers, such as bran, does not modify or even hasten it [31].
We performed a study on this aspect [32], testing the effect on gastrointestinal transit of a balanced
mixture of dietary fibers (Fitomagra Plus, ABOCA), expressly chosen to hasten small intestinal transit
and delay gastric emptying. In a series of 10 patients with a slight overweight the effects of dietary
fibers mixture on gastric emptying and intestinal transit of a meal were investigated with a scintigraphic
method. The values the half gastric emptying time (T1/2) and of the intestinal transit time of a
semiliquid and bromatologically equilibrated meal of 300 kcal, measured without the previous intake of
the preparation were compared with those obtained after the fiber mixture intake in a different day and
the results were statistically compared by using the ANOVA, Wilcoxon and Student t test for paired
data. The intestinal transit was significantly (p<0.05) accelerated by the fiber intake, while gastric
emptying was delayed, but not significantly (Figure 5 ).

Clinical Corollary : Intestine


The effect of the acceleration of intestinal transit may decrease the intestinal absorption of nutrients
[33,34]. This effect is added to the increase in satiety due to decrease in gastric emptying and contributes
with other factors to lose body weight of patients overweight, as it has been demonstrated after a month
intake of the fiber mixture above described [32].
Dietary Fibers and Gut Motility 13

Figure 5. Values (mean + SD) in minutes (min) of gastric emptying (T 1/2) and intestinal transit of a meal with a
scintigraphic technique in basal conditions (black bars-A) and after a dose of dietary fiber mixture (white bars-B).

Note that the intestinal transit was significantly accelerated, whereas the gastric emptying
was delayed, although not significantly.
* = statistically significant [p<0.05] in comparison with the basal period with ANOVA test
" = statistically significant [p<0.05] in comparison with the basal period with Wilcoxon test
^ = statistically significant [p<0.05] in comparison with the basal period with Student t test

However, in patients with a delayed intestinal transit, that in some cases may reach the severity of a
chronic pseudo-obstruction (Figure 4), the continuous intake of a large quantity of dietary fibers, especially
those that delay intestinal transit, may create a problem. In fact, if the peristaltic activity front (phase III) of
the IMMC, that sweeps through the intestine the not digested fibers, is absent or impaired, an accumulation
of fibers may take place in the intestinal lumen, favoring in some cases the functional block of the transit.
In addition, in patients with small intestinal bacterial overgrowth due to intestinal stasis, the abundant
intake of dietary fibers may be responsible of an excessive out of place small intestinal fermentation
with many patho-physiological problems. These range from fat and vitamins malabsorption [35] with
consequent malnutrition, osteoporosis and anemia [36], to a secondary alteration of intestinal motility
[37] and visceral hypersensitivity, through the production of a liposaccharide and short chain fatty acids
[38, 39].

Colon

Dietary fibers affect colonic transit both by increasing fecal mass and through specific effects of the
substances produced during bacterial fermentation. The increase of the endoluminal volume of intestinal
14 Rakesh Sharma, Bharti D Shrinivas
content influences the intestinal peristaltic motor activity and hence the transit especially of the colon.
Many factors contribute to determine the volume of the fecal mass.

The amount of fibers added to the diet is of great importance in increasing the volume of the feces and
hence the velocity of the transit. In fact the addition of 20 g. of psyllium, guar gum or ispaghula
accelerates the total or colonic transit in a patient with constipation, while dosages lower than 5 g. are
ineffective on transit velocity, but nevertheless increase the quantity of feces and the number of
defecations, that become easier through a decrease in fecal consistency [31,40-44]. The increase in fecal
mass is not only due to the quantity of a non fermented fiber that remains in the feces, but also to the “
fecal bulking index” of a fiber [45), that is related to its water holding capacity, the capacity of retain
water into its matrix. The fecal bulking index may differ remarkably among fibers [Table 5], being, for
example, 446 for ispaghula and 3.6 for pectin [45]. This property of some fibers remarkably influences
the fecal mass, and also facilitates their fermentability, as bacteria may penetrate more easily into its
matrix.

The fiber fermentability may contribute, although not strikingly, to increase the fecal mass, by favoring
the bacterial growing [46]. In fact bacteria represent a quote of the volume of the feces that may exceed
the 60% [47-49]. More a fiber is fermentable, larger is the fecal mass produced and the level of
fermentability shows marked differences among fibers (Table 6). The cabbage fiber, for example, the
92% of which is fermentable, increases the fecal mass more than the wheat fiber, that has only the 36%
fermentable [50], whereas the resistant starch which is easily fermented reaches the colon in a
considerable quantity that ranges between 8 and 40 g a day [51] and increases the fecal mass of 2-3 g for
each gram of it [52]..

Finally, the water retained in the lumen by the products of fermentation


(i.e. short chain fatty acids), that exert an osmotic effect, may contribute to increase the volume of feces.
The amount of fecal mass is strictly correlated with intestinal transit. A variation in fecal mass may
influence transit and viceversa [52]. A slow transit may cause an increase in water absorption, hence a
decrease in fecal mass, while an accelerated transit increases the amount of feces.
Some products of fiber fermentation by the colonic flora are able to influence the motor activity of the
colon. In fact, the presence of methane may slow the colonic transit [53], as it stimulates non propulsive
contractions, especially in irritable bowel syndrome (IBS) patients [54], worsening the constipation.
Short chain fatty acids, instead, stimulate propulsive colonic motor activity [55,56] through a release of
5HT and Ach [57]. If the production of short chain fatty acids takes place in the proximal colon, the
effect on colonic transit is more evident. The relationship between short chain fatty acids and motility is
bidirectional: in fact, on one hand, they stimulate transit and, on the other hand, the acceleration of
transit increases the concentration of them in stools [58]. Moreover the fibers may indirectly influence
motility through a stimulation of mucin and gas production.

Mucin is essential for the propulsion of feces and is produced by a diet rich in fibers [59]: The
production of mucin in the small intestine and colon is correlated with the fiber “bulking property”,
while in the caecum it depends on fiber fermentability [60]. The gas produced in consequence of
Dietary Fibers and Gut Motility 15
bacterial fermentation and trapped in the fiber matrix, increases the volume of the intestinal content
leading to a stimulation of motility and transit. The gas represents itself a stimulus for propulsive
motility, when is not absorbed by the mucosa or is consumed by other bacteria through methanogenesis
[61] or acetogenesis [62]. The gut motor activity that determines the transit of gas is somewhat different
from that responsible of the transit of liquids and solids. The gas propulsion takes place more easily in
the erect rather than supine position [63], is independent from the motor activity that propels solid and
liquid contents, as is not carried out by propagated phasic waves, but by tonic contractions [64].
Particularly effective are in the colon the giant propulsive contractions with consequent gas evacuation
[65], probably activated by a reflex response to the distension of the colonic lumen induced by gas [66].
However the motor activity, on one hand, is stimulated by gas, but, on the other hand, influences the gas
absorption by the mucosa, the gas production through fermentation and the gas consumption by bacteria.
The consequences of an increase and decrease of colonic propulsive motility on the colonic gas
absorption are opposite, under the same conditions of gas production and consumption. In fact, if there
is a high propulsive motor activity, the time allowed for gas absorption is less, whereas a slow
propulsion favors a higher gas absorption.

In the absence of methanogenic bacteria, an increase of propulsive motility shortens the time allowed for
fiber fermentation and, consequently, the gas production is lower, whereas, if there is a low propulsive
motility, the fiber fermentation and gas production is increased. In the case of consistent presence of
methanogenic bacteria that take place in 30-50% of the population [67] is necessary to make other
considerations.

If there is an increase of propulsive motor activity, the time available for gas consumption by the
bacteria is reduced and more gas is available in the colon for excretion. On the other hand a decrease of
propulsive motility, with consequent stasis of gas in the lumen, favors a higher gas consumption by
methanogenic bacteria.
In conclusion, the amount of gas present in the colon is the result not only of the production, but also of
the interrelations among motor activity, absorption and consumption.

Clinical Corollary : Colon


The presence of fibers in the gut lumen is most important to maintain a normal colonic motility. In fact
an experiment with a diet devoid of fibers for 27 days impairs the myogenic and neurogenic intestinal
contractions through a decrease of enterochromaffin cells, that are important for the transduction of
mechanical and chemical stimuli in the colonic lumen necessary for the motor activity [68].
16 Rakesh Sharma, Bharti D Shrinivas

Figure 6. Manometric recording of the rectosigmoid tract in a normal subject (top tracing) and in two patients with
constipation (middle and bottom tracings).

In the first patient (1) the stimulation with bisacodyl induces high pressure
contractions, while in the other (2) the drug is completely ineffective (colonic inertia).
(From: Preston JE and Lennard-Jones JE. Dig Dis Sci 1985;30:289).

On the other hand, if the colonic motility is severely impaired for neuromuscular alterations (Figure 6)
with consequent delay in content transit, the addition of a high amount of fibers to the diet further
impairs colonic motility and worsens the symptoms [69]. In fact in 80% of patients with constipation
due to slow colonic transit and in 60% of those with obstructed defecation, the effect of fibers is scarce
or absent [70] with worsening of symptoms [71]. In addition the consumption of a high fiber diet delays
intestinal gas transit, promoting gas retention and the appearance of symptoms due to abdominal
distension in some individuals [72]. In particular the insoluble fibers, as corn and wheat bran, may
Dietary Fibers and Gut Motility 17
worsen the clinical outcome in some cases of IBS [69]. On the other hand a soluble fiber
supplementation, while does not affect the oro-cecal transit, significantly prolongs colonic transit
[73].To date there are few studies about the relationship between viscosity and laxative effect [21], but a
study with soluble fibers (psyllium, ispaghula, etc) demonstrated an improvement of symptoms of
irritable bowel syndrome [69].

EFFECT OF SINGLE FIBERS ON GUT MOTILITY


The effects of some most used dietary fibers on gut motility is summarized in Table 8 and is described
below with more details.

Gums form viscous solutions that delay gastric emptying, intestinal transit and nutrient absorption from
the small bowel [4,11,74] at a dose ≥ 9 g [75], whereas with lower doses other investigators found that
guar gum does not have any influence on gastric emptying and small intestinal transit time with
scintigraphy and breath test [44,76] . In addition, guar gum at a dose of 30 gr accelerates the frequency
of antral contractions and the postprandial pattern of motility in duodenum and jejunum, increases the
jejunal transit time [77] and markedly prolongs the duration of postprandial motor activity in the human
small bowel [30]. Partially hydrolized guar gum decreases colonic transit time [78] .

Glucomannan (Amorphophallus konjac), which is highly viscous, delays gastric emptying and
decreases nutrient absorption [79]. It also increases stool frequency and restores to normal a delayed
oro-cecal transit in patients with constipation in studies with radio-opaque markers and H2 breath test
[80,81].

Psyllium (Plantago psyllium) increases stool frequency, delays gastric emptying and decreases total
intestinal transit time [40,82-84], but does not modify colonic and rectal motor activity [85].

Ispaghula (Plantago ovata) seeds do not influence gastric emptying, small intestine transit and colonic
transit [86], ispaghula husks do not alter total intestinal transit time in IBS patients [87] and oro-cecal
transit time with breath test [31,44], but decreases the colonic transit time with radiopaque markers [42].

Pectin for some investigators delays gastric emptying [11,88-90], whereas for others does not
significantly influence gastric emptying [82,91-93], but decreases total transit time [94].

Table 8. Effect of Some Fibers on Gut Motility

Gastric Intestinal Oro-caecal Colonic Oro-anal


emptying transit transit transit transit
delays delays delays
/ /
Guar gum (dose>5 g) (dose>5 g) (dose>5 g)
no effect no effect no effect / /
18 Rakesh Sharma, Bharti D Shrinivas
(dose<5 g) (dose<5 g) (dose<5 g)
delays hastens
hastens hastens hastens
(coarse) (coarse)
Bran
no effect no effect
/ / /
(fine) (fine)
Lignin / no effect no effect hastens no effect
Cellulose little effect delays hastens /
Hemicellulose / / delays /
Ispaghula no effect no effect / hastens /
Glucomannan delays / hastens / /
Inulin / hastens / / /
Pectin delays * / / / hastens
Psyllium delays / / / hastens
Linseed / hastens / / /
Calcium no effect^ hastens*
/ / /
polycarbophil or hastens§ or delays°
Resistant no effect or
hastens / / /
starch shortens
^ = in man ; § = in dog; * = in IBS constipation; ° = in IBS diarrhea

Linseed (Linum usitatissimum) accelerates intestinal transit [95,96].

Bran has an effect on motility that depends on the dimension of its particles. In fact coarse bran, but not
fine bran, delays gastric emptying [23,31, 93] and accelerates small bowel transit [ 23,31,97], as well as
whole gut transit [98,99]. Wheat bran increased the duration of postprandial pattern of duodenal motility
[77] and decreases the mean retention time in the small intestine and colon [100]. Rice bran and barley
bran flour accelerate total gut transit [101-103].

Lignin has a little effect on gastric emptying and small intestinal transit, but accelerates colonic transit
[11,104].

Cellulose has little influence on gastric emptying and small bowel transit, but accelerates colonic transit
[11,104], increases the duration of postprandial pattern in duodenum and jejunum and markedly
increases jejunal transit time [77].

Hemicellulose does not modify the gastric emptying of lipids [105], but delays gastrointestinal transit
time [106], while purified hemicellulose (xilan) decreases transit time [94].

Inulin, which is soluble but not viscous, increases the frequency and velocity of the activity front of the
interdigestive migrating motor complex [107], likely because it produces short chain fatty acids with
fermentation. Other fructo-oligosaccharides accelerate gastric emptying [108].
Dietary Fibers and Gut Motility 19
Calcium polycarbophil does not delays the gastric emptying of radiolabeled pellets in man [109], but
stimulates a fed-like gastric motility and accelerates the gastric emptying of radiolabeled pellets in dog
[110]. It accelerates colonic transit in constipation of IBS patients [111] and patients with spinal cord
disorders [109,112], while in patients with IBS and diarrhea it prolongs mean colonic transit time
measured with radiopaque markers [111].

Resistant starch hastens gastric emptying [113], does not influence whole gut transit time in man [101]
or slightly shortens transit time in rats [114],while in association with wheat bran further shortens transit
time in humans [115].
The differences observed in the above listed effects on gut motility of some fibers may be due to
differences in methods used for measuring the gut transit, to different fiber dosages and preparations, as
well as to different subjects used for experiments (animals, healthy subjects or patients).

CONCLUSION
The gastrointestinal and colonic motility play a pivotal role in the accomplishment of the physiological
functions of dietary fibers. The fibers influence and in some cases determine the kind of motor activity
of the stomach, small intestine and colon, through their characteristics, such as viscosity, water holding
capacity, fermentability and production with their fermentation of substances acting on gut motility. It is
necessary to know the effect of the fibers in each gut segment, because some fibers go fast through some
bowel segments and slow through others. Consequently the total transit time is not a good predictor of
the rate of transit through particular gut segments.
Besides other beneficial effects, fibers are necessary for a correct gut motility performance, but in some
conditions may be dangerous especially if there is an impairment of gut motor activity. In this case the
beneficial fiber functions are replaced by noxious effects that lead to pathological conditions. This may
happens, for example, in patients with markedly delayed gastric emptying and intestinal transit, as in the
case of gastroparesis and pseudo-obstruction, respectively, as well as in patients with small intestine
bacterial overgrowth and colonic inertia. In these conditions fibers may worsen motor activity and
symptoms, leading to potentially dangerous effects.
For these reasons it is important to keep in mind the reciprocal influences that take place between fibers
and gut motility, when a diet with a high amount of fibers is prescribed to a patient with the above
mentioned motility problems.

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Dietary Fibers and Gut Motility 25
on the digestion and the intestinal absorption of dietary lipids in the rat. Ann Nutr Metab., 33:237-45.
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Foods, Diets and Disease
©
Editor: Rakesh Sharma, Bharati D Shrinivas 2009 Innovations And Solutions, Inc.
___________________________________________________________________________

Lecture 3

PURIFICATION, STRUCTURE AND HEALTH


BENEFITS: FERULOYL ARABINOXYLANS IN FIBERS

Rakesh Sharma, Bharti D Shrinivas

The nature of carbohydrates present in the food is growing field of interest


within the food industry due to the potential of some of them to help prevent
diseases of lifestyle. Non-glycemic carbohydrates, i.e., those carbohydrates (or
their components) that are not absorbed in the small intestine and, therefore,
transit down to become fermented in the colon, have drawn lot of attention. In
fact, food carbohydrates can be broadly classified on the basis of their in vivo
digestibility into digestible and non-digestible carbohydrates (Table1) (Asp,
1996; Englyst et al., 1992). Non-digestible carbohydrates have been
collectively referred to as „dietary fibre‟ (Hipsley, 1953). Some of these
carbohydrates are of particular interest to the food industry for the purpose of
developing „functional foods‟, i.e., foods that are able to exert positive health
effects. Non-digestible oligo/polysaccharides are considered as prebiotics,
which stimulate the growth of bifidobacteria in the colon.

A distinction was established between insoluble DF and soluble DF. The


effects of insoluble DF are of limited interest because of their low
functionality and fermentability (Hsu and Penner, 1989). By contrast, soluble
DF in general has a wide functionality due to its ability to interact with water,
2 Rakesh Sharma, Bharti D Shrinivas

and is almost fully fermented by the large intestine micro flora, bringing about
much desired physiological/metabolic effects (Lopez et al., 1999). Cereals, the
staple food for millions of people across the world, are the chief source of both
soluble and insoluble DF (Plaami, 1997). Arabinoxylans, along with some
amount of -D-glucans, are the major components of soluble DF (Rao and
Muralikrishna, 2004).

Table 1. Classification of Carbohydrates Based on Their in Vivo


Digestibility

Subgroup Components
Glucose, galactose, mannose, fructose
Monosaccharides (ketose), Arabinose, xylose Sorbitol,
mannitol
Digestible Disaccharides Sucrose, maltose, lactose,
Oligosaccharides malto-oligosaccharides
Polysaccharides Starch – amylose and amylopectin
Disaccharides Trehalose
Raffinose, stachyose, verbascose, fructo-
Oligosaccharides
Non- and xylo-oligosacchairdes
digestible Starch - modified and resistant, Non-starch
Polysaccharides – cellulose, ligno-cellulose, arabino-xylans,
mixed glucans, mannans, pectins

DISEASES OF LIFESTYLE/CIVILIZATION – ROLE OF


DIETARY FIBRE
Interest in carbohydrates/polysaccharides is increasing due to the recent
worldwide concern about the continuously increasing rates of many common
diseases, known as diseases of lifestyle/civilization. Some of these common
diseases in western countries are linked to the deficiency of complex
carbohydrates/dietary fibre in food. The list includes obesity, diabetes,
atherosclerosis and chronic heart problems, increased cholesterol in the body,
hypertension, constipation and diverticulosis, colorectal cancer and many
Purification, Structure and Benefits… 3

more. Obesity in particular, is raising in adults and now in children as well


(FAO/WHO, 1998). Although obesity as a significant phenomenon has usually
been associated with developed countries, it is now also on the rise in the
developing countries.
Cereals form the quantitatively most important source of DF. Consuming
cereals and cereal based products are known to have beneficial roles in human
nutrition and health and have been linked to their phytochemical profiles
(Adom and Liu, 2002; Adom et al., 2003; Charalampopoulos et al., 2002;
Mori et al., 1999). After more than 30 years of research into many and varied
claims for its benefits, it is now clear that fibre has uniquely significant
physical/physiological effects. Accumulating evidence favors the view that
increased intake of DF can have positive health effects against chronic
diseases, such as cardiovascular diseases, diverticulosis, diabetes and colon
cancer. Prevention of constipation and regulation of transit time are mainly
caused by the bulking effect of DF. It is also partly fermented in large intestine
by a mixed flora of anaerobic bacteria and most of the physiological effects of
DF are thought to be based on this property (Scheeman, 1998).
A daily intake of approximately 30 g of dietary fibre is encouraged to
promote health benefits associated with fibre. Because of the increased
nutritional awareness, the food industry is facing the challenge of developing
new food products with special health enhancing characteristics
(Charalampopoulos et al., 2002). To meet this challenge, it must identify new
sources of neutraceuticals and other natural and nutritional materials with the
desirable functional characteristics (Izydorczyk et al., 2001). In view of the
therapeutic potential of DF, more fibre incorporated food products are being
developed all over the world. However, consumer acceptability of these
functional foods depends not only on the nutritional property, but also on the
functional and sensory quality. These factors are considered while developing
functional foods.

CHARACTERIZATION OF POLYSACCHARIDES
Simple monosaccharides can be built into giant molecules called complex
polysaccharides that rival DNA and proteins in size and complexity. It‟s a
testament to the importance of sugars that scientists have granted them an
„ome‟ of their own. Just as the „genome‟ and „proteome‟, the „glycome‟ of an
organism or cell encompasses all the sugars it makes. Still in its infancy,
4 Rakesh Sharma, Bharti D Shrinivas

glycomics is slowly revealing its huge cast of sugar-related characters –


structure and their myriad roles. The glycome – study of carbohydrates, of a
single cell type or creature is probably many thousands of times more complex
than the genome (Schmidt, 2002). It is the polysaccharides – their structure or
bonding that makes the characterization very difficult. Even chemically
synthesizing oligosaccharides by capping sugar molecules with „protecting‟
groups at all but one branch point leaves compounds with a mixture of bonds
formed in different orientations, requiring extensive purification procedures
after each new sugar building block is added.
A polysaccharide may contain between ten and a million sugar residues.
Polysaccharides are rarely homogeneous and usually have a very wide
molecular weight distribution; often they are regarded as group of very closely
related molecular species varying in both molecular architecture and size
(„polydisperse‟). Structural characterization of polysaccharides usually
requires extensive purification procedures.

ISOLATION, FRACTIONATION AND PURIFICATION


Characterization of polysaccharides first requires them to be isolated from
biological samples. They may be extracted with various extractants such as
water (for water soluble arabinoxylans and mixed glucans), polar non-aqueous
solvents (for starch and glycogen) (Leach and Schoch, 1962), chelating agents
(for pectins) (Selvendran, 1985), N-methyl morpholine-N-oxide (MMNO, for
cellulose) (Chanzy et al., 1979) and alkali (for hemicellulose A and B)
(Wilkie, 1979). Water extraction at different temperatures can be carried out to
obtain gums and mucilages.
Polysaccharides thus isolated from the biological samples are rarely
homogeneous and require extensive fractionation and purification steps before
proceeding further with structural characterization. Polysaccharides differ in
their molecular size, shape and charge, and can be fractionated using various
methods such as fractional precipitation with solvents (ethanol, acetone), salts
(ammonium sulphate) or methods such as ion exchange/affinity/gel
permeation chromatographies.

HOMOGENEITY CRITERIA
Purification, Structure and Benefits… 5

Polysaccharides are highly complex and diverse, and unlike proteins, they
are heterogeneous in their chemical characteristics like molecular weight and
composition which in turn affects their physical properties. The heterogeneity
arises because their biosynthesis, which is controlled indirectly by
glycosyltransferase genes, unlike direct templates in case of DNA and
proteins. Glycosyltransferases, the enzymes with individual specificities, are
responsible for the transfer of sugar residues from particular glycosyl donor to
the growing polysaccharide chain. Variations in polysaccharide structures may
result from (a) departure from absolute specificity of the transferases, (b)
incomplete formation of segments/side chains and (c) post polymerization
changes. If these variations are continuous with respect to parameters such as
molecular size, proportions of sugar constituents and linkage type, separation
into discrete molecular species will be impossible and the polysaccharide
sample would be called „polydisperse‟. If the heterogeneity lies in their
molecular size, but not in their chemical composition, they are called
„polymolecular‟ (Aspinall, 1980).It is difficult to establish the purity of any
polysaccharide sample unambiguously. Showing the absence of heterogeneity
by as many independent criteria as possible is considered to be sufficient to go
ahead with the structural characterization.
There are number of methods to show the absence of overall heterogeneity
of a polysaccharide sample. Some of them are (a) consistency in chemical
composition and physical properties such as optical rotation and viscosity, (b)
chromatographic methods such as ion exchange, affinity and gel filtration, (c)
ultra centrifugation pattern, (d) electrophoretic methods such as cellulose
acetate and capillary electrophoresis and (e) spectroscopic methods such as IR
and NMR. Purified sample is then subjected to the structural characterization
(Aspinall, 1982).

Structural Characterization

Sugar residues forming the polysaccharide chain may either be in linear or


branched arrangements. They may all be of the same type (homoglycan) or of
different types (heteroglycan). The length of a polymer chain, called degree of
polymerization, is specified by the number of structural units it contains. The
structural units may either in pyranose or in furanose ring form.
The structure of a polysaccharide can be organized into four different
levels similar to that of proteins; they are (a) primary, (b) secondary, (c)
tertiary and (d) quaternary.
6 Rakesh Sharma, Bharti D Shrinivas

The covalent sequence of monomeric units along with the respective


glycosidic linkages is known as „primary structure‟. Depending on the primary
structures, polysaccharide chains may also adopt characteristic shapes such as
ribbons, extended helices and hollow helices, which are known as „secondary
structures‟. Energetically favored interactions between chains of well defined
secondary structures result in ordered organizations, which are known as
„tertiary structures‟. Further associations between well-defined entities result
in higher levels of organizations, known as „quaternary structures‟ (Perez and
Kouwijzer, 1999).
The major problems in the determination of the molecular structure of
complex carbohydrates/polysaccharides are to establish (a) the molecular
weight and nature of constituent sugar residues including their ring size, (b)
the position and anomeric configurations of the inter-glycosidic linkages, (c)
the sequence of residues/linkages and (d) overall arrangement of polymeric
chains.
Structural elucidation of plant polysaccharides is a very tough task due to
their non-periodic repeating units unlike microbial polysaccharides. However,
several methods are available for the determination of the polysaccharide
structure and are broadly categorized into three main classes: (a) chemical, (b)
enzymatic and (c) spectroscopic methods (Aspinall, 1982).

Chemical Methods

Molecular Size
As the polysaccharides are „polydisperse‟ in nature, their molecular size is
represented as average of either weight (Mw) or number (Mn). The
determination of both weight and number average gives an indication of the
molecular size distribution, greater the difference between Mw and Mn, greater
the polydispersity of the sample. Number average can be obtained by
membrane osmometry (Mn > 20 kDa) and vapor pressure osmometry (van
Dam and Prins, 1965) and weight average can be obtained by light scattering
(Manley, 1963). Similarly, weight average can also be obtained by
ultracentrifugation (sedimentation equilibrium or approach to equilibrium).
These methods, however, are based mainly on the theoretically calculated
average values.
Gel filtration chromatography, on the other hand, is a simple and widely
used method to obtain the average molecular weight of the polysaccharide
Purification, Structure and Benefits… 7

sample. The column needs to be pre-calibrated with known molecular weight


markers to determine the molecular weight of the unknown samples.

Sugar Composition
Determination of the sugar composition of the polysaccharides involves
the identification and quantification of sugar constituents. De-polymerization
of the polysaccharide is a prerequisite, for which various methods have been
developed and complete acid hydrolysis is the most common and widely used
one. Aldose containing polysaccharides can be completely hydrolyzed with
minimum loss of constituent sugars either by 0.5 or 1.0 molar sulfuric acid at
100C for about 6 h (Selvendran et al., 1979) or by 1.0 molar trifluoro acetic
acid at 120C for 1 h (Albersheim et al., 1967). However, ketose containing
sugars are very unstable under these conditions and thus mild acid hydrolysis
either by 0.1 molar oxalic acid at 70C for 1 h (Aspinall et al., 1953) or by
0.05 molar sulfuric acid at 80C for 1 h (Codington et al., 1976) is followed
for their de-polymerization. Incomplete hydrolysis takes place when a
polysaccharide contains either amino sugars or uronic acid residues. Amino
sugar containing polysaccharides require stronger acid and they can be
completely hydrolyzed by 4 molar HCl at 100C for about 6 h (Spiro, 1972).
Uronic acid containing polysaccharides undergo decomposition (liberate
carbon dioxide) upon acid hydrolysis by 12% HCl (Whyte and Englar, 1974).
This can be circumvented by reducing the carboxyl group with water soluble
carbodiimide/sodium borohydride mixture followed by acid hydrolysis.
Determining the difference in the sugar composition before and after carboxyl
reduction gives the amount of uronyl residues (Lindberg et al., 1972).
Monosaccharides released upon acid hydrolysis can easily be identified
and quantified either by HPLC of GLC method. HPLC method is non-
destructive, requires no derivatization and sample can be recovered after the
analysis. Separation of individual sugar residues is based either on
cation/anion exchange (water) or on partition (acetonitrile: water)
chromatography. Detection of sugars is done using refractive index (RI)
detector (McGinnis and Fang, 1980). However, low sensitivity of RI detector
requires large amount of sample (in micrograms) for analysis.
GLC is a destructive method and requires sample derivatization. However,
it is much more sensitive than HPLC and requires very less amount of sample
(in nanograms) for analysis. Trimethylsilyl (TMS) ethers, trifluoroacetyl
(TFA) esters and alditol acetates are the most commonly prepared derivatives.
Constituent monosaccharides are reduced with sodium boro-hydride or
8 Rakesh Sharma, Bharti D Shrinivas

deuteride to obtain acyclic form and then acetylated using either acetic
anhydride and pyridine (1:1) (Sawardekar et al., 1965) or acetylated
aldonitrilation (hydroxylamine/pyridine and acetic anhydride, PAAN
derivatives) (Dmitriev et al., 1971). Unlike TMS derivatization, acetylation
eliminates the formation of multiple derivatives when different rings are
formed (pyranose or furanose) or different anomeric forms are generated from
reducing sugars or from equilibrium mixture of methyl glycosides. The acyclic
derivatives can be identified by their retention time and if necessary by mass.
The enantiomeric forms of the constituent sugars can not be distinguised
by the above mentioned methods. Although majority of the sugars are in D
form, sugars such as rhamnose (in pectins) and arabinose (in arabinoxylans)
are in L-form. These enantiomeric forms can be distinguished by converting
into equilibrium mixtures of glycosides of chiral alcohols (+/- 2-butanol or +/-
2-octanol) followed by GC analysis using capillary columns (Leontein et al.,
1978).

Linkage Analysis
Methylation (conversion of all the free hydroxyl groups into methoxyl
groups) is the most versatile and widely used technique for the determination
of linkages in polysaccharides. It gives information on linkage positions, ring
size (pyranose or furanose), non-reducing end groups, and kind and extent of
branching (Hirst and Percival, 1965). The method involves complete
etherification of free/un-substituted hydroxyl groups; i.e., those not involved in
ring formation, inter sugar glycosidic linkages, or carrying substitutions stable
at conditions used for methylation of the polysaccharides and for subsequent
hydrolysis of the methylated derivatives (Lindberg, 1972).
In particular, Hakomori methylation is the one which is most reliable and
extensively used method for the complete methylation of the polysaccharides
(Hakomori, 1964). In this method, polysaccharide is dispersed in dimethyl
sulfoxide (DMSO), treated with sodium methyl sulfinyl methanide (sodium
dimsyl) and then reacted with methyl iodide. Hakomori‟s method is very
effective compared to other methods wherein etherification may not be
achieved in a single step or complete alkoxide formation may not take place.
Haworth (dimethyl sulfate as alkylating agent and aqueous 30% sodium
hydroxide as base), Purdie (methyl iodide as both solvent and alkylating agent
and silver oxide as base) and Kuhn (N,N-dimethyl formamide/DMSO as
dipolar aprotic solvent, methyl iodide/dimethyl sulfate as alkylating agent and
silver/barium oxide as base) methylation are some other methods, which have
restricted use and can be employed when the sample is partially methylated.
Purification, Structure and Benefits… 9

The completeness of methylation can be ascertained either by methoxyl group


determination or by the absence of O-H stretching vibrations in the IR
spectrum.
The characterization of per-methylated polysaccharides requires
identification and quantification of all the sugar derivatives formed upon de-
polymerization and is performed by GLC-MS (Dutton, 1973). Per-methylated
polysaccharides are hydrolyzed and reduced to form acyclic derivatives. Since
per-methylated polysaccharides are less soluble in aqueous solvents, initial
partial hydrolysis is done with organic solvents such as formic acid and then
complete hydrolysis with dilute aqueous acids is performed. Acyclic
derivatives obtained after reduction are acetylated to obtain alditol acetates,
which is the most widely used derivatization method for the characterization
of per-methylated sugars. The mass spectra of per-methylated alditol acetates
are generally simple to interpret, with fragmentation patterns characteristic of
constituent sugars and their substitution pattern. However, methylation
analysis does not give information on stereo-chemical nature (/) of the
constituent sugars (Lonngren and Svensson, 1974).
Primary fragment ions from per-methylated alditol acetates arise by -
cleavage with preferred formation of (a) ions with lower molecular weight, (b)
ions from cleavage between two methoxyl bearing carbon atoms, (c) ions from
cleavage of a methoxyl bearing and an acetoxyl bearing carbon atom with
marked preference for the methoxyl bearing species to carry the positive
charge and (d) ions from very low abundance of cleavage between two
acetoxyl bearing carbon atoms. Primary fragment ions undergo a series of
subsequent elimination reactions to give secondary fragment ions which
include losses by (a) β-elimination of acetic acid (m/e 60) or methanol (m/e
32), (b) -elimination of acetic acid but not methanol and (c) via cyclic
transition states of formaldehyde, methoxy-methyl acetate or acetoxy-methyl
acetate (Jansson et al., 1976).

Oxidation

Characterization of the products obtained from oxidative cleavage of


polysaccharides can give details about the mode of linkage, substitution pattern
and configuration of sugar residues/linkages (/β). Two important methods of
oxidative cleavage are chromium trioxide and periodate oxidations.
10 Rakesh Sharma, Bharti D Shrinivas

Cro3 Oxidation
The configuration of glycosidic linkages in polysaccharides can be
determined by chromium trioxide oxidation, which is shown to preferentially
oxidize -linked polysaccharides over -linked ones. The difference is
attributed to the easy formation of a keto-ester by cleavage at the bridge
oxygen of β-anomeric compounds (Lindberg et al., 1975).

Periodate Oxidation
It is the widely used method for the determination of linkages in
polysaccharides. Glycol cleavage via oxidation by sodium metaperiodate gives
formic acid (usually from triol cleavage in pyranose) or formaldehyde (from
exocyclic diol, CHOH-CH2OH groups) and the oxidant is reduced to iodate. The
liberated products can be estimated by various methods such as titrimetry and
spectrophotometry for the oxidant reduced, acid-base titration for the formic
acid liberated or colorimetry for the formaldehyde formed (Hay et al., 1965).

Smith Degradation

The aldehydes liberated upon periodate oxidation, and the sugar residues in
the polysaccharide, which are resistance to oxidation are reduced with sodium
borohydride and hydrolyzed to obtain monosaccharides along with residual
stubs of oxidized units; either glycerol (from pentitol) or erythritol and threitol
(from 1→4 and 1→6 linked hexitols, respectively) (Goldstein et al., 1965).
Smith degradation products are identified and quantified by GLC-MS.

Oligosaccharide Analysis

Polysaccharides can be partially fragmented/de-polymerized and the


analysis of the oligosaccharides thus obtained can give information regarding
the distribution of side chains (random/uniform or non-random) in turn
providing complete structural information of the parent polysaccharides.
Oligosaccharides can be obtained by several chemical methods such as partial
acid hydrolysis, acetolysis, trifluoroacetolysis, mercaptolysis and
methanolysis. They can be fractionated/purified and characterized by
following similar methods employed for polysaccharides. In particular,
MALDI-TOF-MS and FAB-MS are useful for oligosaccharide
Purification, Structure and Benefits… 11

characterization, providing molecular mass and sequence of the constituent


sugar residues, respectively (York et al., 1990).

Enzymatic Method

Oligosaccharides can be obtained by fragmentation/de-polymerization of


polysaccharides with the use of specific polysaccharide degrading enzymes.
Enzymes cleave the polysaccharides with the varying degree of
polymerization. By characterizing the oligosaccharides released and also the
leftover polysaccharides, one can get information regarding the structure of
parent polysaccharides. Based on the mode of action, polysaccharide
degrading enzymes – glycosidases are classified into two groups: exo- and
endo-glycosidases. Exo-glycosidases act on polysaccharides and release
mono/disaccharide units from the non-reducing terminal, whereas endo-
glycosidases cleave the polysaccharides randomly (at the un-branched regions
of both main and side chains), resulting in the release of oligosaccharides with
varying degree of polymerization. Enzymatic method of obtaining
oligosaccharides has several advantages over chemical method, viz. (a) their
specificity, both to linkage type and substitution pattern, (b) lack of by-
products, (c) high reaction rates and (d) control over the reaction. Various cell
wall polysaccharides such as arabinoxylans (Hoffmann et al., 1992; Subba Rao
and Muralikrishna, 2004) and xyloglucans (Lerouxel et al., 2002) have been
characterized by analyzing the oligosaccharides obtained on enzymatic
method.

Spectroscopic Methods

Spectroscopic methods are much easier to perform compared to chemical


and enzymatic methods of oligo/polysaccharide analysis and they complement
the data obtained from other two methods. Some of the important
spectroscopic methods are: infra red (IR), mass spectrometry (MS), optical
rotatory dispersion (ORD), circular dichroism (CD) and X-ray diffraction and
nuclear magnetic resonance(NMR).

NMR Spectrometry
NMR spectrometry is the rapid and non-destructive method to study the
structure of polysaccharides, requiring no modification or degradation of the
12 Rakesh Sharma, Bharti D Shrinivas

sample. 13C and 1H NMR together can give the details on molecular
complexity and fine structure of the polysaccharides. 13C NMR can give
details about the composition, linkage and conformation of polysaccharides
(Jennings and Smith, 1978) and can also ascertain the purity of the
polysaccharide sample. However, it can not differentiate the enantiomeric
configuration of sugars. Various plant polysaccharides like arabinoxylans
(Hoffmann et al., 1991; Izydorczyk and Biliaderis, 1993; Subba Rao and
Muralikrishna, 2004), mixed glucans (Uzochukwu et al., 2002) and pectins
(Ryden et al., 1989) have been characterized using 13C NMR spectroscopy(
ShyamaPrasad Rao & Muralikrishna 2007).

IR Spectroscopy
Infrared waves are absorbed by the vibrating chemical bonds in the
polysaccharides giving characteristic IR spectra (vibrational) in the frequency
range of 4000 to 400 cm-1. IR spectroscopy can be used for the detection of
functional groups, configuration of sugar residues and to know the substitution
pattern. It is used to characterize arabinoxylans and their oligosaccharides
(Kacurakova et al., 1998).

Mass Spectrometry
Mass spectrometry is the indispensable technique in the characterization
of oligo/polysaccharides. In the conventional mass spectrometry,
polysaccharide sample can not be analyzed directly and hence it is separated
into small molecules/constituent sugar residues and derivatized with
acetylation/alkylation in order to make them volatile. Mass spectrometry is
based on the principle that ions of different mass: charge ratio (m/e) are
separated due to their differential diffraction in the combined electric and
magnetic fields. Chemical ionization and electron ionization are the two
important methods by which ionization can be achieved. In chemical
ionization, molecular ions remain intact and spectra obtained are simple to
interpret. On the other hand, electron ionization may result in complicated
spectra because ions entering the analyzer may get fragmented by the high
energy transferred from the bombarding electrons.
The advent of recent mass spectrometric techniques such as matrix
assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-
TOF-MS) and fast atom bombardment-mass spectrometry (FAB-MS) have
revolutionized the oligo/polysaccharide analysis. These techniques do not need
laborious sample derivatization steps, but provide valuable information on the
molecular mass and sequence of constituent residues. Many plant
Purification, Structure and Benefits… 13

oligosaccharides including arabinoxylans have been characterized using these


techniques (Jacobs et al., 2003; Lerouxel et al., 2002; Subba Rao and
Muralikrishna, 2004).

Cereal Polysaccharides

The major constituent (60 – 80%) of cereals is starch, a storage


polysaccharide, which is made up of two constituents: a linear  1→4 linked
amylose and branched  1→4 linked amylopectin. Apart from starch, cereals
also contain other polysaccharides known as non-starch polysaccharides,
which include cellulose, hemicelluloses, arabinoxylans, 1-3/1-4 -D-glucans,
glucomannans, pectins and arabinogalactans (Fincher, & Stone, 1986;
Izydorczyk, & Biliaderis, 1995). These non-starch polysaccharides mainly
occur in the cell walls, where they play both structural and growth-regulating
role and are divided into two types: „fibrillar‟ and „matrix‟ polysaccharides.
Cellulose, a β 1→4 linked polymer of glucose forms the micro-fibrils in the
cell wall. All other non-starch polysaccharides belong to the „matrix‟
polysaccharide group, are very heterogeneous in structure. They form
complexes with each other and with other cell-wall components such as
cellulose, proteins (extensins, rich in hydroxy proline residues), lignin
(polymer of cinnamyl alcohol) and other phenolic constituents.
Together, alkali extractable matrix polysaccharides have been termed
„hemicelluloses‟ as they were considered to be chemically and structurally
related to cellulose.
The rigidity and strength of the cell wall is related to the integrity of
cellulose/hemicellulose network. During cell growth, however, wall expansion
has been found to be dependent on the enzymatic modification of the
hemicellulosic component (Pauly et al., 2001).

Arabinoxylans/Feruloyl Arabinoxylans

In 1927, non-starchy, gummy polysaccharides were isolated from bread


wheat flours and shown to consist predominantly of pentoses, arabinose and
xylose (Freeman and Gortner, 1932; Hoffman and Gortner, 1927). Similar
polysaccharides were also found in durum wheat, rye and barley, and were
initially referred to as pentosans and later as arabinoxylans. Pentosans, in
general represent a heterogeneous group of polysaccharides which, in addition
14 Rakesh Sharma, Bharti D Shrinivas

to pentose sugars, may also contain hexoses, hexuronic acids and some
proteins, and hence current nomenclature is more structure descriptive,
identifying several polymeric components such as arabinoxylans or
arabinogalactan peptides, depending on the molecular constitution of the
polysaccharides.
Arabinoxylans have been identified in a variety of tissues of the main
cereals of commerce: wheat, rye, barley, oat, rice and sorghum (Fincher and
Stone, 1986) as well as in some other plants: rye grass (Hartley and Jones,
1976), pangola grass (Ford, 1989) and bamboo shoots (Ishii, 1991). Although
arabinoxylans are minor components (but some times up to 10% as in barley
grain) of entire cereal grains, they play important structural and functional role
in plant cell.
Arabinoxylan consists of a linear backbone of β-(1→4)-D-xylopyranosyl
residues, partly substituted with single -L-arabinofuranosyl residues at O-2,
and O-3, or at both O-2 and O-3 positions of the xylose residues (McNeil et
al., 1975; Vietor et al., 1994). The presence of arabinosyl substituents and their
distribution over the xylan backbone affect such arabinoxylan properties as
solubility and interaction with other polymeric cell wall components
(Andrewartha et al., 1979; McNeil et al., 1975) as well as restrict the enzymic
degradation by endoxylanase (Vietor et al., 1994). Some arabinose residues
are covalently linked through ester linkages to ferulic acid (Smith and Hartley,
1983). General structure of feruloyl arabinoxylan is depicted in the Figure 1.
In type II walls, which are present in grasses, arabinoxylans are the major
non-cellulosic polysaccharides in the primary walls. The arabinoxylans in type
II walls have abundant arabinosyl side chains. They are also substituted with
galactose and high amounts of glucuronic acid. In general, arabinoxylans were
divided into water extractable and water un-extractable, based on the
extractability, and this difference largely arises from their degree and pattern
of substitution, feruloylation and non-covalent interactions with other wall
components.
Ferulic acid, a hydroxycinnamic acid is the major bound phenolic acid in
cereals arabinoxylans, and is synthesized by phenylpropanoid pathway. It is
concentrated mainly in the aleurone layer (~ 75%) of the grain and comprises
about 0.5% in wheat and 0.14% in barley grains.
Ferulic acid is a strong antioxidant and known to protect cells from UV
radiation. Ferulic acid groups present in the arabinoxylans, on oxidative
coupling yield diferulates/dimers which link adjacent polymers together,
tightening the structure of the cell wall and thus restricting cell expansion.
Purification, Structure and Benefits… 15

Ferulic acid protects the polysaccharides against enzymic hydrolysis. It also


protects the plants from microbial/pathogen invasion.

Figure 1. General structure of feruloyl arabinoxylan.

Biosynthesis of Arabinoxylan
The cell wall component-polysaccharide biosynthesis takes place in
different sub-cellular compartments. Cellulose and callose are made at the
plasma membrane, whereas pectin and hemicelluloses (arabinoxylans) are
believed to be synthesized in the Golgi apparatus (Carpita and Gibeaut, 1993).
Xylans are common polysaccharides in plant cell walls, particularly in
secondary cell walls where they are deposited as the major non-cellulosic
polysaccharides. The xylans in type II walls, which are present in grasses and
some related plants, have abundant -L-arabinofuranosyl side chains attached
through (1→3) and (1→2) linkages apart from a small amount of glucuronosyl
and other side chains (Aspinall, 1980; McNeil et al., 1984). This type of xylan,
a heteropolysaccharide is known as arabinoxylan.
Heteropolysaccharide biosynthesis can be divided into four steps: chain or
backbone initiation, elongation, side chain addition, and termination and
extracellular deposition (Iiyama et al., 1993; Waldron and Brett, 1985). Our
understanding of these different steps in biosynthesis is still very incomplete.
The main enzymes responsible for heteropolysaccharide biosynthesis are
glycosyltransferases, but only very few genes for these have been identified,
and the enzymes responsible for synthesizing the backbone of xylans are only
partially characterized (Porchia et al., 2002). The backbone-synthesizing
enzymes may belong to the cellulose synthase-like proteins, but this
assumption may be false as it is now known that callose synthase does not
resemble cellulose synthase (Hong et al., 2001).
The biosynthesis of (1→4) linked β-xylosyl backbones in xylans is
catalyzed by β-1,4-xylosyltransferase. This enzyme has been investigated in
many plants, including wheat seedlings wherein the activity was characterized
16 Rakesh Sharma, Bharti D Shrinivas

from microsomal membranes (Porchia and Scheller, 2000). An UDP-D-


glucuronate decarboxylase (E.C. 4.1.1.35) was shown to catalyze the synthesis
of UDP-D-xylose from UDP-D-glucuronate in an essentially irreversible
reaction that is believed to commit glycosyl residues to heteroxylan synthesis
(Zhang et al., 2005).
The addition of side chains to xylans has been less investigated and little
is known about the way in which the different glycosyltransferases interact to
form the complete polysaccharide. A study of glucuronosyltransferase has
shown an interaction with xylosyltransferase (Baydoun et al., 1989). Although
arabinose is a common monosaccharide in plant polysaccharides and
glycoproteins, there are few reports on the arabinosyltransferases involved in
polysaccharide synthesis. Recently, Porchia et al. (2002) reported the presence
of arabinoxylan arabinosyltransferase (AX-AraT) in microsomal and Golgi
membranes isolated from wheat seedlings and showed that AX-AraT is
dependent on the synthesis of unsubstituted xylan acting as acceptor. They
have also demonstrated the formation of a single arabinosylated protein and its
possible role in arabinoxylan biosynthesis.

Biosynthesis of Ferulic Acid


Being a secondary metabolite, biosynthesis of ferulic acid is fairly well
understood. Ferulic acid, a hydroxy-cinnamic acid derivative, is synthesized in
plants via shikimate/phenylpropanoid pathway from phenylalanine or L-
tyrosine. Shikimate/arogenate pathway leads, through phenylalanine, to the
majority of plant phenolics, the phenylpropane (C6-C3) derivatives
(phenylpropanoids). p-Coumaric acid is formed as an intermediate in the
ferulic acid biosynthesis.

Feruloylation of Arabinoxylans
One of the characteristic features of arabinoxylans is their high content of
bound ferulic acid (and small amount of p-coumaric acid), chiefly ester linked
to -L-arabinofuranose usually at O-5 position. The feruloylation and p-
coumaroylation occur on highly specific hydroxyl groups of polysaccharides.
However, there is no complete agreement on to the site of feruloylation of wall
polysaccharides or the nature of the feruloyl donor. Fry and Miller (1989)
administered (3H) arabinose into spinach cultured cells and traced its
incorporation into arabinose units of the major wall polysaccharides. The
authors showed that arabinosylation and feruloylation occurred co-
synthetically and intracellularly. Similarly, Obel et al. (2003) showed the
Purification, Structure and Benefits… 17

intracellular feruloylation of arabinoxylans in wheat suspension-cultured cells.


On the other hand, Yamamoto et al. (1989) suggested that feruloylation site is
located within the matrix of barley coleoptile cell walls.
Meyer et al. (1991) showed that feruloyl-CoA is a donor for feruloylation.
A microsomal preparation from suspension cultured parsely (Petroselinum
crispum) cells was able to transfer ferulic acid from feruloyl-CoA to
uncharacterized endogenous wall polysaccharides. An alternative feruloyl
donor may be the glycosidic ester of ferulic acid (1-O-feruloyl--D-glucose).
Mock and Strack (1993) demonstrated that 1-O-sinapoyl--D-glucose is
formed by UDP-glucose: hydroxycinnamate D-glucosyltransferase (E.C.
2.4.1.120).

OXIDATIVE GELATION IN VIVO


Feruloyl arabinoxylans are known to undergo oxidative phenolic coupling
(dimerization) (Figure 2) reactions in walls; the coupling reactions themselves
in vivo would be remarkably specific. To permit a coupling reaction, feruloyl
groups on the same or different polysaccharide chains must be juxtaposed.
Matrix polysaccharides could be imagined in gelatinous form and they would
have enough mobility to place feruloyl residues in close proximity. But at
present there is no definite proof for this theory. Peroxidases are candidates for
the catalysis of the dehydrogenative dimerization of feruloyl residues in the
cell wall. The peroxidases not only generate free radical intermediates of ester-
linked feruloyl residues, but may also generate the hydrogen peroxide needed
to achieve this from various hydrogen donors. Several mechanisms have been
proposed for hydrogen donor generation. Ogawa et al. (1996) showed that one
of the physiological functions of the cytosolic CuZn-superoxide dismutase is
supplying hydrogen peroxide for lignification.
Obel et al. (2003) have observed the intracellular formation of ferulic acid
dimer, which is limited to 8,5‟-diferlulic acid, while other dimers appeared to
be formed extracellularly in wheat suspension-cultured cells. Similarly, Fry et
al. (2000) reported the intraprotoplasmic and wall-localized formation of
arabinoxylan-bound diferulates and larger ferulate coupling-products in maize
cell-suspension cultures. It is argued that feruloyl arabinoxylans that are cross-
linked before and after secretion are likely to loosen and tighten the cell wall,
respectively and have control on cell expansion.
18 Rakesh Sharma, Bharti D Shrinivas

Figure 2. Covalent diferulate cross-link between arabinoxylan molecules.

FUNCTIONS OF ARABINOXYLANS AND FERULOYL


ARABINOXYLANS IN VIVO
Feruloyl arabinoxylans (feraxans) are the major polysaccharides in the type
II walls, which are present in grasses. With the very complex and diverse
structure, arabinoxylans may have roles in the cross-linking of cellulose
microfibrils and may thereby regulate cell development, expansion and
strengthen the wall by mechanical resistance (Carpita, 1996). These
polysaccharides, by means of oxidative coupling, also become polymerized into
the lignin macromolecules. Such polymerizations decrease wall extensibility and
may ultimately be involved in the control of cell growth. They also limit
biodegradation/digestibility of polysaccharides, thus forming an effective barrier
Purification, Structure and Benefits… 19

against microbial invasion. Feruloyl oligosaccharides are known as signal


molecules between plants and microorganisms (Darvill et al., 1992).

Figure 3. Feraxan – feraxanases system. Each arrow represents a different enzyme:


xylanase (1), xylo-pyranosidase (2), arabino-furanosidase (3), galacto/gluco-
pyranosidases (4), glucuronidase (5), feruloyl esterase (6), p-coumaroyl esterase (7)
and O-acetyl esterase (8).

DEGRADATION OF ARABINOXYLANS AND FERULOYL


ARABINOXYLANS IN VIVO
Feruloyl arabinoxylans (feraxans) (Nishitani and Nevins, 1989) are highly
complex and diverse in structure and therefore require an array of hydrolytic
enzymes for their degradation (Figure 3). Collectively these enzymes are
referred to as feraxanases (Nishitani and Nevins, 1989). Xylanase and feruloyl
esterase are perhaps the key enzymes involved in the biodegradation of
feraxans and they need to act synergistically. Xylanase would break the long-
chain xylans into feruloyl-arabino-xylo-oligosaccharides, which in turn would
be easily accessed by feruloyl esterase for the de-esterification of ferulic acid.
On the other hand ferulic acid esterase may act upon feraxans to cleave the
feruloyl moieties, thus facilitating their degradation by xylanase.
Arabinofuranosidase, xylopyranosidase, glucuronidase, galactosidase and
acetyl esterase are some of the other enzymes in the feraxanase group which
are required for the complete biodegradation of feraxans.
Feraxan biodegradation is supposed to be a constant/continuous process in
the cellular maintenance. However, during seed germination/malting, their
biodegradation is hastened in the endosperm and aleurone cell wall by the
induced feraxanases/xylanolytic enzymes. There are some reports on the in
vivo biodegradation of feraxans during malting/germination of cereals such as
20 Rakesh Sharma, Bharti D Shrinivas

wheat, barley, rye and ragi (Autio et al., 2001; Obel et al., 2002; Rao and
Muralikrishna, 2004; Subba Rao and Muralikrishna, 2004).

Figure 4. One of the (partial) biodegradation pathways for ferulic acid leading to
vanillin via β-oxidation.( Source:- Gasson etal, 1998))

The ferulic acid degradation is not well understood, however, it may take
place by chain shortening via β-oxidation process (Figure 4) (Gasson et al.,
1998) directly analogous to the well known β-oxidation pathway of fatty acids.
Vanillin, a highly valued flavor compound, is the main degradation product of
ferulic acid.

Fine Structure of Arabinoxylans

Although arabinoxylans have been of interest to cereal chemists and


technologists for many years, structural studies initiated in 1951 by Perlin
were taken up only in the 1990s when a number of workers focused on the
detailed structural characteristics of these polysaccharides. General structure
of arabinoxylans is now well known. However, these polymers are highly
heterogeneous in chemical structure and molecular weight. They vary not only
Purification, Structure and Benefits… 21

from source to source, but also in different parts and fractionation and
purification methods employed. This prompts arabinoxylans to be studied
from different cereal sources both from structural and functional viewpoint.
In general, arabinoxylans from various cereals and/or other plants share
the same basic chemical structure. However, they differ in the manner of
substitution of the xylan backbone. The main differences are found in the ratio
of arabinose to xylose, in the relative proportions and sequence of the various
linkages between these two sugars, and in the presence of other substituents.
The ratio of Ara/Xyl in arabinoxylans from wheat endosperm may vary
from 0.50 to 0.71 (Rattan et al., 1994) but it is usually lower than that found in
bran (Shiiba et al., 1993) (figures 5A and 5B). Similarly rye endosperm
arabinoxylans are less substituted (0.48 – 0.55) (Bengtsson and Aman, 1990)
than their bran counterparts (0.78) (Ebringerova et al., 1990). In general rice
(Shibuya and Iwasaki, 1985) and sorghum (Vietor et al., 1994) seem to consist
of more highly branched xylan backbones than those from wheat, rye and
barley (figures 5E and 5F), and they may contain galactose and glucuronic
acid substituents, in addition to the pentose sugars.

Figure 5. Structural models for cereal arabinoxylans. Less branched endosperm/


insoluble (A) and more branched bran/soluble (B) arabinoxylans. Highly branched
22 Rakesh Sharma, Bharti D Shrinivas

(region A) (C) and less branched (region B) (D) arabinoxylans. Less branched wheat
(E) and more branched rice (F) arabinoxylans.

With a relatively low degree of branching, arabinoxylans from wheat, rye


and barley contain a rather high amount of un-substituted Xylp residues and a
relatively low amount of mono-substituted Xylp residues, compared to the
more highly branched arabinoxylans from rice and sorghum. The proportion of
doubly substituted residues seems not to be related to the arabinose to xylose
ratio and varies substantially among various arabinoxylans; highest amount
has been reported for wheat bran arabinoxylans. The presence of O-2 mono-
substituted xylose residues has been verified in all cereal arabinoxylans except
those of rye endosperm. This type of xylose substitution appears to be a
structural feature characteristic especially of barley arabinoxylans; a close to
one ratio of O-3 to O-2 mono-substituted Xylp residues suggest almost equal
distribution of both linkages in the polysaccharide (Vietor et al., 1992).
Cereal arabinoxylans exhibit a high degree of endogenous micro-
heterogeneity. It is, therefore, not possible to assign a single structure to
arabinoxylans. In order to get better insight into the structural characteristics of
individual homogeneous arabinoxylans, several investigators extensively
fractionated arabinoxylans using ethanol or ammonium sulphate graded
precipitation techniques (Gruppen et al., 1992a; Gruppen et al, 1992b;
Izydorczyk and Biliaderis, 1992; Vietor et al., 1992 SubbaRao& Muralikrishna
2004). Increased concentration of ethanol/ammonium sulphate resulted in
arabinoxylan fractions in continuously increasing Ara/Xyl ratios. The higher
degree of branching was also accomplished by variations in the relative
proportions of un-, mono- and di-substituted Xylp residues. Highly substituted
arabinoxylan fractions contained less un-substitued Xylp residues.
The distribution of arabinosyl substituents along the xylan backbone is
probably of greater importance than the degree of substitution itself, since it
affects the conformation (Andrewartha et al., 1979) and the capacity of
arabinoxylans to interact with each other and/or with other polysaccharides.
According to the early work by Perlin and co-workers (Ewald and Perlin,
1959; Goldschmid and Perlin, 1963), wheat endosperm arabinoxylans consist
branched regions where O-3 or O-2,3 substituted xylose residues are separated
by single un-substituted xylose residues. At lengths of approximately 20 – 25
xylose units, relatively smooth domains of at least two to five (and possibly
more) un-substituted xylosyl residues may be present.
Based on ammonium sulphate fractionation and oligosaccharide analysis
upon xylanase hydrolysis, wheat (endosperm) water-soluble arabinoxylans are
Purification, Structure and Benefits… 23

reported to have three structural domains. Region I is highly substituted (more


of O-2,3), and periodate oxidation/Smith degradation studies demonstrated
that substituted xylose residues are present either isolated, in pairs or even as
three contiguous residues, which may in large be limited by steric hindrance.
Region II is similarly substitution, but contains more of O-3 xylose residues.
Region III, which separates highly substituted domains, contains sequence of 2
– 6 or more un-substituted xylose residues. Different fractions differ in the
proportion/ratio of these regions.
Wheat alkali-extractable arabinoxylans differ in their fine structure from
water-soluble arabinoxylans and presumed to have two regions (A and B)
(Figure 5C and 5D). The highly branched region A composed mostly of
repeating tetrameric units of un- and di-substituted xylose residues. This
region also contains some O-2 substituted xylose residues. The less dense
region B, which alternates with region A, includes at least seven contiguous
un-substituted xylose residues.
The structure of arabinoxylans from barley endosperm (Vietor et al, 1992)
was shown to be more regular than that from wheat. The major region, mono-
(enriched with O-2) and di-substituted xylose residues are separated by un-
branched xylose residue, and the clusters are separated by regular un-branched
region of at least four xylose units.
Rye arabinoxylans have a different structure; the major polymer structure
(arabinoxylan I) has xylose chain substituted exclusively at O-3, and minor
polymer (arabinoxylan II) contains di-substituted O-2,3 xylose residues.
Rice and sorghum arabinoxylans are highly substituted and overall they
resemble branched regions of other cereal arabinoxylans. Structural
elucidation purified arabinoxylans isolated from finger millet and its malt by
methylation, GLC–MS, periodate oxidation, Smith degradation, NMR, IR,
optical rotation, and oligosaccharide analysis indicated that the backbone was a
1,4-β-d-xylan, with the majority of the residues substituted at C-3. The major
oligosaccharide generated by endo xylanase treatment was homogeneous with a
molecular weight of 1865 Da corresponding to 14 pentose residues as
determined by MALDI-TOF-MS and gel filtration on Biogel P-2. The structural
analysis of this oligosaccharide showed that it contained 8 xylose and 6
arabinose residues, substituted at C-3 (monosubstituted) and at both C-2 and C-3
(disubstituted SubbaRao & Muralikrishna 2004).
Water-soluble feruloyl arabinoxylans (feraxans), isolated from native and
malted (96 h) rice (Oryza sativa) and ragi (Eleusine coracana) grains, were
fractionated on DEAE-cellulose, followed by purification on Sephacryl S-300
and the homogeneity was ascertained by high performance size exclusion
24 Rakesh Sharma, Bharti D Shrinivas

chromatography, cellulose acetate and capillary electrophoresis. Structural


characterization of the purified polysaccharides by methylation, followed by
GLC–MS, and also by 1H NMR and 13C NMR spectroscopy, indicated very
high branching and presence of high amounts of O-2 substituted xylans.
The amount of O-2, 3 disubstituted xylopyranosyl residues and the
arabinose:xylose ratio was higher in malt feraxans. All feraxan samples
consumed almost equal amounts of Periodate (4.02–4.30 μmol/mg).
High mountf xylose (40%), as identified by Smith degradation, further
substantiated the high branching of feraxans. A model is presented depicting
the structure of water-soluble feraxans from rice and ragi and their changes
upon malting.( Shyama Prasad Rao& Muralikrishna 2007)

PHYSICOCHEMICAL/FUNCTIONAL ROLES OF
ARABINOXYLANS IN RELATION TO FOOD AND NUTRITION
In the past few decades, arabinoxylans have stimulated research interest
since they have been proven to have significant influence on the water balance
(Jelaca and Hlynka, 1971) and rheological properties of dough (Meuser and
Suckow, 1986; Michniewicz et al., 1991), retrogradation of starch (Biliaderis
and Izydorczyk, 1992; Gudmundsson et al., 1991) and bread quality (Delcour
et al., 1991; McCleary, 1986,SubbaRao etal 2004,Shyma prasad Rao etal
2007).
The chemical nature, including the subtle difference in the structure of the
polysaccharides, is important in knowing their exact functional roles. Further,
the multitude of free hydroxyl groups occurring in any polysaccharide allow
for an infinite amount of hydrogen bonding (intra and inter-bonding), which
again influence the physical behavior of the polysaccharides. The distribution
of arabinosyl substituents along the xylan backbone is known to affect the
conformation of arabinoxylans (Andrewartha et al., 1979) and the
intermolecular associations, which in turn have a direct bearing on certain
physical and functional properties of these macromolecules.
Cereal arabinoxylans widely vary in their molecular weight and different
methods of determination of molecular weight may give different values for
the same arabinoxylan population (Fincher and Stone, 1986). Very high
molecular weight of up to 5,000,000 has been reported for barley endosperm
arabinoxylans (MacGregor and Fincher, 1993). The conformation of
arabinoxylans, which can be determined by X-ray diffraction analysis, is
Purification, Structure and Benefits… 25

dependent on substitution patterns. Arabinoxylans are shown to have a 3-fold,


left handed helix and in the solid state they appear as an extended, twisted
ribbon when xylan backbone is un-substituted (Fincher and Stone, 1986). This
conformation is relatively flexible, supported by one H-bond between adjacent
xylose residues and forms aggregates into insoluble complexes, stabilized by
intermolecular H-bonding. Presence of arabinosyl substitution stiffens the
molecule by maintaining the xylan backbone more extended and thus prevents
its aggregation. Flexibility of xylan backbone is limited by the steric
hindrance/interaction of arabinose side groups (Yui et al., 1995).
As a result of their rather stiff conformation, arabinoxylans exhibit very
high viscosity in aqueous solutions, compared to the intrinsic viscosity of other
polysaccharides such as dextran and gum arabica (Fincher ad Stone, 1986). In
general, increased arabinose substitution was associated with increased
asymmetry of arabinoxylan molecules and thus with higher hydrodynamic
volume/viscosity. However, other factors such as xylan chain length, presence
of ferulic acid and specific arrangement of arabinose residues along the xylan
backbone influence this property.
In the presence of free radical-generating agents (e.g. hydrogen
peroxide/peroxidase, ammonium persulphate, ferric chloride, linoleic
acid/lipoxygenase), arabinoxylans are capable of forming three-dimensional
networks (gels or viscous solutions). This unique property, now known as
„oxidative gelation‟ of water extracts of wheat flour was first described by
Durham (1925). A number of factors such as molecular weight and
substitution of the arabinoxylans influence the gelling property. However,
presence of ferulic acid is prerequisite for the gelling ability of the
polysaccharide and numerous hypotheses concerning the mechanism of this
reaction have been developed. Detection of diferulic acid in oxidized
arabinoxylan systems indicates that cross linking occurs through the coupling
of two adjacent ferulic acid residues (Geissmann and Neukom, 1973).
Arabinoxylans are known to influence the quality of bakery products due
to their physicochemical properties like viscosity and water holding capacity
(Izydorczyk and Biliaderis, 1995). They absorb high amounts of water (6 – 8
times their weight) and when added to wheat flour, they compete with other
constituents of dough for water. Studies showed significant increase in the
farinograph water absorption, dough development time and loaf volume when
arabinoxylans are added to the bread dough (Biliaderis et al., 1995; Vanhamel
et al., 1993). However, at very high concentrations, due to the increase in
viscosity, arabinoxylan addition adversely affected the bread quality
(Biliaderis et al., 1995). Arabinoxylans are shown to protect protein foams
26 Rakesh Sharma, Bharti D Shrinivas

against thermal disruption and retain gas in the dough (Hoseney, 1984).
Viscosity of arabinoxylans adds to the strength and elasticity of gluten-starch
films surrounding the gas bubbles and slows down the rate of CO2 diffusion
from dough during baking, affecting firmness and homogeneity of crumb
texture. The presence of minor groups such as feruloyl and acetyl groups in
arabinoxylans do have an affect on their functional properties such as
viscosity,foam stabilization and gelling as revealed by a recent
study(Madhavilatha& Muralikrishna 2009)
Arabinoxylans are now considered to be prebiotics and used especially as
arabinoxylo-oligosaccharides in functional foods for actively managing the
colonic micro-flora with the aim of improving host health. A prebiotic is
defined as „a non-digestible food ingredient that beneficially affects the host
by selectively stimulating the growth and/or activity of one or a limited
number of bacteria in the colon, and thus improves host health‟ (Gibson and
Roberfroid, 1995 Chithra& Muralikrishna 2009)).
They are also emerging as natural antioxidants, immuno-modulators and
components of edible films (ShyamaPrasadRao& Muralikrishna 2006.).

Uses of Ferulic Acid

There have been studies showing potential health benefits of ferulic acid,
such as anti-carcinogenic and anti-inflammatory properties. Ferulic acid is a
strong UV absorber and constitutes the active ingredient in many skin lotions
and sunscreens. It is also part of the gel matrix of wound healing, in a
chemical form similar to the diferulated cross-links between arabinoxylan
polymers in the cell walls. In the food industry, it is extracted from agro-
industrial waste and bio-converted using fungi to vanillin, a much valued
flavor compound. Its ability to inhibit peroxidation of fatty acids finds as
natural food preservative and antioxidant.

Future Perspectives
Dietary fibre is a combination of several polysaccharides varying in their
(a) Composition (b) molecularweight (c) charge(uronicacid) (d) acetyl/feruloyl
groups (e)associated proteins and lipids ( varying with respect to covalent,
hydrogen,hydrophobic and vanderwaal inter actions). Eventhough many
studies have been carried out with respect to their purification,structural
characterization and their combined effect on alleviate disease symptoms
pertaining to diabetus,atherosclerosis and colon cancer, however the same is
not true with respect to individual purified polysaccharides .Various cereal
Purification, Structure and Benefits… 27

brans and pulse husks which are rich in dietary fibre components are the ideal
sources to do in depth work and also to address the various molecular
mechanisms by which the dietary fibre components alleviate disease
symptoms of various diseases .
Dietary fibres and their resultant degradation products such as
oligosacchrides can modulate the beneficial bacterial populations present in
the colon to exert health benefits to the host and they are also responsible to
decrease the growth of harmful bacteria such as Clostridium Perfringens.
Overall the dietary fibre research did not see the end of the tunnel wherein one
can conclusively say that the destination is reached and there is no more path
to traverse. Future research holds lot of promise and many unanswered and
intriguing questions will be addressed with respect to the role of dietary fibre
components and oligosaccharides with and without ferulic acid in health and
disease.

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Food Chem. 50, 6182-6187.
Adom, K. K., Sorrells, M. E. & Liu, R. H. (2003). Phytochemical profiles and
antioxidant activity of wheat varieties. J. Agric. Food Chem. 51, 7825-
7834.
Aman, P. & Bengtsson, S. (1991). Periodate oxidation and degradation studies
on the major water-soluble arabinoxylan in rye grain. Carbohydr. Polym.
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Foods, Diets and Disease
©
Editor: Rakesh Sharma, Bharati D Shrinivas 2009 Innovations And Solutions, Inc.
___________________________________________________________________________

Lecture 4

RHINACANTHUS NASUTUS:
ANTIMUTAGEN PROPERTIES

Rakesh Sharma, Bharti D Shrinivas

ABSTRACT
Rhinacanthus nasutus (Hattkaku-Reishi-Soh, Thong-Pun-Chang) are
widely cultivated in tropical and subtropical regions of South Asia just
like Japanese tea trees and have been used in treatments and preventions
of diverse diseases as a folklore medicines. The phytochemicals of
Rhinacanthus nasutus have been noticed for their healthy effects.
Recently, their phytochemicals have been isolated from various parts such
as leaves, stems, roots and total plants and also their effects Rhinacanthus
nasutus found by researchers. The purpose of this review is to represent
their components and their actions known by now.

Keywords: Rhinacanthus nasutus (Hattkaku-Reishi-Soh, Thong-Pun-Chang),


benzoquinone, naphthoquinones, anthraquinones, anti-mutagenicity

ABBREVIATIONS USED
lawsone (1)
lawson methyl ether (2)
potassium lawson methyl ether (2’)
2 Rakesh Sharma, Bharati D Shrinivas

clotrimazole (3)
chlorhexidine (4)
4-acetonyl-3,5-dimethoxy-p-quinol (5)
β-amyrin (6)
glutinol (7)
lupeol (8)
stigmasterol (9)
sitosterol (β-sitosterol, 10)
2-methoxy-4-propionyl-phenol (11)
umbelliferone (12)
2-methylanthraquinone (13)
2,6-dimethoxybenzoquinone (14)
3,4-Dihydro-3,3-dimethyl-2H-naphtho[2,3-b]pyran-5,10-dione(15)
rhinacanthin-A (16)
rhinacanthin-B (17)
psychorubrin (3-hydroxy-1H-3,4-dihydronaphtho[2,3-c]pyran-5,10-dione, 18)
rhinacanthin C (19)
rhinacanthin D (20)
rhinacanthin-O (21)
rhinacanthin-P (22)
rhinacanthin-G (23)
rhinacanthin-H (24)
rhinacanthin-I (25)
rhinacanthin-J (26)
rhinacanthin-K (27)
rhinacanthin-L (28)
rhinacanthin-M (29)
rhinacanthin-N (30)
rhinacanthin Q (31)
wogonin (32)
adriamycin (33)
oroxylin (34)
(+)-praeruptorin (35)
allantoin (36)
aflatoxin B1 (37)
furylfuramid (38)
ganciclovir (39)
amantadine (40)
rhinacanthin-E (41)
rhinacanthin-F (42)
quercetin (43)
Rhinacanthus Nasutus 3

1. INTRODUCTION
Rhinacanthus nasutus (L.) Kurz of Hattkaku-Reishi-Soh (Japanese name),
Thong-Pun-Chang (China name) and Thong Phan Chang (Thai name) belongs
to Acanthaceae, and is native in tropical and subtropical regions in South-East
Asia such as Taiwan, Southern China, Thailand and India. Rhinacanthus
nasutus (L.) Kurz or Justicia nasutus L. is mainly a shrub around 1-2 m just
like Japanese tea trees (Photo 1).

Photograph 1. Whole plant of Rhinacanthus nasutus.


4 Rakesh Sharma, Bharati D Shrinivas

In these regions, Rhinacanthus nasutus (L.) Kurz has been widely used in
their treatment of hepatitis, diabetes, hypertension, herpes virus infections,
cancer, skin diseases and others since old times [Gotoh et al., 2004; Sendl et
al., 1996; Wu et al., 1998a].
Acanthus mollis L.. of the same family could be found in the wide tropical
regions from Assam or Punjab of India to Sri Lanka (Ceylon) or Singapore.
Surprisingly, Acanthus mollis L.. could be found as an ornamental plant even
in outer cultivations without green house in Tokyo of temperate zone such
Japan (Photo 2).

Photograph 2. Whole plant of Acanthus mollis L.. belonging to Acanthaceae.


Photographed by Noboru Motohashi, 5/20/2009 Wed. at Itabashi Botanical Gardens,
Itabashi-ku, Tokyo.
Rhinacanthus Nasutus 5

Generally, their diverse phytochemicals of functional components could


be widely found in kingdom. Among these phytochemicals, their oxidative
phytochemicals such as carotenoids, flavonoids and anthocyanins could
especially be found highly abundant in human daily fruits and vegetables
[Motohashi, 2008a; Motohashi, 2009b].
Interestingly, Rhinacanthus nasutus (L.) Kurz contains more highly
abundant their quinoid structure molecules such as benzoquinones,
naphthoquinones and anthraquinones when compared to other edible plants
such as fruits and vegetables. Then, the diverse phytochemicals of
Rhinacanthus nasutus (L.) Kurz has been isolated by many researchers with
their great interests. Therefore, the purpose of this review is to reveal their
diverse phytochemicals containing the phytoquinones and healthy effects on
Rhinacanthus nasutus (L.) Kurz.

2. PHYTOCHEMICALS AND HEALTH EFFECTS OF


RHINACANTHUS NASUTUS
Rhinacanthus nasutus has been used as the folkrore medicine in Taiwan
and often administrated in their remedies of mainly hepatitis, diabetes,
hypertension and skin disease.
It has been known that the components of Rhinacanthus nasutus are
mainly generally flavonoids,naphthoquinones, and anthraquinones [Tian-
shung et al., 1995a].

1. Phytochemicals in Leaves of Rhinacanthus Nasutus

(1) Naphthoquinones
In 2000, group of Pharmaceutical Sciences, Prince of Songkla University
in Thailand isolated lawson methyl ether (2) of a derivative of lawsone (1)
from leaves of Rhinacanthus nasutus. In Thailand, Rhinacanthus nasutus has
been used a mouthrinse.
Lawson methyl ether (2) had their antifungal activity with a minimum
inhibitory concentration (MIC) of 512 μg/mL, and also had their antibacterial
activity for Staphylicoccus aureus.
Lawson methyl ether (2) had the very low acute toxicity with 50% lethal
dose (LD50) 70.7 mg/kg by intraperitoneal (ip) administration in mice. It
appeared that an oral administration of 0.5% lawson methyl ether (2) in
6 Rakesh Sharma, Bharati D Shrinivas

sodium carboxymethyl cellulose (CMC) oral base might be stable under a


heating-cooling cycle test.
In antifungal activity against Candida albicans in in vitro test, lawson
methyl ether (2) represented the similar activity when compared to 1.0%
clotrimazole (3) cream [Panichayupakaranant et al., 2000a](Figure 1).
In 2000, the same research group of Pharmaceutical Sciences, Prince of
Songkla University in Thailand studied their chemical stability and skin
irritation to skins on lawson methyl ether (2) in sodium carboxymethyl
cellulose (SCMC) oral base. A mouthrinse lawson methyl ether (2) in SCMC
represented the low acute toxicity with 50% lethal dose (LD50) 70.7 mg/kg by
intraperitoneal (ip) administration in mice.
A mouthrinse of 0.5% lawson methyl ether (2) in sodium carboxymethyl
cellulose (CMC) oral base was stable under a heating-cooling cycle test.
An oral administration of lawson methyl ether (2) in sodium
carboxymethyl cellulose (CMC) oral base did not cause any skin irritation
under both their primary skin irritation test (patch test) and cumulative skin
irritation test.
The solution of potassium lawson methyl ether (2’) produced the erythema
with some papulosquamous in cumulative skin irritation test [Blignaut et al.,
2006; Panichayupakaranant et al., 2002b](Figure 1).
In 2006, a dentistry group of Prince of Songkla University in Thailand
studied the effect of a mouthrinse potassium lawson methyl ether (2’) against
Candida albicans isolated from 51 human immunodeficiency virus/acquired
immunodeficiency syndrome (HIV/AIDS) subjects. Their antifugal activity of
0.5% potassium lawson methyl ether (2’) was compared to the activity of
0.12% and 0.2% chlorhexidine (4), which an antibacterial, effective against a
wide variety of Gram-negative and Gram-positive organisms (Figure 2).

Figure 1. Lawson methyl ether (2) of napthoquinoes in the leaves of Rhinacanthus


nasutus (L.) Kurz] and lawsone (1) of its related napthoquinones, and a mouthrinse
clotrimazole (3)
Rhinacanthus Nasutus 7

Figure 2. Lawson methyl ether (2) of napthoquinoes in the leaves of Rhinacanthus


nasutus (L.) Kurz] and lawsone (1) of its related napthoquinones, and a mouthwash
clorhexidine (4)

Then, The positive culture of Candida albicans was observed in 13 out of


51 plates (25.4%) on 0.12% chlorhexidine (4) mouthwash, in 5 out of 51 plates
(9.8%) on 0.2% chlorhexidine (4) mouthwash and in 4 out of 51 plates (7.8%)
on 0.5% potassium lawson methyl ether (2’).
The mean number of Candida albicans colonies for three mouthwashes of
0.12% chlorhexidine (4), 0.2% chlorhexidine (4) and 0.5% potassium lawson
methyl ether (2’) were 3.08 (range 0-40), 0.35 (range 0-13), and 0.84 (range 0-
24), respectively. Their antifungal activity was statistically found to be
significant different between 0.5% potassium lawson methyl ether (2’) and
0.12% chlorhexidine (4), and between the 0.12% chlorhexidine (4) and 0.2%
chlorhexidine (4). However, there is no difference between antifungal activity
of 0.5% potassium lawson methyl ether (2’) and 0.2% chlorhexidine (4)
[Blignaut et al., 2006; Motohashi, 2009c; Prasirst et al., 2006].
8 Rakesh Sharma, Bharati D Shrinivas

2. Phytochemicals in Leaves and Stems of Rhinacanthus Nasutus

(1) Quinols
In 1995, a group of National Cheng Kung University, Taipei of Taiwan,
isolated a novel 4-acetonyl-3,5-dimethoxy-p-quinol (5) from leaves and stems
of Rhinacanthus nasutus [Tian-shung et al., 1995a](Figure 3).

(2) Triterpenoids
In 1995, a group of National Cheng Kung University, Taipei of Taiwan,
isolated three known triterpenoide ofβ-amyrin (6), glutinol (7) and lupeol (8)
from leaves and stems of Rhinacanthus nasutus [Choudhary et al., 2005; Tian-
shung et al., 1995a](Figure 4).

Figure 3. A novel 4-acetonyl-3,5-dimethoxy-p-quinol (5) in the leaves and stems of


Rhinacanthus nasutus (L.) Kurz]

Figure 4. Three triterpenods of β –amyrin (6), glutinol (7) and lupeol (8) in the leaves
and stems of Rhinacanthus nasutus (L.) Kurz]
Rhinacanthus Nasutus 9

(3) Steroids
In 1995, a group of National Cheng Kung University, Taipei of Taiwan,
isolated two known steroids of stigmasterol (9), sitosterol (β- sitosterol, 10)
and their two derivatives from leaves and stems of Rhinacanthus nasutus
[Tian-shung et al., 1995a](Figure 5).

(4) Benzenoids
In 1995, a group of National Cheng Kung University, Taipei of Taiwan,
isolated 2-methoxy-4-propionyl-phenol (11) of benzenoids from leaves and
stems of Rhinacanthus nasutus [Tian-shung et al., 1995a](Figure 6).

Figure 5. Two steroids of stigmasterol (9) and sitosterol (β-sitosterol, 10) in the leaves
and stems of Rhinacanthus nasutus (L.) Kurz]

Figure 6. A benzonoid of 2-methoxy-4-propionyl-phenol (11) in the leaves and stems


of Rhinacanthus nasutus (L.) Kurz
10 Rakesh Sharma, Bharati D Shrinivas

(5) Coumarins
In 1995, a group of National Cheng Kung University, Taipei of Taiwan,
isolated umbelliferone (12) of coumarins from leaves and stems of
Rhinacanthus nasutus [Tian-shung et al., 1995a](Figure 7).

(6) Anthraquinones
In 1995, a group of National Cheng Kung University, Taipei of Taiwan,
isolated 2-methylanthraquinone (13) of anthraquinones from leaves and stems
of Rhinacanthus nasutus [Tian-shung et al., 1995a](Figure 8).

(7) Benzoquinones
In 1995, a group of National Cheng Kung University, Taipei of Taiwan,
isolated 2,6-dimethoxybenzoquinone (14)of benzoquinones from leaves and
stems of Rhinacanthus nasutus [Tian-shung et al., 1995a](Figure 9).

Figure 7. Umbelliferone (12) of coumarins in the leaves and stems of Rhinacanthus


nasutus (L.) Kurz

Figure 8. An anthraquinoe of 2-methylanthraquinone (13) in the leaves and stems of


Rhinacanthus nasutus (L.) Kurz
Rhinacanthus Nasutus 11

Figure 9. A benzoquinoe of 2,6-dimethoxybenzoquinone (14) in the leaves and stems


of Rhinacanthus nasutus (L.) Kurz

(8)Glycosides
In 1995, a group of National Cheng Kung University in Taipei of Taiwan
isolated four glycosides from leaves and stems of Rhinacanthus nasutus [Tian-
shung et al., 1995a].

Sitosterol-β-D-glucopyranoside
Stigmasterol-β-D-glucopyranoside
3,4-Dimethoxyphenol-β-D-glucopyranoside
3,4,5-Trimethoxyphenol-β-D-glucopyranoside

(9) Carbohydrates
In 1995, a group of National Cheng Kung University in Taipei of Taiwan
isolated methyl-α-D-galactopyranoside from leaves and stems of Rhinacanthus
nasutus [Tian-shung et al., 1995a].

(10) Chlorophyll
In 1995, a group of National Cheng Kung University, Taipei of Taiwan,
isolated methyl phenophorbide-a of chlorophyll from leaves and stems of
Rhinacanthus nasutus [Tian-shung et al., 1995a].

(11) Naphthopyrans
In 1993, a collaboration group with Kasetsart University, Bangkok of
Thailand, isolated 3,4-dihydro-3,3-dimethyl-2H-naphtho[2,3-b]pyran-5,10-
dione(15)of naphthopyrans from leaves and stems of Rhinacanthus nasutus
(Figure 10).
12 Rakesh Sharma, Bharati D Shrinivas

Figure 10. 3,4-Dihydro-3, 3-dimenthyl-2 H-naptho[2, 3-b] pyran-5,10-dione (15) of


napthopyrans in the stems and leaves of Rhinacanthus nasutus (L.) Kurz

The antifungal activity on 3,4-dihydro-3,3-dimethyl-2H-naphtho [2,3-b]


pyran-5,10-dione(15)was determined by observation of their inhibition
effect for spore germination of Pyricularia oryzae. The 50% effective dose
(ED50) value of 3,4-dihydro-3,3-dimethyl-2H-naphtho[2,3-b]pyran-5,10-
dione(15) was 0.4 ppm. The inhibitory value of 3,4-dihydro-3,3-dimethyl-
2H-naphtho[2,3-b]pyran-5,10-dione(15)for rice blast diseases was 82.3% at
100 ppm. It is expected that 3,4-dihydro-3,3-dimethyl-2H-naphtho[2,3-
b]pyran-5,10-dione(15)will be antifungal agents for human, together with
the regions of agrichemicals [Kodama et al., 1993].

(12) Antitumor activity on extracts of leaves and stems of Rhinacanthus


nasutus
In 2004, a collaboration group of medicine of Chiang Mai University of
Thailand and Unversity of Leeds of UK extracted their leaves and stems of
Rhinacanthus nasutus. Generally, it is known that Nitric oxide (NO) produced
from the activated macrophages play a role in both inflammatory processes
and anti-inflammatory processes. Then, it is studied that whether the extracts
from Rhinacanthus nasutus might modulate the production of an oxygen
radical NO and tumor necrosis factor- (TNF- ) using J774.2 mouse
macrophages. Also, it is examined that whether the extracts from
Rhinacanthus nasutus could represent their expression of inducible nitric
oxide synthase (iNOS) which were not controlled by calcium and their
expression of tumor necrosis factor- (TNF- ) genes.
Rhinacanthus Nasutus 13

When the ethanol extracts from Rhinacanthus nasutus were used in


combination with a macrophage activator lipopolysaccharide (LPS) which
enhance their enzyme activity, their productions of the oxygen radical NO was
significantly increased. The secretion of tumor necrosis factor- (TNF- ) was
parallel with their productions of the oxygen radical NO. The increases of
secretion of tumor necrosis factor- (TNF- ) were also associated with their
elevation of tumor necrosis factor- messenger RNA (TNF-α mRNA). These
results mean that their extracts from Rhinacanthus nasutus could either
increase or decrease their productions of the oxygen radical NO by the
increase of macrophages. It suggests that these effects might be mainly
mediated through the effect by the expression of tumor necrosis factor-
(TNF- ) [Punturee et al., 2004a].

3. Phytochemicals in Roots of Rhinacanthus Nasutus

In 2006, a group of Tropical Medicine, Mahidol University, Bangkok in


Thailand, tried methanol extracts from roots of Rhinacanthus nasutus and
examined their antivirus effects on two formulations of tablet with the extract
at 5% and 10% concentration. Their mosquito larvicidal activity of root
extracts from Rhinacanthus nasutus against Ades aegypti which mediate the
yellow fever virus with 5-10% lethal percentage and the chikungunya virus
with severe arthralgia, or against Culex quinquefasciatus (Anopheles spp.) of
mosquito which mediate the yellow fiber virus, dengue virus, chikungunya
virus was studied.
First, their 50% lethal concentration (LC 50) values, mosquito larvicidal
activity against Ades aegypti by two Rhinacanthus nasutus root methanol
extracts at 5% and 10% concentration in water, were 13.6 mg/L and 14.2
mg/L, respectively, and these are not significant activity.
While their 50% lethal concentration (LC 50) values, mosquito larvicidal
activity against Culex quinquefasciatus by two Rhinacanthus nasutus root
methanol extracts at 5% and 10% concentration in water, were 18.7 mg/L and
17.3 mg/L, respectively, and similarly in the Ades aegypti, these also are not
significant activity. There is no any observations on their larval mortality in
the two control groups such as lactone solution and dechlorinated water.
Second, their toxicity of two Rhinacanthus nasutus root methanol extracts
at 5% and 10% concentration was tested by Poecilia reticulata of female and
male guppies belong to Poeciliidae (Family), Poecilia (Genus).
14 Rakesh Sharma, Bharati D Shrinivas

Then, their 50% lethal concentration (LC50) values after 48 against


female guppy by two Rhinacanthus nasutus root methanol extracts at 5% and
10% concentration in water, were 105.2 mg/L and 110.8 mg/L, respectively.
Following, their 50% lethal concentration (LC50) values after 48 against
male guppy by two Rhinacanthus nasutus root methanol extracts at 5% and
10% concentration in water, were 99.1 mg/L and 103.4 mg/L, respectively.
Interestingly, these tests, the female and male guppies were almost
sensitive to the same dose against the Rhinacanthus nasutus root methanol
extracts. There is no any observations on their larval mortality in the two
control groups such as lactone solution and dechlorinated water.
In these two acute-toxiciy bioassays, 50% lethal concentration (LC50)
values after 48 hours exposures against guppies by the Rhinacanthus nasutus
root methanol extracts at 5% and 10% concentration in water were 5-fold to
10-fold higher than the LC50 of Rhinacanthus nasutus root methanol extracts
against mosquito larvae. Therefore, it suggested that Rhinacanthus nasutus
root methanol extracts might be safe for both human and fishes, and moreover,
these extracts might be effective for prevention against mosquitoes which
mediate the mosquito-related diseases such as yellow fiber [Rongsriyam et al.,
2006].

(1) Naphthoquinones
In 1988, a group of Applied Chemistry, Province College of Arts and
Sciences, Taichung Hsien of Taiwan, isolated two novel naphthoquinones of
rhinacanthin-A (16) and rhinacanthin-B (17) from the roots of Rhinacanthus
nasutus [Tian-Shung et al., 1988b](Figure 11).
In 1972, a group of NIH in USA represented that rhinacanthin-B (17) had
significantly 50% effective dose (ED50=3.0μg/mL against KB cells in tissue
culture assay. However, rhinacanthin-A (16) exhibited no cytotoxicity for KB
cells [Geran et al., 1972].
The significant difference of their cytotoxicity between rhinacanthin-A
(16) and rhinacanthin-B (17) also could be supported rhinacanthin-B (17) has
a lipophilic site in the molecule. Therefore, the lipophilic site of rhinacanthin-
B (17) might play an important role to contribution of the lipophilicity to their
enhanced cytotoxicity. Similarly, this could be also observed in the case of a
cytotoxic psychorubrin (3-hydroxy-1H-3,4-dihydronaphtho[2,3-c]pyran-5,10-
dione, 18) of naphthoquinones from Psychotria rubra [Hayashi et al.,
1987](Figure 11).
Rhinacanthus Nasutus 15

Figure 11. Two novel napthoqionones of rhinacanthin-A (16) and rhinacanthin-B (17)
from the roots of Rhinacanthus nasutus and psychorubrin (3-hydroxy-1H-3,4-
dihydronaptho[2, 3-c]pyran-5,10-dione, 18)
16 Rakesh Sharma, Bharati D Shrinivas

Figure 12. Rhinacanthin-A(16), rhinacanthin-B (17), rhinacanthin-C (19) and


rhinacanthin-D (20) of napthoquinones in the roots of Rhinacanthus nasutus (L.) Kurz
Rhinacanthus Nasutus 17

Figure 12. (Continued 1) Rhinacanthin-O (21), rhinacanthin-P (22) of two


dimethyldihydropyrano-1,4-naphthoquinones and rhinacanthin-G (23), rhinacanthin-H
(24), rhinacanthin-I (25), rhinacanthin-J (26), rhinacanthin-K (27), rhinacanthin-L (28),
rhinacanthin-M (29), rhinacanthin-N (30) of eight 2-hydroxy-1,4-naphthoquinones in
the roots of Rhinacanthus nasutus (L.) Kurz)
18 Rakesh Sharma, Bharati D Shrinivas

Figure 13. Psychorubrin (3-hydroxy-1 H-3,4-dihydronaptho [2,3-c] pyran-5, 10-dione,


18) of napthoquinones

In 1998, a group of National Cheng Kung University, Tainan in Taiwan,


isolated four known rhinacanthins such as rhinacanthin-A (16)], rhinacanthin-
B (17), rhinacanthin-C (19) and rhinacanthin-D (20), two novel
dimethyldihydropyrano-1,4-naphthoquinones such as rhinacanthin-O (21) and
rhinacanthin-P (22), and eight novel 2-hydroxy-1,4-naphthoquinones such as
rhinacanthin-G (23), rhinacanthin-H (24), rhinacanthin-I (25), rhinacanthin-J
(26), rhinacanthin-K (27), rhinacanthin-L (28) and rhinacanthin-M (29),
rhinacanthin-N (30) [Tian-Shung et al., 1988b; Wu et al., 1998a](Figure
12)(Figure 12(continued 1).
Interestingly, in 1987, a group of School of Pharmacy, University of North
Carolina, Chapel Hill, confirmed that psychorubrin(18)of a new
naphthoquinone from the alcoholic extract of Psychotria rubra, represented
the significant cytotoxicity for KB cells (ED50=3.0 g/mL). However, when a
hydrophilic hydroxy group was present in such compounds, their reduced in
vitro activity was significantly observed [Hayashi et al., 1987].
In 1998, a group of Chemistry, National Cheng Kung University, Tainan
in Taiwan, isolated a novel rhinacanthin-Q (31) of novel naphthoquinones and
ten known rhinacanthins such as rhinacanthin-A (16),rhinacanthin-B
(17),rhinacanthin-C (19), rhinacanthin-D (20),rhinacanthin-G (23),
rhinacanthin-H (24), rhinacanthin-I (25),rhinacanthin-K (27),rhinacanthin-
M (29) and rhinacanthin-N (30) [Wu et al., 1998b](Figure 14).
Rhinacanthus Nasutus 19

Figure 14. New rhinacanthin-Q (31) and 10 unknown napthoquinonesin the roots of
Rhinacanthus nasutus (L.) Kurz]

In 1998, the same group of Chemistry, National Cheng Kung University,


Tainan in Taiwan, studied their cytotoxic evaluation against five carcinogenic
cell strains such as KB cell of human oral epidermoid carcinoma, P388 murine
leukemia cells, A549 cells of carcinomic human alveolar basal epithelial cells,
HT-29 human intestinal cancer cells and human promyelocytic leukemia cells
(HL-60) on these eleven naphthoquinones (16, 17, 19, 20, 23, 24, 25, 27, 29,
30, 31). Among these five carcinogenic cell strains used in this evaluation, all
20 Rakesh Sharma, Bharati D Shrinivas

1,4 naphthoquinones (16, 17, 19, 20, 23, 24, 25, 27, 29, 30, 31) showed their
significant cytotoxicity against four carcinogenic cell strains of P388 murine
leukemia cells, A549 cells of carcinomic human alveolar basal epithelial cells,
HT-29 human intestinal cancer cells and human promyelocytic leukemia cells
(HL-60), excepting only KB cell of human oral epidermoid carcinoma.
Generally, it is known that when agglutinins such as collagen, and
thrombin were added to normal platelet, the direct agglutination is happened
and a platelet aggregation is observed with their decrease of turbidity.
It is also know that arachidonic acid of straight chain unsaturated fatty
acids bond with peroxisome proliferator-activated receptor (PPAR) in a
nuclear receptor of platelet and activate, and finally inhibit their cell
differentiation. Similarly, it is thought that arachidonic acid also might form
their direct agglutination of platelet.
From these facts, their antiplatelet aggregation activity was examined on
nine rhinacanthins of 1,4-naphthoquinones such as rhinacanthin-A (16),
rhinacanthin-B (17), rhinacanthin-C (19), rhinacanthin-G (23), rhinacanthin-H
(24), rhinacanthin-I (25), rhinacanthin-K (27), rhinacanthin-M (29),
rhinacanthin-Q (31) at each 100μg/mL concentration. These nine rhinacanthins
used in this study showed antiplatelet aggregation activity in wide ranges from
rhinacanthin-B (17, 7% inhibition) to rhinacanthin-A (16, 100%
inhibition),rhinacanthin-C (19, 100 inhibition) and rhinacanthin-M (29,
100% inhibition) against arachidonic acid (100 μM)-induced rabbit platelet
aggregation.
Similarly, their antiplatelet aggregation activity by rhinacanthins against
collagen (100 μM)-induced rabbit platelet aggregation were in wide ranges
from rhinacanthin-M (29, 5% inhibition) to rhinacanthin-A (16, 100%
inhibition),rhinacanthin-B (17, 100% inhibition) and rhinacanthin-M (29,
100% inhibition).
Surprisingly, rhinacanthin-B (17, 100% inhibition) only showed
antiplatelet-activating factor (anti-PAF) at 2 ng/mL concentration. Then, it is
thought that rhinacanthis such as rhinacanthin-B (17) might decompose
platelet-activating-factor (PAF) acetylhydrolase, which exists in plasma and
which happens allergy diseases such as allergic airway disease and
anaphylaxis, or inflammation [Wu et al., 1998b].
In 2004, a group of Kesetsart University, Bangkok in Thailand, examined
their antitumor activity against three cancer cell lines of KB human
epiderimoid carcinoma, HeLa human cervical carcinoma and HepG2 human
hepatocellular carcinoma and antivirus activity against Vero cell line with their
higher sensitivity as many human viruses among the African green monkey
Rhinacanthus Nasutus 21

kidney cells by three rhinacanthis such as rhinacanthin-M (29), rhinacanthin-N


(30)] and rhinacanthin-Q (31), using adriamycin (33) as control (Figure 15).

Figure 15. The cytotoxicity of rhinacanthin-M (29), rhinacanthin-N (30), rhinacanthin-


Q (31) in the roots of Rhinacanthin nasutus (L.) Kurz, and adriamycin (33) of a control
in biossay.
22 Rakesh Sharma, Bharati D Shrinivas

First, among three rhinacanthins (29, 30, 31), their 50% median inhibition
concentration (IC50) against KB human epiderimoid carcinoma was
rhinacanthin-M (29) (IC50=1.53 μM), rhinacanthin-N (30) (IC50<0.22 μM),
rhinacanthin-Q (31) (IC50=0.35 μM) and adriamycin (33)(IC50=0.033 μM),
respectively.
Second, among three rhinacanthins (29, 30, 31), their 50% median
inhibition concentration (IC50) against HeLa human cervical carcinoma was
rhinacanthin-M (29) (IC50=3.02 μM), rhinacanthin-N (30) (IC50<0.30 μM),
rhinacanthin-Q (31) (IC50=1.09 μM) and adriamycin (33) (IC50=0.33 μM),
respectively.
Third, among three rhinacanthins (29, 30, 31), their 50% median
inhibition concentration (IC50) against HepG2 human hepatocellular carcinoma
was rhinacanthin-M (29) (IC50=4.85 μM), rhinacanthin-N (30) (IC50=0.38
μM), rhinacanthin-Q (31) (IC50=0.97 μM) and adriamycin (33) (IC50=0.40
μM), respectively.
Finally, among three rhinacanthins (29, 30, 31), their 50% median
inhibition concentration (IC50) against Vero cell line was rhinacanthin-M (29)
(IC50=48.64 μM), rhinacanthin-N (30) (IC50=12.65 μM), rhinacanthin-Q (31)
(IC50=32.70 μM) and adriamycin (33) (IC50=23.94 μM), respectively.
From these four cytotoxicity examinations, two rhinacanthins such as
rhinacanthin-N (30) (IC50=12.65 μM) and rhinacanthin-Q (31) (IC50=32.70
μM) could be expected as higher antitumor and antivirus agents by further
modifications of their rhinacanthins.
Additionally, the group synthesized their derivatives of three
rhinacanthins(29, 30, 31)and examined the similar biological tests
[Kongkathip et al., 2004].
In 2006, a collaboration group of National Cancer Institute, Bangkok in
Thailand, examined their induction of apoptosis against HeLaS3 human
cervical carcinoma cells by TUNEL method on three rhinacanthins such as
rhinacanthin-C (19), rhinacanthin-N (30) and rhinacanthin-Q (31) from the
roots of Rhinacanthus nasutus (L.) Kurz.
First, their increase of the carcinoma cells was dose-dependently inhibited
when these three rhinacanthins of rhinacanthin-C (19), rhinacanthin-N (30)
and rhinacanthin-Q (31) are added to HeLaS3 human cervical carcinoma cells.
At the same time, their productions of caspase-3 in HeLaS3 human cervical
carcinoma cells also increased dose-dependently. Among three rhinacanthins,
rhinacanthin-N (30) showed significantly antitumor activity
Second, the breakage of DNA fragmentation of HeLaS3 cells was
observed as an apparent morphological change by agarose gel electrophoresis.
Rhinacanthus Nasutus 23

From these facts, their inhibitions of the increase against HeLaS3 human
cervical carcinoma cells by three rhinacanthins of rhinacanthin-C (19),
rhinacanthin-N (30) and rhinacanthin-Q (31) were caused by apoptosis. It
seems that activation of caspase-3 might activation of caspase apperas to be
directly responsible for many of molecular and structural changes in apoptosis.
Then, it is thought that these rhinacanthins might induce the apoptosis against
HeLaS3 human cervical carcinoma cells by mediating caspase-3. Therefore, it
is expected that these rhinacanthins will be one of novel anticancer drugs in
the near future [Siripong et al., 2006a](Figure 16).

Figure 16. Rhinacanthin-C (19), rhinacanthin-N (30) and rhinacanthin-Q (31) in the
roots of Rhinacanthin nasutus (L.) Kurz]
24 Rakesh Sharma, Bharati D Shrinivas

Further, in 2006, a collaboration group of National Cancer Institute,


Bangkok in Thailand, examined their anticancer effect by liposome injection
of three rhinacanthins of rhinacanthin-C (19), rhinacanthin-N (30) and
rhinacanthin-Q (31) from the roots of Rhinacanthus nasutus (L.) Kurz. In
2006, the same research group have already confirmed the induction of
apoptosis against HeLaS3 human cervical carcinoma cells by three same
rhinacanthis [Siripong et al., 2006a].However, these antitumor avtivity of
three rhinancanthins is limited in only water medium. Therefore, their
liposomalization enabled the injection of three rhinacanthins and the
rhinacanthins could be expected to transfer to lipoproteins in the bloodstream.
First, the 50% lethal dose(LC50)against HeLaS3 human cervical
carcinoma cells) by their liposomal formulation of three rhinacanthins of
rhinacanthin-C (19), rhinacanthin-N (30) and rhinacanthin-Q (31) was 32 μM,
17 μM and 70 μM, after 24 hour injection, 19 μM, 17 μM and 52 μM after 48
hour injection, and 2.7 μM, 2.0 μM and 5.0 μM after 72 hour injection,
respectively.
Second, three rhinacanthins of rhinacanthin-C (19), rhinacanthin-N (30)
and rhinacanthin-Q (31) were intraperitoneally (i.p.) administrated the Meth-A
murine sarcoma ascites cells to male BALB/c mice. After 8-10 days, their
increased ascites cells were taken. These ascites cells were implanted
subcutaneously (s.c) to five week-old male BALB/c mice. At the same time,
5.0 mg/kg/day of three rhinacanthins of rhinacanthin-C (19), rhinacanthin-N
(30) and rhinacanthin-Q (31) were intraperitoneally (i.p.) administrated to five
week-old male BALB/c mice fro 10 days. Among three rhinacanthins,
rhinacanthin N (30) inhibited significantly the growth of the solid tumor.
From these facts, it suggests that rhinacanthin N (30) could inhibit
apparently the growth of tumors in in vitro examination. Therefore, the
liposome with the injectable formation of higher hydrophobic drugs might be
expected their higher effective anticancer drugs, just like a sample as the
liposome of hydrophobic rhinacanthin N (30)[Siripong et al., 2006b]
(Figure 16).

(2) Triterpenoids
In 1988, a collaboration group of Applied Chemistry of Province College
of Arts and Sciences, Taichung Hsien in Taiwan, isolated lupeol (8) of
triterpenoids from the roots of Rhinacanthus nasutus (L.) Kurz [Tian-Shung et
al., 1988b](Figure 17).
Rhinacanthus Nasutus 25

In 1998, the group of National Cheng Kung University, Tainan in Taiwan,


also isolated lupeol (8) from the roots of Rhinacanthus nasutus (L.) Kurz [Wu
et al., 1998a](Figure 17).

(3) Steroids
In 1988, a group of Applied Chemistry, Province College of Arts and
Sciences, Taichung in Taiwan, isolated two known steroids such as
stigmasterol (9) and sitosterol (β- sitosterol (10)), and also the glucosides of
two their steroid from the roots of Rhinacanthus nasutus (L.) Kurz [Tian-
Shung et al., 1988b](Figure 18).

Figure 17. Lupeol (4) of triterpenoids in the roots of Rhinacanthus nasutus (L.) Kurz

Figure 18. Two steroids of stigmasterol (9) and sitosterol (β -sitosterol, 10) in the roots
of Rhinacanthus nasutus (L.) Kurz
26 Rakesh Sharma, Bharati D Shrinivas

(4) Flavonoids
In 1998, a group of National Cheng Kung University, Tainan in Taiwan,
isolated two known antioxidative flavonoids such as wogonin (32) and
oroxylin (34) from the roots of Rhinacanthus nasutus (L.) Kurz [Shih et al.,
2009; Wu et al., 1998a](Figure 19).
In 1998, the same group of Chemistry, National Cheng Kung University,
Tainan in Taiwan, studied their cytotoxic evaluation against five carcinogenic
cell strains such as KB cell of human oral epidermoid carcinoma, P388 murine
leukemia cells, A549 cells of carcinomic human alveolar basal epithelial cells,
HT-29 human intestinal cancer cells and human promyelocytic leukemia cells
(HL-60) on an oxidative flavonoid wogonin (32).
Among these five carcinogenic cell strains used in this evaluation,
wogonin (32) showed their significant cytotoxicity against four carcinogenic
cell strains of P388 murine leukemia cells, A549 cells of carcinomic human
alveolar basal epithelial cells, HT-29 human intestinal cancer cells and human
promyelocytic leukemia cells (HL-60), excepting only KB cell of human oral
epidermoid carcinoma.
Generally, it is known that when agglutinins such as collagen, and
thrombin were added to normal platelet, the direct agglutination is happened
and a platelet aggregation is observed with their decrease of turbidity.
It is also know that arachidonic acid of straight chain unsaturated fatty
acids bond with peroxisome proliferator-activated receptor (PPAR) in a
nuclear receptor of platelet and activate, and finally inhibit their cell
differentiation. Similarly, it is thought that arachidonic acid also might form
their direct agglutination of platelet.
From these facts, their antiplatelet aggregation activity was examined on
wogonin (32) at 100μg/mL concentration. Wogonin (32) used in this study
showed antiplatelet aggregation activity (100% inhibition) against arachidonic
acid (100 μM)-induced rabbit platelet aggregation.
Similarly, their antiplatelet aggregation activity by Wogonin (32) against
collagen (100 μM)-induced rabbit platelet aggregation showed the 73%
inhibition [Wu et al., 1998b].

(5) Coumarins
In 1998, a group of Chemistry of National Cheng Kung University
isolated a (+)-praeruptorin (35) of coumarins from the roots of Rhinacanthus
nasutus [Wu et al., 1998a](Figure 20).
Rhinacanthus Nasutus 27

Figure 19. Wogonin (32) and oroxylin (34) of two flavonoids in the roots of
Rhinacanthus nasutus (L.) Kurz

Figure 20. (+)-praeruptorin (35) of coumarins from the root of Rhinacanthjus nasutus

(6) Allantoin
In 1998, a group of National Cheng Kung University, Tainan in Taiwan,
isolated allantoin (36) of the diureide of glyoxylic acid as a metabolite of
purines from the roots of Rhinacanthus nasutus (Figure 21).
28 Rakesh Sharma, Bharati D Shrinivas

Figure 21. Allantoin (36) of purine metabolite in the roots of Rhinacanthus nasutus
(L.) Kurz

Widely, allantoin (36) could find in many plants and also in allantoic fluid
and fetal urine. Interestingly, allantoin (36) could be found as a urinary
excretion product of purine metabolism in most mammals, however, allantoin
(36) could not find in man or the higher apes. Allantoin (36) also could be
chemically synthetized by the oxidation of uric acid. Allantoin (36) had once
used to encourage the epithelial formation in mounds and ulcers, and also in
osteomyelitis [Wu et al., 1998a].

(7) Antimutagenicity on extracts of the roots of Rhinacanthus nasutus


In 1990, a group of National Cancer Institute, Bangkok in Thailand,
examined their mutagenicity (carcinogenicity) by Ames test on the extracts
from the roots of Rhinacanthus nasutus (Honma et al., 1999).
First, the mutagenic potential on the extracts from the roots of
Rhinacanthus nasutus was tested. For the test of whether the extracts from the
roots of Rhinacanthus have their mutagenicity or have not, their mutagenicity
test was examined against two Salmonella typhimurium tester strains such as
TA98 and A100 together with their PCB-induced S-9 mix, which the
mutagenic polychlorinated biphenyls (PCBs) were added in a single lot of
post-mitochondrial supernatant fractions of rat liver homogenates (S-9 mix) of
a rat liver homogenate. By the test, the extracts from the roots of Rhinacanthus
nasutus showed no mutagenicity against two Salmonella typhimurium of
TA98 and A100.
Second, the antimutagenicity of the extracts from the roots of
Rhinacanthus nasutus was examined by Ames test.
Generally, it is known that the domestic fowl and other animals fed with
their infected peanut meal to aflatoxin B1 (AFB1, 37) might die by the cause
of afflatoxicosis, hepatocellular carcinoma, or cholangiocarcinoma, and has
Rhinacanthus Nasutus 29

also been implicated as a cause of human hepatic carrcinoma. Furylfuramid


(AF-2, 38) was also known to be their higher positive reverse mutation.
Therefore, two standard mutagens such as aflatoxin B1 (AFB1, 37) and
furylfuramid (AF-2, 38) were used on the antimutagenicity by the extracts
from the roots of Rhinacanthus nasutus (Figure 22). First, their mutagenicity
against Salmonella typhimurium tester strarin TA100 was examined by adding
two hexane and chloroform extracts from the roots of Rhinacanthus nasutus to
AFB1 (37) S-9 mix, or alone S-9 mix. Then, both of two hexane and
chloroform extracts from the roots of Rhinacanthus nasutus showed
significantly their antimutagenicity of Salmonella typhimurium strarin TA100
by aflatoxin B1 (AFB1, 37) of an indirect mutagen. However, methanol
extract from the roots of Rhinacanthus nasutus was not antimutagenic on
Salmonella typhimurium TA100 by aflatoxin B1 (AFB1, 37). Second, three
methanol, hexane and chloroform extracts from the roots of Rhinacanthus
nasutus were examined on their antimutagenicity of Salmonella typhimurium
TA100 in S-9 mix with furylfuramid (AF-2, 38) and alone S-9 mix. Then, all
three methanol, hexane and chloroform extracts from the roots of
Rhinacanthus nasutus were not antimutagenic on Salmonella typhimurium
TA100 against furylfuramid (AF-2, 38) of a direct mutagen. Additionally, the
methanol extract from the roots of Rhinacanthus nasutus was no
antimutagenic on Salmonella typhimurium TA100 against both two aflatoxin
B1 (AFB1, 37) and furylfuramid (AF-2, 38) [Rojanapo et al., 1990].
Third, their extracts from the roots of Rhinacanthus nasutus were
examined on the enzyme inhibition of rat liver aniline hydroxylase. Aniline
hydroxylase is one of oxygenases, which a drug-metabolizing, cytochrome P-
450 enzyme which catalyzes the hydroxylation of aniline to hydroxyaniline in
the presence of reduced flavoprotein and molecular oxygen. For example, aryl
4-hydroxylase catalizes aniline to aminophenol.

Figure 22. Two mutagens of aflatoxin B1 (AFB1, 37) and furylfuramid (AF-2, 38)
30 Rakesh Sharma, Bharati D Shrinivas

The enzyme inhibitory effects by their extract from the roots of


Rhinacanthus nasutus were measured by using the PCB-induced S-9 fractions
as the source of aniline hydroxylase. Among three extracts of methanol,
hexane and chloroform extracts, the chloroform extract showed the highest
inhibition effect against aniline hydroxylase, followed by hexane extract.
However, the methanol extract was no inhibitory against aniline hydroxylase.
It suggests that from these three facts of Ames test and their enzyme
inhibitory effects of rat liver aniline hydroxylase, their qualitative and
quantative inhibition of mutagenicity by their extract from the roots of
Rhinacanthus nasutus might be greatly corresponding to their enzyme
inhibition of the rat liver aniline hydroxylase [Rojanapo et al., 1990].

4. Phytochemicals in Leaves and Roots of Rhinacanthus Nasutus

(1) Naphthoquinones
In 2004, a collaboration group of Kobe University isolated rhinacanthin-C
(19) of naphthoquinones from the leaves and roots of Rhinacanthus nasutus
[Gotoh et al., 2004; Sendl et al., 1996](Figure 23).
When rhinacanthin-C (19) containing in ethanol extract (500 mg/kg/day,
pa administration) from the roots of Rhinacanthus nasutus and water extract
(500 mg/kg/day, pa injection) from the leaves of Rhinacanthus nasutus and
mitomycin C (MMC) (1 mg/kg/day, ip administration) have been
administrated to sarcoma 180 mice for 14 days, their inhibition ratio % (ir %)
were significantly 52.5, 44.2 and 52.7%, respectively [Gotoh et al., 2004].

Figure 23. Rhinacanthin-C (19) of napthoquinones in the leaves and roots of


Rhinacanthus nasutus (L.) Kurz
Rhinacanthus Nasutus 31

5. Phytochemicals of Whole Plant of Rhinacanthus Nasutus

In 2005, a group of Medicine of Chiang Mai University, Chiang Mai in


Thailand, examined the influence of cell-mediated and humoral immune
response by extracts of whole plants of Rhinacanthus nasutus.
First, the water and ethanol extracts of whole plants of Rhinacanthus
nasutus influenced significantly on the lymphocytes of human peripheral
blood mononuclear cells (PBMCs) which are widely distributed in bloods and
enhanced their activity of the NK cells. Moreover, the extracts of whole plants
of Rhinacanthus nasutus enhanced their increases and productions of their
interleukin 2 (IL-2) which induced their production of interferon- (IF- )
having the antitumor effects, and also enhanced their increases and
productions of their tumor necrosis factor-α (TNF-α) which cause their
necrosis against solid tumor cells.
Second, the water and ethanol extracts of whole plants of Rhinacanthus
nasutus enhanced only the secondary antibody response against BALB/c mice
are useful for research into both cancer and immunology (100 mg/kg body
weight) [Punturee et al., 2005b].

(1) Naphthoquinones
In 1996, a group of Shaman Pharmaceuticals, California in USA, isolated
two novel naphthoquinones such as rhinacanthin-C (19) and rhinacanthin-D
(20) from whole plant of Rhinacanthus nasutus [Sendl et al., 1996](Figure 24).
Two rhinacanthin-C (19) and rhinacanthin-D (20) showed high antivirus
activity. For example, their median inhibition concentration (IC50) of
rhinacanthin-C (19), rhinacanthin-D (20) and a derivative of acyclovir,
ganciclovir (39) with in vitro activity against human herpesviruses and used
for the treatment of cytomegalovirus infections were 0.56μg/mL, 0.75μg/mL
and 1000μg/mL, respectively and showed their significant antivirus activity
against human CMN virus (hCMV)(Figure 25).
Similarly, their median inhibition concentration (IC50) of rhinacanthin-C
(19), rhinacanthin-D (20) and amantadine (40) (Figure 25) of an antiviral
compounds used in the prophylaxis and management of type A influenza also
were 0.2μg/mL,0.78μg/mL and >56μg/mL, respectively. Their antivirus
effects against type A influenza by rhinacanthin-C (19) and rhinacanthin-D
(20) were higher than that of amantadine (40) [Sendl et al., 1996].
32 Rakesh Sharma, Bharati D Shrinivas

Figure 24. Rhinacanthin-C (19) and rhinacanthin-D (20) of two novel napthoquinones
in the whole plants of Rhinacanthus nasutus (L.) Kurz]

Figure 25. Ganciclovir (39) of anti-human herpes virus agent and amantadine (40) of
anti-influenza type A
Rhinacanthus Nasutus 33

Figure 26. Rhinacanthin-E (41) and rhinacanthin-F (42) of two novel lignans in the
whole plant of Rhinacanthus nasutus (L.) Kurz, and amantadine (40) of antiviral
(Influenza A) agent

(2) Anti-influenza virus


In 1997, group of Sharnan Pharmaceuticals of South San Francisco in
California USA, isolated two novel rhinacanthins (lignans) such as
rhinacanthin-E (41) and rhinacanthin-F (42) from whole plant of Rhinacanthus
nasutus and examined their antivirus activity of rhinacanthin-E (41) and
rhinacanthin-F (42) by anti-Flu-A cytopathic effect assay (CPE) in in vitro
assay.
Their antiviral activity (μg/mL) against Influenza virus type A by
rhinacanthin-E (41) was 7.4μg/mL at 50% effective concentration (EC50),
102μg/mL at 50% inhibitory concentration (IC50)and 15 at selective index
(IC50/EC50, SI), respectively.
Similarly, Their antiviral activity (μg/mL) against Influenza virus type A
by rhinacanthin-E (41) was 3.1μg/mL at 50% effective concentration (EC50),
21μg/mL at 50% inhibitory concentration (IC50)and 6.8 at selective index
34 Rakesh Sharma, Bharati D Shrinivas

(IC50/EC50, SI), respectively. Then, the two rhinacanthins (41, 42) showed
significantly their antiviral activity.
Their antiviral activity (μg/mL) against Influenza virus type A by a control
amantadine (40) which used in the prevention and treatment of type A
influenza was 0.054μg/mL at 50% effective concentration (EC50), 56μg/mL at
50% inhibitory concentration (IC50)and 1000 at selective index (IC50/EC50, SI),
respectively [Kernan et al., 1997](Figure 26).

6. Phytochemicals from Flowers of Rhinacanthus Nasutus

(1) Flavonols
In 1981, a group of Chemistry Department of University of Madras,
Tiruchirapalli in India, isolated a yellow pigment quercetin (43) of oxidative
flavonols from fresh flowers of Rhinacanthus nasutus [Subramanian et al.,
1981] (Figure 27).

Figure 27. Quercetin (43) of flavonols in the flowers of Rhincanthus nasutus (L.) Kurz
Rhinacanthus Nasutus 35

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Wu, TS; Hsu, HC; Wu, PL; Teng, CM; Wu, YC. Rhinacanthin-Q, a
naphthoquinone from Rhinacanthus nasutus and its biological activity.
Phytochemistry 49, 2001-2003, 1998b.
Foods, Diets and Disease
©
Editor: Rakesh Sharma, Bharati D Shrinivas 2009 Innovations And Solutions, Inc.
___________________________________________________________________________

Lecture 5

DIETARY FIBERS IN CULTURED CELLS


EXPERIMENTAL IN VITRO DIGESTION AND FERMENTATION MODELS OF FIBER
HEALTH EFFECTS

Rakesh Sharma, Bharati D Shrinivas

ABSTRACT
To study health effects of different foodstuffs and nutrients, e.g.
dietary fibres, on the large intestine, it is a prerequisite to obtain samples
that resemble contents of the gut after digestion of the respective
foodstuffs. Since the content of the human large bowel is inaccessible for
routine investigations, several different in vitro systems have been
established to simulate digestion and bacterial fermentation in the human
gastrointestinal tract. The simplest form of these in vitro models is the
batch style fermentation using defined populations of bacteria or faecal
material to simulate digestive processes in the large intestine. More
sophisticated models involve multistage systems simulating the whole
gastrointestinal tract, including mouth, stomach as well as small and large
intestine. In vitro fermentation models have been extensively used to
analyse the metabolites generated by digestion and fermentation of
different dietary fibres and other foodstuffs and how these metabolites
influence the gut microbiota. Additionally these in vitro fermentation
systems have been also used to produce samples resembling gut contents
and to analyse possible health effects of these samples in in vitro cell
culture studies. Since dietary fibres reach the colon undigested, simple
2 Rakesh Sharma, Bharati D Shrinivas

batch fermentation models of the large intestine are sufficient to study


possible health effects of dietary fibres on cultured cell lines. Dietary
fibres are recognised for their potential to prevent cancer, e.g. colorectal
cancers. Therefore a lot of research has been done to analyse effects of
fermentation supernatants from dietary fibres on colon cancer prevention
using different colon cell models. Chemical analyses of fermentation
supernatants obtained by in vitro fermentation of dietary fibres, provided
information on the respective concentrations of different components, e.g.
short chain fatty acids (SCFA). Synthetic mixtures of the different
components were then used to investigate which components are
especially active. Dietary fibres are typically ingredients of foodstuffs
that have numerous other ingredients. To analyse possible synergistic
effects of dietary fibres and these ingredients, more complex in vitro
systems, taking into account earlier stages of digestion, e.g. mouth,
stomach or small intestine, have to be used. These multi-stage
fermentation systems have therefore been modified to be used in cell
culture studies. Such models will improve studies analysing the effects of
foodstuffs on prevention of colon cancer and gut health in general. The
following chapter summarises recent progressions on the use of in vitro
models to study health effects of dietary fibres and other nutrients using
in vitro colon cell models.

INTRODUCTION
The gastrointestinal tract (GIT) is one of the major organ systems of
multicellular animals. It is important for food uptake, digestion and extraction
of energy and nutrients as well as excretion of the remaining waste. In humans
the GIT can be roughly divided into the upper and lower gastrointestinal tract.
Mouth, pharynx, oesophagus and stomach make up the upper GIT, whereas
the lower GIT is comprised of small intestine, large intestine and anus. The
small intestine can be structurally divided into duodenum, jejunum and ileum.
In the small intestine the majority of digestion takes place and nutrients reach
the blood stream after absorption by the epithelium. Caecum, colon and
rectum form the large intestine. The colon consists of the ascending,
transverse, descending, and sigmoid part. It is important for storage and
disposal of waste as well as maintenance of water balance. Furthermore the
colon hosts the majority of the gut flora. The most important function of the
gut flora is the fermentation of dietary fibre, resulting in short chain fatty acids
(SCFA), mainly acetate, propionate and butyrate and gases (e.g. CO2, CH4,
H2). Additionally colonic microorganisms also play a part in vitamin synthesis
Digestion and Fermentation Models… 3

and in absorption of calcium, magnesium and iron. Diverse functions are


attributed to SCFA and have been reviewed in great detail before (Topping
and Clifton, 2001). To name a few, SCFA can stimulate growth of beneficial
bacteria, reduce the growth of pathogenic bacteria and constitute the major
energy source for small intestine and colon. In general, increased levels of
SCFA are associated with improved gut health (Wong et al., 2006). Major
health issues concerning the gastrointestinal tract are obstipation,
inflammatory bowel disease and colorectal cancer. All of these diseases can be
influenced by modulation of the fermentation profile, which is affected by the
composition of the colonic microflora (Rastall, 2004; Guarner and
Malagelada, 2003). In this regard the use of probiotics as functional foods has
gained major interest by nutritionists and clinicians (Saxelin et al., 2005).
Probiotic bacteria are resistant towards the acidic environment of the stomach
and are therefore able to colonize the colon after oral ingestion. After
colonisation they confer a health benefit for their host. In addition to their role
in stimulation of the immune system (Delcenserie et al., 2008), which has
implications for gut health in general, a possible preventive effect of probiotics
against colorectal cancer is discussed (de LeBlanc et al., 2007). The growth of
probiotic bacteria can be selectively stimulated by prebiotics. Prebiotics are
polysaccharides which meet certain criteria, namely (1) resistance to gastric
acidity, to hydrolysis by mammalian enzymes, and to gastrointestinal
absorption; (2) fermentation by intestinal microflora; and (3) selective
stimulation of the growth and/or activity of those intestinal bacteria that
contribute to health and well-being (Gibson et al., 2004). Due to these
properties prebiotics can generally improve gut health and they are specifically
important for the prevention of colorectal cancer (Lim et al., 2005). One of the
major mechanisms of colon cancer prevention by prebiotics seems to be the
increase in SCFA, especially butyrate, and this may be the basis of preventive
effects of dietary fibre which has been suggested by some, but not all
epidemiologic studies (Bingham et al., 2003). On the molecular level,
selective inhibition of proliferation, induction of differentiation, and apoptosis
in colon cancer cells may account for the chemopreventive potential of SCFA
(Kles and Chang, 2006). Colorectal cancer is a global health issue, because it
is one of the most frequent cancers worldwide. The formation of colorectal
cancer is promoted by frequent exposition to carcinogens which are ingested
with the food and therefore contained in faeces. These carcinogens can reach
the faeces directly through the GIT or indirectly after elimination via bile
acids. Detoxification of carcinogens, bulking of stool, reducing faecal transit
time, reducing the exposition of the colonic epithelium to carcinogens, and
4 Rakesh Sharma, Bharati D Shrinivas

reducing growth/inducing death of initiated cells are thus the most important
mechanisms of colorectal cancer prevention by nutrition, especially dietary
fibres (Surh, 2003). Next to nutritional factors, physical activity is the most
important factor in reducing risk for colorectal cancer, as has been recently
highlighted by an expert report of the American Institute for Cancer Research
and the World Cancer Research Fund (WCRF/AICR, 2007).

HOW TO STUDY EFFECTS ON GUT HEALTH?


Given the significance of dietary fibres and other nutritional and
environmental factors on gut health, it is important to find suitable approaches
to analyse those effects under physiological conditions. In this regard human
studies analysing the effects of dietary fibres on colonic health can be
considered the most relevant approach. However, access to human materials
like gut contents and tissues is usually restricted for practical and ethical
reasons. Hence, varying methodologies have been used to analyse the
digestion and fermentation of dietary fibres and possible consequences.
Macfarlane and colleagues (Macfarlane et al., 1992) analysed products from
bacterial fermentation in the colon in sudden death victims within 3 hours of
death. A more applicable approach is the determination of fermentation
products in faecal samples, but this approach is also limited, because the
majority of SCFA is absorbed in the intestine. Studies using ileostomy patients
are therefore a valuable alternative to study intestinal absorption or excretion
of nutrients (Knudsen and Hessov, 1995). Because of the restrictions in human
studies, a lot of research on the digestion of dietary fibres has been done in
animal studies (Bach Knudsen and Hansen, 1991; Elsden et al., 1946). On the
one hand animal studies offer the possibility of controlled diets, direct
accessibility to gut contents and tissues as well as direct administration of
drugs or toxins. On the other hand rodents, which are used in many of the
studies, are coprophages displaying a different physiology of digestion than
humans, whereas pigs, which have also been used in a number of animal
studies, display a structurally different digestive tract with a different
distribution of gut bacteria. As a result of these methodical limitations diverse
models simulating the digestion of the human GIT in vitro have been
developed within the last two decades. These models are powerful tools to
study processes of digestion and fermentation as well as effects on colonic
bacterial profiles. Their main advantage is that they allow the addition and
Digestion and Fermentation Models… 5

removal of samples at practically any step of human digestion as well as


modulation of specific factors, like pH, faecal transit time, or addition of
selected strains of bacteria to study the effects of these factors on digestion and
fermentation. The different in vitro digestion and fermentation models will be
introduced in the following paragraphs, focussing on the basic principles and
applications.

OVERVIEW OF IN VITRO FERMENTATION SYSTEMS TO


STUDY GUT FUNCTION
In vitro systems simulating the human digestive tract range from simple
batch fermentations to elaborate computer-controlled multi-stage continuous
systems. Based on the question being addressed, the whole GIT, from mouth
to colon, or selected parts of the GIT can be simulated. Barry et al. (1995),
Lebet et al.( 1998a) and Olano-Martin et al. (2000) developed simple batch
approaches simulating the colon to characterize prebiotic effects of different
polysaccharides. All three models use faecal slurries from healthy human
donors as bacterial source and incubate them with different carbohydrates
under anaerobic conditions. The models differed in the media, duration of
incubations or amount of substrate used. Determined endpoints to analyse the
fermentation profile typically include quantification of modified SCFA
concentrations, consumption of carbohydrates, gas production, pH values, bile
acids or monitoring populations of predominant gut bacterial groups. To
validate physiological relevance of the methods a comparison of the results to
in vivo data (animal or human if available) is indicated. Thus, Barry et al.
(1995) demonstrated that SCFA concentrations obtained from their batch
system were in close correspondence to the data obtained from in vivo studies
using rats. Instead of using complex bacterial mixtures (i.e. faecal slurries), it
is also possible to use specific bacterial strains.

In general, static batch systems are inexpensive and allow a large number
of substrates to be tested. Therefore these methods represent a simple way to
analyse digestion of single carbohydrates. However, they have some
drawbacks, when digestion of more complex food matrices shall be
investigated. For example, Lebet et al. (1998b) showed that high
concentrations of starch mask the fermentation patterns of fibre fractions.
They therefore expanded the fermentation system and included an in vitro
6 Rakesh Sharma, Bharati D Shrinivas

digestion procedure before the in vitro fermentation. The in vitro digestion


included incubations with -amylase, porcine pepsin and porcine pancreatin as
well as a dialysis step to remove small molecular metabolites. Hence, this in
vitro digestion simulates processes occurring in the early GIT, namely mouth,
stomach and small intestine. By this modification of the model they were able
to remove the majority of starch and obtained unmasked results for different
fibre fractions. For the analysis of complex food matrices pre-digestion steps
are therefore indicated. Another drawback of the colon models introduced
above is the fact that they neglect regional differences within the colon, are
static and pH values are uncontrolled. In consequence long-term fermentations
with these models are not possible, because physiological conditions are not
maintained possibly resulting in unphysiological bacterial profiles. To study
long-term effects on bacterial profiles, multi-stage continuous models are thus
required. A number of such models have been developed including models of
the stomach and small intestine (Minekus et al., 1995a), the colon (Gibson et
al., 1988; Macfarlane et al., 1989; Minekus et al., 1999) as well as small
intestine and colon (Molly et al., 1993). A recent review gives an excellent
overview of the different models (Macfarlane and Macfarlane, 2007).

The Simulated Human Intestinal Microbial Ecosystem (SHIME) model


established in 1993 by Molly et al. (1993) is a complex 5-step multi-chamber
system that is increasingly used to simulate human GIT. It consists of 5 vessels
corresponding to different parts of the GIT, namely duodenum/jejunum, ileum,
caecum/ascending colon, transverse colon, and descending colon. The vessels
are connected via pumps to transport contents between the vessels. In addition,
a pH electrode and pH control unit is attached to each vessel and ports for
output of medium and sampling of headspace and liquid phase gas are part of
each vessel. This setup enables simulation of varying conditions in the small
intestine and colon, i.e. continuous movement of contents, different and
controlled pH conditions, different retention times, different media and
different sample volumes. Additionally liquid and gaseous samples can be
taken from each vessel at any time to analyze metabolites and bacterial
profiles. After inoculation with faecal suspensions bacteria were grown for six
weeks, allowing long-term studies on bacterial profiles. The determined
bacterial profiles were comparable to in vivo data. After selection of an
appropriate culture medium the concentrations of SCFA after digestion of a
human western diet suspension (15% protein, 20% fat, 45% carbohydrate)
corresponded fairly well to in vivo data. Since absorption of metabolites is not
simulated by the SHIME model, analyses of the digestion process are not
Digestion and Fermentation Models… 7

possible and the system is limited to study microbial interactions. However,


since different substances can be added to the system (e.g. bile acids, drugs,
probiotics), it is a powerful tool to study influence of those substances on
human gut microflora. The model was thus used to address various interesting
questions. For example, the prebiotic potential of chicory inulin was confirmed
using this system, as digestion of inulin resulted in a significant increase in
bifidobacteria (Van de Wiele et al., 2004). The addition of Lactobacillus
acidophilus 74-2, which is used in probiotic products, and
fructooligosaccharide to the second vessel (duodenum/jejunum) of the SHIME
increased the amounts of bifidobacteria and SCFA (especially butyrate and
propionate) in the colon (vessel 4-6) demonstrating probiotic activity of this
strain (Gmeiner et al., 2000). Furthermore analysis of soygerm powder
increased bacterial fermentation resulting in substantial metabolism of free
isoflavones and suggesting for the first time a possible beneficial effect of
soygerm powder on gut health.

Very interesting dynamic continuous multi-stage models were developed


by scientist at the TNO Nutrition and Food Research Institute in Zeist,
Netherlands. These models are computer-controlled and thus probably the
most sophisticated so far. Two different models were established namely TIM-
1 (TNO intestinal model 1) (Minekus et al., 1995b) and TIM-2 (Minekus et al.,
1999), corresponding to stomach/small intestine and colon, respectively. The
technology of the two systems is very complex and a detailed description is
beyond the scope of this article. However, flexible walls of the reaction
container to which a pressure can be applied enabling the simulation of
peristaltic motions are one of the special features. Additionally, both models
contain hollow-fiber membranes to remove small molecules and simulate
absorption by the intestinal epithelium. A unique feature of the TIM models is
a connected computer with specifically developed software which is based on
parameters from in vivo data. This computer controls quantity and duration of
the meals, pH values, secretion rates, water absorption, and transit time. The
physiological relevance was analyzed in comparison to available in vivo data
and it was demonstrated that the models can accurately reproduce
physiological parameters like meal size and duration, peristaltic motions, pH,
gastric and intestinal secretions, gastrointestinal transit and absorption of
digested products and water. Therefore, the TIM models are potent tools to
investigate digestion of nutrients and even complete meals, the fate of ingested
medicines, survival of ingested microorganisms and effects on the colonic
microflora in vitro under nearly physiological conditions. Physiological
8 Rakesh Sharma, Bharati D Shrinivas

processes that cannot be simulated are active transport and feedback


mechanisms of the intestinal epithelium. In addition the instruments are highly
complex and thus comparably expensive. TIM models were for example used
to analyze metabolism of the glucosinolate sinigrin (Krul et al., 2002), effects
of bile on survival of lactic acid bacteria (Marteau et al., 1997) or the
availability of heterocyclic aromatic amines in the GIT.

Since all of the different models simulating digestion of the human GIT
have advantages and disadvantages, the selection of a suitable model
decisively depends on the question being addressed (and of course on the
availability of the respective method). In some instances it can be useful to
study effects using more than one model. Olano-Martin et al. used a batch
culture fermentation and a three-stage continuous culture system of the colon
(simulating the proximal, transverse and distal parts of the colon) to analyze in
depth the fermentability of dextran, oligodextran and maltodextrin (Olano-
Martin et al., 2000). They were able to demonstrate the prebiotic properties of
the analyzed carbohydrates. A direct comparison of the TIM-1 model with a
three-step batch culture simulating digestion in mouth, stomach and small
intestine revealed different digestibility of resistant starch (RS) (Fassler et al.,
2006b). Depending on the type of RS fermented, the models were either
comparable or gave different results, but the authors were not able to conclude
which model reflected the in vivo situation more closely. However, results
from both models were in general analogous to data obtained from ileostomy
patients. In another study by the same authors (Fassler et al., 2006a)
fermentability of different resistant starch preparations in the TIM-2 model
and a static batch fermentation were compared and a substantial differences in
the concentrations of SCFA and a higher reproducibility of results obtained
with the batch culture was found. The authors concluded that “the dynamic
model simulates the behavior of the human colon in vivo more precisely
whereas the batch model is better for screening of compounds”. On the other
hand, they also highlighted that direct comparison of the two methods is
difficult, because they differ in a variety of parameters.

In summary, different models are available to simulate digestion and


fermentation by the human GIT and the colonic microflora. All methods have
advantages and disadvantages, but all are useful tools to investigate processes
in the GIT. The selection of a suitable model strongly depends on the aim of
the study.
Digestion and Fermentation Models… 9

EFFECTS OF FERMENTATION SAMPLES ON CULTURED


COLON CELLS
As explained above, in vitro simulation of digestion as well as
fermentation is important to analyse digestibility of foods and nutrients,
determination of metabolites or influences of varying factors or substances on
colonic microflora. Therefore these methods are important to study effects on
gut health in general. Nonetheless, to determine in detail how the intestinal
epithelium is influenced by the different metabolites or differences in
fermentation profiles, it is necessary to study the effects of fermentation
samples in cell culture experiments using colon cells. This approach is
relatively new and the number of publications is sparse. However the studies
that are available have yielded highly interesting results and will be introduced
in the following section.
In general incubation of cultured colon cells with complex fermentation
samples were used to analyse uptake of nutrients, effects on membrane
integrity, antioxidative effects, cytotoxic effects or effects on gene expression
and signal transduction. Many studies have analysed potential of different
foods to prevent the formation of colorectal cancer, i.e. the chemopreventive
potential (Beyer-Sehlmeyer et al., 2003). Since many endpoints relevant to
colorectal cancer, e.g. antioxidative capacity are also relevant for other
diseases, statements about gut health in general can be often derived from such
studies. Other studies dealt with the uptake of nutrients. Salovaara et al. (2003)
treated colon adenocarcinoma cells (Caco-2) with TIM-1 samples obtained
from digestion of vegetables with white or whole meal bread. They analysed
effects of different transit times on iron metabolism and demonstrated that iron
absorption from white bread samples increased with prolonged transit times.
Many studies investigated the effects of prebiotics and prebiotics on cultured
cells. Commane et al. used different prebiotic carbohydrates and incubated
them with different probiotic bacterial strains in a batch-style in vitro
fermentation (Commane et al., 2005). Fermentation samples increased the
strength of tight junctions as measured by trans-epithelial electrical resistance
measurements with Caco-2 cells. Since disruption of tight junctions is
involved in promotion of colorectal cancer, this finding suggests protective
effects. Furthermore some of the samples were able to protect against the
negative effects of deoxycholic acid, a potentially carcinogenic secondary bile
acid, upon tight junction integrity. Batch-fermented samples of RS also
increased membrane integrity of Caco-2 cells (Fassler et al., 2007).
10 Rakesh Sharma, Bharati D Shrinivas

Additionally the same samples displayed anti-genotoxic activity against H2O2.


Wheat-bran derived arabinoxylans fermented with faecal suspensions in vitro
protected HT29 colon cancer cells against DNA damage induced by 4-
hydroxynonenal (HNE) and enhanced the activity of Glutathione-S-
Transferases (GSTs) which are important for detoxification of carcinogens
(Glei et al., 2006). However, a control (fermentation without arabinoxylans)
was comparably effective. In another study HT29 and Caco-2 cells were
incubated with fermentation samples obtained by fermentation of Synergy1
(inulin enriched with oligofructoses, Beneo-Orafti) using a three-stage
fermentation model of the colon. The samples modulated different tumour
markers, i.e. cell survival, differentiation, tumour progression and invasive
growth. The sample from the gut model vessel representing the distal colon,
was most effective for all parameters, probably on account of higher butyrate
concentrations (Klinder et al., 2004).

These are some examples for studies analysing the effects of complex
fermentation samples on colon cancer cells. It is also possible to investigate
which specific compounds of the complex samples are responsible for
certain effects, by comparing effects of the complex samples with single
compounds or mixtures of several compounds in corresponding
concentrations. This was done in a study using fermentation samples of
various dietary fibre sources and samples of butyrate alone and SCFA
(acetate, propionate and butyrate) in the same concentrations found in the
fermentation samples. All of the employed samples had significant inhibitory
effects on the growth of HT29 cells. Inhibition of cell growth was strongest
in complex fermentation samples and butyrate alone was the least effective
(Beyer-Sehlmeyer et al., 2003). This result suggests additive or synergistic
effects of the complex samples and SCFA mixtures, respectively. GST
activity was only induced by butyrate and chemoresistance towards HNE
was caused by selected SCFA mixtures, but not all corresponding
fermentation samples. All of the studies mentioned before used colon cancer
cells, but to study effects on the healthy epithelium, primary cells isolated
from healthy colon tissues need to be used. While primary colon cells are
difficult to cultivate and typically die after short times, a method has been
recently described incubating whole epithelial tissue stripes from clinical
colon samples which were viable in vitro for more than 12 hours (Sauer et
al., 2007). These tissue stripes were incubated with Synergy1 fermented by
gut bacteria. The samples were not cytotoxic for primary cells, but increased
metabolic activity, pointing to trophic effects. Effects on gene expression
Digestion and Fermentation Models… 11

were analysed using a cDNA macroarray and real-time qPCR. Expression of


GSTM2 and GSTM5 was increased, but the activity of total GSTs was not
modulated. The induction of GST gene expression by the fermentation
samples may protect the cells from carcinogenic compounds (Sauer et al.,
2007).

Whereas most of the studies used dietary fibres/prebiotics as substrate


for the fermentation reaction, it is also possible to analyse the effects of other
nutritional matrices. For example, fermentation samples from apple juice
extracts (AEs) decreased the levels of reactive oxygen species in Caco-2
cells. However the unfermented AEs were more effective, indicating that
active compounds may have been degraded by the fermentation procedure
(Bellion et al., 2008). In another study polyphenolic apple extracts (PAE)
were fermented in vitro with human faecal flora, resulting in increased levels
of SCFA and degradation of polyphenols. The apple extracts reduced cell
growth of HT29 cells and the colon adenoma cell line LT97, pointing at
chemoprotective properties of apple extracts (Veeriah et al., 2007). The two
latter studies used static batch fermentation models of the colon to digest
apples. This is a simple approach to obtain first hints on effects of fermented
apple extracts. However, since many of the apple ingredients would be
degraded in vivo and not reach the colon, a more sophisticated simulation of
digestion, including simulation of mouth, stomach and small intestine would
be more appropriate for such substrates. A problem in this regard seems to
be the presence of cytotoxic compounds in samples from simulation of small
intestine. Both samples obtained by digestion of RS with TIM-1 (Fassler et
al., 2007) and samples from the SHIME model (De Boever et al., 2000)
supplemented with oxgall, a dry mixture of the salts of the gall of the ox,
displayed cytotoxic effects on Caco-2 or Hela cells, respectively. In the latter
study the effects were caused by high concentrations of oxgall. Our group
has recently adapted an in vitro simulation of the GIT (including mouth,
stomach, small intestine and colon) which can be used in cell culture
experiments (unpublished). The method to simulate digestion of mouth,
stomach and small intestine, includes a dialysis step to simulate absorption
of small molecules, and is based on the model established by Aura et al.
(1999). Samples using the original protocol were cytotoxic on different
cultured colon cell lines. This cytotoxic effect was avoided by reducing the
bile acid concentrations. The modified protocol is currently successfully
used to study effects of different nutrients and foodstuffs on
chemoprevention and general markers of gut health in colon cells.
12 Rakesh Sharma, Bharati D Shrinivas

CONCLUSION
The human GIT is of major significance for uptake and digestion of
nutrients, energy supply and excretion. It is continuously exposed to
xenobiotics and endogenous metabolites which can cause colorectal cancer
or other diseases of the colon like inflammatory bowel disease. A key
influence comes from the colonic microbiota. The composition and
metabolism of those bacteria can modulate the fermentation profile and
thus improve gut health. Because of its important role in the physiology of
humans, it is of high interest to find ways to investigate the processes
occurring in the intestine. Since tissues and gut contents from humans are
not accessible and animal studies are not always representative, because of
a different physiology, in vitro simulations of the human GIT are powerful
tools to analyse digestion and the colonic microflora. A number of
different models is available, each one with advantages and disadvantages.
Simple batch techniques are inexpensive and allow testing of a large
number of samples. However, especially for the digestion of complex food
matrices and to analyse long-term effects on gut bacteria, pH controlled
multi-stage models are of advantage. They better reflect the physiology of
the GIT, because the pH value, transit time and addition of different me dia
or bacteria can be controlled, absorption is simulated and they take
regional differences of the GIT into account. Nonetheless all models are
helpful to gain information of digestive processes, but the appropriate
method has to be selected and the selection depends on the aim of the
study. To analyse the effects of the complex fermentation samples on the
intestinal epithelium different cell culture models can be used. Cell culture
studies have been used to demonstrate anti-carcinogenic and anti-oxidative
properties of different foodstuffs and nutrients e.g. dietary fibres. This
approach is therefore very useful to study how complex fermentation
samples interact with the cells of the epithelium and modulate molecular
processes in the epithelial cells resulting in protection against colorectal
cancer and improvement of colonic health. A further progression of the
methods involved will certainly increase our understanding of the
physiology and pathology of the GIT.
Digestion and Fermentation Models… 13

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Foods, Diets and Disease
©
Editor: Rakesh Sharma, Bharati D Shrinivas 2009 Innovations And Solutions, Inc.
___________________________________________________________________________

Lecture 6

BAMBOO SHOOTS AS DIETARY FIBRES

Rakesh Sharma, Bharati D Shrinivas

ABSTRACT
Bamboo is a grass which belongs to the same family as our staple
cereal crops and has multifarious uses in industry besides being used by
rural people for food, housing and other domestic purposes. A little
known fact is the edible characteristics of its juvenile shoots in the form
of fresh, fermented and canned form. Use of juvenile, fresh and soft
bamboo shoots as vegetable, and in the form of fermented food produced
by traditional and industrial methods is well known, more particularly in
bamboo growing Asian countries. Commercially, bamboo shoots are
available in canned form, though fresh shoot is far superior in taste and
texture. Bamboo shoots are considered as a health food as they are
endowed with health enhancing properties being rich in nutrient
components mainly proteins, carbohydrates, minerals, fibre, amino acids
and vitamins, are low in fat and sugar and have no cholesterols. The
shoots contain phytosterols and a high amount of fibre due to which they
are labeled as nutraceuticals or natural medicines. Dietary fibre contents
are cellulose, hemicelluloses and lignin. There are two broad types of
dietary fibre described according to its solubility: insoluble and soluble.
Nutrient detergent fibre (NDF) determines the indigestible component of
the plant material. It consists of hemicellulose, cellulose and lignin. Acid
detergent fibre (ADF) primarily represents cellulose and lignin. ADF is
often used to calculate digestibility, while NDF is often used to predict
intake potential. The major function of dietary fibre is to reduce the time
2 Rakesh Sharma, Bharati D Shrinivas

of release of ingested food in colon. The roughage is aids in the digestive


process and elimination of waste It also functions for holding water and
acts like sponge in alimentary canal. The content of dietary fibre and its
components in bamboo shoots increases with age, but as the tissue grows
old, the shoots become inedible. With fermentation and canning also,
their content increases in the shoots. Whereas, the fermented shoots have
a lesser amount of ADF, the canned as well as the fresh shoots have
nearly equal amount of ADF. Lignin content in both the fresh and canned
shoots was less than the fermented shoots. The canned shoots have a
comparatively higher content of hemicelluloses than the fermented as
well as the fresh shoots. The fermented shoots have higher amounts of
cellulose than the fresh shoots while canned shoots have lower amount of
cellulose than both the fresh and fermented shoot. The dietary fibre
content in the fresh juvenile shoots ranges between 2.255-4.490 g/100g
fresh weight and reaches upto 13.840/100gm fresh weight in 10 days old
emerged shoots of some bamboo species. The recommended level of
fibre for adults is 25-30 g a day, in combination with at least 2 litres of
fluid to ensure thorough digestion. Foods and food products that contain
6g fibre per 100g or 100 ml are labeled as a „high fibre‟ food. Thus,
bamboo shoots can be considered as „fibre rich‟ foods and can meet the
daily requirement of fibre in the diet. German and US companies Qualicel
and Vitacel market fibre additives in white powder form with at least
95% fibre and bamboos being the fastest growing plants, can provide raw
material for production of such fibre additives.

INTRODUCTION
Bamboos are plants of global interest because of their distinctive life
form, ecological importance and the wide range of uses and values they have
for humans [Bystriakova et al., 2004]. At least one third of the human race
uses bamboo in one way or another. They are gaining increased attention as
an alternative crop with multiple uses and benefits providing human beings
with various living resources. They are intermingled with the tradition and
culture of rural and tribal populations from times immemorial due to which
they have been variously called as „The Cradle to Coffin Plant‟, „The Poor
man‟s Timber‟, „Friend of the People‟, „Green Gasoline‟, „The Plant with
Thousand Faces‟ and „ The Green Gold‟. This green gold is sufficiently
cheap and plentiful to meet the vast needs of human populace from the
“child‟s cradle to the dead man‟s bier”. As a renewable natural resource, it
plays a major role in the livelihood of rural people and is an integral part of
our cultural, social and economic conditions [Tewari, 1988; Madhab, 2003].
Bamboo Shoots 3

Because of its multifarious utility, both in the traditional way for the rural
people as well as in modern society, bamboo is becoming a very important
plant worldwide. There are more than 1500 different documented traditional
uses of bamboo [INBAR, 1997; Shrestha, 1999]. Bamboos provide food,
shelter, medicine, raw materials for construction, wood substitute and paper
and pulp industry. They are also used for making furniture, handicrafts,
containers, tool handles, poles, musical instruments, bows and arrows, boats,
rafts, fishing poles etc. In addition, because of their characteristic growth
habits, bamboos have enormous potential for alleviating many environmental
problems like soil erosion control, water conservation, land rehabilitation
and carbon sequestration [Benzhi et al., 2005]. However, a lesser known use
of bamboos is the edible value of its shoots which are excellent vegetables
with tender sprouts and have a good taste. Bamboo shoots are highly popular
in Asian cuisine, particularly in countries like China, Japan, Korea, Taiwan,
Thailand and Philippines. Juvenile bamboo shoots, in addition to being
delicious, are rich in nutrient components, mainly proteins, carbohydrates,
minerals and fibre, are low in fat and sugar and have no cholesterols.
Moreover, they are rich in vitamins and amino acids. Young edible shoots
are harvested during the monsoons when the shoots just emerge from the
ground (Figure 1a,b), and the typical “shooting season” of a species rarely
exceeds two months. For consumption, hard sheaths are removed and thin
slices are prepared (Figure 1. c-e). Fresh shoots have a crisp, crunchy and
sweet flavour imparting a unique taste and are mostly used in soup, salads,
pickles, spring rolls or other stewed and dried dishes. These are also used as
an extender because it takes on the flavour of the ingredients in which it is
cooked. The most common preparation involves boiling the shoots in stocks,
soups or salted water for use in assorted dishes. The shoots are not only used
as vegetables, but are also processed and preserved in many forms such as
dried, fermented, salinised, pickled, water soaked and canned shoots.
Bamboo shoots are gastronomic treats whether used fresh or in fermented or
roasted form. Being endowed with many health enhancing properties,
bamboo shoots are projected as a new health food.
4 Rakesh Sharma, Bharati D Shrinivas

Figure 1. a. Plant morphology; b. emerging juvenile shoot; c. shoots with sheaths and
with sheaths removed; d. juvenile shoots cut into half; e. sliced shoots.

NUTRITIONAL VALUE OF BAMBOO SHOOTS


The criterion for healthy nutrition, in so far as food intake is concerned,
constantly evolves with an understanding of food-health relationship.
Bamboo Shoots 5

Recently, the research and exploration of bamboo as a food commodity has


witnessed tremendous activity. Due to this, major advances have been made in
fresh shoot production and processing and analysis of nutrient component of
edible shoots. This is because bamboo shoots are endowed with health
enhancing properties being rich in nutrients with a high content of protein,
amino acids, carbohydrate, minerals and several vitamins. They also contain
phytosterols and high amount of fibre that are labeled as “nutraceuticals” or
“natural medicines”. Phytosterols have cholesterol lowering activity. Bamboos
grow well without the application of fertilizers or chemicals and are hence are
more likely to be free from pollution. Moreover, the shoots are well protected
from pollutants by several layers of sheaths. Bamboo shoots are a good source
of edible fibre (6-8%) which helps in lowering cholesterol level in the blood.
Fat content is extremely low in bamboo shoots (2.46%) which are, therefore,
very good for weight conscious people. The high cellulosic content of bamboo
shoots stimulates appetite. Being crisp, crunchy, tender with a sweet flavour,
the shoots have a unique and delicious taste that functions as an appetizer.

Bamboo Shoots as a Source of Dietary Fibre

Bamboo shoots are a rich source of dietary fibre. Dietary fibre is an


important part of a health promoting diet and consists of the remnants of
edible plant cells, polysaccharides, lignin and associated substances resistant
to digestion by the alimentary enzymes of humans (DeVries et al. l999). It
identifies a macroconstituent of foods that includes cellulose,
hemicellulose,,lignin, gums, modified celluloses, mucilages, oligosaccharides,
and pectins and associated minor substances, such as waxes, cutin, and
suberin. They pass through the stomach and small intestine undigested and
reach the large intestine virtually unchanged (Lee and Prosky, 1995; Trowell
and Burkitt, 1986). Most other nutrients are digested and are being used in
other parts of the body by this stage. During its passage through the large
intestine some components of dietary fibre are broken down to varying
degrees and absorbed by the body; the remaining components are excreted in
the faeces. The analysis of dietary fibre in food is very complex and only a
limited number of foods have been examined in detail. Dietary fibre from
different foods, and even different samples of the same food, contain varying
quantities of the components that collectively make up dietary fibre. Each of
these components has different biological properties and it is frequently not
clear which of these is most beneficial. Dietary fiber has been one of the most
6 Rakesh Sharma, Bharati D Shrinivas

enduring dietary interests worldwide and has been a topic of considerable


volume of medical research and is recommended as part of the treatment and
prevention of a number of diseases. The major function of dietary fibre is to
reduce the time of release of ingested food in colon. It is known to be
associated with reduced incidence of coronary heart diseases. It also functions
for holding water and acts like sponge in alimentary canal. So the food with
high dietary fibre is recommended for diet and is good for health. This
roughage is essential for the mechanism of digestion and elimination of waste
[Gopalan et al., 1971]. In hypercholesterolemic man, a diet rich in dietary fibre
can significantly lower plasma total cholesterol and low density lipoproteins
cholesterol and thus potentially lower the risk of coronary heart diseases
[Whyte et al., 1992; Gold et al, 1999]. There is increasing epidemiological
evidence that population groups which consume reasonable amounts of dietary
fiber (20 – 35g / day) have lower risk of a number of chronic diet-related
diseases such as diverticular disease, coronary heart disease, obesity, type 2
diabetes mellitus, gall stone, colonic carcinoma, hyperlipidaemia, constipation,
haemorrhoids and irritable bowel syndrome [Cummings et al, 1997]. Due to
these qualities, bamboo shoots have been projected as a health food.
The fibre content in bamboo shoots can be classified accordingly as
Nutrient detergent fibre (NDF) which determines the indigestible component
of the plant material consisting of hemicellulose, cellulose and lignin [Van
Soest, 1978] and Acid detergent fibre (ADF), primarily representing cellulose
and lignin. ADF is often used to calculate digestibility, while NDF is often
used to predict intake potential. Sixteen species of bamboos viz. Bambusa
bambos (Linn.) Voss, B. kingiana Gamble, B. nutans Wall ex. Munro, B.
polymorpha Munro, B. tulda Roxb., B vulgaris var. vulgaris Schrad.,
Dendrocalamus asper (Schultes f.) Becker ex Heyne, D. brandisii (Munro)
Kurz., D. giganteus Munro, D. hamiltonii Nees & Arn. ex Munro, D.
membranaceus Munro, D. strictus (Roxb.) Nees, Gigantochloa albociliata
(Munro) Kurz, G. rostrata Wong, Thyrsostachys oliveri Gamble and T.
siamensis Gamble were selected and analyzed for the dietary fibre content of
their fresh edible juvenile shoots. The juvenile shoots of all the species were
collected at the stage when their tips were just emerging above the ground.
The fermented and canned shoots of Dendrocalamus giganteus available in the
local market were also analyzed for the same component as in the case of fresh
juvenile shoots. The results of dietary fibre content and its components of the
bamboo species is presented below.
Bamboo Shoots 7

Table 1. Dietary Fiber (NDF) and Its Components in the Freshly Emerged
Juvenile Shoots of Various Species (G/100g Fresh Weight).

Sl Names Hemi-
NDF ADF Lignin Cellulose
No. of species cellulose
Bambusa 3.535 2.810 1.460 0.725 1.350
1.
bambos ±0.015 ±0.010 ±0.030 ±0.005 ±0.001
4.490 3.190 2.010 1.300 1.180
2. B. kingiana
±0.060 ±0.043 ±0.020 ±0.017 ±0.023
B. nutans 2.275 1.240 0.430 1.034 0.710
3.
±0.005 ±0.050 ±0.010 ±0.995 ±0.040
B.
3.815 2.290 1.300 1.515 0.990
4. polymorpha
±0.055 ±0.012 ±0.009 ±0.043 ±0.003
B. tulda 3.970 3.360 2.300 0.609 1.060
5.
±0.020 ±0.031 ±0.010 ±0.989 ±0.021
B. vulgaris 4.240 3.280 2.400 0.959 0.780
6.
var. vulgaris ±0.010 ±0.022 ±0.011 ±0.988 ±0.011
Dendrocalam 3.540 3.000 1.260 0.475 1.740
7.
us asper ±0.065 ±0.014 ±0.010 ±0.054 ±0.004
4.027 3.060 2.010 0.967 1.050
8. D. brandisii
±0.087 ±0.061 ±0.013 ±0.026 ±0.048
2.645 2.150 0.560 0.495 1.589
9. D. giganteus
±0.025 ±0.009 ±0.010 ±0.016 ±0.999
D. hamiltonii 3.900 3.230 2.170 0.670 1.060
10.
±0.030 ±0.026 ±0.017 ±0.004 ±0.009
D.
2.905 1.400 0.870 1.425 0.609
11. membranaceu
±0.055 ±0.011 ±0.024 ±0.044 ±0.992
s
D. strictus 2.255 1.380 0.640 0.844 0.939
12.
±0.005 ±0.020 ±0.028 ±0.985 ±0.992
Gigantochloa 4.150 3.750 2.700 0.400 1.050
13.
albociliata ±0.106 ±0.054 ±0.050 ±0.052 ±0.004
4.200 3.320 1.820 0.930 1.500
14. G. rostrata
±0.090 ±0.023 ±0.021 ±0.067 ±0.002
Thyrsostachys 3.910 2.840 1.280 1.069 1.560
15.
oliveri ±0.023 ±0.040 ±0.010 ±0.983 ±0.030
3.715 2.150 1.200 1.565 1.250
16. T. siamensis
±0.054 ±0.014 ±0.009 ±0.040 ±0.005
The variation in the content of cellulose in D. giganteus and T. oliveri shoots was
insignificant (Table 1).
8 Rakesh Sharma, Bharati D Shrinivas

Figure 2. Comparison of NDF content (g/100g fresh weight) in the freshly emerged
juvenile shoots of various species.

Figure 3. Comparison of ADF content (g/100g fresh weight) in the freshly emerged
juvenile shoots of various species.

Dietary Fibre (NDF)


The NDF content in juvenile shoots ranged between 2.255 - 4.490 g/100 g
fresh weight (Table 1). The highest fibre content was found in B. kingiana
Bamboo Shoots 9

(4.490 g/100g fresh weight) shoots while the lowest was found in those of D.
strictus (2.255 g/100 g fresh weight) (Table 1 and Figure 2). The shoots of B.
vulgaris var. vulgaris, D. brandisii, G. albociliata and G. rostrata had also
high amount of dietary fibre (Table 1 and Figure 2).

Acid Detergent Fibre (ADF)


The content of ADF ranged from 1.240-3.750 g/100 g fresh weight (Table
1) where the highest value was recorded in G. albociliata (3.750 g/100 g fresh
weight) and the lowest in B. nutans (1.240 g/100 g fresh weight) (Table 1 and
Figure 3). The shoots of B. tulda, B. vulgaris var. vulgaris, D. hamiltonii and G.
rostrata have nearly equal amount of ADF% (Table 1). Likewise, the variation
in the shoots of B. bambos and T. oliveri was insignificant (Figure 3).

Lignin
The content of lignin in the shoots ranged from 0.430-2.700 g/100 g fresh
weight (Table 1). G. albociliata has the highest amount of lignin content
(2.700 g/100 g fresh weight) and that of B. nutans has the lowest (0.430 g/100
g fresh weight) (Table 1 and Figure 4). The shoots of D. giganteus, D.
membranaceus and D. strictus have low content of lignin (< 1 g/100 g fresh
weight) (Table 1). The variation in lignin content in B. polymorpha, D. asper
and T siamensis was insignificant (Table 1 and Figure 4). Lignin content in B.
kingiana and D. brandisii was found equal, each having 2.010 g/100 g fresh
weight (Table 1 and Figure 4).
10 Rakesh Sharma, Bharati D Shrinivas

Figure 4. Comparison of lignin content (g/100g fresh weight) in the freshly emerged
juvenile shoots of various species.

Figure 5. Comparison of hemicellulose content (g/100g fresh weight) in the freshly


emerged juvenile shoots of various species.

Hemicellulose
Hemicellulose content in the shoots varied from 0.400-1.565 g/100 g fresh
weight (Table 1). The highest hemicellulose content was present in T.
siamensis (1.565 g/100 g fresh weight) and the lowest in the shoots of G.
albociliata (0.400 g/100 g fresh weight) (Table 1 and Figure 5). B. vulgaris
var. vulgaris and D. brandisii had almost equal content (Table 1).

Cellulose
The shoots have cellulose content varying from 0.609-1.740 g/100 g fresh
weight (Table 1) with the maximum cellulose content in D. asper (1.740 g/100
g fresh weight) and the minimum in D. membranaceus (0.609 g/100 g fresh
weight) (Table 1 and Figure 6). B. nutans, B. polymorpha, B. vulgaris var.
vulgaris and D. strictus have low cellulose contents with their values being
lesser than 1 g/100 g fresh weight of the shoots (Table 6). The shoots of B.
Bamboo Shoots 11

tulda and D. hamiltonii have identical amounts of cellulose (Table 1 and


Figure 6).
Table 2. Dietary Fiber (NDF) and Its Components in the Freshly Emerged Juvenile (Jv) and 10 Days Old Emerged
(Old) Shoots of the Five Species (G/100g Fresh Weight)

Names NDF ADF Lignin Hemicellulose Cellulose


Sl No.
of species jv old jv old jv old jv old jv old
Bambusa 3.535 9.640 2.810 7.560 1.460 4.802 0.725 2.080 1.350 2.758
1.
bambos ±0.015 ±0.105 ±0.010 ±0.092 ±0.030 ±0.041 ±0.005 ±0.013 ±0.001 ±0.051
3.970 10.690 3.360 8.206 2.300 5.542 0.609 2.484 1.060 2.754
2. B. tulda
±0.020 ±0.102 ±0.031 ±0.089 ±0.010 ±0.056 ±0.989 ±0.013 ±0.021 ±0.033
3.540 10.850 3.000 8.270 1.260 5.680 0.475 2.580 1.740 2.690
3. D. aspe
±0.065 ±0.023 ±0.014 ±0.010 ±0.010 ±0.008 ±0.054 ±0.013 ±0.004 ±0.002
2.645 13.840 2.150 9.520 0.560 6.320 0.495 4.320 1.589 3.200
4. D. giganteus
±0.025 ±0.041 ±0.009 ±0.021 ±0.010 ±0.014 ±0.016 ±0.020 ±0.999 ±0.007
3.900 8.200 3.230 6.105 2.170 3.890 0.670 2.095 1.060 2.095
5. D .hamiltonii
±0.030 ±0.020 ±0.026 ±0.012 ±0.017 ±0.009 ±0.004 ±0.008 ±0.009 ±0.003
± indicates standard deviation
14 Rakesh Sharma, Bharti D Shrinivas

Figure 6. Comparison of cellulose content (g/100g fresh weight) in the freshly


emerged juvenile shoots of various species.

Table 3. Dietary Fibre (NDF) and Its Components in the Freshly Emerged
Juvenile, 10 Days Old Emerged, Fermented and Canned (Non-Salted)
Shoots of D. Giganteus

Hemi-
Nature of shoots NDF ADF Lignin Cellulose
cellulose
Freshly emerged 2.645 2.150 0.560 0.495 1.589
juvenile shoots ±0.025 ±0.009 ±0.010 ±0.016 ±0.999
10 days old emerged 13.840 9.520 6.320 4.320 3.200
shoots ±0.041 ±0.021 ±0.014 ±0.020 ±0.007
4.180 3.280 1.398 0.900 1.882
Fermented shoots
±0.104 ±0.076 ±0.042 ±0.028 ±0.034
Canned shoots (non- 3.040 2.020 0.780 1.020 1.240
salted) ±0.108 ±0.095 ±0.038 ±0.013 ±0.057

The bamboo shoots vary in their dietary fibre quantity even within same
species with the variation in age of the shoots and the way in which the shoots
were preserved (Table 2 and 3). The increase in dietary fibre content and its
components in the shoots increases with age [Nirmala et al., 2007]. With
Bamboo Shoots 15

fermentation and canning also, their content increases in the shoots. Whereas,
the fermented shoots have a lesser amount of ADF, the canned as well as the
fresh shoots have nearly equal amount of ADF. Lignin content in both the
fresh and canned shoots was less than the fermented shoots. The canned shoots
have a comparatively higher content of hemicelluloses than the fermented as
well as the fresh shoots. The fermented shoots have higher amounts of
cellulose than the fresh shoots while canned shoots have lower amount of
cellulose than both the fresh and fermented shoot.
Among some selected species of bamboo shoots, the dietary fibre content
in the fresh juvenile shoots range between 2.255-4.490 g/100g fresh weight
with the highest fibre content in Bambusa kingiana (4.490 g/100 g fresh
weight) and the lowest in D. strictus (2.255 g/100 g fresh weight). The fibre
content in the 10 days old emerged shoots increased more than 3 times to those
present in their respective freshly emerged juvenile shoots (Table 2). There
was significant increase of fibre components (ADF, lignin, hemicellulose and
cellulose) in the old emerged shoots of all the five species. High content of
upto 40% cellulose stimulates appetite. Being nutritious, crisp, crunchy and
with a sweet flavor, the shoots have a unique and delicious taste that functions
as an appetizer. Foods and food products that contain 6g fibre per 100g or 100
ml may be labeled as a „high fibre‟ food and the recommended level of fibre
for adults is 25-30 g a day, in combination with at least 2 litres of fluid to
ensure thorough digestion. Thus, bamboo shoots can be considered as „fibre
rich‟ foods and can meet the daily requirement of fibre in the diet.

CONCLUSION
Fibre has been associated with a number of health benefits such as a faster
“transit time” i.e. the time it takes for the body's waste to be moved out of the
body, reduced exposure of the body to carcinogens or cancer causing
components in food and fluid, bowel protection, more increased fermentation
by the microflora or “bugs” in the bowel and increased amounts of a
compound called butyrate, the preferred energy source for cells called
colonocyctes. It decreases the fat products and promotes the peristalsis of
intestine, which has the effect to prevent colon cancer [Cummings et al, 1997].
Bamboo shoots, due to their high nutrient content, are finding a new place in
the spectrum of plants, fibre and foods used to enhance the quality of life.
Tremendous progress has been made in fresh shoot production, processing and
16 Rakesh Sharma, Bharati D Srinivas

utilization of edible bamboos, exploitation of bamboo beverage, extraction and


utilization of effective components from bamboo hydrolysate. Nearly 90
bamboo shoot based industries exist in Zhejiang province of China, and there
are 25 canning industries in Prachinburi province of Thailand [Thammincha,
1988]. Canned bamboo shoots occupy an important place in Thailand‟s global
trade providing an export earning of about US$ 30 million annually. In
contrast to being their routine component of everyday meals of Asian people,
in the western countries bamboo shoots are available only in canned form,
imported from other countries. This pattern is however, now gradually
changing and in Europe, shoots are available, though on a small scale from
groves planted near Bordeaux in France, Carasco in Italy and New South
Wales in Australia. But, since the local produce is not sufficient to meet the
market demand, bamboo shoots in Western markets and Australia are usually
imported in processed form. Bamboo shoots with its 6-8% fibre content can be
considered as “fibre rich” food and can meet the daily requirement of fibre in
the diet. German and US companies Qualicel and Vitacel market fibre
additives in white powder form with at least 95% fibre and bamboos being the
fastest growing plants, can provide raw material for production of such fibre
additives. Hence, efforts need to be made to popularize the consumption of
bamboo shoots from the health perspective.

REFERENCES
Benzhi, Z., Maoyi, F., Xiaosheng, Y. & Zhengcai, L. (2005). Ecological
function of bamboo forest: Research and Application. Journal of Forestry
Research, 16(2), 143-147.
Bystriakova, N., Kapos, V. & Lysenko, I. (2004). Bamboo Bioiversity- Africa,
Madagascar and the Americas. UNEP-WCMC/INBAR, A Bandson
production, UK.
Cummings, J. H., Roberfroid, M. B. & Anderson, H. (1997). A new look at
dietary carbohydrates: Chemistry, Physiology and Health. European
Journal of Clinical Nutrition, 51, 417-423.
DeVries, J. W., Prosky, L., Li, B. & Cho, S. (1999). A Historical Perspective
on Defining Dietary Fiber. Cereal Foods World, 44(5),367-369.
Gold, S. P., Man, I. L. Kader, A. A. & Keintz, C. (1999). Influence of
production, handling and storage on phyto nutrient content of foods.
Nutrition Reviews, 57 (9), S46 – S52.
Bamboo Shoots 17

Gopalan, C., Ramasastri, B. V. & Balasubramanian, S. C. (1971). Nutritive


value of Indian foods. ICMR Publ., Hyderabad, India.
INBAR. (1997). The plant with thousand faces. 5, 13.
Lee, S. & Prosky, L. (1995). International survey on dietary fibre definition,
analysis and reference materials. Journal of Association of Official
Analytical Chemists International. 78, 22- 36.
Madhab, J. (2003). The Green Gold: Under Exploited Wealth of the North-
East India. Dialogue, 5 (2), 45-52.
Nirmala, C., David, E. & Sharma, M. L. (2007). Changes in nutritive
components during ageing of emerging juvenile bamboo shoots.
International Journal of Food Sciences and Nutrition, 58(8), 612-618.
Shrestha, K. (1999). Distribution and status of bamboos in Nepal. In: Bamboo-
Conservation, Diversity, Ecogeography, Resource, Utilization and
Technology (eds. Rao, A.N. and Rao, V.R.). Proceedings of a training
course cum workshop, 10-17 May, 1998, Kunming and Xishuangbanna,
Yunnan, China and IPGRI-APO, Serdang, Malaysia.
Tewari, D. N. (1988). Bamboo as poverty allevator. Indian Forester, 114 (10),
610-612.
Thammincha, S. (1988). Some aspects of bamboo production and marketing.
In Bamboo: Current Research (eds. Rao, I.V.R., Gnanaharan, R. and
Sastry, C.B.), Proc. International Bamboo Workshop, Cochin, 14-18,
November, 1988, KFRI and IDRI, Canada, 1988, 320-326.
Trowell, H. & Burkitt, D. (1986). Physiological role of dietary fibre: a ten
year review. Journal of Dentistry for Children. 53, 444-447.
Van Soest, P. J. (1978). Component analysis of fibre in foods: Summary and
recommendations. American Journal of Clinical Nutrition, 31 (10), 75.
Whyte, D. L., McArthur, R., Topping, D. & Nestel P. (1992). Oat bran lowers
plasma cholesterol levels in mildly hypercholesterolemic men. Journal of
the American Dietetic Association, 92 (4), 446-449.
Foods, Diets and Disease
©
Editor: Rakesh Sharma, Bharati D Shrinivas 2009 Innovations And Solutions, Inc.
___________________________________________________________________________

Lecture 7

TROPICAL AND TEMPERATE FRUITS

Rakesh Sharma, Bharati D Shrinivas

Epidemiological studies provide evidence that plant-based foods play a


crucial role in the prevention of chronic diseases. The association between
dietary vegetable intake and chronic diseases is mainly attributed, along with
the dietary fiber constituent, to a wide range of plant secondary compounds
called phytochemicals. Fiber-rich foods are very good sources of these
phytochemicals, which include polyphenolics, carotenoids, plant sterols and
lignans. These so-called co-passengers, or co-travelers, of dietary fiber may
contribute to the nutritional benefits of fiber-rich food and are an essential part
of the healthful dietary fiber complex. Fruits are rich sources of various
vitamins, minerals and dietary fiber required by the human body for optimal
health. In addition, fruits are diverse in antioxidant composition and
antioxidant activity. The objective of this chapter is to highlight the potential
of tropical and temperate fruits as sources of dietary fiber with associated
antioxidant compounds.

INTRODUCTION
Epidemiological studies evidence that plant-based foods play a crucial role
in the prevention of chronic diseases. Specifically, consumption of fruit and
vegetables has been strongly associated with a decreased risk of disease by
2 Rakesh Sharma, Bharati D Shrinivas

modulating DNA damage, lipoprotein and protein oxidation, platelet


aggregation, leukocyte adhesion and vascular function 1-3. The association
between dietary vegetable intake and chronic diseases is mainly attributed,
along with the dietary fiber constituent, to a wide range of plant secondary
compounds called phytochemicals 4-7. Fiber-rich foods are very good
sources of these phytochemicals which include polyphenolics, carotenoids,
plant sterols and lignans. These so-called co-passengers, or co-travelers, of
dietary fiber contribute to the nutritional benefits of fiber-rich food and are an
essential part of the healthful dietary fiber complex 8.
Fruits are rich sources of various vitamins, minerals and dietary fiber
required by the human body for optimal health. In addition, fruits are diverse
in antioxidant composition and antioxidant activity 9. Either fruits from the
temperate zone, usually characterized by a large edible portion, or fruits from
tropical and subtropical regions, with relatively high ratios of non-edible
portion such as peels, seeds and stones, are rich in these antioxidant bioactive
compounds 10.
Reactive oxygen species (ROS) are generated in humans under
physiological conditions as a result of normal intracellular metabolism in
mitochondria and peroxisomes, as well as from a variety of cytosolic enzyme
systems. Oxidative stress arises from an imbalance between the production of
ROS and the natural antioxidant defense mechanisms of the body in favor of
the former 11-13. Increasing evidence suggests that inflammatory and
oxidative stress play a pivotal role in the development of certain degenerative
diseases associated with aging 14,15. Thus, the ability of exogenous
antioxidants from dietary intake to scavenge ROS in the human body—and
thereby to decrease the amount of free radical damage to biological molecules
like lipids, proteins and DNA—may be one of the protective mechanisms of
foods of plant origin.

TROPICAL AND TEMPERATE FRUITS AS A SOURCE OF


DIETARY FIBER AND BIOACTIVE COMPOUNDS
Some recent studies have pointed out that fiber derived from fruits may
possess associated bioactive compounds, and thus may be partially responsible
for the health benefits of high fruit intake 8. The selection of suitable sources
Tropical and Temperate Fruits 3

to provide new dietary fiber products with high antioxidant capacity derived
from natural associated compounds could be an appropriate tool with which to
achieve a better antioxidant status along with the recommended higher dietary
fiber intake 16. The objective of this chapter is to highlight the potential of
selected fruits as sources of dietary fiber with associated antioxidant
compounds.

Apple (Malus domestica Borkh., Rosaceae)

Apples have been characterized as a good dietary source of polyphenols,


along with dietary fiber. Polyphenol compounds are predominantly localized
in the peel. The major compounds isolated and identified included catechin,
hydroxycinammates, phloretin glycosides, quercetin glycosides and pro-
cyanidins 17,18. The relationship of hydroxycinnamic acids in the peel and
the peeled fruit of Golden delicious varieties have been studied in some detail
19. A significantly higher content in ferulic acid (16.76%), -coumaric acid
(29.50%) and caffeic acid (23.27%) in the peel than in peeled apples was
reported. Also, total polyphenols were significantly higher in peels (1.11 
0.12 g/100 g) than in peeled apples (0.69  0.07 g/100 g) (19). These authors
measured the content of total dietary fiber (AOAC method) in fractions (both
peeled and peels) of fresh apple fruits. Results showed significantly lower
dietary fiber (both soluble an insoluble) in peeled apples than in the peels. In a
second stage, Gorinstein and co-workers 20 evaluated the influence of peel
and peeled apple fruit on plasma antioxidant capacity on rats. A significant
increase in the plasma antioxidant activity in the group fed with whole apple in
comparison to the control group was found. In this research, when antioxidant
status was evaluated in plasma, an increase in the TRAP value and a decrease
in the biomarker of lipid oxidation, malondialdehyde—measured spectropho-
tometrically as thiobarbituric reactive substances—was found. It is worth to
mention that these authors stated that the correlation between dietary fiber and
its antioxidant activity by TRAP assay was very poor (R2 = 0.3267).
Interestingly, the effect of baking on dietary fiber, phenolics and total
antioxidant capacity was investigated using a model system of muffins
incorporated with dried apple skin powder (ASP) as a value-added food
ingredient. As a result, the total dietary fibre content, total phenolic content
and total antioxidant capacity of muffins were positively correlated to the
amount of ASP incorporated into muffins 21.
4 Rakesh Sharma, Bharati D Shrinivas

Citrus Fruits

Citrus fruits have a high content of phenolics, dietary fiber, ascorbic acid,
and trace elements. In order to define the antioxidant potential of certain citrus
fruits and to propose the most preferable one for dietary prevention of
cardiovascular and other diseases, a systematically comparison of peel and
pulp of grapefruit (Citrus paradise, Macfad), oranges (Citrus sinensis, L.) and
lemons (Citrus limonis, Osbeck ) was done 22. The range of the total dietary
fiber content was 2.43–2.54 and 1.29–1.34 g/100 g fresh citric fruits for peels
and peeled fruits, respectively. No significantly difference was found among
the citrus fruits tested. The contents of total soluble, and insoluble dietary fiber
in peels were significantly higher than in peeled fruits. The peeled lemons,
oranges and grapefruits contained 164, 154 and 135 mg/100g, and their peels
190, 179 and 155 mg/100g of total polyphenols, respectively. The content of
total polyphenols in peeled lemons and their peels was significantly higher
than in peeled oranges and grapefruits and their peels, respectively.

Grape (Vitis sp., Vitaceae)

Apart from oranges, grapes are the world´s largest fruit crop. About 80%
of the total crop is used in wine making and pomace (grape skins and seeds)
represents approximately 20% of the weight of grapes processed 23.
Dietary fiber is a major fraction in many by-products from the agricultural
industry 23. Grape pomace is a good source of dietary fiber and polyphenolic
associated compounds 24. The phenolic composition of grape pomace varies
considerably, depending on grape, variety and technology of wine making
(specifically, duration of pomace content, cell disruption, during crushing, and
pressing) 25. The antioxidant capacity of grape pomace 25,26 has led to the
development of a new concept of antioxidant dietary fiber. Grape pomace is
shown as a product that combines in a single material the physiological effects
of both dietary fiber and antioxidants 27. This natural product should have a
content of dietary fiber of up to 50% (dry matter basis), and one gram exerts
the same radical scavenging activity as 50 mg of DL--tocopherol (DPPH
assay). More recently, the dietary fibre content and antioxidant activity of two
by-products of the vinification process—pomace and stem—of Manto Negro
Tropical and Temperate Fruits 5

red grape was measured. Both by-products presented high contents of total
dietary fibre, comprising three-fourths of the total dry matter; remarkably, the
stem by-product showed large amounts of extractable polyphenols (11.6%).
The free radical-scavenging capacity of the former by-products was
determined using the DPPH method, resulting in EC50 values of 0.46 g dm/g
DPPH (stem) and 1.41 g dm/g DPPH (pomace) 28.

Pear (Pyrus communis L., Rosaceae)

To underline the importance of using whole fruit, Gorinstein and co-


workers 26 assessed the bioactivity of pears using in vitro and in vivo
models. In line with the findings of other authors 29,30, the content of
dietary fiber, phenolic acids, flavonoids and total polyphenol were
significantly higher in peels than in pulps. The assays used to evaluate the
potential antioxidant activity—inhibition of linoleic acid oxidation measured
by the bleaching of -carotene as indicator, and scavenging of nitric oxide and
DPPH- radicals—by pear fractions exerted similar trends. An inhibition
percentage of 65.3%, 66.0% and 53.6% was found in the case of peels,
respectively; whereas 24.5%, 20.7% and 9.9% was found in the case of pulps,
respectively. That is, the antioxidant potential was significantly higher in peels
than in pear pulp. In both fractions, a correlation was found between the
antioxidant potentials and the total polyphenol content. On the other hand,
dietary fiber content was higher in pear peel than in pulp. In contrast, a poor
correlation was observed between the nitric oxide values and the content of
total dietary fiber (R2 = 0.2937). In a model rat, diets supplemented with pear
peel exerted a significantly higher positive influence on plasma antioxidant
capacity (TRAP and malondialdehyde values) of rats than diets with fruit pulp.

Peach (Prunus persica Batsch., Rosaceae)

Goristein’s group 20,21 studied the dietary fiber content and related
polyphenols in the pulp and peel fractions of peaches (Catherina variety), and
compared it with their radical scavenging measured by the TRAP assay. The
content of soluble and insoluble dietary fiber in pulp was lower than in peel in
6 Rakesh Sharma, Bharati D Shrinivas

fresh peaches. In both fractions, insoluble was higher than soluble fraction. In
general, hydroxicinammic acids showed higher significant content in peel
from peach than in peeled peaches, i.e., 34.96% for -coumaric acid and
25.06% for caffeic acid, whereas no difference between both fractions was
observed in the case of ferulic acid. TRAP value was higher in peel than in
peeled fraction.

Guava (Psidium guava L., Myrtaceae)

The results of recent research 15 indicated that pulp and peel fractions of
guava showed high content of dietary fiber (48.6–49.4%) and total
polyphenols (2.6–7.8%). The antioxidant activity of enriched polyphenol
extracts from these fractions was studied, measuring scavenging of DPPH
radical and protection towards Cu-induced oxidation of in vitro human low-
density lipoprotein. Also, reducing ability was tested using ferric reducing
antioxidant power assay (FRAP). All fractions tested showed a remarkable
antioxidant capacity, and its activity was correlated with the corresponding
total phenolic content. Peel fractions, with higher content of polyphenol than
pulp, showed a better antioxidant capacity in the three models. Guava fruit was
proposed as a useful ingredient rich in dietary fiber with associated antioxidant
compounds. A high correlation among the assays tested and the amount of
total polyphenols was found. It is worth mentioning that because guava is one
of the best sources of vitamin C among fruits, it is likely that vitamin C
contributed to a relatively high extent to the antioxidant activity measured in
the hydrosoluble extracts in the three models tested.

Mango (Mangifera indica L., Anacardiaceae)

Mango is one of the most important tropical fruits. This fruit has gained
increasing relevance in the European market 24. The peel proportions in
mango fruit range from 20% to 30% of the whole fruit. Recently, the need has
been stressed to develop technological processes for the preparation of high
dietary fiber powders from orange or mango fruits. Specifically, the washing
and drying processes are the two steps that could influence to a greater extent
the losses of the associated bioactive compounds 31. Larrauri and co-
workers 29 evaluated the antioxidant activity of powdered dietary fiber
Tropical and Temperate Fruits 7

samples obtained from mango peels. This product was obtained from fresh
mango peels by successively wet-milling, washing with water and a drying
process. In addition, soluble sugars were removed to increase soluble dietary
fiber content and total polyphenols. As a result, an insoluble and soluble
dietary fiber content of 43.4% and 28.1%, respectively, and a content of
polyphenols of 7.0%—dry matter basis—were given. At a concentration of
0.05%, the inhibition of temperature-induced oxidation of linoleic of mango
peel dietary fiber was 0.75 times as effective as 2-terc-butyl-4-hydroxianysol
and, 3.4 times higher than that of DL--tocopherol.
Another approach deals with the incorporation of mango peel powder in
soft dough biscuits to improve dietary fiber content and antioxidant properties
as nutraceutical properties of the biscuits. The results indicated that wheat
flour incorporated with mango peel powder (10%) yielded dietary fiber-
enriched biscuits with improved antioxidant properties 32.

Pineapple (Ananas comosus L. Merr., Bromeliaceae)

A dietary fiber powder obtained from pineapple fruit shell has been
characterized as having a high total dietary fiber content (70.6%) 33. The
insoluble fraction amounted to 99% of the total dietary fiber. At a
concentration of 0.5 of powdered sample/100 mL in the final mixture,
pineapple fibers showed higher antioxidant activity (86.7%) than orange peel
fiber (34.6%) in the inhibition of linoleic acid oxidation. Myricetin is the
major polyphenol identified (59% of total polyphenols) in pineapple fiber,
which could be responsible for the antioxidant activity. To get a balanced fiber
composition, pineapple fiber could be mixed with another dietary fiber product
with a high soluble dietary fiber fraction.

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[24] Larrauri, JA; Sánchez-Moreno, C & Saura-Calixto, F. (1988). Effect of
temperature on the free radical scavenging capacity of extracts from red
and white grape pomace peels. J Agric Food Chem, 46, 2694-2697.
[25] Saura-Calixto, F. (1988). Antioxidant dietary fiber product: A new
concept and a potential food ingredient. J Agric Food Chem, 46, 4303-
4306.
[26] Leontowicz, M; Gorinstein, S; Leontowitcz, H; Krzeminski, R; Lojek,
A; Katrich, E; Cíz, M; Martín-Belloso, O; Soliva-Fortuny, R; Haruenkit,
10 Rakesh Sharma, Bharati D Shrinivas

R & Trakhtenberg, S. (2003). Apple and pear peel and pulp ant their
influence on plasma lipids and antioxidant potentials in rats fed
cholesterol-containing diets. J Agric Food Chem, 51, 5780-5785.
[27] Bocco, A, Cuvelier, M-E; Richard, H & Berset, C. (1998). Antioxidant
activity and phenolic composition of citrus peel and seed extracts.
J Agric Food Chem, 46, 2123-2129.
[28] Llobera, A & Cañellas, J. (2007). Food Chem, 101, 659-666. Dietary
fibre content and antioxidant activity of Manto Negro red grape (Vitis
vinifera): pomace and stem.
[29] Larrauri, JA; Rupérez, P & Saura-Calixto, F. (1997). Mango peels fibres
with antioxidant activity. Z Lebensm Unters Fosrch A, 205, 39-42.
[30] Mahattanatawee, K; Manthey, G; Luzio, G; Talcott, ST; Goodner, K &
Baldwin, EA. (2006). Total antioxidant activity and fiber content of
select Florida-grown tropical fruits. J Agric Food Chem, 54, 7355-7363.
[31] Larrauri, JA. (1999). New approaches in the preparation of high dietary
fibre powders from fruit by-products. Trends Food Sci Technol, 10, 3-9.
[32] Ajila, CM; Leelavathi, K & Prasada Rao, UJS. (2008). Improvement of
dietary fiber content and antioxidant properties in soft dough biscuits
with the incorporation of mango peel powder. J Cereal Sci, 48,319-326.
[33] Larrauri, JA; Rupérez, P & Saura-Calixto, F. (1997). Pineapple shell as a
source of dietary fiber with associated polyphenols. J Agric Food Chem,
45, 4028-4031.
Foods, Diets and Disease
©
Editor: Rakesh Sharma, Bharati D shrinivas 2009 Innovations And Solutions, Inc.
___________________________________________________________________________

Lecture 8

RELATIONSHIP OF PHYSICAL ACTIVITY AND


DIETARY FIBER CONSUMPTION

Rakesh Sharma, Bharati D Shrinivas

ABSTRACT
Background
Increasing fruit and vegetable intake in the general population is one
of the major concerns and aims of health promotion programs around the
world. The teenage years are an important transition period from
childhood to adulthood, when patterns of behavior and lifestyle choices
are developing that will affect their current and future health. The heavy
consumption of fast food and lack of adequate fruit and vegetable intake
among adolescents in most developed countries are of increasing concern.
Objective
The study aims to examine the fruit and vegetable consumption,
physical activity levels and body mass index among teenagers.
Methodology
A total of 203 adolescents (115 males and 88 females, mean age 13.5
years) from a secondary school participated in the study, in which their
body mass index, dietary habits and physical exercise pattern were
recorded.
Results
Participants’ intake of fruits and vegetables was inadequate, with half
consuming ≤ 1 serving of fruit per day and the great majority (90%)
consuming < 3 servings of vegetables per day. The reported reasons for
the low consumption of fruits and vegetables included dislike of these
foods, (47%), lack of availability of fruits and vegetables at home (25%),
and habitually dining out (28%). Physical activity levels were far from
2 Rakesh Sharma, Bharati D Shrinivas

optimal, with almost 50% of the participants not performing any form of
exercise during the previous seven days, and only 28% having done some
form of exercise during the week prior. Only 22% reported doing
moderate amounts of exercise.

Conclusion
In light of the inadequate consumption of fruits and vegetables, low
physical activity level, and high prevalence of overweight and obesity
found in this study, educational initiatives are urgently needed to
encourage teenagers in Hong Kong to adopt a healthier diet and more
active lifestyle.

INTRODUCTION
Diet is one of the most potent tools for the prevention of certain types of
cancer (US Department of Health and Human Services, 2000; Willet, 2000),
preventing or reversing non-communicable diseases such as diabetes, and
improving cancer survival (Physicians Committee for Responsible Medicine,
2008). It is noted that lifelong dietary preferences are formed based on the
eating patterns that are established in youth.
However, the dietary pattern of teenagers is found to be inadequate to
meet the dietary recommendations for health. Youngsters in the United States
and in Hong Kong tend to eat snack food products that are high in sugar or fat
but low in disease-preventing ingredients such as fruits and vegetables (Krebs-
Smith et al., 1996; Peterson and Sigman-Grant, 1997; Tse & Benzie, 2006).
The food preferences of the younger population are far from optimal, with
consumption of desserts and snacks (such as cakes and ice-cream) four times
or more per day, and fried food also being eaten four times or more per day
(Lee & Tsang, 2004).
It is estimated that 19% of gastrointestinal cancers, 31% of ischemic heart
disease and 11% of stroke worldwide is related to inadequate intake of fruits
and vegetables (World Health Organization, 2003). In this regard, it is
worrying that no US state reportedly achieves the national targets for fruit and
vegetable consumption as recommended by WHO (World Health
Organization, 2003; Center for Disease Control, 2008a). Likewise, even
though the traditional Chinese diet is regarded as a healthy one, people in
Hong Kong have been found to consume inadequate amounts of fruits and
vegetables (Lam, Chan and Ho, 2002).
Fruit and Vegetable Consumption and Physical Activity 3

Sedentary lifestyles have also become a concern, with more than 50% of
adults in the US not doing enough physical activity and 24% of them inactive
for most of the day. The population in Hong Kong is also found to be
physically inactive. It is reported that adults spend an average of 2.4 hours per
day watching television, while teenagers spend 3 hours per day watching
television (Lam, Chan and Ho, 2002; The Boys’ and Girls Club Association of
Hong Kong, 2003).
Laziness and lack of interest in exercise are the main causes for physical
inactivity (Center for Health Protection, 2005). It is worrying to find that
teenagers in Hong Kong are physically inactive and exercise less than their
counterparts in other developed countries (Hui et al., 2001). Secondary school
children were found to spend too much time on computers, homework and
other relevant activities, with the amount of time spent on sports and exercise
low (Lam, Chan and Ho, 2002).
The teenage years are an important transition period from childhood to
adulthood, when patterns of behavior and lifestyle choices are developing that
will affect their current and future health (Centers for Disease Control and
Prevention, 2008a). The objective of the present study was to examine health
status, dietary habits and physical activity among teenagers in Hong Kong.
The findings will give more insight for planning and implementing nutrition
and lifestyle education programs to modulate dietary and behavioral changes
for health promotion and lowering the risk of age-related disease.

MATERIALS AND METHODS


Research Design

An experimental pre-post study design was selected, at the school level.


After gaining ethical approval from the Ethics Committee of the University,
teenagers in a secondary school in Hong Kong were approached and invited to
join our study.

Sample Description

The secondary school was located in Shatin, a residential district in Hong


Kong in which 76% of the population is aged below 65. Shatin is an urban
4 Rakesh Sharma, Bharati D Shrinivas

district with public and private housing estates. The majority of the residents
were middle class with a satisfactory socio-economic status.

Procedure

The researcher and research assistants visited the school and performed a
health assessment for the participants, including body weight, height, body fat
content, and body mass index (BMI), and subjects completed a questionnaire
regarding their dietary habits and physical exercise pattern. The questionnaire
was developed by the research team. To address the content validity, five
experts, including two dietitians, two physiotherapists and one occupational
therapist, reviewed the questionnaire; the content validity index was 0.804.
The test-retest reliability was also established (r = 0.861) by repeat testing
among 10 secondary two students over two weeks.

RESULTS
Characteristics of the Participants

The study population included a total of 203 teens in year 8 of school


education. There were 115 males and 88 females (male: 56.7%; female:
43.3%), and the mean age was 13.5 years.

Body Mass Index

Table 1 shows the Body Mass Index (BMI), calculated using the Asian
standard (Choo, 2002). It was found that 17% of the participants were
overweight or obese, while body fat content was found to be slightly high to
high in 26% of the participants. No significant difference was found between
boys and girls in their BMI, yet more boys than girls were found to have
slightly high or high body fat content, and this result was statistically
significant (p<0.05). Please see that table 1.
Fruit and Vegetable Consumption and Physical Activity 5

Table 1. Body Mass Index and Fat Composition of the Participants


(n=203)

Overall % % by Gender p valuea


Boys Girls
n=115 n=88
BMI
Underweight 12.3 10.4 14.8 0.27
Normal 70.9 73.0 68.2
Overweight 8.4 6.1 11.4
Obese 8.4 10.5 5.6
Fat composition
Low 15.3 1.7 33.0 0.00*
Normal 58.6 65.2 50.0
Slightly high 15.8 20.9 9.1
High 10.3 12.2 8.0
a
Chi-square test was used to compare boys’ and girls’ data. A p value of < 0.05 was
considered statistically significant.

Dietary Patterns

Intake of fruits and vegetables was inadequate among the participants. As


shown in Table 2, around half of the participants (49.8%) consumed ≤ 1
serving of fruit per day and most of them (90.1%) consumed < 3 servings of
vegetables per day. The reported reasons for the inadequate consumption of
fruits and vegetables included dislike of eating fruits and vegetables (46.7%),
lack of availability of fruits and vegetables at home (25%), and habitually
dining out (28.3%).

In terms of intake of fried food and desserts, the majority of the participants
ate less than one serving per day. The overall intake for less healthy food (fish
balls, candies/chocolates, French fries, and ice-cream) as snacks was higher than
that of healthy food choices (low fat biscuits, bread, fruits).
6 Rakesh Sharma, Bharati D Shrinivas

Table 2. Dietary Habits Among Participants, Overall and by Gender


(n=203)

Overall (%) % by Gender p valuea


Male n=115 Female n=88
Fruit consumption per day
None 2.0 1.9 2.1 0.87
≦ 1 serving 47.8 49.6 45.3
2-3 servings 45.2 44.6 45.8
> 3 servings 4.4 2.3 2.1
Vegetable consumption per day
None 0.4 0.0 1.1 0.71
< 3 servings 89.7 89.6 89.8
3-5 servings 7.9 7.8 8.0
> 5 servings 2.0 2.6 1.1
Fried food consumption per day
< Once 49.5 48.2 51.2 0.66
Once 34.0 34.2 33.7
Twice 14.0 14.0 14.0
Dessert consumption per day
< Once 33 37.7 29.5 0.62
Once 37.4 38.3 36.4
Twice 24.6 22.6 27.3
> Three times 4.9 3.5 6.8
a
Chi-square test was used to compare boys’ and girls’ data. A p value of < 0.05 was
considered statistically significant.

Table 3. Snack Preferences among Participants (n=203)

Overall (%) % by Gender p valuea


Male Female
n=115 n=88
Preferred Snacks
a. Healthy food
Biscuits 6.8 6.3 6.2 0.68
Sponge cakes/bread 7.1 7.4 8.1
Fruit 12.8 12.4 13.2
b. Less healthy food
Fish balls 16.8 16.4 16.7 0.77
Fruit and Vegetable Consumption and Physical Activity 7

Candies/chocolates 16 17.4 17.3


Fries/potato chips 23.7 27.1 18.3
Ice-cream 16.8 18.0 16.2
Snacks Expected to be Sold in Tuck Shop
a. Healthy food
Sushi 32.2 34.8 32.2 0.46
Wheat bread 5.1 4.1 5.3
Fruit 12.1 13.2 10.1
b. Less healthy food
Cup noodles 7.3 8.3 7.6 0.55
Fish balls 13.7 15.2 13.1
Fried chicken wings/legs 12.7 13.4 12.9
Ice-cream 16.9 15.0 14.8
a
Chi-square test was used to compare boys’ and girls’ data. A p value of < 0.05 was
considered statistically significant.

No significant difference was found between boys and girls in their fruit
and vegetable consumption, intake of fried food and desserts, preference for
snacks and expectations regarding items sold in the school tuck shop (p<0.05).
(tables 2 & 3 near here)

Physical Activity

Table 4 shows the total hours reportedly spent performing exercise and
sitting (being sedentary) in the previous seven days. It was found that about
half of the participants had not performed exercise of any form in the previous
seven days, and 40% of them seldom or rarely walked up the stairs. No
significant difference was found between boys and girls in the frequency of
doing exercise, indicating that all participants had been quite physically
inactive over the previous seven days (p>0.05). Also, the average sitting
(sedentary) hours per day in the previous seven days was around 9 hours,
although boys were found to be less sedentary than girls (p<0.05).
(table 4 & 5 near here)
8 Rakesh Sharma, Bharati D Shrinivas

Table 4. Physical Activity Habits Among Participants (n=203)

Overall (%) % by Gender p valuea


Male n=115 Female n= 88
% Reporting Doing Exercise During Previous Seven Days
None 48.8 47.2 47.6 0.50
< 3.5 23.6 23.7 29.9
≧ 3.5 27.6 29.1 22.5
Daily Physical Activity Reported
a. Walking up stairs instead of taking the elevator
Always 12.8 16.5 14.8 0.00*
Sometimes 42.5 38.3 47.7
Seldom 26.6 28.7 23.9
Rarely 15.2 16.5 13.8
b. Doing housework
Always 12.8 7.8 19.3 0.00*
Sometimes 50.2 46.6 55.7
Seldom 28.1 32.2 22.7
Rarely 8.9 13.9 1.3
c. Declining transportation or increasing walking time to destination
Always 12.8 11.3 14.8 0.00*
Sometimes 30.0 22.1 26.1
Seldom 32.0 27.8 37.5
Rarely 25.1 27.8 21.6
a
Chi-square test was used to compare boys’ and girls’ data. A p value of < 0.05 was
considered statistically significant.

Table 5. Participants’ Daily Average Hours of Sitting During the Previous


Seven Days (n=203)

Hours Per Day (Mean ± SD)


Sitting Duration Per Day in Previous Seven Days p valuea
Total 8.95 ± 4.77 0.000*
Male (n=115) 7.40 ±3.93
Female (n=88) 10.98 ± 5.03
a
Paired t test was used to compare boys’ and girls’ data. A p value of < 0.05 was
considered statistically significant.
Fruit and Vegetable Consumption and Physical Activity 9

DISCUSSION
The present study showed inadequate intake of fruits and vegetables, as
well as less healthy snack choices, among the majority of participants. Also,
the prevalence of overweight and obesity was quite high (17.5%) and around
one quarter had high body fat content. It was also worrying to find that
physical activity levels were far from optimal, with almost half the participants
not performing any form of exercise and less than one third having done some
form of exercise during the previous week. These findings are consistent with
previously reported dietary habits, prevalence of obesity and physical activity
levels among teenagers in Hong Kong and worldwide (World Health
Organization, 2000; Department of Health of Hong Kong, 2002; Center for
Disease Control and Prevention, 2008b; Centers for Disease Control and
Prevention, 2008c).
The finding of inadequate consumption of fruits and vegetables indicates
the need for nutrition education. People are born with great sensitivity to bitter
tastes and so children tend to avoid foods with bitter flavors. Examples of
such foods include cabbage, broccoli, spinach, Brussels sprouts, and grapefruit
juice. Nevertheless, these foods, as well as many other fruits and vegetables,
contain phytochemicals that may reduce the risk of cancer and other diseases
(DeBruyne, Pinna & Whitney, 2008). The information regarding fruits and
vegetables and their role in cancer prevention needs to get across to teenagers
and their parents in order to encourage them to make healthier choices of food
and consume more fruits and vegetables.
Adult behavior patterns and lifestyle choices are developed in the teenage
years, and will subsequently affect their current and future health (Centers for
Disease Control and Prevention, 2008a). It is therefore important to
disseminate information regarding optimum dietary habits and physical
activity among teenagers. In the Ottawa Charter (1986), health promotion is
defined as the process of enabling people to increase control over and to
improve their health (Bhuyan, 2004). This means that health should be seen in
the context of people’s lives, and as a resource for life rather than an ‘ideal’
state free of disease. To enable us to increase control and to improve our
health, strategies including building public policy, creating supportive
environments, strengthening community action are needed, as well as
developing personal skills via health education programs and activities
(Bhuyan, 2004). Nutrition behavior is a complex process. Thus, a holistic
10 Rakesh Sharma, Bharati D Shrinivas

health education and health promotion approach would appear to be essential


in successfully implementing nutrition education programs.
It is important to create a supportive environment in the school grounds by
making healthy foods easily available, having strategically placed information
regarding nutritional quality and food labels, providing incentives to promote
dietary behavior change, and providing easy access to sound nutrition advice
via professional websites. Teenagers’ independent purchases of snack foods
are very much influenced by environmental factors such as availability,
advertising and marketing strategies (Hill et al., 1998). In this regard, school
tuck shops should provide more healthy food, such as fruits, and reduce their
stock of unhealthy food, including salt- and fat-laden food such as deep-fried
chicken wings, chicken legs and potato chips.

CONCLUSION
Most of the healthy teenagers who participated in the present study had a
normal BMI, but a high tendency to being overweight and obese was seen in
the group. Also, their largely inactive, sedentary lifestyle implies the need for
improved health behavior. It is important to provide nutrition education in the
school environment. It would be beneficial to integrate structured and
culturally suitable dietary and lifestyle programs into the secondary school
curriculum. Likewise, an environment more supportive of health-promoting
lifestyles among students should be provided by making available the
resources and opportunities for increasing physical activity, enforcing healthy
dietary guidelines in the school cafeteria, and providing proper weight
management measures in the school grounds.

REFERENCES
Bhuyan, K. K. (2004). Health promotion through self-care and community
participation: elements of a proposed programme in the developing
countries. BMC Public Health, 4, (11).
Center for Disease Control and Prevention. (2008a). Chronic Disease
Prevention. [Cited 11 August, 2008.] Available from URL:
http://www.cdc.gov/ node.do/id/0900f3ec801e457a.
Fruit and Vegetable Consumption and Physical Activity 11

Center for Disease Control and Prevention. (2008b). Chronic Disease


Prevention. [Cited 11 August, 2008.] Available from URL:
http://www.cdc.gov/nccdphp/
Centers for Disease Control and Prevention. (2008c). Overweight and Obesity.
[Cited 11 August, 2008.] Available from URL: http://www.cdc.gov/
nccdphp/dnpa/obesity/index.htm
Center for Health Protection. (2005). A Qualitative Study on Dietary and
Exercise Practices of People in Hong Kong (Main Report). [Cited 8
October, 2008]. Available from URL: http://www.cheu.gov.hk/files/
professional/Focus_group_EN.pdf
Choo, V. (2002). WHO reassesses appropriate body-mass index for Asian
populations. The Lancet, 20, 360(9328) 235.
DeBruyne, L. K., Pinna, K. & Whitney, E. (2008). Nutrition and diet therapy.
principles and practice. Australia: Thomson.
Department of Health. (2002). Prevalence of Childhood Obesity in Secondary
School, 1997-2002. Hong Kong: Department of Health.
Hill, L., Casswell, S., Maskill, C., Jones, S. & Wyllie, A. (1998). Fruit and
vegetables as adolescent food choices in New Zealand. Health Promot
International, 13(1), 55-66.
Hui, S.C., Chan, C. M., Wong, S. H. S., Ha, A. S. C. & Hong, Y. (2001).
Physical activity levels of Chinese youths and its association with physical
fitness and demographic variables: The Hong Kong youth fitness study.
Research Quarterly for Exercise and Sport, 72(Supplement), A92-3.
Krebs-smith, S. M., Cook, A., Subar, A. F., Cleveland, L., Friday, J. & Kahle,
L. L. (1996). Fruit and vegetable intakes of children and adolescents in the
US. Archives of Pediatrics & Adolescent Medicine, 150 81-86.
Lam, T. H., Chan, B. & Ho, S. Y. (2002). A Report on the Healthy Living
Follow-up Survey 2001. Hong Kong.
Lee, A. & Tsang, C. K. (2004). Youth risk behaviour in a Chinese population:
a territory-wide youth risk behavioural surveillance in Hong Kong. Public
Health 2004, 118, 88-95.
Peterson, S. & Sigman-Grant, M. (1997). Impact of adopting lower-fat food
choices on nutrient intake of American children. Pediatrics, 100 E4.
Physicians Committee for Responsible Medicine. Nutrition Education
Curriculum. [Cited 11 August 2008.] Available from URL:
http://www.pcrm.org/resch/ edresources/nutr_curr/.
Tse, M. Y. M. & Benzie, I. F. F. (2006). Personal perspective of a group of
nursing students in Hong Kong on diet and health: implication for nurse
education. Asian Journal of Nursing, 9 (2), 74-77.
12 Rakesh Sharma, Bharati D Shrinivas

The Boys’ and Girls’ Club Association of Hong Kong. (2003). Taipei, Hong
Kong and Shanghai Children’s Living Survey. Hong Kong: The Boys’ and
Girls’ Clubs Association of Hong Kong.
US Department of Health and Human Services, eds. (2000). Healthy People
2010: Understanding and Improving Health. Washington, DC: US
Government Printing Office.
Willett, W. C. (2000). Diet and cancer. Oncologist, 5, 393-404.
World Health Organization. (2000). Obesity: Preventing and managing the
global epidemic report of a WHO consultation. WHO Technical Report
894. Geneva: WHO.
World Health Organization. (2003). Diet, Nutrition and the Prevention of
Chronic Diseases. Geneva: WHO.
World Health Organization. (2006). Ottawa Charter for Health Promotion,
1986.
[Cited 11 August 2008.] Available from URL: http://www.euro.who.int/
AboutWHO/Policy/20010827_2.
Foods, Diets and Disease
©
Ed:Rakesh Sharma, Bharati D Shrinivas 2009 Innovations And Solutions, Inc.
___________________________________________________________________________

Lecture 9

CARDIOVASCULAR PREVENTION BY SOLUBLE FIBER


(PLANTAGO OVATA HUSK):
PLASMA TRIGLYCERIDES AND APOLIPOPROTEIN B TO APOLIPOPROTEIN A-I
RATIO IN MEN

Rakesh Sharma, Bharati D Shrinivas

ABSTRACT
Background: New dietary strategies to reduce cardiovascular disease (CVD) risk include, as part
of secondary prevention, the addition of fiber to the diet.

Objective: To study the effect of treatment with soluble-fiber derived from Plantago ovata (Po)
husk on lipids, in 28 men with CVD and plasma low density lipoprotein (LDL)-cholesterol
concentration ≤3.35 mmol/L (≤130 mg/dL). A low-saturated-fat diet supplemented with 10.5 g/d
Po husk was consumed for 8 weeks, under controlled conditions.
Results: Following Po husk treatment, plasma apolipoprotein (apo) A-I increased 4.3% (P<0.01
and plasma triglycerides decreased 6.7% (P<0.02), the apo B 100/ apo A-I ratio decreased 4.7%
(P<0.02) from (mean ±SD) 0.77±0.10 to 0.73±0.19.

Conclusion: In secondary prevention of CVD, Po husk treatment induces a reduction in the


apoB/apoA-I ratio, a measure that is identified as a strong new risk factor for CVD and which is a
target for lipid-lowering therapy; the lower the apoB/apoA-I ratio, the lower is the risk. No effect
on plasma LDL cholesterol was observed. Possible Po husk modes-of-action will be proposed.

INTRODUCTION
Dietary fiber intake can reduce the risk of cardiovascular disease (CVD)(1) by acting, in part,
on plasma lipid concentrations. Several studies have shown that soluble fibers are more
effective in lowering blood cholesterol than are insoluble fibers(2).
2 Rakesh Sharma, Bharati D Shrinivas
Current advice on lowering cholesterol concentrations and reducing CVD risk by therapeutic
lifestyle changes include increasing the amount of dietary fiber consumption, specifically of
soluble (viscous) fiber, to 10–25 g/d(3-6). Thus, an increased soluble fiber intake, within a
therapeutic lifestyle, takes on an essential modality in the clinical management of CVD risk
reduction(3-6).
Differences in genetic background are known to affect lipid metabolism and the response to
diet(7,8). Identifying common variations in genes involved in the intestinal absorption of lipids
and, hence, in the dietary response to soluble fiber is an attractive goal.
We investigated Plantago ovata (Po) husk, ((Madaus, S.A., Barcelona, Spain), a source of
natural, concentrated, soluble fiber obtained from the outer membranous green envelope of the
Po seed. Po husk and Po seeds, the latter often used as a source of insoluble fiber for
experimental control purposes in trials, are well-tolerated, safe, and effective bulk laxatives(9).
The purpose of our study was to assess, in a crossover design protocol, the effects of 10.5 g/d
soluble Po husk fiber compared to 10.5 g/d of an equivalent insoluble fiber added as
therapeutic measures to a diet low in saturated fat and cholesterol. The target variables
measured were plasma lipids, lipoproteins and apolipoproteins in patients with established
coronary heart disease but with plasma low density lipoprotein (LDL)-cholesterol ≤3.35
mmol/L (130 mg/dL)(9). The potential interactions with genes involved in the response to
dietary fiber therapy were explored, as well.
The insoluble fiber used as control additive was hemicellulose and lignin Klason obtained
from Po seeds (Madaus S.A., Barcelona, Spain). The two products are obtained from the same
Po plant; Po husk consisting only of the epidermis and collapsed adjacent layers removed from
the dried ripe Po seeds(9).
In a controlled, single-blind, crossover study consisting of a 4 week dietary adaptation period,
28 patients were randomly assigned to two different fiber-supplement periods of 8 weeks each.
We incorporated a washout period of 8 weeks between the 1st and 2nd periods of the study in
order to test possible interactions between treatment and sequence order (carry-over effect)(9).
In our study in men with CVD following a low saturated-fat low-cholesterol diet, the
incorporation of Po husk significantly reduced plasma triglyceride concentrations by 6.7%, apo
B:apo A-I ratio by 4.7% and increased the apo A-I concentration by 4.3%(9).
The plasma triglyceride reduction observed with the Po husk treatment was approximately half
that of the reduction (between 10% and 16%) observed with statin therapies (the most widely
used LDL-cholesterol-lowering drugs) and was similar to that of ezetimibe (7% triglyceride
reduction). With this modest
hypotriglyceridemic effect, Po husk can be considered as an adjuvant treatment in patients
with moderate hypertriglyceridemia(10).
The Apo B/apo A-I ratio has been shown to be the best marker of atherogenic and anti-
atherogenic particles in plasma. The lower the apoB/apoA-I ratio means the lower CVD risk.
The INTERHEART study showed this ratio to be a marker of risk of myocardial infarction,
irrespective of the geographic regions of the populations studied(11). Recently, the apo B/apo
Beneficial Effects of Soluble Fiber 3
(12)
A-I ratio was linked to the risk of fatal stroke in a similar manner to that of myocardial
infarction, and other ischemic events(12).
Po husk significantly increased high density lipoprotein (HDL)-cholesterol concentrations by
6.7% relative to the insoluble fiber (9). This finding is of considerable clinical relevance
because studies on the secondary prevention of CVD have shown that the recommended LDL-
cholesterol lowering diets (low in saturated fat and cholesterol) have, as well, the detrimental
effect of decreasing HDL-cholesterol concentrations.
In our study, Po husk did not produce the LDL-cholesterol-lowering effect observed with other
soluble fibers. This could have been due to several factors. For example, our patients
consumed a more palatable and more tolerable low dose (10.5 g/d) than did the subjects in
another study in which the dose of the same Po husk preparation was higher (14 g/d)(13).
Reports indicate that the initial concentration of cholesterol is predictive of the subsequent
reduction in cholesterol concentrations induced by some soluble fibers(14). In our study, the
low-moderate basal LDL-cholesterol concentration of the patients (mean: 3.04 mmol/L;
maximum: 3.36 mmol/L) was highly predictive of the failure of dietary Po husk
supplementation to lower LDL cholesterol concentrations.
Of note is the finding that both fibers (soluble and insoluble) significantly reduced the patients’
waist circumference and waist-to-hip ratio. This aspect has not been previously reported.
Hence, supplementing a low-fat diet with fiber may have an additional benefit on CVD risk
factors by reducing abdominal fat without appearing to have a significant effect on the BMI(9).
A limitation of the present study is, probably, the small sample size of our study population.
Also, an influence of the genes studied such as fatty acid binding protein (FABP-2) as well as
apo A-IV, and apo E genes and their variants on the response to the dietary fiber intervention
cannot be ruled out(9). However, following the Po husk treatment, the carriers of the Thr54
allele of FABP-2 had significantly higher HDL cholesterol (8%) than did those who had the
insoluble fiber added to the diet.
Our results indicate that soluble Po husk treatment induced a more favorable effect on the
lipoprotein profile (i.e. reduction in CVD risk factors) than did a comparable insoluble fiber.
The lowering of plasma triglycerides by Po husk has been observed in obese Zucker rats(15).
The variables such as higher systolic blood pressure, plasma concentrations of triglycerides,
total cholesterol, FFA, glucose, insulin, and TNF-α, and the hypoadinectinemia that occurred
in obese Zucker rats consuming the control diet for 25 weeks, were significantly improved in
those fed the 3.5% Po husk fiber-supplemented diet. These data indicate that treatment of a Po
husk-supplemented diet prevents endothelial dysfunction, hypertension, and obesity
development, while ameliorating dyslipidemia and abnormal plasma concentrations of
adiponectin and TNF- α in obese Zucker rats(15). The mode of action of soluble fiber remains
unclear. Diet and its nutrients can modify the expression of genes involved in CVD(7,8).
Therefore, an assessment of how dietary components such as fiber can affect the expression of
genes and proteins involved in CVD is a relevant clinical issue and would elucidate biological
action mechanism of the nutrient. The effect of soluble fiber on lipid metabolism in intestinal
4 Rakesh Sharma, Bharati D Shrinivas
cells has been poorly studied(16). Short chain fatty acids (SCFAs) produced by anaerobic
bacterial intestinal fermentation of soluble fiber are proposed as regulating lipid metabolism in
the intestine. However, the exact mechanism of action of SCFAs in the intestine remains
unknown. The presence of SCFAs in the colon as well as the small intestine has been well
documented(17-21). Intestinal anaerobic bacterial fermentation of fiber produces mainly acetate
(Ac), propionate (Pr) and butyrate (Bu) in a quasi-constant proportion, and with a molar
relation of 60:25:15(21). Following the consumption of a high fiber diet, 90–95% of the SCFAs
produced are absorbed by the human colon(22).
To improve the knowledge of health-benefit mechanisms of SCFAs, we studied the effects
of SCFAs on gene expression in a human enterocyte cell line (Caco-2/TC-7)(16). The
transcriptional profiles of Caco-2/TC-7 cells following SCFA exposure in vitro were evaluated
with a whole genome array (AB Human Genome Survey Array). The SCFAs used were
acetate (Ac), propionate (Pr), and butyrate (Bu) at different concentrations ranging from 2 to
20 mM. SCFA concentrations have been described as ranging from 1 to 13 mM from the
jejunum to the ileum(17) while other reports indicate a luminal range of Bu between 10 and 20
mM in the intestinal lumen(18). Total RNA was then isolated for microarray and quantitative
real-time PCR analysis. Our results showed that treatment of human enterocytes with Pr and
Bu has an impact on a wide variety of genes. These genes were classified according to the
PANTHER classification system, and the results showed that 7 and 9 biological processes
were significantly affected by Pr and Bu, respectively. Further, the results from the
classification of genes differentially expressed by Pr and Bu showed that 4 and 11 metabolic
pathways, respectively, were significantly altered; including the intestinal cholesterol
biosynthesis pathway. The differential array expression analysis showed that 9 genes of this
cholesterol biosynthesis pathway were down-regulated (Figure 1). These results were validated
by real-time PCR.
Mevalonate formation from acetyl CoA is the first fundamental step of cholesterol
biosynthesis. At this step, Pr and Bu decreased the expression of 3-hydroxy-3-methylglutaryl-
CoA (HMGCoA) synthetase 2 by 2.9-fold and 4.3-fold, respectively, compared to controls. In
addition, both fatty acids decreased by 1.5-fold the expression of 3-hydroxy-3-methylglutaryl-
CoA reductase (HMGCoAR), the key enzyme in the cholesterol synthesis process. In the
second fundamental step of this pathway, which is the transformation of mevalonate into
squalene, Bu decreased the expression of isopentenyl diphosphate isomerase (IDI1) by 3.6-
fold, the expression of dimethylallyl/geranyl trans-transferase (FDPS) by 3.2-fold, and the
expression of the farnesyl-diphosphatase farnesyltransferase (FDFT1) by 2.3-fold.
In the last fundamental step in the process, which is the transformation of squalene into
cholesterol, Pr and Bu decreased the expression of lanosterol 5-desaturase (SC5DL) by 2.4-
fold and 2.5-fold, respectively, and the expression of methylsterol mono-oxygenase
(SC4MOL) by 7.8-fold and 9.8-fold, respectively. Moreover, Pr decreased the expression of
the peroximal trans-2-enoyl-CoA reductase (PECR) by 2.2-fold.
Beneficial Effects of Soluble Fiber 5

Figure 1. Cholesterol biosynthesis pathway. Downregulated genes are represented in grey boxes.
6 Rakesh Sharma, Bharati D Shrinivas
We selected FDPS, SC4MOL, and HMGCoAR for validation by Real-Time PCR. In addition,
we also selected SQLE because it is a novel target in the design of new hypocholesterolemic
drugs.
The effects of Pr and Bu on the expression of FDPS, SC4MOL, HMGCoAR, and SQLE genes
were evaluated by real-time PCR. We showed that treatment of TC-7 enterocytes with Pr and
Bu decreased, with variable intensities, the mRNA levels of all the genes tested. Pr and Bu had
a dose-dependent effect on FDPS mRNA levels. Further, the inhibitory effects of Pr and Bu on
the expression of HMGCoAR were 16% and 33% respectively. For SC4MOL and SQLE, the
inhibitory effect of Pr and Bu showed variable intensity. Our results are in agreement with a
previous study in which SCFAs suppress cholesterol synthesis in the intestine(23). However,
from our results we are unable to conclude a definitive mechanism for the cholesterol-reducing
effect of soluble fiber.
Our in vitro study enabled us to identify a wide variety of biological processes and metabolic
pathways affected by the SCFAs tested. Importantly, our results show that the overall effect of
Pr and Bu is to down-regulate the expression of 9 key genes involved in intestinal cholesterol
biosynthesis and, hence, to inhibit this pathway. Bu appears to be the SCFA with the most
important impact on enterocyte gene expression.
Of interest in future studies would be gene expression after treatment with SCFAs obtained
from the in vitro fermentation of soluble fiber, in an intestinal fermentation model system. In
vitro fermentation is a non-invasive and time-efficient method used to assess fiber
fermentability in vivo.
These studies need to be conducted with triacylglycerol and apo A-I because both are, at least
in part, produced in intestinal cells and are modified by Po treatment.
Increasing evidence indicates that genetic factors influence the relationship between dietary
components and disease risk, and that various protective and risk factors affect the incidence of
disease(7,8).
It is now possible to visualize differences between the genetic profiles of individuals at the
molecular level and to begin to understand how they relate to differences between individuals’
responses to physiological factors at the level of the whole organism.
Also, new techniques can be applied to gain an understanding of the way differences between
other aspects of an individual’s molecular biology affect the response to physiological factors.
These techniques are known collectively as “omics”. The study of the total genetic makeup
(the genome) known as genomics, and the analysis of the total metabolic profile (the
metabolome) defined as metabolomics, can provide ways to understand the influence of
dietary components on disease. Also, transcriptomics and proteomics are the terms that
describe the study of the total gene expression and the total protein complement of an
organism, respectively (7,8).
Our transcriptomic approach with respect to intestinal cells suggests that a relationship could
exist between fiber, plasma lipids, and atherosclerosis. However, the mechanisms involved
remain unknown.
Beneficial Effects of Soluble Fiber 7
Dietary fiber treatment may reduce the risk of CVD, but the processes involved would need to
be elucidated by “omics” in order to clarify the new mechanisms of action of fiber. In the
future, the situation will arise in which it would be possible for individuals to make truly
informed choices regarding which foods provide the best opportunities for health, well-being
and reduced risk of disease.
Finally, it is important to focus on the overall quality of the diet, which includes fiber and other
individual foods or nutrients, such that there is a balance in energy intake and expenditure,
while enhancing regular physical activity to maintain health.

REFERENCES
[1] Pereira, MA; O’Reilly, E; Augusstsson, K; et al. (2004). Dietary fiber and risk of
coronary heart disease. A pooled analysis of cohort studies. Arch Intern Med., 164:370-6.
[2] Petchetti, L; Frishman, WH; Petrillo, R & Raju, K. (2007). Nutriceuticals in
cardiovascular disease: psyllium. Cardiol Rev, 15;116-22.
[3] Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in
Adults. (2001). Executive summary of the third report of the National Cholesterol Education
Program (NCEP). JAMA, 285:2486-97.
[4] Grundy, SM; Cleeman, JI; Bairey Merz, N; et al. (2004). Implications of recent clinical
trials for the National Cholesterol Education Program Adult Treatment Panel III Guidelines. J
Am Coll Cardiol, 44:720-32.
[5] Lichtenstein, AH; Appel, LJ; Brands, M; et al. (2006). Diet and lifestyle
recommendations revision 2006: a scientific statement from the American Heart Association
Nutrition Committee. Circulation, 114:82-96.
[6] Graham, I; Atar, D; Borch-Johnsen, K; et al. (2007). European guidelines on
cardiovascular disease prevention in clinical practice: executive summary. Eur Heart J,
28:2375-414.
[7] Subbiah MT. (2008). Understanding the nutrigenomic definitions and concepts at the
food-genome junction. OMICS, 12:229-35.
[8] Ordovas, JM & Tai, ES. (2008). Why study gene–environment interactions? Curr Opin
Lipidol, 19:158-67.
[9] Solà, R; Godàs, G; Ribalta, J; et al. (2007). Effects of soluble fiber (Plantago ovata husk)
on plasma lipids, lipoproteins, and apolipoproteins in men with ischemic heart disease. Am J
Clin Nutr, 85:1157-63.
[10] Brunzell JD. (2007). Hypertriglyceridemia. N Engl J Med, 357:1009-17.
[11] Yusuf, S; Hawken, S; Ounpuu, S; et al. (2004). Effect of potentially modifiable risk
factors associated with myocardial infarction in 52 countries (the INTERHEART study): case-
control study. Lancet, 364:937-52.
8 Rakesh Sharma, Bharati D Shrinivas
[12] Walldius, G; Aastveit, AH & Jungner, I. (2006). Stroke mortality and the apoB/apoA-I
ratio: results of the AMORIS prospective study. J Intern Med, 259:259-66.
[13] Sierra, M; García, JJ; Fernandez, N; et al. (2002). Farmafibra Group. Therapeutic effects
of psyllium in type 2 diabetic patients. Eur J Clin Nutr, 56:830-42.
[14] Brown, L; Rosner, B; Willett, WW & Sacks, FM. (1999). Cholesterol-lowering effects of
dietary fiber: a meta-analysis. Am J Clin Nutr, 69:30-42.
[15] Galisteo, M; Sánchez, M; Vera, R; et al. (2005). A diet supplemented with husks of
Plantago ovata reduces the development of endothelial dysfunction, hypertension, and obesity
by affecting adiponectin and TNF-alpha in obese Zucker rats. J Nutr, 135:2399-404.
[16] Alvaro, A: Solà, R; Rosales, R; et al. (2008). Gene expression analysis of a human
enterocyte cell line reveals down regulation of cholesterol biosynthesis in response to short-
chain fatty acids. IUBMB Life, 60:757-64.
[17] Cummings, JH; Pomare, EW; Branch, WJ; et al. (1987). Short chain fatty acids in human
large intestine, portal, hepatic and venous blood. Gut, 28:1221-7.
[18] Mortensen, PB & Clausen, MR. (1996). Short-chain fatty acids in the human colon:
relation to gastrointestinal health and disease. Scand J Gastroenterol, 216 (suppl.):132-48.
[19] Roy, CC; Kien, CL; Bouthillier, L; et al. (2006). Short-chainfatty acids: ready for prime
time? Nutr Clin Pract, 21:351-66.
[20] Wong, JM; de Souza, R; Kendall, CW; et al. (2006). Colonic health: fermentation and
short chain fatty acids. J Clin Gastroenterol, 40:235-43.
[21] Garcia Peris, P; Breton Lesmes, I; de la Cuerda Compes, C; Camblor Alvarez, M &
Colonic metabolism of fiber. (2002). Nutr Hosp, 17(Suppl 2):11-6.
[22] Schmitt, MG Jr; Soergel, KH; Wood, CM; et al. (1977). Absorption of short-chain fatty
acids from the human ileum. Am J Dig Dis, 22:340-4.
[23] Hara, H; Haga, S; Aoyama, Y; et al. (1999). Short-chain fatty acids suppress cholesterol
synthesis in rat liver and intestine. J Nutr, 129:
942-8.
Foods, Diets and Disease
Rakesh Sharma, Bharati D Shrinivas © 2009 Innovations And Solutions, Inc.
___________________________________________________________________________

Chapter 10

PROCESSING TECHNIQUES AND THEIR


EFFECT ON FRUIT PHYTOCHEMICALS

Rakesh Sharma, Bharati D Shrinivas

1. ABSTRACT
Fruits are an excellent source of essential vitamins, minerals, and
dietary fiber in the human diet. They are also a rich source of secondary
metabolites that proving to play an important role in protection against
numerous chronic diseases. These substances are almost ubiquitous in
plant-derived foods and inherently have more subtle effects than
nutrients. Carotenoids and flavonoids, the mostly spread secondary
metabolites in fruits, have received much attention over the past decade
due to their putative health-protective effects. A significant portion of the
fruits consumed are processed and many of the processed products are
stored in a variety of packaging materials for extended periods of time
prior to consumption. Bioactive compounds that are naturally present in
fruits may undergo transformations during food processing that neither
decrease their nutritional value nor bioactive value but may increase it by
favoring their absorption and metabolism in the human body. Thus, in
this chapter there is a significant need to understand how the different
processing methods used in the food industry may modify their contents,
structure, and biological activity in humans.

Key words: Fruits; Phytochemicals; Food processing


2 Rakesh Sharma, Bharati D Shrinivas

2. INTODUCTION
Broadly speaking, nutrients are classified into two groups, namely
macronutrients (also called energy producing nutrients or energy-yielding
nutrients) and micronutrients (which are characterized by their essentiality for
human health and the low quantities in which they need to be ingested).
Energy-producing nutrients include carbohydrates, fats, and proteins.
Micronutrients often refer to vitamins and minerals. Phytochemicals, also
called bioactive compounds, are substances present in foods in low levels that
may have a role in health maintenance in humans. Fruits have proved to be
essential for a balanced diet. This is believed to be mainly due to their content
of vitamins, fibers, and phytochemicals, the latter being responsible in part for
the antioxidant properties of fruits and foods of fruit origin. Manufacturing
processes are changing the nutritional properties of some foods. For instance,
partial hydrogenation of vegetable oil results in the formation of trans-fatty
acids, and heat treatment of protein solutions in an alkali environment results
in the formation of lysinoalanine. Both of these have been shown to have
detrimental health effects. On the other hand, some nutrients and bioactive
compounds that are naturally present in fruits may undergo transformations
during food processing that neither decrease their nutritional value nor
bioactive value but may increase it by favoring their absorption and
metabolism in the human body. Thus, in this chapter there is a significant need
to understand how the different processing methods used in the food industry
may modify their contents, structure, and biological activity in humans.

3. IMPORTANCE OF FOOD COMPOSITION DATABASES FOR


DIETARY RECOMMENDATION
Information on the retention of phytochemicals in processed foods is
much needed to update food composition tables, commonly used for
epidemiological and nutritional studies. In addition to compositional data on
proximate (water, protein, total lipid, total carbohydrate, and ash), minerals,
vitamins, lipid components, and amino acids databases have been developed
for dietary phytochemicals namely carotenoids and flavonoids (flavonols,
flavones, flavanones, flavan-3-ols and anthocyanidins), including a separate
Processing Techniques 3

database for proanthocyanidins. Documentation of the effects of processing on


bioactive phytochemicals would enable better interpretation of data with
respect to dietary habits and human health, which is a critical step for
formulating dietary recommendations. A greater understanding of such issues
would also be of great interest to food processors who wish to retain or
possibly boost levels of health-promoting compounds during manufacturing,
as well as substantiate health claims for phytochemicals present in their
products. Information on nutrient content of processed fruit is also of great
interest to consumers. Consumers are becoming more aware of health benefits
of fruits and are interested in knowing how fresh and processed products differ
in nutritional quality. Unfortunately, a common perception among consumers
is that processed fruits are inferior in nutritional quality compared to fresh
products. Although fruit nutritional quality can be compromised during
processing, there are instances where chemical reactions, interactions between
components, and release of bound components during processing can lead to
an increase in nutritional quality. Clearly, more information is needed on this
topic in order to better inform consumers about dietary choices. The goal of
this chapter is to provide a comprehensive review of the effects of thermal and
non-thermal processing techniques, freezing, packaging, and storage on
flavonoids, ascorbic acid and carotenoids, the major phytochemicals found in
fruits. The potential effects of processing unit operations on phytochemicals
will also be discussed and potential strategies to prevent losses identified
(Tomas Barberan and Gil, 2008).

4. EFFECTS OF COMPOUND SOLUBILITY, CELLULAR AND


STRUCTURAL LOCALIZATION ON PHYTOCHEMICAL
LOSSES
Phytochemical structure, which influences solubility, and cellular
localization of phytochemicals are two critical factors influencing their
retention during processing (Table 1). The relatively polar phytochemicals
including various classes of phenolics (hydroxybenzoic and hydroxycinnamic
acids) and flavonoids (primarily anthocyanins, flavonols, flavones,
procyanidins) are readily leached into water during blanching and syrups/
brines during canning. In contrast carotenoids are well retained during
blanching and canning due to their non-polar nature and resistance to leaching,
although losses can occur due to thermal degradation and oxidation.
4 Rakesh Sharma, Bharati D Shrinivas

Localization of phytochemicals within the plant cell can also influence losses
during processing. Phenolics compartmentalized in the vacuole and apoplast
are readily lost as a result of membrane disruption following thermal
treatments, while cell wall bound phenolics resist leaching and may be more
easily extracted or bioavailable after processing due to tissue softening
(Dewanto et al., 2002a). The carotenoids are contained within chloroplasts or
chromoplasts in membrane-protein complexes. After thermal processing,
carotenoids are more easily extracted from plant tissues due to tissue softening
and/or destruction of the membrane-protein complex. This phenomenon is
commonly cited as one of the reasons why many processed fruits have higher
levels of carotenoids than their fresh counterparts (Shi and Maguer, 2000;
Dewanto et al., 2002b), although other factors such as leaching of soluble
solids into the liquid canning medium and inactivation of carotene oxidizing
enzymes may also explain the apparent increase (Sa and Rodriguez-Amaya,
2004).

Table 1. Properties of Phytochemicals in Plant Tissues


(Adapted From Kalt, 2005)

Feature Carotenoids Phenolics


Numerous, for example, Numerous, for example, phe-
lutein, lycopene, nolic acids, hydrozycinna-
Subgroups
α- carotene, β-carotene, mates, flavonoids inc. flavor-
zeaxanthin; β-cryptoxanthin nols, catechins, anthocyanins
Solubility Lipid Water
Associated with membrane
Cellular Dissolved in vacuole and
protein, complexes in
Localization apoplast
chloroplast or chromoplast
Anthocyanins preferentially
Some types (for example,
in peel; proanthocyanidins
Structural tomato lycopene) preferen-
in peel and seed;
Localizati tially in surface tissues like
hydroxycinnamates in flesh;
peel and outer pericarp
ellagitannins in seeds
Processing Techniques 5

5. EFFECTS OF PROCESSING UNIT OPERATIONS ON


PHYTOCHEMICAL LOSSES
There are many potential sites where phytochemical losses can occur
during canning operations (Table 2). Raw materials must be washed
thoroughly to remove dirt and debris prior to blanching and minor losses of
water soluble phytochemicals may occur if epidermal tissues have been
damaged. In fruit canning where blanching is typically not employed due to
the delicate nature of fruit tissue, the heat-labile enzyme polyphenol oxidase
can cause considerable losses of chlorogenic acid and flavonoids prior to its
inactivation through pasteurization. Physical removal of tissues such as skins
and seeds during processing can have a substantial effect on phytochemical
retention. In many fruits, carotenoids and flavonoids located predominantly in
epidermal tissues are removed by peeling operations (manual or lye assisted)
prior to processing, which can greatly reduce concentrations of the compounds
in processed products. In a study on peaches, manual and lye-assisted peeling
methods resulted in 1.5 and two-fold reductions in total phenolics, respectively
(Asami et al., 2003b). Removal of seeds prior to processing can also result in
losses of phenolics residing in seeds such as procyanidins and ellagitannins.
Significant quantities of lycopene and flavonols in tomatoes can be lost during
peeling since the skin contains about three-fold higher levels of lycopene than
the whole fruit and 98% of the flavonols are located in the skin (Stewart et al.,
2000).
The byproducts of fruit processing represent promising sources of
compounds with bioactive properties that could be exploited in the
development of novel food ingredients and dietary supplements (Schieber et
al., 2001). The size and type of fruit product (whole, halved, quartered, diced,
sliced, julienne, puree) may influence phytochemical retention due to variation
in the surface area of abraded surfaces. Although a study has not been
conducted comparing phytochemical retention in different piece sizes of fruits,
it is likely that the higher number of disrupted cells and larger surface areas of
small pieces would promote greater leaching of water-soluble phytochemicals.
Fruit purees are distinctly different from other canned products in that only a
small amount of water is added to control product consistency. Greater
retention of water-soluble phytochemicals in purees in comparison to products
canned in water or syrup would be expected since in purees the compounds are
trapped within the product matrix, whereas extensive leaching can occur in
products canned in aqueous media. The composition of brine is another
6 Rakesh Sharma, Bharati D Shrinivas

important consideration, since specific phenolic compounds can precipitate


when exposed to acidic conditions. The type of retort method used for
sterilization can also impact the phytochemical content of canned fruits.
In a static system cans or jars remain stationary throughout the
sterilization process resulting in slow heat penetration throughout the can and
lengthy process times. In more elaborate rotary processing systems, the cans or
jars may be rotated end-over-end or allowed to rotate on their axes during the
sterilization process. The agitation incurred via rotary processing increases
heat penetration throughout the can and greatly reduces process time, enabling
greater color and texture retention (Abbatemarco and Ramaswamy, 1994). In
addition to the retort method, the sterilization time and temperature can
influence the retention of phytochemicals. The degradation of anthocyanins
(Ahmed et al., 2004; Kirca et al., 2006) and carotenoids (Shi et al., 2003)
follow first-order reaction kinetics, hence high temperature short-time
processing is recommended for maximizing the retention of anthocyanins and
carotenoids (Lin and Chen, 2005) in foods. Although losses of naturally
occurring antioxidant phytochemicals in fruits occur during sterilization, the
formation of Maillard reaction products can result in elevated antioxidant
capacity (Nicoli et al., 1999). The antioxidant activity of Maillard reaction
products has been attributed to the high molecular weight brown melanoidin
compounds formed in the advanced stages of the reaction (Anese et al., 1999).

Table 2. Potential Sites and Processing Variables Influencing the


Phytochemical Content of Processed Fruits

Process unit operation Variables influencing phytochemical content


Washing Spray vs immersion
Water vs steam
Blanching
Time and temperature
Peeling Amount of tissue removed
Particle size reduction Degree of exposed surface area
Screening Physical removal of skins and seeds
Fruit/vegetable to brine ratio
Brining
Brine composition
Static vs agitated
Thermal processing
Time and temperature
(adapted from Hager and Howard, 2006)
Processing Techniques 7

6. THERMAL PROCESSING TECHNIQUES INFLUENCING


PHYTOCHEMICAL CONTENT OF FRUIT
6.1 Survey of Industrial Processes

The greatest quantity of processed fruit is preserved by heat treatment. A


wide range of practical and theoretical knowledge is needed for an industrial
process, as Figure 1 illustrates. Food processing involves the fields of
microbiology, plant biology, thermophysics, food rheology and chemistry,
packaging technique, unit operations, reactor techniques, construction and
materials science, machinery, and electrophysics. The most important factors
(besides the nature of the raw material and type of product) for constructing a
plant are as follows:

type and size of the container (e.g., from small cans up to large tanks),
and
mode of heat treatment (e.g., batch or continuous pasteurization of
closed containers; full aseptic process or some combination;
temperature and pressure above or below 100°C and absolute pressure
of 100 kPa).

In order to preserve the color of processed fruit, selection of fruit with


optimal pigmentation is recommended. Undesirable compounds such as
melanoidins and melanins are formed in browning reactions. The major
pigments in fruits are chlorophylls, carotenoids, and anthocyanins.
Chlorophylls are lipid soluble pigments that disappear during fruit ripening.
Carotenoids are subdivided into xanthophylls and carotenes. Xanthophylls are
yellow (as in Golden Delicious apples and bananas), and carotenes are red
(such as lycopene, the major pigment in watermelon and pink grapefruit flesh)
and orange (the ß-carotene found in orange and apricots). Anthocyanins are
water soluble pigments responsible for the red, blue, and purple of many fruits,
flowers, and vegetables. All these pigments related to color are destroyed
during thermal processing. The chlorophylls are degraded to brown
pheophytins (Diane, 2005), the carotenoids are converted to epoxides, and the
anthocyanins are rapidly degraded. During heat processing, anthocyanins react
quite readily with the metal walls of nonlacquered cans. Thus, it is necessary
to lacquer the cans to protect both the product and the can, as the color will
usually pass out into the syrup during processing. There are two types of
8 Rakesh Sharma, Bharati D Shrinivas

discoloration associated with anthocyanins. The first type occurs when


leucoanthocyanins are converted to anthocyanins during canning. This causes
the characteristic pink discoloration of pears and the excess red color in
peaches. Other fruits that undergo pink discoloration include guava, lychee,
and banana. The second type involves enzymic browning, which can be
prevented by blanching and the addition of ascorbic acid, citric acid, and malic
acid. Heat processing may also cause the formation of various zinc complexes
such as in canned kiwifruit slices (Cano and Marin, 1992).

(adapted from Hui et al., 2006)

Figure 1. Co-operation between science and technology for achievements in heat


treatment processes.
Processing Techniques 9

6.2 Effects of Thermal on Carotenoids

In most studies investigating processing effects on carotenoid retention


and isomerization the sterilization process has been delivered in a still or static
mode. The high temperatures required to achieve commercial sterility of low
acid canned fruits (>120°C) generally do not result in significant losses of
carotenoids, but significant trans to cis-isomerization of carotenoids is evident
The percent increase in total cis isomers on a percent basis as a result of
thermal processing occurred in tomatoes (18%), and peaches (10%) (Lessin et
al., 1997). In various commercial tomato products total cis isomers of
lycopene increased 3.6% to 6% in response to thermal processing (Nguyen and
Schwartz, 1998). In addition to process duration and temperature, food matrix
components such as oil or fat have been shown to increase lycopene
isomerization in tomatoes (Schierle et al., 1996). The major cis isomer of the
provitamin A carotenoids in thermally processed red, yellow, and orange fruits
was 13-cis, with much lower amounts of 9-cis and 15-cis isomers. In thermally
processed tomato products the major cis isomer of lycopene is 5-cis lycopene
followed by smaller quantities of 9-cis and 13-cis lycopene and other
unidentified cis isomers (Schierle et al., 1996).
Carotenoids are well retained in fruit that receive a less severe thermal
treatment (pasteurization), with negligible losses reported for mango slices and
puree and papaya puree (Rodriguez Amaya, 1999). The increased levels of
carotenoids in fruits after canning have generally been attributed to several
factors including an unaccounted loss of soluble solids into the canning medium,
inactivation of enzyme systems that oxidize carotenes during extraction of fresh
samples, and increased extraction efficiency due to tissue softening and
disruption of carotenoid–protein complexes and carotenoid–dietary fiber
interactions. To assess the true retention of carotenoids in thermally processed
fruits it is important to correct for weight changes during processing in order to
account for losses of soluble solids and water. New extraction techniques for
fresh samples are needed that result in rapid inactivation of carotene oxidizing
enzymes and release of carotenoids bound to the sample matrices.

6.3 Effects of Thermal Processing on Flavonoids and Phenolic


Contents

The flavonoid content of fruits is affected by unit operations such as


peeling, chopping, and blanching as well as heating and cooking method. In
10 Rakesh Sharma, Bharati D Shrinivas

addition to processing factors, the retention of flavonoids and other


phytochemicals is significantly influenced by leaching of compounds in the
heating medium. Several studies have reported on flavonoid retention of fruits
processed under commercial conditions and results indicate that thermal
processing may enhance, reduce, or cause no change in flavonoids compared
to those in fresh samples. The influence of phytonutrient solubility on
retention during processing was demonstrated in a study on pasteurized yellow
banana pepper rings (Lee and Howard, 1999). After processing, only 37% of
ascorbic acid was retained, while 55% of quercetin and luteolin were retained,
and capsaicinoids were fully retained. Hence, the polarity of the compounds
(ascorbic acid > flavonoids > capsaicinoids) influenced their diffusion and
solubility into the brine during processing. The effects of three different
methods of processing tomatoes into sauce and paste on flavonoid content
were reported by Re et al. (2002). Only 9% of naringenin was retained after
processing tomatoes into sauce regardless of the processing method used,
whereas 71% and 75% of rutin were retained by super cold break (65°C under
vacuum) and hot break (90°C) methods, respectively, compared to only 48%
retained by the cold break (65°C) method.
The effects of three different time–temperature process conditions (101°C
for 40 min, 104°C for 10 min, and 110°C for 2.4 min) on the total phenolic
and procyanidin contents in clingstone peaches were reported by Asami et al.
(2003b). Peaches processed at 104°C for 10 min lost 21% of total phenolics,
those processed at 110°C for 2.4 min lost 11% of total phenolics, while those
processed at 101°C for 40 min retained comparable levels of total phenolics as
the raw material. Peaches processed at 104°C for 10 min were analyzed for
procyanidins with results compared to levels in frozen fruit. Marked
reductions, 49% and 88% of procyanidin monomers and dimers, respectively,
and a complete loss of procyanidin trimers through heptamers occurred in
thermally processed peaches. In a follow-up study, the authors determined that
loss of procyanidins in peaches during thermal processing was the result of
leaching from the fruit into the syrup (Hong et al., 2004).
Several studies have reported changes in anthocyanins and other
polyphenolics during canning of highly pigmented fruit. The total anthocyanin
and total phenolic contents of pitted Bing cherries canned in light syrup did
not change appreciably during canning, but approximately 50% of the
anthocyanins and phenolics were transferred from the fruit into the syrup
(Chaovanalikit and Wrolstad, 2004a). In another study involving canned Bing
cherries increased levels of anthocyanins, total phenolics, hydroxycinnamates,
epicatechin, and flavonol glycosides were observed after canning, which was
Processing Techniques 11

attributed to increased extraction efficiency of the softened fruit


(Chaovanalikit and Wrolstad, 2004b). Consistent with results from the first
study, approximately 50% of the polyphenolics were leached into the syrup
during canning. The anthocyanin content of fresh and frozen plums decreased
17% and 37%, respectively, during canning (Weinert et al., 1990a). During
canning decreased levels of anthocyanins in the skin were accompanied by
increased levels in the flesh and syrup, with uniformly distributed levels
obtained in the skin, flesh, and syrup after one week of storage. In a follow-up
study Weinert et al. (1990b) reported that anthocyanins losses during canning
of plums were not associated with changes in polymer concentration, and
suggested the losses were due to thermal destruction, irreversible binding, or
oxidation.
The effects of processing unit operations on the total phenolic and total
anthocyanin content of strawberry puree were studied by Klopotek et al.
(2005). The mashing step resulted in a 15% loss in total phenolics, but an
apparent 20% increase in total anthocyanins, which was thought to be the
result of enhanced extractability. After pasteurization an additional 11% of
total phenolics were lost, while total anthocyanin levels remained unchanged.
Vacuum treated purees had similar levels of total phenolics and total
anthocyanins than non-vacuum treated purees, indicating that the loss of
phenolics was not related to oxidation. Strawberry processing to produce jams
decreased the total ellagic acid content by 20% and the flavonoids by 15–20%
(Hakkinnen et al., 2000). Aguilar-Rosas et al., (2007) who observed that, the
high temperature sort time (HTST) treatment of apple juice caused a
considerable lost of phenols (32.2%) when compared with the PEF treatment,
which only caused a 14.49% reduction. Spanos and Wrolstad (1992) reported
that total phenol concentration is reduced up to 50% in apple juice pasteurized
thermally at 80°C for 15 min. Gardner et al., (2000) observed also
considerable losses in phenolics in apple juice pasteurized by thermal means.

6.4 Effects of Drying Processing on Phytochemical Content of


Fruit

6.4.1 Fruit Drying


Fruit drying has a long tradition. Inhabitants living close to the
Mediterranean Sea and in the Near East traded fruits that had been dried in the
open sun. Dried fruit is a delicacy, because of the nutritive value (66– 90%
carbohydrate) and shelf life. For example, inhabitants of hillside villages
12 Rakesh Sharma, Bharati D Shrinivas

isolated from the outside world by snow ate diets consisting primarily of seeds
and dried fruits. Today, the production of dried fruits is widespread. Nearly
half of the dried fruits in the international market are raisins, followed by
dates, prunes, figs, apricots, peaches, apples, pears, and other fruits.
Significant amounts of sour cherries, cherries, pineapples, and bananas are
also dried. Fruit may be dried as a whole (e.g., grapes, various berries, apricot,
plum, etc.), in sliced form (e.g., banana, mango, papaya, kiwi, etc.), in puree
form (e.g., mango, apricot, etc.), as leather, or as a powder by spray or drum
drying. Depending on the physical form of the fruit (e.g., whole, paste, slices),
different types of dryers must be used for drying. Figure 2 illustrates the wide
assortment of dryers that may be found in practice for drying of fruits. The
selection of fruit for drying depends on local circumstances and customs. For
example, in the Middle East, lemons with thin peel are dried whole. The taste
and aroma are preserved in the brownish inner fleshy part, which remains soft.
In the United States, blackberries, cowberries (ligonberries), and grapes are
dried, while in Spain, red grapes are dried. Apricots, dates, plums, and tropical
fruits are dried in the sun in several countries, while apples, pears, prunes, and
peaches are dried by artificial means. Dryers with natural air ventilation were
used in the 19th century in California for apple drying or to finish products
that have previously been dried by the sun. Fruits dried in the sun or in dryers
with natural air ventilation are referred to as “evaporated fruit,” while fruits
dried in dryers with artificial ventilation are described as “dehydrated fruits.”
The residual moisture content varies from small (3–8%) to large (16–18%)
amounts, according to the type of fruit. Significant amounts are packaged in
small portions (200–1000 g) in manufacturing plants. Often, countries
importing dried fruit repackage it to meet the needs of consumers and large
kitchens.

Figure 2. Various types of dryers for drying of fruit.


Processing Techniques 13

Fruit mixtures are widely consumed both in the United States and Europe.
Fruit is packed as a mixture or each component is packed separately in a
transparent, appealing packaging. Well-known components are round slices of
apples, apricots and peaches, pear halves, prunes, sour cherries, and dates.
Often, walnuts and almonds are also added to the mixture. Dried fruit is
widely used by the confectionery, baking, and sweets industries. Soup
manufacturing plants use dried fruits in the various sauces, garnishments,
puddings, and ice powders, and food for infants and children. Dried fruits are
used in various teas, e.g., rose hips, and by the distilling industry (dried
prunes, apricots). Applications include fruit powders processed from juices or
pulps that dissolve quickly. The development of the fruit powders was possible
through processing, which preserves color and flavor (vacuum drying,
lyophilization, and swelling). Artificial drying made it economically possible
to use raw materials at competitive prices and of high quality; examples are
apples, prunes, and rose hips. Various milling procedures make it possible to
dry highly valuable berries with soft flesh (strawberries, raspberries) and
mature stone-fruits (apricots, peaches) (Hui et al., 2006).

6.4.2 Effects Of Dehydration On Flavonoids And Phenolic Compounds


Dehydration is one of the oldest methods for preserving foods and is still
widely used in commercial manufacturing of dried fruit products. It is well
known that drying methods employing high temperatures and long drying
times result in thermal degradation of heat sensitive nutrients, including
carotenoids and flavonoids, which can adversely affect the color of dehydrated
products. Several different dehydration techniques available for preserving
fruits vary significantly in both drying temperature and duration. Solar drying
(SD) and conventional hot-air drying (HAD) methods require high
temperatures and long durations, parameters that are detrimental to the texture,
color, flavor, and nutritional quality of the product. Conversely, freeze drying
(FD) of foods, although expensive compared to other dehydration methods, is
a relatively benign method that minimizes thermal damage and generally
results in excellent retention of color, flavor, texture, and nutritional quality.
Vacuum microwave drying (VMD) has recently been promoted as an
alternative method to improve the quality of dehydrated products. By
combining the positive effects associated with vacuum (lower drying
temperature and rapid mass transfer) with those of microwave heating (rapid
energy transfer), products can be dried rapidly at lower temperatures.
Additionally, VMD reduces the exposure of nutrients to oxygen, a critical step
in minimizing oxidation of pigments responsible for acceptable product color.
14 Rakesh Sharma, Bharati D Shrinivas

The effects of different dehydration methods on the retention of


carotenoids and flavonoids in fruits have been studied. The studies indicate
that phenolic compounds are much more susceptible to thermal degradation
during dehydration than carotenoids, and that FD and VMD result in greater
retention of the compounds than HAD. In a study of Saskatoon berries, the
retention of total anthocyanins in FD, VMD, and HAD samples dried to
similar water activity levels of 73%, 49%, and, 18%, respectively, as
compared with levels found in fresh frozen berries (Kwok et al., 2004).
Different classes of phenolic compounds show marked differences in their
retention during dehydration, which appears to be related to their susceptibility
to enzymatic oxidation. Changes in the phenolic composition of raisins
subjected to three different dehydration treatments – sun-dried, dipped in hot
water and dried (dipped), and dipped in hot water, treated with sulfur dioxide
and dried (golden) – were compared with fresh and frozen Thompson seedless
grapes (Karadeniz et al., 2000). Procyanidins and flavan-3-ols were
completely degraded in all raisin samples, only 10% of the two major
hydroxycinnamic acids (caftaric and coutaric acids) were retained, while
approximately 38% of the major flavonols (quercetin and kaempferol
glycosides) were retained. Golden raisins retained higher levels of caftaric and
coutaric acids and had lower levels of oxidized cinnamic acids than the sun-
dried and dipped samples, suggesting that the sulfur dioxide treatment
ameliorated oxidation. The retention of flavonols among the three treatments
varied, with golden raisins retaining higher levels of one quercetin glycoside
but lower levels of rutin and two kaempferol glycosides than the sun-dried and
dipped raisins. Ferreira et al. (2002) studied the effect of sun drying on the
phenolic composition and content of a Portuguese pear (var. Bartolomeu).
Compared to fresh fruit only 4%, 9%, and 32% of hydroxycinnamic acids,
monomeric catechins, and procyanidins, respectively, were retained in the
dried fruit. Arbutin was the only phenolic compound that was not degraded
during sun drying presumably due to the low affinity of polyphenol oxidase
(PPO) for the compound. Results from this study also indicated that the loss of
large molecular weight procyanidins during sun drying was in part due to
irreversible binding to cell wall polysaccharides, a phenomenon that may
explain the sensorial loss of astringency in sun dried pears.
Shi et al. (1999) measured lycopene degradation and isomerization in
tomatoes subjected to osmotic-vacuum drying, vacuum drying, and HAD.
Osmotic-vacuum drying resulted in greater retention and less isomerization of
lycopene than the vacuum and air drying methods. This was explained by the
protective role of sugar present on the tomato surface in preventing oxygen
Processing Techniques 15

from penetrating and oxidizing lycopene. HAD tomatoes retained the least
amount of lycopene and contained the highest level of cis-isomers due to the
adverse effects of heat and oxygen.

6.5 Effects of Microwave Heating on Carotenoids, Phenolic


Acids and Flavonoids

Heating rate remains one of the major limitations for the optimization of
conventional thermal processes in which the heat is transferred through both
conduction and convection, although advanced equipment such as rotary retorts
and scraped-surface exchangers, etc., have been developed. Microwave heating
is one of the volumetric heating methods that has the potential to lead to a
quantum change in the ability of the food processor to achieve the heating rates
necessary to deliver profiles that could improve current UHT process routes
(Mullin, 1995). Microwaves used in the food industry for heating are of ISM
(industrial, scientific and medical) frequencies (2450 or 900 MHz,
corresponding to 12 or 34 cm in wavelength). In this frequency range the
dielectric heating mechanism dominates up to moderated temperatures. Polar
molecules, the dominant water try to align themselves with the rapidly changing
direction of the electric field. The energy to achieve this alignment is taken from
the electric field. When the field changes direction, the molecule “relaxes” and
the energy previously absorbed is dissipated into the surroundings, that is,
directly inside the food. This means that the water content of the food is an
important factor in the microwave heating performance of foods.
Microwave heating has been widely applied in industrial food applications
such as defrosting or thawing of frozen foods, drying, blanching, and
pasteurization. Sterilization using microwaves has been investigated for many
years but the commercial introduction of this technique has only come about in
the last few years in Europe and Japan. Microwave pasteurization and
sterilization promise to give very quick heat processing that should lead to small
quality changes due to the thermal treatment according to the HTST principle.
However, it has turned out that very high requirements of heating uniformity
must be met in order to fulfill these quality advantages (Ohlsson, 1991).
Microwave heating is reported to have varying effects on the retention of
carotenoids and phenolics. De Ancos et al. (1999) studied the effects of
microwave heating on carotenoid, chlorophyll, and anthocyanin contents of
fruit purees. Generally, microwave heating produced minor modifications of
the qualitative and quantitative composition of carotenoids in papaya and
16 Rakesh Sharma, Bharati D Shrinivas

anthocyanins in strawberry purees, but resulted in extensive losses of


chlorophyll a and b and xanthophylls in kiwi puree. In a study on apple
mashes, juice from four heat treatments (40°C, 50°C, 60°C, and 70°C) in a
2450 MHz microwave oven at 1500 W of Fuji and McIntosh apple mashes
were compared to juice from unheated mash. Microwave heat treatment of the
mash increased extraction of phenolics and flavonoids from apple mash and
resulted in juice with increased concentrations of total phenolics and
flavonoids. Therefore, microwave heating of apple mash before juice
extraction resulted in a high quality juice with increased phenolic and
flavonoid content as well as increased juice yield (Gerard and Roberts 2004).
Chlorogenic acid concentration increased 2–3 times after the microwave
heating of Idared apple puree, in comparison with control samples. Microwave
energy has the advantage of heating solids rapidly and uniformly, thus
inactivating the enzymes more quickly; it minimizes phenolic oxidation. The
addition of ascorbic acid and microwave heating significantly increased not
only chlorogenic acid concentrations, but also polymeric procyanidin
concentration from 145 mg/kg to 404 mg/kg and 620 mg/kg, respectively. (Jan
Oszmianski et al., 2008)

7. NON-THERMAL PROCESSING TECHNIQUES


INFLUENCING PHYTOCHEMICAL CONTENT OF FRUIT
Thermal processing is the most common method for extending the shelf
life of fruit products, by inactivating microorganisms and enzymes. However,
thermal processing can diminish the sensory and nutritional qualities of juices
(Pedro Elez-Martinez, 2007; Braddock, 1999).Consumer requirements for
foods are constantly changing. Today consumers demand foods that are both
fresh and natural. Therefore the steps used to process foods should be designed
to preserve their natural quality. Hence non-thermal processing techniques
such as high-pressure processing (HPP) and pulsed electric field (PEF) have
been attracting more attention from food scientists and engineers in recent
years not only because of their food preservation capabilities but also because
of their potential to achieve some interesting functional effects.
Processing Techniques 17

7.1 Effects of High-Pressure Processing on Carotenoids and


Flavonoids

High-pressure processing (HPP) is an excellent alternative to thermal


processing of fruits as pressures commonly used affect primarily covalent
bonds allowing for inactivation of microorganisms and enzymes, without
adverse effects on flavor and nutritional quality. Homogeneous foods are most
amenable to HPP, thus most of the research has focused on juices, purees, and
soups. Most studies consistently show that HPP does not significantly alter
levels of bioactive compounds or antioxidant activity of fruits. In a study of
tomato puree HPP (400 MPa/25°C/15 min) treated purees retained much
higher levels of individual and total carotenoids than purees subjected to low
(70°C/30 sec) and high (90°C/1 min) pasteurization treatments (Sanchez-
Moreno et al., 2006). In this as well as other studies of tomato (Sanchez-
Moreno et al., 2004) and persimmon (De Ancos et al., 2000) purees HPP
treatment increased the amount of extractable carotenoids compared with raw
purees. The enhanced extraction of carotenoids by HPP may be due to several
factors including membrane alteration, disruption of carotene–protein
complexes, and alteration of macromolecular structures such as proteins and
cell wall carbohydrates. Likewise, the carotenoid contents of a mixed juice
(orange, lemon, and carrot) and carrot juice (Butz et al., 2003), tomato
homogenate (Butz et al., 2002), and orange juice (Bull et al., 2004) were
unaffected by ultra high pressure treatments ˃600 MPa. The effects of HPP
treatments on flavonoids have received little attention. Anthocyanins in
strawberry jam are reported to be well retained after HPP processing compared
with heat-processed jam, but anthocyanins in HPP-treated jam were more
susceptible to degradation during storage (Gimenez et al., 2001). The greater
instability of anthocyanins during storage of HPP-treated jams as opposed to
heat-processed jams is most likely the result of residual enzymatic activities
(peroxidase, polyphenol oxidase, and ß-glucosidase) that are readily destroyed
by heat during traditional jam processing.

7.2 Effects Of Pulsed Electric Field Processing on Ascorbic Acid,


Carotenoids and Flavonoids

The pulsed electric field (PEF) process is a new and innovative non-
thermal minimal processing technology that is used as an alternative
preservation process for fruit juices. The aim of this technology is to inactivate
18 Rakesh Sharma, Bharati D Shrinivas

microorganisms and to decrease the activity of enzymes in order to increase


the shelf life of food products without undesirable heat and chemical effects.
The theoretical basis of PEF technology is the use of an external electric field
to destabilize cell membranes and form one or more pores in them. PEF
technology applies high voltage pulses (generally 20– 80 kV/cm) for very
short time (µs to ms), producing PEFs between two electrodes. This technique
is very similar to electroporation, used in cell biology and genetic
manipulation of cells. But, in the case of foods, the applied pulses are shorter
and much more intense. The aim of the application of high voltage pulses to
foods is not only to disrupt temporarily the cell membranes of
microorganisms. However, in this process, the microorganisms are also killed
or their numbers are drastically decreased by irreversible disruption of cell
membranes. PEF has been mainly applied to preserve the quality of foods,
such as to improve the shelf-life of bread, milk, orange juice, apple juice and
liquid eggs (Hui et al., 2006).
A high retention of vitamin C content in orange juice was observed after
PEF-processing with maximum values of 98.2% for PEF-treated orange juice.
Retention of this vitamin after PEF treatment was always above 87.5% for
orange juice, working with 35 kV/cm during 1000 µs at 200 Hz with
monopolar pulses of 4 µs. Min, et al. (2003a) reported no differences between
fresh orange juice and PEF-treated orange juice when they processed orange
juice at 40 kV/cm for 97 µs at 2000 Hz with bipolar pulses of 2.6 µs and a
maximum temperature of 45 °C. On the other hand, after processing orange
juice by PEF with bipolar pulses of 4 µs at 800 Hz and 35 kV/cm during 750
µs (maximum temperature of 50 °C), a vitamin C retention of 93% was
observed (Sanchez-Moreno et al., 2005). Evrendilek et al. (2000) reported that
PEF-processing of apple juice did not alter the natural vitamin C of the juice
(35 kV/cm, 94 µs, 952 Hz, monopolar pulses of 1.92 µs, maximum
temperature 38 °C). Min et al. (2003b) did not observed differences in vitamin
C retention between fresh and PEF-processed tomato juice at 40 kV/cm for 57
µs with bipolar pulses of 2 µs and a maximum temperature of 45 °C. When a
thermal pasteurization (90 °C, 1 min) was applied to orange juice, the retention
levels of vitamin C were 82.4%. Min, et al. (2003a, 2003b), and Sanchez-
Moreno et al. (2005) also reported higher levels of vitamin C retention in
orange and tomato juices treated by PEF compared with those processed by
thermal treatment. High temperatures led to a loss of vitamin C because heat is
known to speed the oxidation process of ascorbic acid (Gahler et al., 2003).
Moreover, the depletion of vitamin C in fresh juices is also attributed to
Processing Techniques 19

oxidative enzyme reactions promoted by ascorbate oxidase and peroxidase


(Davey et al., 2000).
Gemma et al., (2009) who found that watermelon juices treated at 25
kV/cm for 50 µs at 50 Hz using mono- or bipolar 1-µs pulses exhibited the
highest vitamin C retention (96.4–99.9%). On the other hand, vitamin C loss
was higher than 50% when PEF treatment was set up at 35 kV/cm for 2050 µs
at 250 Hz applying mono- or bipolar 7-µs pulses. Such severe conditions seem
to greater affect vitamin C retention in watermelon juice than in other juices
such as orange, orange–carrot or strawberry juices, which exhibited retention
of vitamin C above 80% (Odriozola-Serrano et al., 2009). Applying the same
PEF conditions, differences in vitamin C retention among PEF-treated juices
could be due to their different pH, since more acidic conditions are known to
stabilise vitamin C. Increased vitamin C retention of PEF-treated fruit juices in
monopolar mode may be related to inactivation of enzymes that catalyse
vitamin C oxidation. In PEF-treated orange juices, enzymes such as peroxidase
were more inactivated with monopolar pulses than with bipolar pulses (Elez-
Martinez et al., 2006). Loss of vitamin C in watermelon juice was accelerated
when increasing severity of PEF treatments. In accordance, the lower the
electric field strength, the treatment time, the pulse frequency or the pulse
width, the higher the vitamin C retention in orange, tomato and strawberry
juices. Lycopene retention in PEF-processed watermelon juice ranged from
87.6% to 121.2%. The content achieved with PEF treatments set up at 35
kV/cm with pulses of 250 Hz was slightly higher than that of untreated
samples. The application of 35 kV/cm at low frequency led to a decrease in the
lycopene content of treated watermelon juice of up to 10–12% compared to the
fresh fruit juice. Maximal lycopene content of 114% in watermelon juice was
achieved with 7-µs bipolar pulses for 1050 µs at 35 kV/cm and frequencies
ranging from 200 to 250 Hz (Odriozola-Serrano et al., 2009). Cortés et al.,
(2006) observed that the carotenoid concentration in orange juice rose slightly
after applying intense PEF treatments of 35 and 40 kV/ cm for 30–240 µs.

8. EFFECT OF FREEZING PROCESSING, FROZEN STORAGE


AND THAWING ON BIOCHEMICAL CHANGES

Freezing is one of the best methods for long-term storage of fruits.


Freezing preserves the original color, flavor, and nutritive value of most fruits.
Fresh fruits, when harvested, continue to undergo chemical, biochemical, and
20 Rakesh Sharma, Bharati D Shrinivas

physical changes, which can cause deterioration reactions such as senescence,


enzymatic decay, chemical decay, and microbial growth. The freezing process
reduces the rate of these degradation reactions and inhibits the microbiological
activity. However, it should be recognized that a number of physical,
chemical, and biochemical reactions can still occur and many will be
accentuated when recommended conditions of handling, production, and
storage are not maintained. Although few microorganisms grow below −10°C,
it should be recognized that freezing and frozen storage is not a reliable
biocide. The production of safe frozen fruits requires the same maximum
attention to good manufacturing practices (GMP) and hazard analysis critical
control points (HACCP) principles as those used in fresh products. The quality
of the frozen fruits is very dependent on other factors such as the type of fruit,
varietal characteristics, stage of maturity, pretreatments, type of pack, and the
rate of freezing. The freezing process reduces the fruit temperature to a storage
level (−18°C) and maintaining this temperature allows the preservation of the
frozen product for 1 year or more. Fruits are frozen in different shapes and
styles: whole, halves, slices, cubes, in sugar syrup, with dry sugar, with no
sugar added, or as juices, purees, or concentrates, depending on the industrial
end-use (Hui et al., 2006).

8.1 Freezing Principles

The freezing process reduces food temperature until its thermal center
(food location with the highest temperature at the end of freezing) reaches
−18°C, with the consequent crystallization of water, the main component of
plant tissues. Water in fruit and fruit products constitute 85–90% of their total
composition. Crystallization of water during freezing reduces water activity
(aw) in these tissues and consequently produces a decline in chemical and
biochemical reactions and microbial growth. Freezing also involves the use of
low temperatures and reactions take place at slower rates as temperature is
reduced. The study of temperature changes during freezing is basic to an
understanding of how products are processed. Figure 3 shows typical freezing
curves at different freezing rates. When the product is cooling down to 0°C,
ice begins to develop (see section A–S, Figure 3). The exact temperature for
the formation of first ice crystal depends on the type of product and is a
consequence of the constituents concentration independent of water content;
for example, fruits with high water content (≈90%) have a freezing point
below −2°C or −3°C, while meat with less water content (≈70%) has a
Processing Techniques 21

freezing point of −1°C; the main difference being the high sugar and organic
acid concentration in fruits. Ice formation takes place after the product reaches
a temperature below its freezing point (−5°C to −9°C) for only a few seconds.
This process is known as super-cooling (position S in Fig. 3).
After that, due to heat release during the first ice formation, the
temperature increases until the freezing point is reached (position B in Fig. 3).
Section B–C in Fig. 3 corresponds to the freezing of most of the tissue water at
a temperature that is practically constant, with a negative slope from a decline
of the freezing point due to solute concentration. The increase of solute
concentration as freezing progresses causes the unfrozen portion to undergo
marked changes in such physical properties as ionic strength, pH, and
viscosity. This increases the risk of enzymatic and chemical reactions, e.g.,
enzymatic browning or oxidation–reduction, with adverse effects on frozen
fruit quality. A short B–C section increases the quality of frozen fruit. This
means that a fast rate freezing produces a better quality frozen fruit (see curves
b and c of Figure 3). Section C–D corresponds with the cooling of the product
until the storage temperature, with an important increase of solute
concentration in the unfrozen portion. Below −40°C, new ice formed is
undetected. Up to 10% of the water can be unfrozen, mainly joined to protein
or polysaccharide macromolecular structures that take part in the physical and
biochemical reactions. In frozen foods the relationship between the frozen
water and the residual solution is dependent on the temperature and the initial
solute concentration. The presence of ice, and an increase in solute
concentration, has a significant effect on the reactions and state of the fruit
matrix. The concentration of the solute increases as freezing progresses; and
thus, solute concentration of the unfrozen matrix can leach out of the cellular
structures causing loss of turgor and internal damage. Solute-induced damage
can occur whether freezing is fast or slow, and cryoprotectants, such as sugars,
are usually added to aqueous solution to reduce the cell damage
(Rahman, 1999).

8.2 Plant Cell Structure

Understanding the effect of freezing on fruit requires a short review of


plant cell structure. Plant cells are surrounded by a membrane and interspersed
with extensive membrane systems that structure the interior of the cell into
numerous compartments. The plasmalemma or plasma membrane encloses the
plasma of the cell and is the interface between the cell and the extracellular
22 Rakesh Sharma, Bharati D Shrinivas

surroundings. Contrary to animal cells, plant cells are almost always


surrounded by a cell wall and many of them contain a special group of
organelles inside the plastids (chloroplasts, leucoplasts, amyloplasts, or
chromoplasts). An important property of the plant cell is its extensive vacuole.
It is located in the center of the cell and makes up the largest part of the cells
volume and is responsible for the turgor. It helps to maintain the high osmotic
pressure of the cell and the content of different compounds in the cell, among
which are inorganic ions, organic acids, sugars, amino acids, lipids,
oligosaccharides, tannins, anthocyanins, flavonoids, and more. Vacuoles are
surrounded by a special type of membrane, the tonoplast. The cell wall of
plants consists of several stacked cellulose microfibrils embedded in a
polysaccharide matrix able to store water thereby increasing the cell volume
(hydration and absorption). According to their capacity to bind or store water,
the polysaccharides involved in the matrix can be classified as follows:
pectin>hemicellulose>cellulose>lignin. Pectins are mainly polygalacturonic
acids with differing degrees of G-galactosyl, L-arabinosyl or L-rhanmosyl
residue and are predominant in the middle lamella, the layer between cells.
The deesterification process of pectin is related to the softness of fruit tissues
during ripening and processing (Hui et al., 2006).
Processing Techniques 23

Figure 3. Typical freezing curves of foods at different rates: (a) very slow; (b) fast; and
(c) very fast (Fennema, 1976).

8.3 Color Changes

Color is the most important quality characteristic of fruits because it is the


first attribute perceived by the consumers and is the basis for judging the product
acceptability. The most important color changes in fruits are related to chemical,
biochemical, and physicochemical mechanisms: (a) breakdown of cellular
chloroplasts and chromoplasts, (b) changes in natural pigments (chlorophylls,
carotenoids, and anthocyanins), and (c) development of enzymatic browning.
Mechanical damage (ice crystals and volume expansion) caused by the freezing
process can disintegrate the fragile membrane of chloroplasts and chromoplasts,
releasing chlorophylls and carotenoids, and facilitating their oxidative or
enzymatic degradation. Also, volume expansion increases the loss of
anthocyanins by lixiviation due to disruption of cell vacuoles.

(I) Chlorophylls
Chlorophylls are the green pigment of vegetables and fruits, and their
structures are composed of tetrapyrroles with a magnesium ion at their center.
Freezing and frozen storage of fruits cause a green color loss due to
degradation of chlorophylls (a and b) and transformation in pheophytins,
which transfers a brownish color to the plant product (Cano, 1996). One
example is kiwi-fruit slices that show a decrease in chlorophyll concentration
between 40% and 60%, depending on cultivar, after freezing and frozen
storage at −20°C for 300 days (Cano et al., 1993).
24 Rakesh Sharma, Bharati D Shrinivas

Figure 4. Pathways of chlorophyll degradation. (adapted from Heaton et al., 1996)

Different mechanisms can cause chlorophyll degradation; loss of Mg due


to heat and/or acid, which transforms chlorophylls into pheophytins; or loss of
the phytol group through the action of the enzyme chlorophyllase, which
transforms chlorophyll into pheophorbide. Loss of the carbomethoxy group
may also occur and pyropheophytin and pyropheophorbide can be formed
(Figure 4) (Heaton et al., 1996). Acids, temperature, light, oxygen, and
enzymes easily destroy the chlorophylls. Thus, blanching (temperature/time),
storage (temperature/time), and acidity are the important factors to be
controlled during processing in order to preserve chlorophylls. Other
chlorophyll degradation mechanism can cause degradation by the action of
peroxides, formed in the fruit tissue due to the oxidation reaction of
polyunsaturated fatty acids catalyzed by the enzyme LOX. An important
quality parameter employed to determine the shelf life of frozen green fruits is
the formation of pheophytins from chlorophylls. As different types of enzymes
can be involved in chlorophyll degradation (LOX, POD, and chlorophyllase),
blanching and addition of inorganic salts such as sodium or potassium chloride
and sodium or potassium sulphate are efficient treatments to preserve green
color (Cano et al., 1993).

(II) Carotenoids
Carotenoids are among the most abundant pigment in plant products and
are responsible for the yellow, orange, and red color of most of the fruits. All
of them are tetraterpenes and contain 40 carbon atoms in eight isoprenes
Processing Techniques 25

residues. ß-carotene and lutein are the carotenoids present in most of the fruits.
Important sources of these pigments are as follows:

ß-cryptoxanthin: oranges
lycopene: tomatoes, watermelon, papaya and persimmon
α-carotene: banana and avocado
zeaxanthin: orange and peach

Carotenoids are affected by pH, enzymatic activity, light, and oxidation


associated with the conjugated double bond system. The chemical changes
occurring in carotenoids during processing have been reviewed by several
authors (Rodriguez-Amaya, 1997). The main degradation reaction that
damages carotenoid compounds is isomerization. Most plants appear to
produce mainly trans forms of carotenoids but with increased temperature, the
presence of light, and catalysts such as acids, isomerization to the cis forms
increases, and the biological activity is dramatically reduced. However, heat
treatments of products rich in carotenoids reduce the degradation of
carotenoids because of the inactivation of enzymes LOX and POD. Blanching
fruits before freezing could be efficient in the preservation of carotenoids due
to enzyme inactivation. Although most carotenoids are heat resistant, some
carotenoids, such as epoxycarotenoids, could be affected. Carotenoids are fat-
soluble pigments and breakdown of chromoplasts, by heat treatment or
mechanical damage, improves their extraction with organic solvents and
bioavailability but not their loss by lixiviation (Hof et al., 2000). Freezing
without protector pretreatment slightly decreases total carotenoid
concentration (20%) of some fruits rich in carotenoids, such as mango and
papaya. But after 12 months of frozen storage at −18°C, an important decrease
of total carotenoid concentration (between 40% and 65%) occurred, although
the carotenoid profilewas unchanged (Cano et al., 1996). Similar results have
been found with frozen tomato cubes. A pronounced stability of total
carotenoids, ß-carotene, and lycopene was recorded up to the 3rd month of
storage. But after 12 months of storage at −20°C, the losses of carotenoids
reached 36%, of ß-carotene 51%, and of lycopene 48% (Lisiewska and
Kmiecik, 2000). Freezing and frozen storage could affect the carotenoid
structure and concentration depending on the type of fruit and cultivar (pH,
fats, antioxidants, etc.) and the processing conditions (temperature, time, light,
oxygen, etc.) (Rodriguez-Amaya, 1997).
26 Rakesh Sharma, Bharati D Shrinivas

(III) Anthocyanins
Anthocyanins are one class of flavonoid compounds, which are widely
distributed plant polyphenols, and are responsible for the pink, red, purple, or
blue hue of a great number of fruits (grape, plum, strawberry, raspberry,
blackberry, cherry, and other types of berries). They are water-soluble
flavonoid derivatives, which can be glycosylated and acylated. The effect of
freezing, frozen storage, and thawing in different fruits rich in anthocyanins
pigments have been reviewed by Skrede (1996). Anthocyanins in cherryfruit
underwent pronounced degradation during storage at −23°C (87% after 6
months), but they are relatively stable at −70°C storage (Chaovanalikt and
Wrolstad, 2004a). But in raspberry fruit, the stability of anthocyanins to
freezing and frozen storage depends on the seasonal period of harvest. Spring
cultivars were practically unaffected by freezing and frozen storage for 1 year
at −20°C, but autumn cultivars showed a decreasing trend in total anthocyanin
content (4–17%)(De Ancos et al., 2000b). In general, the freezing process does
not affect the level of anthocyanins in raspberry fruit (Mullen et al., 2002).
Authors explain degradation of anthocyanins during frozen storage by
different chemical or biochemical mechanisms. Anthocyanins are water-
soluble pigments located in the vacuoles of cell and are easily lost by
lixiviation when the cell membranes break down. Also oxidation can play an
important role in anthocyanin degradation catalyzed by light. PPO and POD
enzymatic activities have been related to anthocyanin degradation. Thus,
frozen–thawed cherry discoloration disappeared when the fruits were blanched
before freezing. In slightly acidic aqueous solution at ambient temperatures,
anthocyanins exist as essentially four species in equilibrium. These are the
blue quinoidal base (A), the red flavylium cation (AH+), the colourless
hemiacetal base (B), and the colourless chalcone form (C) (Figure 5). The
changes in pH during processing can affect anthocyanin stability. Maintenance
of red fruit requires an acid medium (pH < 3.5). The flavylium cation structure
of anthocyanins transfers a red color to the fruit. But an increase in pH value
produces a change from red to blue until the product is colorless, a
consequence of transforming flavylium cation into a neutral structure (Figure
5). The loss of characteristic red color can also be produced by formation of
the anthocyanin complex with different products present in the fruit matrix:
ascorbic acid, acetaldehyde, proteins, leucoanthocyanins, phenols, quinones,
metals (Fe3+ and Al3+), hydrogen peroxide, etc. (Escribano-Bailon et al.,
1996).
Processing Techniques 27

Figure 5. Structural transformations of anthocyanins (anthocyanidin-3-glucosides). R3


and R5 are normally H, OH or OCH3 moieties; GL represents a glycosyl moiety
(adapted from Dangles and Brouillard, 1992)

(IV) Enzymatic Browning


Browning usually occurs in certain fruits during handling, processing, and
storage. Browning in fruit is caused by enzymatic oxidation of phenolic
compounds by PPO (Martinez and Whitaker, 1995). PPO catalyzes either one
or two reactions involving molecular oxygen. The first type of reaction is
hydroxylation of monophenols, leading to formation of o-hydroxy compounds.
The second type of reaction is oxidation of o-hydroxy compounds to quinones
that are transformed into polymeric brown pigment. Freezing, frozen storage,
and thawing of fruits, like mangoes, peaches, bananas, apples, apricots, etc.,
quickly develop color changes that result in nonreversible browning or
darkening of the tissues. Freezing does not inactivate enzymes; however, some
enzyme activity is slowed during frozen storage. Browning by PPO can be
prevented by the addition of sulfites, ascorbic acid, citric acid, cysteine, and
others. Selection of varieties with low PPO activity could help to control
browning in frozen–thawed fruits (Cano et al., 1998).

8.4 Effect of Freezing Processing on Vitamin C, Carotenoids and


Phenolic Compounds
28 Rakesh Sharma, Bharati D Shrinivas

(I) Vitamin C
Freezing processes have only a slight effect on the initial vitamin C
content of fruit (Cano and Marin, 1992). The destruction of vitamin C
(ascorbic acid) occurs during freezing and frozen storage, and this parameter
has been employed to limit the frozen storage period of frozen fruit. The main
cause of loss of vitamin C is the action of the enzyme ascorbate oxidase. If
pretreatments or freezing processes do not destroy this enzyme, it is
continuously active during the frozen storage. Vitamin C degradation depends
on different factors, such as time–temperature conditions, type of fruit, variety,
pretreatments, type of package, freezing process, etc. (Skrede, 1996). Thus as
the frozen storage temperature decreases, higher vitamin C retention is
achieved for different fruits like berries, citrus, tomato, etc. (Skrede, 1996;
Lisiewska and Kmiecik, 2000). Also, significantly different vitamin C
retention values have been achieved between varieties of fruits such as
raspberry (De Ancos et al., 2000c), mango, and kiwi (Cano and Marin, 1992),
which were frozen and stored under the same conditions. Vitamin C stability
in freezing and frozen storage of strawberries seems to be more dependent on
storage temperature than on the type of freezing process. Nonstatistical
differences were observed between strawberries processed by fast rate freezing
(at −20°C) and quick rate freezing (at −50°C to –100°C), but great loss was
shown between strawberries stored at −18°C and −24°C (Sahari et al., 2004).

(II) Provitamin A and Antioxidant Carotenoids


Some carotenoids, like ß-carotene, α-carotene, and ß-cryptoxanthin, are
recognized as precursors of vitamin A. This provitamin A carotenoids, in
addition to lycopene and lutein, constitute the group of antioxidant
carotenoids. The prevailing opinion is that freezing and frozen storage do not
prevent degradation of carotenoids. The content of ß-carotene, and
consequently the provitamin A value, was decreased during frozen storage of
mango, kiwi (Cano and Marin, 1992), papaya (Cano, 1996), and tomato
(Lisiewska and Kmiecik, 2000). The losses were mainly due to the activity of
enzymes (POD, LOX, and CAT), particularly during frozen storage in an
oxygen environment. Lycopene, a characteristic carotenoid in tomato fruit, has
been recognized as a powerful antioxidant (Lavelli et al., 2000). After 3
months of frozen storage (−20°C and−30°C), great stability of lycopene was
recorded. After this period, slow losses occurred, the rate being faster at the
higher storage temperature. After 12 months at −20°C and −30°C, the
lycopene content was 48% and 26%, respectively, lower than that in the raw
material (Lisiewska and Kmiecik, 2000). Other authors have reported an
Processing Techniques 29

increase in the extraction of lycopene after 1 month of frozen storage, although


after 3 and 6 months the loss of lycopene concentration was significantly
higher than 40% (Urbanyi and Horti, 1989). Papaya fruit could be an
important source of lycopene, but freezing and frozen storage at −20°C during
12 months produced a significant loss of lycopene concentration (34%) in
frozen papaya slices (Cano, 1996).

(III) Phenolic Compounds


The freezing process does not modify either total phenolic content or
ellagic acid concentration in raspberry fruit. There is an increasing interest in
ellagic acid, a dimeric derivative of gallic acid, due to its anticarcinogenic and
antioxidant effects. Although frozen storage produces a slight decrease in
ellagic acid content because of PPO enzyme activity, frozen storage is a good
methodology to preserve phenolic compounds during long term periods (De
Ancos et al., 2000c). Asami et al. (2003b) evaluated the effects of storage at
refrigeration and frozen temperatures on the concentration of total phenolics in
clingstone peaches. Maturity stage III peaches of the Ross variety were peeled,
pitted, sliced, and frozen at –12°C for a period of 3 months. There appeared to
be a statistically significant increase (P < 0.05) in total phenolic content
following freezing, and this higher content was retained after 2 and 3 months
of frozen storage. It was postulated that the freezing process may have resulted
in cellular disruption and more facilitated extraction of phenolics.
The effect of freezing and frozen storage on raspberry phytochemicals and
volatiles was the subject of two manuscripts by De Ancos and colleagues (De
Ancos et al., 2000a, 2000b). These authors compared two early-season and
two late-season raspberry cultivars and found differential effects of freezing.
In the early-season cultivars, freezing resulted in increased anthocyanin
content, while in the late-season cultivars, which initially had higher
concentrations of anthocyanins, freezing caused an overall reduction. The
authors suggested that the preservation of anthocyanins during freezing
depends on the pH of the fruit, organic acid content, sugar concentration,
initial anthocyanin concentration, and initial cyaniding-3-glucoside content.
They did not find a relationship between polyphenol oxidase activity and
anthocyanin content. De Ancos et al., (2000b) also found that freezing had a
slight effect on ellagic acid, vitamin C, and total phenolics, depending on the
raspberry cultivar. Free radical scavenging capacity was decreased as a result
of the freezing process, anywhere from 4 to 26%, again related to cultivar.
Frozen storage of raspberries at –20°C for a 1-year period did not appear to
affect total phenolics or free radical scavenging capacity, but did cause a
30 Rakesh Sharma, Bharati D Shrinivas

decline in ellagic acid vitamin C. In another study of the effects of freezing on


raspberry phenolics, ellagitannins, flavonoids, and antioxidant capacity
(Mullen et al., 2002), these authors found that the antioxidant capacity of the
fruit and vitamin C levels were not affected by freezing. The raspberry cultivar
used in this study differed from those evaluated by de Ancos, however, and
this may have affected the results. Freezing preservation of fruit is less
destructive toward some antioxidant compounds, in particular total phenolics
and ascorbic acid, than other means of preservation. One illustration of this is
a recent publication (Asami et al., 2003a) in which Marionberries,
strawberries, and corn were preserved using freezing, freeze-drying, and air-
drying methods. The highest levels of both total phenolics and ascorbic acid
(reduced form) were consistently found in the extractions of frozen samples,
followed by those of freeze-dried and then air-dried samples. Freezing may
cause some damage to cell structure, and application of a drying procedure
following freezing, even though this is under vacuum at reduced temperatures,
may result in even greater losses of beneficial nutrients. Air-drying at
temperatures above 60°C may result in oxidative condensation or
decomposition of thermolabile compounds, such as (+)-catechin and ascorbic
acid. Therefore, the presence of total phenolics and ascorbic acid in the air-
dried products was lower than that in either frozen or freeze-dried products.

8.5 Effect of Thawing on Phytochemical Content

The quality of the original fruit, preserved by freezing, is retained by


quick thawing at low temperature in controlled conditions. During incorrect
thawing, chemical and physical damage and microorganism contamination can
also occur. Fruit products exhibit large losses of ascorbic acid (up to 40%) and
color changes when thawed for an unusually long period, e.g., 24 h at room
temperature. Good results in terms of vitamin C and anthocyanins retention
(90%) were achieved by thawing small frozen fruits such as bilberry,
raspberry, black currant, red currant, and strawberry at room temperature (18–
20°C /6–7 h), in a refrigerator (2–4°C /18 h), or in a microwave oven. Color
and ascorbic acid retention of fruit was equally affected by thawing
temperature and time. Thorough thawing must be determined by taking into
account the size of the fruit and/or the type of packaging (Kmiecik et al.,
1995).
In relation to the control samples (frozen and thawed by traditional
methods) freezing in liquid nitrogen and thawing in microwave oven allowed
Processing Techniques 31

an increase of the retention of polyphenols in all strawberry varieties (1.7–


25.7%). The difference between effects of the frozen strawberry preparation
and thawing methods explained 68.9% of the total variation in principal
component. The samples which were thawed in microwave oven had a much
higher phenolic content (anthocyanins, proanthocyanins, (+)-catechin and
ellagic acid) than samples which was thawed during 20 h at 20 °C. Probably
enzymatic reaction could take place in destroyed strawberry tissues during
long thawing process. To achieve a higher content of phenolic compounds in
strawberries, they should be thawed by faster method in microwave oven (Jan
Oszmianski et al., 2009).
Yurena et al.,( 2006) who found that, the relative to the content before
freezing, differed depending on the thawing method (84 ± 7%, 91 ± 9% and 97
± 6% when thawing at room temperature, in the refrigerator and in the
microwave oven, respectively). There were no significant differences in the
initial content and the values measured after microwave thawing. Thawing at
room temperature resulted (for both standard solutions and extract) in lower
AA content than thawing in the microwave oven. Microwave thawing was
chosen on the basis of these results and because it was the most practical
method for routine analysis.

9. PACKAGING, STORAGE AND HANDLING PROCEDURES


INFLUENCING PHYTOCHEMICAL CONTENT OF FRUIT
9.1 Packaging Materials Used for Processed Fruits

Thermally processed fruits have traditionally been packed in metal


containers and glass jars. Products stored in glass and metal containers
generally maintain their nutritional and organoleptic quality for extended
periods of time due to the durability and excellent oxygen and moisture barrier
properties of the containers. Plastic packaging materials have recently become
more popular due to their light weight, unbreakable nature, and convenience
features. Driven by advances in aseptic processing, fruit purees (baby foods)
are now available in reclosable, multilayer, barrier-plastic cups that are
microwaveable. Plastic polyethylene terephthalate (PET) containers are also
becoming more popular for high acid fruit products and many of the containers
provide convenience for the consumer through easy dispensing designs.
Although the convenience afforded by these products is unquestioned,
32 Rakesh Sharma, Bharati D Shrinivas

information is lacking on how processing and storage of fruits in plastic


containers affect the retention of phytochemicals. Storage stability of oxygen-
sensitive phytochemicals may be an important issue since the plastic
packaging materials are not completely oxygen impermeable.

9.2 Storage Factors Influencing Retention of Phytochemicals

The stability of carotenoids and phenolics in processed fruits during


storage is influenced by temperature, light, oxygen, and chemical interactions,
which may or may not be oxidative in nature. The package plays an integral
role in protecting food constituents from the adverse effects of oxygen and
light, which is especially important for dehydrated powders that are highly
prone to oxidation. Although thermally processed fruits are typically stored at
ambient temperature, storage at refrigerated temperatures can result in greater
retention of color and phytochemicals.

(I) Cold Storage


At optimum cold storage conditions vitamin C content is decreasing;
whereas most phenolics, carotenoids, glucosinolates and dietary fibers are
relatively stable (Table 3). Deviations from optimum conditions may indeed
affect the contents of health-related constituents. Suboptimum temperature and
humidity usually give rise to enhanced rates of breakdown due to increased
metabolism leading to faster maturation and senescence. Some constituents,
such as phenolics, can increase their content under dehydration (without
change in total amount) or when exposed to visible or UV radiation (with
increased total amount). Except for vitamin C and phenolics, storage effects on
nutrients and health-promoting phytochemicals have not been investigated to
any great extent (Bengtsson, 2008). The biosynthesis of anthocyanins in red
small fruits and berries tends to continue after harvest and during storage.
Increased content of anthocyanins during cold storage has been recorded in
strawberries, blueberries, grapes and pomegranates (Tomás-Barberán and
Espín, 2001).

(II) Controlled Atmosphere (Ca) Storage


CA is a technologically advanced storage method for which temperature,
humidity and the levels of oxygen and carbon dioxide are precisely controlled.
Low oxygen concentration in the atmosphere during storage leads to reduced
Processing Techniques 33

rate of respiration with a delayed onset of senescence. This has been exploited
commercially for suitable species of fruit using special storage facilities.
In addition to typically 1–2 kPa concentration of O2, the CA contains CO2
at an elevated level, for instance 2–6 kPa. The exact concentrations used
depend upon the product. CA storage is an extension of cold storage; thus an
optimum temperature is also a prerequisite for optimum product quality. In
general, CA storage retards the loss of health-related constituents compared to
cold storage in air (Table 3). Some constituents can also increase their content
during CA storage, for example some glucosinolates and phenolics. Controlled
atmosphere (CA) storage of strawberry fruit did not affect anthocyanin content
in external tissues but decreased anthocyanin content in internal tissues
(Holcroft and Kader, 1999).
Table 3. General Effects of Storage on Contents of Health-Related Constituents of Fruits and Vegetables

Optimal Suboptimal Incident Reduced Elevated Elevated


Dehydration
temp. temp. light O2 CO2 O2
Slower
Vitamin C Decrease Decrease Decrease Decrease
decrease
Phenolics, Stable or Stable or
Stable Decrease Increase Variable
fruits increase increase
Phenolics, Stable or Stable or
Increase Increase Variable Increase Increase
berries decrease decrease
Carotenoids,
Variable Variable
fruits
Stable or
Glucosinolates Decrease Variable Increase Decrease
Decrease
Dietary fibre Stable Variable
(adapted from Bengtsson et al., 2008)
Processing Techniques and Their Effect on Fruit Phytochemicals 35

(III) Modified Atmosphere Packaging (Map)


Modified atmosphere packaging is an extension of CA to small packages
for retail, the main difference being that the gas composition is not controlled
by external systems. The atmosphere composition inside the closed package
changes with time and is dependent on uptake and release of gases by the
food, as well as on transmission of gases through the package. The effects of
MAP on health-related constituents are very similar to those of CA storage,
when similar atmosphere compositions are compared (Table 3).
In a study on fresh-cut jackfruit bulbs, it was observed that the total
phenolic (TP) content decreased during storage of fresh-cut jackfruit bulbs in
dip-pretreated as well as in untreated bulbs. Dip pretreatment coupled with
MAP showed a significantly (p < 0.05) lower TP loss as compared to untreated
samples which recorded a higher degree of degradation after just 7 days. The
percentage phenolics loss in the pretreated samples was found to be 7–15%, as
compared to 15–26% in the case of untreated samples kept under MA
conditions (Alok Saxena, 2009). Alasalvar et al. (2005) reported that storage
under low O2 atmosphere could reduce the accumulation of TP in shredded
orange compared to those stored under air and high O2 conditions. Cocci et al.,
2006), who reported a restricted degradation in TP, due to the reducing action
of AA added in the dip pretreatment given to fresh-cut apple stored under MA
conditions. The decrease in TP during storage of fresh-cut commodities could
be attributed to enzymatic degradation by peroxidase (POD) and PPO
activities. The loss in total flavonoids content was found to be 8–20% in the
case of pretreated MA packaged samples as compared to a significantly higher
loss of 20– 33% in the control samples under different MA conditions. Total
carotenoids content was observed to decrease from the 7th day onwards in the
control samples. The overall retention of total carotenoids was found to be in
the range of 40–57%, in the case of pretreated samples, whilst the control ones
showed a significantly lower retention (5–39%) under the various MA
conditions during storage for 35 days (Alok Saxena, 2009). The pretreated
samples kept under MA conditions showed a significantly (p < 0.05) higher
retention of AA (56–69%), as against 10–49% in the case of untreated
samples. Reports exist about higher retention of AA in fresh-cut commodities
subjected to MAP (Odriozola-Serrano et al., 2008). Lower respiratory activity
could be attributed to higher retention of AA content, due to restriction in
enzymatic oxidation of AA into dehydroascorbic acid, through headspace
oxygen in the MA-packaged samples during storage.
36 Rakesh Sharma, Bharati D Shrinivas

9.3 Influnence of Storage and Handling on Health- Related


Properties of Fruit

(I) Vitamin C
Vitamin C as L-ascorbic acid (AA) and its oxidation product L-
dehydroascorbic acid (DHA) is present in all plants. The variation in content
between various plant foods and within species is very large (Davey et al.,
2000). L-dehydroascorbic acid is present in most fruits in small amounts,
usually about 10% of the AA level, but it can increase during storage due to
oxidation of AA. The storage stability of AA seems to be related to the initial
level (Lee and Kader, 2000). The percentage losses are larger with a lower
starting value. After cold storage for several months the AA level of apples is
halved or more. Davey and Keulemans (2004) found among 31 Belgian apple
cultivars that cultivars low in vitamin C had the largest losses during storage
(both for three months at 1°C and for 10 days at room temperature) and these
cultivars were also the ones with the worst storage outcome.
The effect of storage temperature (4°C) on the AA stability was studied by
Silvia Tavarini et al., (2008) in two standard solutions (50 mg/l AA in water
and 3% MPA–8% acetic acid, n = 3) and a ripe banana extract spiked with 50
mg/l AA (n = 3), which was selected as the extract model because it had the
highest complexity among the extracts. After 24 h, the stability of AA kept at
4°C was 95 ± 5% from the initial AA content; after 4 days it was 75 ± 4% and
after 8 days 51 ± 3% (Yurena et al., 2006). In kiwi fruit, the AA significantly
decreased after 6 months of cool storage and slightly increased again after a
week to ambient temperature.

(II) Phenolics
Apple is one of the most studied fruits with regard to phenolic content.
The phenolic content in apple has been repeatedly reported to be relatively
stable during long-term storage at low temperature (<4°C) in both normal air
and CA with reduced O2 and increased CO2 levels (Awad and De Jager, 2003)
and no changes in the phenolic content of „Aroma‟ apples were detected after
storage for 10 days in normal air at 10°C (Hagen et al., 2007). MacLean et al.
(2006) observed a small decrease in anthocyanins and increase of chlorogenic
acid, but no change in the overall content of phenolic compounds in „Red
Delicious‟ apples during 120 days of cold storage at 0–1°C and eight days of
shelf-life at room temperature. In contrast, Leja et al. (2003) found a
considerable increase in the total phenolic content of „Jonagold‟ and
„Sampion‟ apples stored in normal air or CA (2% O2 + 2% CO2) for 120 days
Processing Techniques 37

at 1°C followed by an additional seven days at 16°C. A slight decrease in


anthocyanin content was observed in the apples stored in normal air, but not in
CA. In „Rocha‟ pears stored in air or CA (2 kPa O2 + 0–5 kPa CO2) at 2°C for
four months, the content of hydroxycinnamic acid derivatives and flavonols
were stable, the level of flavan-3-ols decreased and the concentration of
arbutin increased as time elapsed. No differences in the effects of storage
atmospheres were observed (Galvis-Sánchez et al., 2006). In a study about
fresh-cut fruits versus whole fruits during storage, Gil et al., (2006) found that
during 9 days of storage no significant changes occurred in phenol content in
kiwifruits and no difference was determined between slices and whole fruits.
Generally, phenol content may either increase or decrease in fruits and
vegetables depend on the storage conditions (Kalt, 2005).
No changes in the levels of anthocyanins, flavonols or
hydroxycinnamates, but a slight increase of total phenolics, were observed in
red raspberries stored at 4°C for three days and then at 18°C for 24 hours,
mimicking the route of fresh fruit after harvest to the supermarket and onto the
consumer‟s table (Mullen et al., 2002). The effect of temperature on the rate of
anthocyanin formation during storage may depend on the maturity of the fruits
at harvest (Kalt et al., 2003). CA storage, typically used with low O2 and high
CO2 concentrations, may reduce the anthocyanin pigmentation in red fruits
(Tomás-Barberán and Espín, 2001), but was found to have little or no effect on
the phenolic content in blueberries (Schotsmans et al., 2007) and cranberries
(Gunes et al., 2002). No changes in the content of phenolic compounds in
„Superior seedless‟ table grapes were detected after storage under passive
MAP for seven days at 0°C followed by four days at 8°C, but slight decreases
were registered after two days of simulated shelf-life at 20°C (Artés-
Hernández et al., 2006). Elevated levels of O2 have been found to induce
accumulation of phenolic compounds during the initial stage of storage of
strawberries, but it may also promote the oxidation of phenolic compounds
upon prolonged storage treatments (Zheng et al., 2007). In highbush blueberry,
total phenolics and total anthocyanins as well as individual phenolic
compounds were markedly increased by 60–100 kPa O2 treatments compared
to 40 kPa O2 or normal air during 35 days of storage at 5°C (Zheng et al.,
2003). During the 6 months storage the content of polyphenols in frozen
control strawberries underwent decreases, which varied between 17.8% for cv.
Senga Sengana and 36.6% for cv. Kent (Jan Oszmianski et al., 2009). Total
phenolic compounds increased continuously in berries stored at 10°C and 5°C.
However, strawberry fruit stored at 0°C maintained a constant value of total
phenolic compounds during the storage period. Both temperature and storage
38 Rakesh Sharma, Bharati D Shrinivas

time had a significant effect (p˃0.05) on total phenolic compounds of


strawberry fruits (Fernando et al., 2004). A study involving the effects of
blanching and processing on antioxidant capacity and phenolic content of
peach puree reported that a major loss of antioxidant capacity occurred in
pasteurized peach puree stored at 40°C for four weeks, which reflected major
losses of chlorogenic acid (Talcott et al., 2000). Hydroxycinnamates,
epicatechin, and flavonol glycosides are also subject to losses during storage.
Bing cherries stored at 2°C for five months lost 8%, 20%, and 16% of
hydroxycinnamates, epicatechin, and flavonol glycosides, respectively, while
fruit stored at 22°C lost 30% and 16% of hydroxycinnamates and epicatechin,
but an apparent 9% increase in flavonol glycosides was observed
(Chaovanalikit and Wrolstad, 2004b).

(III) Carotenoids And Anthocyanins


Mature green mangoes ripened at 20°C will accumulate carotenoids after
about one week and this is irrespective of prior hot water treatment (Talcott et
al., 2005). When the mangoes were initially held at 5°C and then at 20°C, the
carotene increase and the ripening were delayed. Wright and Kader (1997)
studied changes in quality, retinol equivalents, and individual provitamin A
carotenoids in fresh-cut Fay Elberta peaches held for 7 days at 5°C in air or
CA. They concluded that the limit of shelf life was reached before major
losses of carotenoids were observed. In a study about carotenoids changes
after storage of Hayward kiwifruit, Silvia Tavarini et al., (2008) found that the
carotenoid content in kiwifruit increased after 2 months of cool storage and
after a week to ambient temperature, whereas after 6 months of cool storage,
fruits harvested at showed a significant decrease in carotenoid content.
Although carotenoid levels can be compromised during thermal processing
due to oxidation and isomerization, they are generally well preserved during
storage of canned products, primarily due to their non-polar nature and
resistance to leaching into the canning medium. Provitamin A activity was also
well retained in clingstone peaches stored for 18 months at ambient
temperature. Storage temperature can significantly affect carotene retention in
canned fruits. Canned peaches, peas, plums, spinach, and tomatoes stored at 10
and 18.3°C retained higher levels of carotenes over 24 months storage than
products stored at 26.7°C. The type of packaging container is reported to
influence retention of carotenoids during long-term storage of mango slices
and puree (Godoy and Rodriguez-Amaya, 1987). No appreciable loss of β-
carotene occurred in mango slices packed in lacquered (epoxy) or plain tin-
plate cans during 10 months storage at ambient conditions, but significant
Processing Techniques 39

losses were observed after 14 months (50%) and 24 months (84%) of storage.
Similar losses of β-carotene were observed in mango puree stored long-term in
lacquered epoxy cans and bottles, but β- carotene showed a greater propensity
to degrade when stored in bottles. Carotenoids in dehydrated fruit powders are
prone to oxidation during long-term storage and require special packaging
materials to exclude light and oxygen or gas flushing treatments completely to
exclude oxygen. Low temperature during storage is, however, known to
reduce the rate of anthocyanin formation. Increased levels of anthocyanins
under storage at higher temperatures (15–25°C) have been reported for
strawberries (Cordenunsi et al., 2005), blueberries (Kalt et al., 2003;
Schotsmans et al., 2007), cherries (Gonçalves et al., 2004) and cranberries
(Wang and Stretch, 2001).
During storage at 30 °C, significant changes were observed in the
concentrations of procyanidins and cyanidin- 3-galactoside of all the apple
purees. After 6 months of storage, cyanidin-3-galactoside was not detected in
any samples. Anthocyanins are very sensitive to heat degradation (Garcia-
Viguera et al., 1999). The procyanidins were more stable than were
anthocyanins during puree storage. An increasing degree of procyanidin
polymerization (DP) was observed during puree storage. Procyanidins in apple
purees are better protected than are those in apple juice concentrate.
Bengoechea et al. (1997) detected procyanidins in purees, but not in
concentrates. During manufacturing of apple juice concentrate, they could
polymerize to form insoluble forms and precipitate out, yielding lees or
sediment. The procyanidins in purees are protected because of their ability to
bind with cell-wall polysaccharides. (Guyot et al., 1998). Spanos et al. (1990)
reported that apple juice stored for 9 months at 25 °C, of concentrates, showed
36% degradation of hydroxycinnamic acids, 60% degradation of quercetin and
phloretin glycosides, and total loss of procyanidins. After 6 months of storage
at 30°C, the apple purees had a maximal 26% degradation of chlorogenic acid
and 18% degradation of phloretin-2 -glucoside. Anthocyanin content
decreased in strawberry fruit stored at 0°C and 5°C during the first 5 days.
Meanwhile, anthocyanin content in fruit stored at 10°C increased gradually
during the storage period and reached its highest values near the end of the
storage period (Fernando et al., 2004). Ochoa et al. (1999) reported that
storage temperature markedly affected the color and anthocyanin content of
raspberry pulp, with product stored at 4°C for 90 days retaining much higher
levels of total anthocyanins than product stored at 20°C or 37°C. Fruits packed
in liquid canning medium experience leaching of water-soluble
phytochemicals during storage. In canned peaches stored for three months
40 Rakesh Sharma, Bharati D Shrinivas

procyanidin monomers, dimers, trimers, and tetramers decreased by 10, 16, 45,
and 80%, respectively, with complete loss of pentamers through to octamers
(Hong et al., 2004). Analysis of the canning syrup revealed that losses of
procyanidins from the fruit during storage were mostly accounted for by
migration into the syrup. Substantial losses of anthocyanins can also occur
during storage of canned fruit, with losses being temperature dependent.
Anthocyanin losses in canned Bing cherries stored for five months in syrup at
2°C and 22°C were 12% and 42%, respectively (Chaovanalikit and Wrolstad,
2004b), while anthocyanin losses in canned plums stored for 42 days at 4°C
and 30°C were 13.5% and 46%, respectively (Weinert et al., 1990a).

10. FUTURE TRENDS


In a hungry and increasingly competitive world, reducing phytochemical
food losses is a worthy agricultural goal. Most of the studies involving the
effects of processing techniques on fruit phyochemicals have been conducted
in pilot plants, which most likely do not accurately reflect actual losses that
occur during commercial manufacturing. Due to the continuous nature of unit
operations used in commercial production, the losses of phytochemicals may
be ameliorated since the products are processed rapidly and steps are taken to
minimize oxygen in the product prior to thermal treatments. Studies conducted
in commercial settings are needed in order to assess the true retention of
phytochemicals. Although kinetic data for time–temperature effects on
carotenoid and anthocyanin degradation are available, this important
information is lacking for other important phenolic compounds commonly
found in fruit such as flavan-3-ols, procyanidins, flavonols, phenolic acids, and
hydroxycinnamates. Generally, retention of maximum levels of antioxidants
through application of appropriate food technology processes is an appropriate
goal. However, few data exist on the nutritional value of specific non-nutrient
antioxidants include flavonoids, polyphenols, and terpenes. Assuming such
data are generated, there will be a need to adapt processes to maximise their
retention. Innovative processing methods that minimize the amount of canning
media or prevent the migration of phenolics into the canning media are
needed. Information is also lacking about how new plastic packing materials
(multilayer barrier-plastic cups and PET) impact the processing and storage
stability of phytochemicals. Studies are needed to determine if greater losses
of phytochemicals occur in these materials compared to products processed
and stored in traditional glass and metal containers. Additional studies
Processing Techniques 41

involving optimization of non-thermal processes such as high pressure, pulsed


electric field, and microwave processing or their combinations are needed to
validate quality and phytochemical retention. PEF-processing has good
prospects for use in the food industry as an alternative to thermal
pasteurization, in order to achieve healthy foods. There is also a need for
standardized methods to calculate the true retention of phytochemicals. These
methods should include calculation of the phytochemical in a known weight of
food before and after processing in order to account for solids losses that occur
during processing. Almost all the studies published on minimally processed
fruits and vegetables have been market quality studies. Except for irradiation
studies, published data on nutrient content and nutrient retention of minimally
processed foods are generally sparse and are needed. The potential safety
considerations that may result from application of minimal processes also need
to be understood.

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Foods, Diets and Disease
©
Editors: Rakesh Sharma, Bharati D Shrinivas 2009 Innovations And Solutions, Inc.
___________________________________________________________________________

Lecture 11

GRAFTING OF BIOFIBERS
CARBOXYLIC ACID ACTION AT COLD PLASMA CONDITIONS

Rakesh Sharma, Bharati D Shrinivas

ABSTRACT
Physical and biochemical functionalisation of bast fibers are ways to improve
thermo- and moisture regulation, anti-bacterial anti-allergies, hygiene, creating “smart”
textile. Enhancing natural properties of vegetable fibers is an intermediary step in the
obtaining of new products with special applications. The vegetable fibers are
biodegradable, can be recycled, and in natural state are highly polar and hydrophilic. To
improve the properties of the cellulosic fibers, the chemical and/or physico – chemical
modifications were applied. The surface esterification of the natural polymer with acids
can be carried out to obtain biodegradable materials, novel fibres with tailored
functionalities for special applications.
In this paper, starting from Spanish broom (Spartium junceum, syn. Genista juncea)
fibers, under action of cold plasma discharges, and using different kinds of carboxylic
acids, cellulose esters with short and long side chains have been synthesized.
The new grafted polymers were characterized by FT – IR spectroscopy (ATR), XPS
and SEM in order to assess the existence of incorporated functional groups. The thermal
characterization of the obtained fibres reveals their particular behaviour.

INTRODUCTION
The development of synthetic materials (e.g. plastics) at the beginning of the 20th century
has caused the steady replacement of bio-based products. As a result of this change in raw
material utilization, combined with an enormous increase in energy and chemical demand, the
world is now facing an ecological crisis. This crisis will greatly intensify with the expected
growth in demand for industrial products in developing countries. It has been estimated that
global industrial output will be five to ten times that of world production in 1987, when the
Rakesh Sharma, Bharati D Shrinivas

world population stabilizes some time in the 21st century. Thus, the world community is
facing a challenge of having to decrease pollution levels while at the same time, significantly
increasing industrial output. Such predictions have led to a number of political initiatives,
including support for enhanced industrial use of renewable resources (e.g. biomass) at the
expense of non-renewable resources (plastic, glass fibres etc.). Plant fibres may therefore face
a renaissance, not only for past uses, but also for the manufacture of three-dimensional
products by hot-pressing of fibre mats or by extrusion or injection moulding of plant fibres in
combination with plastic [1-4]. A classification of the plant fibres is given below – Scheme 1
[5].

Scheme 1. Classification of the plant fibres.

The applications of the plant fibres are very diverse such as: technical yarns, mechanical
bonded nonwovens, various fields of application as reinforcing fibre, friction linings, paper
production. Automotive components including natural fibres are currently being used by the
following vehicle manufactures (as: Fiat, Ford, Mercedes Benz, Opel, Peugeot, Renault).
Plant fibres have also found application in production of cement-based composites. A number
of alternative crops have been identified particularly for southern Europe, while some
progress has been made, as further production, processing and market development is
required for large scale commercialization. A range of harvesting processes are being
developed for each crop but harvesting cost and efficacy is still a limiting factor to economic
production of high quality fibre. Much of the benefits of products derived from plant fibres
are built on biodegradability. These benefits must be seen to be maintained in all new
products.
Spanish Broom a member of the Pea family (Leguminosae), and known botanically as
Spartium junceum (, syn. Genista juncea), also known as Weaver's Broom, is a perennial,
leguminous shrub. It is a native of the Mediterranean region and the Canary Islands, in
southern Europe, southwest Asia and northwest Africa, where it is found in sunny sites,
usually on dry, sandy soils and is often cultivated. Spanish broom is a handsome shrub with
long switch-like green few-leaved or leafless branches and large yellow sweet-scented
papilionaceous flowers. Spanish Broom typically grows to 2-4 m tall, rarely 5 m, with main
stems up to 5 cm thick, rarely 10 cm. It has thick, somewhat succulent grey-green rush-like
shoots with very sparse small deciduous leaves 1-3 cm long and 2-4 mm broad. The whole
plant, but especially the flower shoots and seeds have a bitter taste and tonic and diuretic
Grafting of Biofibres with Carboxylic Acids 3

properties, and were formerly used medicinally. The fibres of the young stems were used in
making nets, carpets, mats, baskets, etc. The stem fibres are a hemp substitute being used
mainly for coarse fabrics, cordage and paper. The stems are very pliable and can be used in
basketry. It is also used for stuffing pillows etc., and for making paper. The smaller stems are
used in basket making. The branches are often made into brooms. A yellow dye is obtained
from the flowers. An essential oil is obtained from the flowers; it is used in perfumery [6].
During the last years, an increasing awareness of the public opinion about environmental
and health problems pushed towards the utilization of natural raw materials, drawing the
attention to industrial fibre crops. Industries all over the European Community are looking for
raw material for replacing artificial fibres in composite materials to alleviate problems related
with composite materials disposal at the end of the technical life. In Europe the use of
natural fibres in the automotive industry in 1999 was about 21,300 tones and in 2000
about 28,300 tones. In 2005 the use of natural fibres was about 70,000 tones and in 2010
could increase to more than 100,000 tones of natural fibres for the effect of EU end-of-
life vehicle directive than influence this development. The sources of raw material used in
composites for automotive industry are mainly represented by flax, hemp, jute, kenaf, sisal
and coconut fibres. Recently there has been a revival of interest in Spanish Broom as a
possible source of natural fibre in automotive industry. Natural abundance, much higher
strength per unit weight than most inorganic fillers, lower density and their biodegradable
nature make natural fillers attractive as reinforcements of engineering polymer systems [7 –
9]. Spanish Broom cortical fibres are multiple elementary fibres (ultimates) arranged in
bundles. The elementary fibres are bound together by lignin. A thick secondary cell wall
indicates high cellulose content. The diameter of ultimates varies from 5-10 μm while the
diameter of the whole bundle is about 50 μm. The values in tensile strength and elastic
modulus are promising supporting the hypothesis that these fibres can be a potential
replacement for man made fibres in composite materials. Spanish broom (Spartium junceum)
fibres are new biofibres used as such or in composites [10]. The improvement of the
properties is necessary in most purposes. Chemical processes of esterification with these acids
are developed in special solvents for cellulose, in the presence of acid chlorides, pyridine and
trifluoroacetic acid at very long reaction times. In these cases the pollution is high. [11] To
avoid these drawbacks, a physical way of functionalization of vegetable fibre is proposed
[12]. A highly hydrophobic product with retention of fibrous structure was obtained by the he
reactions with higher saturated fatty acids (C10–C18) yielded lower DS values but still
comparable hydrophobicity [13].
Possible ways of Spanish broom valorization are given in Scheme 2 [14, 15]. Enhanced
fibre properties for improving the properties of new products and applications can be obtained
by: new processing and production concepts include the development of environmentally
friendly and energy-efficient processing and surface modification of fibres, yarns and fabrics.
Creation of new natural polymer surfaces by plasma grafting and the deposition of thin film
coatings by plasma assisted processes are applied in order to: improve: hydrophilisation,
dyeing, reactive dyeing, printing, increase of the flame retardance and thermal insulation
properties, increase abrasion resistance, to obtain higher conductivity, barrier layers to
chemicals, UV protection, etc. use of various gases, mixtures of gases and monomers in vapor
form and solutions, new softeners, to remove the impurities (reduction of chemical
Rakesh Sharma, Bharati D Shrinivas

pretreatments) which are summarized in the Scheme 3. Special applications are also for:
specific protein attachment [16], biomolecule (e.g. heparin) immobilization [17], improved
cell attachment and spreading [18, 19] reduction of calcium carbonate nucleation [20]
siloxane coating [21] to create smart clothes / wearable computing [22] elaboration of
technologies for nano and micro coatings on textiles, etc.

Scheme 2. Possible ways of Spanish broom (Spartium junceum, syn. Genista juncea) valorization.

Scheme 3. Low temperature plasma: effects on textile.

Several low temperature plasma systems are known as : low pressure plasma systems
(glow discharge) and atmospheric plasma systems (corona discharge, dielectric barrier
discharge, glow discharge) The last one can be integrated into the production line
manufacturing processes and also offers some advantages such as sample size is unlimited,
Grafting of Biofibres with Carboxylic Acids 5

are running at lower temperatures, [23, 24] and secondary reaction are avoided. Use of
various gases, mixtures of gases and monomers in vapor form and solutions can be used [25 -
29]. Composites of natural fibers and thermo-plastics can be combined to form new enhanced
materials. One of the problems involved in this type of composites is the formation of
chemical bonds between the fibers and the polymers at the interface. Low energy glow
discharge plasmas are used to functionalize cellulose fibers implanting polystyrene between
the fibers and the matrix that improve the adhesion of both components, the adhesion in the
fiber-matrix interface increasing with time in the first 4 min of treatment [30]. Methods of
textile functionalization by plasma treatment include: a) plasma polymerization using
different reactive gases, monomers or prepolymers, mixture of gases and monomers; b)
plasma activation which mainly involves incorporation of new functional groups, which can
be achieved by treatment with solutions and physical vapor deposition. Different procedures
have been used to modify the cellulosic fibres from jute [31], wood fibres [32] cotton fabrics
[33] for which the effect of corona discharge consists in the removal of impurities (reduction
of chemical pretreatments), improvement of mercerisation process and dye uniformity in
continuous dyeing, improvement of rubbing fastness of pigment printing and also it can
influence the chemical finishing (softening, crease recovery, flame retardancy) and physical
properties of linen fabrics and make raw cotton hydrophilic. The uniformity of dyeing is
similar when the wetting agent in the recipe is replaced by a corona treatment. The authors
claim that by corona treatment the wetting agents in different operations can be avoided, for
example, in desizing, mercerization, bleaching and dyeing, higher number of barium is
obtained in cotton mercerization, higher yield, penetration and rubbing fastness are obtained
in pigment printing. In easy-care finishing, higher crease angle recovery, lower formaldehyde
release, as well hydrophilisation is obtained.
This paper deals with plasma grafting of Spanish broom (Spartium junceum, syn. Genista
juncea) fibres with several acids in order to establish the optima conditions for their
modification and obtaining of new improvements.

1. EXPERIMENTAL
Materials and Methods

The composition of the Spanish broom (Spartium junceum) fibres undergone to plasma
treatment and those modified were globally characterized by FT-IR spectroscopy.
The FT-IR spectra have been recorded by means of a DIGILAB Scimitar Series FT-IR
spectrometer (USA) at 4 cm-1 resolution. Five recordings were performed for each sample and
the evaluations were made on the average spectrum obtained from these five recordings. FT-
IR spectra are recorded in KBr pellets. Processing of the spectra was done by means of
Grams/32 program (Galactic Industry Corp.).
The FT-IR spectra of the sample under study are given in Figure 1 and the assignment of
the bands is presented in Table 1.
Rakesh Sharma, Bharati D Shrinivas

0.3
Absorbance, a.u.

0.2

0.1

0.0
4000 3500 3000 2000 1500 1000
-1
Wavenumber, cm

Figure 1. FT-IR spectra of the fibres from Spanish broom (Spartium junceum) fibres.

Table 1. Assignment of the FT-IR bands from the spectrum of Spanish broom (Spartium
junceum) fibres
Band position (cm-1) Band assignment
O(2)H…O(6) intramolecular H – bonds in cellulose, OH intermolecular, H – bond in the
3418 vs
10Ī plane
3277 sh O(6)H…O(3) intermolecular H bonds in cellulose
2913 s Asymmetric CH valence vibration
2

2861 sh Symmetric CH valence vibration


2
C=O stretch in unconjugated ketone, carbonyl and in ester groups (frequently of
1723 sh
carbohydrate origin)
1639 s Protein impurity and water associated with lignin
1503 sh C=C stretching of the aromatic ring (G)CH deformation
CH in pyran ring symmetric scissoring; OH in plane bending in cellulose I and cellulose
1453 s 2
II
1429 s H-O-C in plane bending of alcohol groups
1373 s CH bending in cellulose I and cellulose II
1320 s CH wagging in cellulose I and cellulose II
2
1277 m CH bending in cellulose I and cellulose II
1239 m OH in plane deformation, also COOH
1202 sh OH in plane bending in cellulose I and cellulose II
1160 s C – O – C asymmetric stretching in cellulose I and cellulose II
1114 s Ring asymmetric stretching in cellulose I and cellulose II
1057 s C – O valence vibration mainly from C(3) – O(3)H
1031 s Stretching C – O in cellulose I and cellulose II
896 w Anomere C – groups, C – H deformation, ring valence vibration
1
Grafting of Biofibres with Carboxylic Acids 7

In spectrum are identified all the bands of the main components mentioned in literature
cellulose, lignin, pentosans, and extractives and also traces of amine groups [34].
A qualitative appreciation of the relative content of cellulose and lignin in these samples
can be obtained from the ratios of the integral adsorption which are presented in Table 2.
These ratios are proportional with lignin content which lied between 25 and 30 % [35, 36].

Table 2. Lignin/Carbohydrates ratio from FT-IR integral absorbances

Sample Relative intensities of aromatic skeletal vibration


I /I I /I I /I
1505 1738 1505 1375 1505 1158
Height Area Height Area Height Area
Spanish broom fibres 0.88 0.55 0.38 0.24 0.44 0.40

According to the literature data, the chemical composition of the whole stem outlined a
high content of cellulose (67-76 %) while lignin (13-22%), pentosans (4-5%) and extractives
(6-7%) were low [37].
The following carboxylic acids have been used: butyric acid, oleic acid, olive oil,
sunflower oil, lactic and polylactic acids.
Butyric acid was purchased from Merck, its purity is >99% being grade for synthesis;
Lactic acid was also purchased from Merck, its purity was between 88 – 92 %;
Olive oil contains between 55.0 - 83.0 oleic acid, 3.5 - 21.0 % linoleic acid and 7.5 - 20.0
palmitic acid, 0.3 - 3.5 % palmitoleic acid, 0.5 - 5.0 stearic acid and others in amounts less
than 1%;
Oleic acid from sunflower oil which belongs to the vegetable oils group with a high
content in mono and polyunsaturated acids of about 94 ,90% ( 23.7 % oleic acid and 59.8 %
linoleic acid) and saturated acids 11,30% (mainly palmitic and stearic). It contains 99,90 %
lipids, trigliceride 99,20%, moisture content 0,1% [38].
Polylactic acid was laboratory synthesized and it has a number average molecular weight
of Mn = 2000.

Procedure of Grafting by Cold Plasma

The experimental set - up for cold plasma fibers‟ grafting is presented in figure 2. In a
typical experiment, after several washing cycles with inert gas (nitrogen) from the gas
metallic reservoir (6), into the cylindrical shaped vacuum plasma reactor (1), the working
pressure was established (0.3 mm Hg) and then the R.F. power was transferred to the reactor
through the semi cylindrical, external, silver-coated electrodes (8). The R.F. power was
dissipated to the electrodes from a R.F. generator (11) with the possibility of generating 50 -
300 W. The samples were deposited on special glass support for fibres exposure to grafting
(12). The Spanish broom fibres have been treated in plasma at a P=300 W, frequency 13.56
MHz, pressure 0.3 mm Hg for 5 or 10 minutes. Before treatment the samples were
impregnated with solutions of acids as: butyric acid, sunflower oil and olive oil (solutions 20
% in acetone) or with lactic and polylactic (Mn = 2000) acids (solution 4 % in ethanol).
Rakesh Sharma, Bharati D Shrinivas

Figure 2: Experimental set-up for fibers‟ grafting: 1 – vacuum plasma reactor; 2 – close glass vacuum
system; 3 – vacuum gauge; 4 – vacuum pump; 5 and 7 – glass valves; 6 – monomer flask (only for
distilled monomers in plasma medium, not for impregnated samples); 8 – semicylindrical external
silver coated electrodes; 9 – glass support for samples; 10 – central monomer‟s admission glass tube; 11
– HF generator (13.56 MHz); 12- special support for fibres exposure to grafting.

Unbounded acid from the treated samples was extracted for 6 h in a Soxhlet extractor.
The treated fibres were dried and analysed.
After plasma treatment all modified fibres become much softer comparatively with
untreated fibre.

2. INVESTIGATION METHODS:
FT-IR spectroscopy – see above.
2.1. XPS Analysis of the Fibres Surface

XPS spectra have been recorded by means of an Axis-Ultra de Kratos (UK) instrument
equipped with an electrostatic analyzer with a great radius and a detection system having 8
channels. Two X-rays sources are used one double of Al-Mg without monochromator and one
of Al with monochromator. The system is also provided with a source of electrons of low
energy in order to neutralize the electrostatic charge which will appear on the samples when
they are exposed to the monochromatic X-rays beam. The spectrometer operates in a high
vacuum of 5·10-9 mm Hg. The XPS chamber of analysis is connected with a
Grafting of Biofibres with Carboxylic Acids 9

transport/preparation chamber having multiple uses allowing rapid introduction of the


samples in the position suitable for analysis.
The samples in quartz crucibles have been introduced in XPS chamber and a pressure of
1-3·10-7 mm Hg was realized which is also necessary for the XDR source running.
The XPS spectra were acquired with an Al source of 300 watts power. For the elemental
composition determination the recording of the total spectra was done with an energy of
analyzer of 160 eV, with step size of 1 eV, lens act in hybrid mode which assures the
maximum sensitivity. The spectra of high resolution for chemical analysis have been recorded
using an energy of 20 eV, step size having 50 meV (lens in hybrid mode).
X-rays diffraction (XRD) analysis was done using a Bruker diffractometer equipped with
a Kristalloflex 760 sealed-tube copper anode generator, operated at 40 kV and 40 mA, and a
two-dimensional position-sensitive wire-grid detector (Bruker AXS) pressured with xenon
gas. Collimation was effected by a graphite monochromator with a 0.8-mm pinhole sample-
to-detector distance = 9 cm. Samples were placed in sealed Mark-Röhrchen glass capillaries
(Charles Supper) of 1.0 mm inner diameter. Scans: 1200 s (1200 scans)
SEM images have been taken with a scanning electron microscope TESLA BS 301
(Acceleration potential 10 kV).
Differential scanning calorimetry (DSC).Thermal characterization was performed using a
Mettler Toledo DSC 823e differential scanning calorimeter (DSC), calibrated with indium and
flushed with nitrogen. The heating and cooling scans were performed at 10°C/min, on 5 to 15
mg of sample packed into standard aluminium pans. First-order transition temperatures are
given by the peak values.
Thermogravimetry. Thermogravimetric analysis (TGA) was carried out under constant
nitrogen flow (200 ml/min) at a heating rate of 10 °C/min using a Mettler Toledo TGA/SDTA
851 balance. The heating scans were performed on 10 to 15 mg of sample. The kinetic
parameters have been evaluated by integral and differential methods. The kinetic parameters
have been evaluated by integral or differential methods using VERSATILE commercial
program which gives kinetic parameters by various methods (see below).

3. RESULTS AND DISCUSSION


3.1. FT-IR Spectra Results

FT-IR spectra of the studied samples are given in Figures 3. The spectra of the treated
samples are different from that of untreated fibre and they are particular for each acid used for
grafting. In all cases the evidence of the ester bonds in plasma grafted samples is clear at 1740
and 1610 cm-1. Bands shift and splits are also found at 2850 cm-1 and 2925 cm-1 (Figures 3 a
and b).
A very different spectrum shows the sample grafted with polylactic acid. Supplementary
bands being present at: 3020 and 1500 cm-1 and the band at 1750 cm-1 found in other samples
is shifted to 1725 cm-1. On the basis of these results it can appreciate that the grafting took
place with a high yield.
Rakesh Sharma, Bharati D Shrinivas

a) b)

c) d)

Figure 3. FTIR spectra of Spanish broom (Spartium junceum) fibres untreated and treated with different
carboxylic acids.

XPS Results
The measurement of binding energy (BE) can be used to characterize materials. The
observed BE depend on the specific environment where the functional groups are located,
most changes being within  0.2 eV of variation [39, 40]. The C1s BE is observed to increase
monotonically with the number of oxygen atoms bonded to carbon, that is C-C < C-O < C=O
< O-C=O < O-(C=O)O- consistent with that the carbon becomes more positively charged
with increasing number of oxygen atoms bonded to carbon.
The XPS intensity (integrated area under the photoelectron peak) is proportional to the
atom quantity in the detected volume, therefore by integrating the area under a given peak and
correcting for its ionization cross section, quantitative elemental analysis of the material can
be made [41, 42]. This requires an algorithm for peak fitting, which should include all
characteristic elements for a photoelectron peak, i.e. the peak height, width, shape. The
relatively simple shape of a photoelectron peak, due to the one electron process involved,
allows in most cases the deconvolution of complex experimental peaks. To extract
Grafting of Biofibres with Carboxylic Acids 11

quantitative information from the XPS spectra, the area and the BE of each subpeak for a
given orbital, e.g. C1s, must be determined. Typically, the spacing between subpeaks is
similar or inferior to observed peak widths (1 eV). Thus, it is rare when individual subpeaks
are completely separated in an experimental spectrum. This requires the use of a peak fitting
procedure. Quantities in such procedures, performed using appropriate software, include the
background, peak shape (Gaussian, Lorentzian, asymmetric, or mixtures thereof), peak
position, and peak height and peak width.
In the XPS spectra of organic compounds, peaks corresponding to carbon atoms in
different chemical environments every so often exhibit almost equal binding energies, and
they cannot be correctly separated using a standard mathematical procedure, especially in
substances with unknown chemical structures of the surface [43]. For example, in examining
substances containing functional groups like those below:

 C  OH COC COC

Peaks due to these groups in C1s spectra exhibit almost equal binding energies (about
286.5 eV) and, hence, cannot be separated using mathematical treatment. All the above is also
true in the determination of functional groups by other XPS spectra such as O1s or N1s.
This problem became more severe once those modification techniques were developed to
tailor the polymer surface properties, the chemical derivatization in XPS being firstly used in
the fundamental research of phenomena that occur in surface polymer layers under exposure
to plasma and other modifying agents.
XPS results for untreated and Spanish broom fibres grafted in cold plasma conditions are
presented in Figures 4 and Tables 3 and 4.
The main bands in XPS spectra – Figure 4a are as it is expected those of carbon and
oxygen. Each sample contains in traces some impurities.
The Si2p and Ca 2p bands are present in almost all spectra, this element being contained
in raw material. Si might be from the holder of the samples. The unmodified fibres also
contain traces of nitrogen and chlorine which are also find in plasma treated fibres with lactic
acid and oleic acid. The Zn2p band was found in the fibres treated with butyric acid while the
sample treated with polylactic acid contains manganese.
Rakesh Sharma, Bharati D Shrinivas

unmodified Butyric acid 10 min

Oleic acid 10 min Olive oil 10 min

Polylactic acid Lactic acid

Figure 4a. XPS spectra of the studied samples.


Grafting of Biofibres with Carboxylic Acids 13

Untreated sample Butyric acid

Oleic acid 10 min Olive oil

Lactic Acid Polylactic acid

Figure 4b. Deconvoluted carbon bands of XPS spectra of the studied samples
Rakesh Sharma, Bharati D Shrinivas

Untreated sample Butyric acid 5 min

Oleic acid 5 min Olive oil

Lactic acid Polylactic acid

Figure 4c. Deconvoluted Oxygen bands of the XPS spectra of the studied sample.
Grafting of Biofibres with Carboxylic Acids 15

Concerning the bands position, it can be observed a shift in C1s band for sample treated
with lactic, oleic and olive oil, while O1s band is shifted to lower binding energy only in the
spectra of sample treated with lactic acid – Table 3. The percentage area of the main elements
takes values particular for each treated samples. The C1s area increases in the following
order: untreated fibre < lactic acid < butyric acid < polylactic acid < oleic acid < olive oil cold
plasma treated sample. The O1s area has an opposite variation.
This is an evident proof that the grafting reaction took place. The most evident change in
the fibre structure at least at their surfaces is in the number and type of carbon atoms, as it
appears from deconvoluted carbon bands in Figures 4b. The untreated fibres and butyric acid
treated sample have four types of carbon atoms, while the other treated samples have at least
six types of carbon atoms. The oxygen deconvoluted bands – Figure 4c – are much simpler
being evidenced two types of oxygen atoms in untreated fibre and butyric acid or lactic acid
treated fibres, while the oleic acid-, olive oil- and polylactic acid grafted fibres exhibit three
kinds of oxygen atoms.
The assignment of bonds which belongs these atoms is given in Table 4 and it was made
according to the literature data [44 – 47].

Table 3. Band position and percentage area of total area for untreated and
cold plasma treated samples
Untreated Butyric acid Oleic acid Olive oil Lactic acid Polyactic acid
Band Band position/% Band Band Band Band position/%
Atom position/% of total area position/% of position/% of position/% of total area
of total area total area total area of total area
C1s 282.91/67.6 282.91/74.08 281.91/87.7 281.91/89.90 281.91/70.43 282.91/ 82.36
O1s 529.91/31.3 529.91/24.58 529.91/11.5 529.91/9.32 528.91/28.10 529.91 /16.80
N1s 397.91/0.4 397.91/0.57 396.91/0.3 396.91/0.44
Si2p 99.91/0.4 99.91 /0.42 98.91/0.3 98.91/ 0.64 96.91/ 0.32 99.91/ 0.32
Cl2p 197.91/0.1 194.91/0.13 193.91/0.1 196/ 0.06
Ca2p 3442.9/0.2 344.91/0.21 342.91/0.08 343/ 0.04 344.91/0.29
Zn2p 1020.91/0.06

Table 4. Results of C1s and O1s high resolution spectral fitting (%)

Groups Untreated Butyric Oleic acid Olive oil Lactic Polyactic


acid acid acid

Carbon atom containing group

Positions of the carbon atoms in deconvoluted curves


C-C (285.0eV) 285.00 285.00 285.00 285.00 285.00 285.00
C-O-C (286.6 286.63 286.55 285.88 285.68 286.15 285.85
eV) 287.99 287.97 286.78 286.40 286.85 286.73
O-C-O (288 eV) 287.17
289.13 287.76 287.91 288.00 288.52
289.17 289.25 289.22 289.21 289.32
O-C=O (288.7
Rakesh Sharma, Bharati D Shrinivas

eV) 290.43 289.87 290.1 290.46


O-CO-O (290.4
eV)
C1(C-C;C-H) 32.34 44.63 47.58 30.36 37.26 27.23
C2(C-O-) 51.57 41.62 17.73 17.29 26.32 20.87
C3 (O-C- 13.45 10.03 4.89 4.01 11.75 15.36
O;C=O)
C4 (O-C=O) 2.65 3.72 4.42 4.40 7.01 5.28

Oxygen atom containing group


Position of the oxygen containing groups in deconvoluted curves
532.61 532.81 532.79
532.93 532.89 533.82 535.55 533.02
533.55 533.79
534.16 534.10 534.10
535.02 535.26
O1 (OC=O) 78.41 90.04 45.18 39.36 94.00 35.87
O2 (C-O) 21.59 9.96 43.30 48.31 6.00 37.98
O3 - - 11.52 12.33 - 26.15
 C1 is a carbon bonded only to another carbon (C-C) or a hydrogen atom (C-H);
 C2 – atom singly bonded to a oxygen atom (C-O-), other than a carbonyl atom;
 C3 – a carbon atom single bonded of two oxygen atoms (-O-C-O-) or to a single
carbonyl atom (-C=O);
 C4 – a carbon atom single bonded to an oxygen atom and to a carbonyl oxygen atom
(-O-C=O). C4 group (-O-C=O) is very low in untreated sample (2.65 %) and
increases in treated sample as a result of ester bonds appearance. In the same time the
content of C2 group (C-O- from C-O-H) decreases from the same reason. The results
are in good agreement with FTIR measurements.
 O1 is a oxygen atom linked to a carbon atom by a double bond, and also an oxygen
single linked (O-C=O);
 O2 – an oxygen atom linked to a carbon atom by single bond (C-O- );
 O3 – oxygen atom from supplementary oxidation processes.

Taking into account a content of hydroxyl groups in the superficial layers of the cellulose
fibers (up to ~ 10 nm depth) of 51,57 % and also the variation of the C2 groups (C-O- from
C-OH) which decrease from 51.57 % to 16.13 % and the increase of the C4 (-O-C=O) groups
from 2.65 % to 7.01 % can be estimate a degree of grafting of superficial layers which
depends on the type of carboxylic acid employed as it appears in Table 5. . Some errors in
XPS results for non-smooth topographies samples as in our case (see SEM results) can
appear, but they do not affect the find results, because the modifications are very clear.
Grafting of Biofibres with Carboxylic Acids 17

Table 5. Degree of grafting of the Spanish broom fibres with various carboxylic acids

Fibre grafted with: Degree of grafting (%) O/C atomic ratio


Untreated 0 0.463
Butyric acid 19.3 0.332
Oleic acid 53.6 0.131
Olive oil 45.8 0.104
Lactic acid 68.8 0.399
Polilactic acid 62.8 0.204

The highest degree of grafting was achieved with lactic acid and this decreases in the
order: lactic acid > polilactic acid > oleic acid (sunflower oil) > olive oil > butyric acid.
In cold plasma conditions the grafting can take place both with unsaturated and saturated
carboxylic acids, therefore the fibres grafted with sunflower and olive oil should have both
chains with oleic acid, linoleic acid , palmitic and stearic.
The formation of other kinds of bonds is also possible, taking in the view the diversity of
the active species possible to be formed in plasma conditions.
Comparing the values of the O/C atomic ratio it could appreciate that the grafting of the
lactic, polylactic and butyric acids do not change significantly the surface composition while
the other can be bonded by different kinds of bonds which lead to decrease of the oxygen
percentage on the surface.
The morphological aspects of the grafted samples have been studied by XRD and SEM.

XRD Results
X-ray diffraction (XRD) techniques were used in the study of the effect of grafting on
crystallinity. Generally XRD patterns of the cold plasma treated samples are similar with that
of the untreated fibre – Figure 5.
Rakesh Sharma, Bharati D Shrinivas

Untreated

Butyric acid grafted

Oleic acid grafted


Grafting of Biofibres with Carboxylic Acids 19

Olive oil grafted

Lactic acid grafted


Rakesh Sharma, Bharati D Shrinivas

Polylactic acid grafted

Figure 5. XDR patterns of the untreated and cold plasma treated Spanish broom (Spartium junceum,
syn. Genista juncea) fibres.

The crystalline peaks are found in the untreated sample at 2 theta degree of: 3; 15.8; 22.5;
29 and 34 and they correspond to the Iα - cellulose crystalline structure [48, 49]. The same
peaks characterize the crystalline fraction of all cold plasma grafted samples but they are
shifted to higher 2 theta degree and they are much wider and the rings become much diffuse
and for the fibres grafted with oleic acid, olive oil and lactic acid some rings can not be
distinguish. This observation is in accordance with the data obtained for other cellulose esters
[50]. It was shown that X-ray diffraction analysis indicates distinct crystal patterns for these
crystalline cellulose esters, and differential thermal analysis shows strong melting peaks. X-
ray diffraction analysis of secondary cellulose esters, that is, esters having a substantially
lower degree of esterification, shows very diffuse patterns which are only slightly indicative
of crystalline structure. On the other hand, differential thermal analysis, shows strong
endothermic peaks which appear to indicate melting of crystalline material.
A small peak appears at 38 2 theta degree in the samples treated with butyric acid, oleic
acid, lactic acid or polylactic acid, probably because of some changes in crystallinity network.
The crystallinity degree was evaluated as the ratio between the area under the crystalline
peaks and the area under the amorphous halo which is a broad hump in the XRD pattern –
Table 6. The decrease of the crystallinity degree after grafting with carboxylic acids is much
important for the fibres grafted with oleic and lactic acid, therefore even the bulk morphology
of the Spanish broom fibre is affected by grafting.
Grafting of Biofibres with Carboxylic Acids 21

Table 6. XDR data for untreated and grafted Spanish Broom fibres with carboxylic
acids
Spanish Treated
Treated Treated Treated Treated
broom polylactic
butyric acid oleic acid olive oil lactic acid
untreated acid
Crystallinity
55.8 45.53 39.52 42.41 32.86 40.54
index (%)

SEM Results

Untreated sample X 810


(1 cm = 10 μm)

Butyric acid grafted fibers (10 min) X 810 (1 cm = 10 μm)


Rakesh Sharma, Bharati D Shrinivas

Oleic acid grafted fibers (10 min) X 410


(1 cm = 20 μm)

Olive oil grafted fibers (10 min) X 430


(1 cm = 22 μm)
Grafting of Biofibres with Carboxylic Acids 23

Acid polylactic grafted fibers (10 min) X 810


(1 cm = 10 μm)

Lactic acid grafted fibers (10 min) X 430


( 1 cm = 22 μm)

Figure 6. SEM micrographs of the untreated and grafted Spanish broom (Spartium junceum, syn.
Genista juncea) fibres under cold plasma conditions.
Rakesh Sharma, Bharati D Shrinivas

The existence of the graft layers on the surfaces of the Spanish broom fibers is clearly
evidenced by scanning electron microscopy (SEM) (Figures 6). Important morphological
differences can be observed between the support and the grafted derivatives in all cases. It can
be noticed that the aspect of the carboxylic acids grafted fibers are totally different from the
surface of the control fibers (support). On the surfaces of the fibres grafted with oleic and
olive oil some irregularities looking as depositions on particles are observed, while the fibres
grafted with butyric acid, lactic and polylactic acid show a uniform aspect, the fibres becomes
voluminous and fibrils seems to be expanded and distinct.

Thermal Properties

DSC Results
Two important aspects can be followed by DSC study of the cellulosic materials:
influence of the substituents on thermal characteristics and because of their hydrophilicity the
interaction with absorbed water.
Strong correlationship between the melting point and the length of substituents at the
secondary hydroxyl groups at C2 and C3 positions, have been found for the cellulose fatty
acid heteroesters (cellulose propanoate diacetate, cellulose butanoate diacetate, cellulose
acetate dipropanoate, cellulose butanoate dipropanoate, cellulose acetate dibutanoate, and
cellulose propanoate dibutanoate) [51].
Hatakeyama et al. found that vaporization peak is split into two peaks, one is at around
60°C and the other is at around 120°C. The high temperature vaporization peak is related with
the structural change of amorphous chains of cellulose by desorption of bound water [52, 53].
Cieśla et al., established that the profiles of thermal effects depend on water content, time of
conditioning, film pretreatment etc [54].
Both heating – Figure 7 - and cooling – Figure 8 - cycles have been applied from -50 –
250 oC to the untreated and grafted fibres. The DSC curves recorded by heating show two or
three peaks. The process with peak temperature around 100 oC can be due to release of the
absorbed water. A large amount of non-freezable strongly bounded water was also detected.
Cellulose absorbs water and the structural changes appear on ordering of polymer
fraction. Due different strengths of water binding in the case of the grafted fibres the
characteristic temperatures and the enthalpies vary with nature of grafts. Differences between
interactions of particular cellulose fibres with water can be detected during the first, the
second and the third heating. Because of water loss in the first run, in the next runs its
quantity (and enthalpy) decreases and some temperatures are changed.
Comparing the data of Table 7 of the grafted fibres with those of untreated one the
following conclusions can be draw: the first peak is shifted to lower temperature after grafting
but its enthalpy is generally higher than of untreated fibre. Some peaks at negative
temperatures are present in the DSC curve of fibres grafted with oleic and olive oil. The
biggest difference between peak temperatures of the peak of absorbed water release is found
for samples grafted with olive oil, lactic and polylactic acid. This should means that water is
stronger bonded in grafted fibres than in untreated one but in the lower amount as was found
also by TG/DTG (see below).
Grafting of Biofibres with Carboxylic Acids 25

a) 8

5
Exo

3'

-50 0 50 100 150 200


Temperature (°C)

Figure 7. DSC thermograms of the treated and untreated samples obtained by heating at 10°C/min [with
8 = untreated; 7 = treated with oleic acid; 6 = treated with butyric acid; 5 = treated with lactic acid; 4 =
treated with polylactic acid; 3 = treated with olive oil (first heating); 3‟ = treated with olive oil (second
heating)].

Similar values have been obtained for cellulose esters with linear aliphatic acyl
substituents ranging in size from C[12] (lauric acid) to C[20] (eicosanoic acid). A series of
transitions that represented motion by both ester substituents and cellulosic main chain. Broad
crystallization and melting transitions attributed to side-chain crystallinity were observed in
the range between -19 and +55°C. Melting temperature values of 96°C and 107°C [55].
As concerns the variation of the temperatures of the second peak which is probably a
decomposition/melting step expecting fibres grafted with oleic acid, all other grafted fibres
exhibit lower decomposition/melting temperatures and also lower enthalpy of this process, the
most important decrease being found for fibres grafted with olive oil and lactic acid (which
acts probably as plasticizer).
Rakesh Sharma, Bharati D Shrinivas

Table 7. Characteristic temperatures and enthalpies of the processes evidenced in DSC


curves recorded by heating of the studied samples

Sample Peak 1 Peak 2


Tonset, Tpeak, Tendset, H(J/g) Tonset, Tpeak, Tendset, H(J/g)
(oC) (oC) (oC) (oC) (oC) (oC)
Untreated 94.3 149.6 191.0 -59.63 179.5 186.4 198.5 -32.85
-46.5 -18.2 17.7 -10.5
Butyric 1.84 45.62 76.72 -3.48 144,7 164.4 172.7 -0.53
acid 56.0 99.9 138.9 -107.2
94.06 130.9 178.1 -93.75
-32.8 -10.6 3.9 -0.54
Oleic acid
36.3 47.6 56.4 -1.14

99.05 132.8 172.4 -57.6 198.1 202.8 216.0 -1.32

-41.47 2.08 15.82 -4.81


Olive oil
44.32 106.49 153.1 -68.07 121.7 139.7 173.8 -3.28
Lactic acid 59.1 104.4 147.1 -79.9 154.2 160.1 168.1 -1.57
Polylactic 59.26 111.91 158.07 -92.76 163.58 169.47 179.68 -0.91
acid

The DSC curves recorded by cooling are particular for each fibre studied and there are
big differences in respect with the untreated sample. The DSC curves of the untreated sample
and that of the fibres grafted with butyric acid and polylactic acid show two exothermic
processes with peak temperature at 38 and 181 oC, the temperatures of the grafted fibre being
close to those of the untreated sample, that should means that this grafts do not influence
these crystallization processes.
The crystallization process which occurs in the temperature range 50 – 200 oC is also
present in the DSC curves of fibres grafted with oleic acid but it occurs at lower temperature.
The fibres grafted with oleic acid, lactic acid and polylactic acid shows an exothermic process
around 100 oC while the fibre grafted with olive oil and oleic acid show a crystallization peak
at low temperatures at around -20 oC which could be due to the frozen bonded water.
Grafting of Biofibres with Carboxylic Acids 27

a) b)

8 3
Exo

-50 0 50 100 150 200 -50 0 50 100 150 200 250


Temperature (°C)

Figure 8. DSC curves of the treated and untreated samples obtained by cooling at 10°C/min of a)
untreated sample (8); treated with oleic acid (7) and b) treated with olive oil (3).

Table 8. Characteristic temperatures and enthalpies of the processes evidenced in DSC


curves recorded by cooling of the studied samples

Sample Peak 1 Peak 2


Tonset, Tpeak, Tendset, H(J/g) Tonset, Tpeak, Tendset, H(J/g)
(oC) (oC) (oC) (oC) (oC) (oC)
Untreated 48.39 38.42 1.89 0.77 190.6 181.23 171.1 2.16
Butyric 55.1 33.3 21.9 0.54 198.4 193.6 182.9 0.27
acid
Oleic acid -14.75 -24.12 27.6 1.46 202.3 195.2 184.8 0.32

39.5 29.3 11.5 0.35

116.4 110.97 98.7 0.43

Olive oil -3.4 -18.4 -43.87 4.22 220.8 215.2 201.94 2.08
Lactic acid 13.06 10.7 8.22 0.78 194.5 191.5 185.4 0.26
106.2 100.06 89.9
Polylactic 46.09 35.1 21.7 0.13 202.1 193.5 173.99 0.76
acid 131.5 117.9 99.2 0.13

The sorbed water molecules are directly bound to the hydrophilic site to form non-
freezable water. Then, beyond a certain water content threshold, the sorbed water molecules
Rakesh Sharma, Bharati D Shrinivas

become freezable, but with a melting point lower than 0°C, due to their location in the second
hydration layer [56, 57].
The peak at high temperature seems to be due to of a reversible process because it
appears both during heating and cooling at close temperature. This is very difficult to be
explained. It is possible as some substituents to act as plasticizer as was found by Aranishi et
al., for a melt spun fiber comprising a thermoplastic cellulose mixed ester composition
containing as plasticizer polylactic acid [58], and for cellulose oligomers with n>20: (cotton,
wood, paper), a melting temperature > 250 oC is appreciated, but all time this process is
accompanied by carbonization. For Eastman cellulose acetate a glass transition temperature is
found at 180-186 oC [59] while the melting range lies between 230-250 oC. Therefore the first
type of transition associated with other phenomena could be much probable in our case.

TG Results
There is a great number of studies thermal degradation of cellulose and cellulose esters
such as cellulose acetates, nitrate, cellulose phosphate [60]; cellulose benzoate, cellulose
succinate and cellulose cinnamate [61] esters with fluorine-containing substituents [62]
cellulose fibers partially esterified with some long chain organic acids such as: undecylenic
acid; undecanoic acid; oleic acid and stearic acid [63] and a particular behaviour was found in
each case.
The TG/DTG curves of under study samples are given in Figure 9 and characteristics
thermogravimetric data are summarized in Table 9.

Figure 9. TG/DTG curves of Spanish broom fibres untreated and grafted with different carboxylic acids
in cold plasma conditions.

In the first thermogravimetric step, all characteristic temperatures for grafted fibres are
superior those of untreated Spanish broom fibres and mass loss is lower. This process could
attributed to water loss and should means that after grafting the bonding with water is
stronger, but the absorbed amount is lower. In the second thermogravimetric step onset
temperatures for all grafted fibres are higher than that of untreated one, while peak
temperatures are almost unchanged. This step corresponds to decomposition and the shift of
the onset temperatures to high temperature indicates an increase in thermal stability at least at
Grafting of Biofibres with Carboxylic Acids 29

the fibres surface. This process is very complex showing many inflexions which appear at
low temperatures for grafted fibres, indicating some changes in reaction mechanism as was
previously shown [64].

Table 9. Thermogravimetric data of Spanish broom fibres untreated and functionalized


with different carboxylic acids

Thermogravimetric Spanish Treated Treated Treated Treated Treated


Characteristic broom butyric oleic olive oil lactic polylactic
untreated acid acid acid acid
Peak I
Tonset (oC) 22,5 24.9 25.1 23.5 23.9 23.6
Tpeak (oC) 49,0 49.0 53.8 49.0 49.0 55.2
Tendset (oC) 118,3 118.3 114.6 126.3 115.6 117.4
w (%) 5,4 5.0 3.5 3.9 3 2.7
Peak 2
Tonset (oC) 177,2 182.2 184.3 198.3 179.9 180.0
358,5 358.5 359.5 358.5 358.5 358.5
Tpeak (oC) (i) 409,0 (i) 405.7 (i) 406.5 (i) 406.4 (i) 406.4 (i) 406.4
(i) 449,8 (i) 449.2 (i) 444.5 (i) 457.2 (i) 449.1
Tendset (oC) 550,6 547.9 578.2 529.2 534.5 542.6
w (%) 75,2 77.3 79.9 79.8 76.8 78.4
i – temperature at inflexion point of DTG curve.

To have many information about the involved processes the overall kinetic parameters
have been evaluated – Table 10, using both differential and integral methods coupled in the
commercial computing program VERSATILE.

Table 10. Kinetic parameters (E - overall activation energy and n - reaction order) of
thermal decomposition (second step) of Spanish broom fibres untreated and grafted
with different carboxylic acids

Sample Spanish Treated Treated Treated Treated Treated


broom butyric acid oleic acid olive oil lactic acid polylactic
untreated acid
Methods of evaluation E, E, E, E, E,
E, kJ/mol; n
kJ/mol; n kJ/mol; n kJ/mol; n kJ/mol; n kJ/mol; n
Coats – Redfern [65] 89.18; 1.7 87.75; 1.6 85.52; 1.5 106.95; 1.7 88.99; 1.7 87.08; 1.6
Flynn-Wall [66] 94.38 ; 1.7 93.02; 1.6 90.92; 1.5 111.30; 1.7 91.06; 1.6 92.40; 1.6
van Krevelen [67] 131.09; 2.0 129.76; 1.9 121.88; 1.7 156.50; 2.0 127.70; 1.9 124.74; 1.8
Urbanovici-Segal [68] 90.53; 1.7 89.13; 1.6 86.89; 1.5 108.13; 1.7 90.25; 1.7 88.44; 1.6
Achar [69] 71.44; 1.3 72.98; 1.3 67.75; 1.1 91.46; 1.4 75.09; 1.4 77.48; 1.4
Freeman Caroll [70] 75.54; - 70.01; - 65.30; 90.91; - 66.98; - 64.44; -
Piloyan 71] 62.97 - 64.25; - 66.25; 83.98; - 60.31; - 62.90; -

Significant changes in the kinetic parameters are obtained only for fibre grafted with
olive oil which contains 55.0 - 83.0 oleic acid, 3.5 - 21.0 % linoleic acid.
Rakesh Sharma, Bharati D Shrinivas

To explain the important changes in properties of the Spanish broom fibres after plasma
treatment, the plasma action must be considered. Plasma aided synthesis (deposition and
grafting reactions) involves fragmentation of plasma gases and reorganization of the resulting
neutral and charged species, inside and outside the plasma area, into nonvolatile, high-
molecular-weight structures. Recombination mechanisms developed in the absence of plasma
(outside the plasma zone) usually lead to the incorporation of less fragmented building blocks
into the nascent macromolecular structures [72-75].
Graft-polymerization reactions initiated from plasma-activated polymer surfaces involve
two distinctive and consecutive processes: implantation onto polymer substrate of active sites,
like free radicals or reactive functionalities followed by the initiation of conventional graft-
polymerization reactions in situ or ex-situ conditions depending on the stability of the active
sites. Supramolecular structure of polysaccharides controlled by intramolecular and
intermolecular hydrogen-bonding (conformation of macromolecules), existence of crystalline
and amorphous zones, exerts a significant influence on both their physical and chemical
properties. Conventional modification techniques performed on lignocellulosics to make them
compatible with other polymers or to enhance their surface properties by grafting with some
additives (such fatty acids) alter significantly the supramolecular order of the macromolecular
networks, diminishing their inherent physicochemical characteristics. Cold plasma
environments offer a unique way for modifying these materials without altering the bulk
structures and characteristics. Intrinsic properties can be preserved in this way, materials with
advanced performances and tailored properties can be obtained. Peroxide functionalities are
possible to be formed and they can strongly interact with the counterpart on surface.
The interaction of the active species of a plasma with polymer surfaces involves electron
mediated processes and positive ion-induced reactions. The last leads through neutralization
reactions. To energy concentrations localized on macromolecular chains (electronically
excited states) which can promote hemolytic bond cleavages leding to the formation of free-
radical sites. These reactive centers lead to a large variety of functionnalization mechanisms
depending on the reaction environments under in situ or ex situ plasma conditions. Several
possible ways of grafting are given in Scheme 4.
Grafting of Biofibres with Carboxylic Acids 31

6
* CH OH CH OOC-R
2 2
5C O 5C
O O
* O O O
C* C H C
4 OH 4 OH
1 1 H
H
3 C* C* H
3C C
6 CH OH Active sites at
2
2 different carbon atoms H OH R OH
5C O 6
O O + R-COOH
N2
C H C or other different ways
4 OH plasma CH OH with various probabilities
H 1 H 2
3C 2
C Fragmentation
5C O
H O CH OH
OH O 2
C H C 5C
4 OH * O
1 H O O
H
3C C* C H C (R)
2 4 OH
H OH 1 H
R'COOH 3 C CH
2
H (OOCR)
OH

Scheme 4. Suggested reaction mechanism for nitrogen plasma induced molecular activation,
fragmentation and grafting of the Spanish broom (Spartium junceum, syn. Genista juncea) fibres.

The trapped free radicals can “survive” in the polymer matrix and their intensities vary
significantly with the nature of fibres and plasma gases. A good correlation has been found
between free-radical concentrations and ex situ plasma surface oxidation reactions. As it was
mentioned the characteristic cellulose peaks are: C-OH, C-O-C (286.6 eV) and O-C-O (288
eV) and also the existence of O-C=O (288.7 eV) and O-CO-O (290.4 eV) can be noticed. The
formation of new functionalities (O-C=O and O-CO-O) are possible through the cleavage of
C1-C2 bonds of the pyranosic ring.
It was demonstrated that all possible four hydroxyalkyl radicals (1,2, 3) are generated as
primary structures, through the hydrogen abstraction mechanism, but with a preference at C2
and C5 carbon atoms. Plasma irradiation produces preferentially the alkoxy-alkyl radicals at
C1 of the glucose units.

CONCLUSIONS
The research was concentrated to obtain fibres with new or significantly improved
properties, with tailored functionalities for special applications starting from natural fibres
based on concept of development of environmentally friendly and energy-efficient processing
by surface modification of fibres, yarns and fabrics.
New fibres with enhanced properties based on existing natural compounds have been
obtained by plasma grafting of the Spanish broom (Spartium junceum, syn. Genista juncea)
fibres with carboxylic acids. The heterogeneous esterification reaction with five different
acids was realized in mild conditions with a high yield. The characterization carried out with
Rakesh Sharma, Bharati D Shrinivas

X-ray photoelectron spectroscopy (XPS), XRD, SEM, differential scanning calorimetry and
thermogravimetry showed that the individual fibers were covered with the corresponding
esters by partial degrees of substitution of the cellulose and that the surface degree of
substitution of the cellulose fiber was higher than for the bulk, showing that the esterification
reaction was a surface phenomenon, mainly in cold plasma conditions.
New biofibres obtained could be processed in products which should improve comfort
performance and enhance micro-climate of bed rooms. Develop additional properties
enhancing natural properties of vegetable fibres lead to promotion of fibers with lower
ecological impact, promotion of integrated European production chain
These fibers could be also useful as reinforcements in various composites (mainly
containing polyolefins).

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In: Food, Diet and Disease Copyright 2009 Innovations And Solutions, Inc. Florida USA 1
Editors: Rakesh Sharma, Bharati D Shrinivas
Open Access

Bioactive Foods and Nutraceutical Supplementation Criteria in


Cardiovascular Protection

Rakesh Sharma1,*, Bharati D Shrinivas*

1
Department of Bioengineering, Florida State University & Tallahassee Community College, Tallahassee, FL 32304, USA

Abstract: Nutraceuticals are natural and commonly found in bioactive foods or whole plants to keep energy balance
in the body and promise substantial therapeutic value in cardioprotection. Major cardioprotective bioactive foods and
nutraceuticals are now part of nutrition supplements at nonprescription counters and their self-prescription is increased at
large scale. The literature suggests the growing use of new bioactive foods and nutraceuticals in cardioprotection and
management. The biochemical mechanisms of nutraceutical action in cardioprotection are poorly reported. The literature
indicates the success of fish oils, nutaceuticals in vegetable fat-free diets and restricted life style to enhance cardioprotec-
tion. The present paper highlights the need and benefits of newly introduced bioactive foods and revisits the cardioprotec-
tive mechanisms of bioactive foods and nutraceuticals. Broadly cardioprotective nutraceuticals are polyunsaturated fatty
acids, antioxidants, omega-3 fatty acids, vitamins, minerals and dietary fibers. Most of the nutraceuticals act as biochemi-
cal metabolites by direct intervention in intermediary lipid metabolism or regulating proteins of vascular system responsi-
ble of ‘cardiovascular incapability’.
Keywords: Cardioprotection, immunity, nutraceuticals, metabolites, diet, fat, nutraceutical supplementation.

INTRODUCTION from sports nutrition bar makers to soy burger manufacturers


– bioactive foods are poised to undergo very rapid growth in
The term “bioactive foods” was first defined as “foods,
the coming years. Bioactive foods are designed basically
food ingredients or dietary supplements that demonstrate
meeting four consumer demands: taste, convenience, simple
specific health or medical benefits including the prevention
proposition and price. A successful bioactive food product's
and treatment of disease beyond basic nutritional functions”.
bioactive role needs its perceptible health benefit. If a health
Now bioactive foods and nutraceuticals have emerged as
benefit is clearly understandable, or if the health benefit is
potential supplements in cardiovascular and cancer preven-
clearly perceptible - such as weight loss or stress reduction,
tive natural sources from food [1].
or can be easily measured - such as a product that reduces
Why new bioactive foods are important? Bioactive foods cholesterol, then the product is much more likely to succeed.
are fortified with nutrients to make them more usable within Now interest is growing for use of bioactive foods in cancer
daily recommended allowences (RDA). These nutrients are prevention. Recently JIVA™ a bioactive food made of res-
rich in vitamins, minerals and nutraceuticals or any food or veratrol combined with garlic has been advocated as poten-
part of a food that provides health or disease prevention tial cardiovascular prevention formula [2]. Similar bioactive
benefits with higher nutrition values. Bioactive foods are foods are in market by Kellogg Co.-cholesterol-reducing
served as cuisine line of frozen foods on shelf such as Cam- foods; Ensemble products - psyllium high fiber reducing
den(balanced meal program) for hypertension, high choles- cholesterol; Johnson & Johnson -cholesterol-lowering mar-
terol, or adult-onset diabetes; Tropicana Products, orange garine, Benecol®; Balance Bar Company, Nestlé, Vevey,
juice; Procter & Gamble's FruitCal® -calcium citrate malate Switzerland – PowerBar. These bioactive foods work on
etc. Growth in the bioactive food market has also rocked principle that cancer and heart disease are concerns of fa-
new combinatorial chemistry and profoundly accelerated the tigue/energy and stress. Tropicana Ultimate Smoothie com-
pace of discovery of new bioactive foods such as new high- bined with Galaxy's soy, rice and oats Veggie Milk® base
oleic soybean with no trans fatty acids that reduces heart with Tropicana's fruit juices. It was estimated that major
diseases. benefactors were subjects with heart disease (75%), cancer
Both food industries and pharmaceutical industries have (81%) including breast cancer (48%), colon cancer (37%)
roped up to use bioactive food, pharmaceutical and nutrition and prostate cancer (25%) using bioactive foods.[3]. The
products - from drinkable yogurt to mainstream designer bioactive foods as part of daily diet and lifestyle guidelines
bone, heart, and digestive health foods to calcium chews, for prevention of coronary artery disease (CAD) have been
evidenced as a major interest during the last few decades.
The concept of bioactive foods was initially considered
*Address correspondence to this author at the Department of Engineering as natural foods to provide energy as recommended daily
and Technology, Florida State University, Tallahassee, Florida 32304 USA
32304, USA;*Zoology Deptt. RTM University of Nagpur, Maharastra, India requirement in the body to maintain health and lipid lower-
E-mails: bharuds312@gmail.com and rksz2004@yahoo.com ing till year 1990. Later the importance of nutraceuticals was

1876-3960/09 2009 Innovations And Solutions


2 Sharma,Shrinivas

realized as beneficial in different cardiovascular disorders reported as active natural compounds. Majority of cardiovas-
with growing use of the nutraceuticals as self prescription in cular prevention evidence comes from clinical trials and
cardiovascular and developmental conditions in the last dec- animal studies [5, 6]. Self-described testimonies of nutraceu-
ade. In new era of 21st century showed enormous growing tical medicine and its success accrued over years in favor of
awareness of nutraceuticals as emerging potent therapeutic cardiovascular protection by lycopene, glucans(for cardio-
supplements with accepted concept of nutraceutical medicine vascular disease), noni Morinda citrifolia (for relief blood
as new branch of ‘complementary and alternative medi- pressure, muscle pain) [7, 8]. Recently, the antiarrythmic
cine’(CAM). In last three decades, national and federal bod- effect of quercetin, lipid lowering effect of fish oils and
ies accepted nutraceuticals as possible neutraceutical therapy beneficial effect of magnesium were reported including them
in main stream of medical education and health. The as cardioprotective food supplements [9-11].
healthcare industry demonstrated the shift of growing popu- Dilemma of Nutraceuticals in Cardioprotection
lation from medical treatment of dreaded cardiac arrest to-
wards non-prescription nutraceuticals as self-medication in Nutraceuticals may act as essential nutrient, as drug like,
acute coronary syndrome and coronary heart disease man- as regulatory biochemical metabolite and as phytohormone
agement and prevention of stroke. The best examples to in the body as shown in Table 1. Most of the side effects of
mention are Atkin’s diet for lipid lowering and CoQ10 for nutraceuticals remain undocumented and unnoticed. Re-
reducing thrombosis. The growing awareness of nutraceuti- cently, some prominent evidences are reported in favor of
cal benefits and shift of healthcare economics in favor of cardiovascular disease inhibitory metabolic activity of nu-
nutraceuticals brought neutraceutical medicine in spotlight of traceuticals in the human body:
government health policy on systematic use of nutraceuticals
1. Nutraceuticals may act as essential amino acid drug like
in cardiac protection and control of various coronary heart
essential nutrients. For example, tryptophan is needed for
and cardiovascular diseases. In last sixteen years, National
protein synthesis at low dose in humans [12].
Heart Lung Institute (NHLI) and other global efforts have
documented the fact sheets and several health documents on 2. The nutraceutical preparations containing phytosterols
nutraceuticals in cardiovascular disease control. The major are effective in lowering LDL cholesterol [13].
efforts were devoted in investigation of cardioprotective ef- 3. Bovine milk fat globule act as anticardiovascular disease,
fect of active nutraceutical component(s) on reduced athero- anticholesterolemic, coronary heart disease [14].
sclerosis plaque development, reduced coronary occlusion,
cholesterol desaturation to result the reduced risk of cardiac 4. The phytonutrients prevent myocardial cell proliferation
arrest, heart failure and reduced risk of atherosclerosis and and play significant role in the prevention of chronic de-
hypertension in initial stages. In last two decades the use of generative diseases. Notable examples are ginseng, spi-
nutraceuticals in prevention and disease control has been rulina, gingko biloba, amino acids, glucosamine, chon-
extended further as protective nutrition supplementation pol- droitin and Aegle marmelos. Herbal and medicinal plants
icy of center of disease control (CDC) under its independent have shown significant inhibition of cell inflammation
supervision. However, mechanisms still remain unproven [15]. Phytoesterogens play role in reducing myocardial
and unvalidated but practice of newly discovered nutraceuti- necrosis.
cals as food supplements in cardioprevention is acceptable. 5. Vitamin C, vitamin E, β -Carotene, lycopene (caro-
tenoids), lipoic Acid, glutathione(thiols) play role in car-
What are Cardioprotective Nutraceuticals in Bioactive diovascular disease prevention and inhibition of necrosis;
Foods? Co-Enzyme Q-10, super oxide dismastase (enzyme), se-
Nutraceuticals are natural bioactive chemical compounds lenium, copper, manganese, zinc (minerals) act as anti-
common in bioactive foods as products supplied from nutri- cardiovascular disease in cardiac cells[16].
tion industries. Nutraceuticals have value in health promot- 7. Polyunsaturated fatty acids (PUFA) such as safflower oil,
ing, disease preventing or semi-medicinal properties. Nu- corn oil, soybean oil, mustard oil, evening primrose oil,
traceuticals are found as natural products from (a) the food flax oil, hemp seeds, borage seeds showed protective ef-
industry, (b) the herbal and dietary supplement, (c) pharma- fects in heart disease and stroke, inflammatory arthritis,
ceutical industry, and (d) the newly emerged bioengineered inflammatory bowel disease, asthma, cardiovascular dis-
microorganisms, agroproducts or active biomolecules. It may ease [17].
range from isolated nutrients, herbal products, dietary sup-
plements and diets to genetically engineered “custom” foods 8. Dietary fibers such as oats, dried beans, legumes, chicory
and processed products such as cereals, soups and beverages. as water soluble fibers, apple, orange, apricot, plum, pine
Chemically the nutraceuticals may be classified as apple contain 18-30% fiber contents. The fish oils and
isoprenoid derivatives (terpenoids, carotenoids, saponins, the vegetable sources such as cabbage, carrot, lettuce, on-
tocotrienols, tocopherols, terpenes), phenolic compounds ion, tomato containing 9 to 12 % fiber contents showed
(couramines, tannins, ligrins, anthrocynins, isoflavones, fla- antioxidant and myocardial cell proliferation inhibitory
vonones, flavanoids), carbohydrate derivatives (ascorbic properties [18].
acid, oligosaccharides, non-starch PS), fatty acid and struc- 9. Wild foods are other major source of nutraceuticals and
tural lipids (n-3 PUFA, CLA, MUFA, sphingolipids, phytoesterogens. Most of the wild plants, wild mush-
lecithins), amino acid derivatives (amino acids, allyl-S rooms, wild fungi, wild vegetables, wild nuts, wild fruits
compounds, capsaicnoids, isothiocyanates, indols, folate, and wild flowers as whole are considered as potential
choline), microbes (probiotics, prebiotics) and minerals natural therapy alternatives rich in long chain omega 3
( Ca, Zn, Cu, K, Se) [4]. However, the nutraceuticals were fatty acids [19, 20].
Bioactive Foods and Nutraceutical Supplementation Food, Diet and Disease 3

Table 1. Examples of Nutraceuticals are Shown with Their Benefits in Different Cardiovascular Diseases and Mechanism of
Cardioprotective Action in the Body. The Structure of Active Nutraceuticals are Shown with Mechanism and Their
Structure with Formula in Chemical Nomenclature
4 Innovations And Solutions, Inc. Sharma, Shrinivas
Bioactive Foods and Nutraceutical Supplementation Food, Diet and Disease 5

a-linolenic acid + antioxidant (20:3, n-3) or ALA


Eicosapentanoic acid ++ antioxidant (20:5; n-3) or EPA
Docosahexanoic acid ++ antioxidant (20:6; n-3) or DHA

10. Soy isoflavones, genistien, lycopene have emerged as prevention of endothelial dysfunction [24, 30]. However,
established cardioprotective nutraceuticals. The eicosap- there is no guideline about the type of oil and type of nuts
entaenoic and docosahexaenoic acids reduce the oxidi- depending upon the omega-3 fat and monounsaturated fatty
zability and thrombogenicity [21] acid (MUFA) content of these foods. While foods and bever-
ages with added sugars and refined starches as well as excess
What Remains Still to Solve the Cardioprotection by of w-6, total and saturated fat and trans fatty acids, may be
Nutraceuticals? proinflammatory, increased intake of w-3 fatty acid and
MUFA may be protective against surge of TNF-alpha,IL-
The major issues that remain unsolved are the nutraceuti-
6,IL-18 and adhesion molecules like VCAM-1(vascular cell
cal side effects, dosage and mechanism, follow up conse-
quences and mandatory guide lines of usage. The diet and adhsion molecule-1)and IVAM-1 caused by high glycemic,
rapidly absorbed proinflammatory foods [27-32] (71-76).
lifestyle guidelines for prevention of coronary artery disease
These foods are known to initiate a proinflammatory milieu
(CAD) have been evidenced as a major interest during the
in the body which is similar to that of AMI, causing further
last few decades. Recommendations of the American heart
increase in complications among these patients.
Association (AHA 2007) have been reformed for better un-
derstanding, based on new scientific evidences after publica- In keeping these facts in mind, it is necessary to identify
tion of guidelines in 2007 [22]. However, none of these the concrete evidences of cardioprotective mechanism in
guidelines emphasize the role of diet in patients with acute both animals and clinical trials under controlled conditions
myocardial infarction (AMI) and stroke. Patients presenting with through investigations, careful nutrition formula design
with AMI are highly motivated to follow the advice of cardi- and success rate vs fallacies of earlier clinical experiences in
ologist due to serious AMI condition. AMI is associated with favor of nutraceuticals in public use.
hyperglycemia, hyperinsulinemia, hypertriglyceridemia, free
radical stress, rise in free fatty acid and pro-inflammatory Animal Studies
cytokines, leading to endothelial dysfunction. There happens
an acute generation of proinflammatory milieu among AMI A large volume of literature is available on nutraceutical
patients which is known to cause disruption of atheroma inhibitory effect on cardiovascular disease cell growth based
plaque, resulting into re-infarction and death [24-29]. The on observations of cultured cardiovascular disease cell pro-
synergy of these mechanisms in chronic disease is not clear liferation, enhanced apoptosis, antioxidant action etc. Still
in order to decide the intervention by nutraceuticals such as attempts are in the direction of morphological, cytomorphic,
walnuts, ginko, vegetables [23-29]. Most American experts histopathology evidences of nutraceutical induced lipid inhi-
very deligently advise dietary patterns; including grains, bition and thrombosis by using 3D localized molecular imag-
vegetables, fruits, nuts, seeds and legumes, fat and oils based ing techniques. Previous studies on micro-MRI and immu-
on research studies. Most of the times, the side effects of nostaining suggested the reduced apoptosis in experimental
newly introduced products in market are not documented rat, mice, rabbit, porcine, dogs experimental models [33].
such as no recommendation for refined starches in the Major evidence was the reduced oxidative stress, slowed
6 Innovations And Solutions, Inc Sharma,Shrinivas

down apoptosis, reduced proliferation, less plaque size, less >300 gm/week reduced non-fatal coronary syndrome
necrosis and poor atherosclerosis growth in treated groups (CARDIO2000 study) [49]; 3. transcription of the positively
[34]. The mechanism of these nutraceuticals are still not regulated genes (alpha-myosin heavy chain (MHC) and cal-
established and it remains to investigate more scientifically cium ATPase, SERCA2) both downregulate the expression
diet controlled experimental methods [35-37]. Moreover the of negatively regulated genes (beta-MHC and phospholam-
beneficial effects of nutraceuticals in experimental animals ban) to increase cardiac contractile performance. There is
were reviewed and two third literature reports on possibility of nutraceutical protection to repair cardiac con-
nutraceuticals are documented on experimental animal tractility and improved ejection time (LVET) [49, 50]; 4.
cardiovascular disease studies as either reviews or animal improved cardiac output, reduced cardiac preload (low renin
bench experiments on cardiovascular disease prevention. state and decreased erythropoietin secretion), increased vas-
The clinical evidence of nutraceutical cardiovascular disease cular resistance, bradycardia, slightly depressed myocardial
prevention success is still based on biochemical mechanisms contractility and some increase in LV mass [51]; 5. IF chan-
of nutrients in diets reported over several decades. Some nel, L-type and T-type calcium channel, potassium channel
mechanisms of nutraceutical action are reported as immune and the ryanodine channel contribute to pacemaker functions
modulatory, induced apoptosis, removal of free radicals, and heart rate [52]; 6. dyslipidemia due to total cholesterol
and low density lipoproteins (LDL) cholesterol, triglyc-
inhibited cell proliferation, inhibited necrosis. New ayurved
erides, very low density lipoproteins (VLDL), intermediate-
(Indian traditional medicine) concepts are also emerging as
density lipoproteins, apoprotein A-1 and apoprotein B are
powerful nutraceuticals in cardiovascular disease prevention
observed as well [53, 54]; 7. cholesteryl ester transfer protein
[38]. The growing literature on mechanism of nutraceutical
and hepatic lipase, increased levels of high-density lipopro-
action in the cardiovascular disease is supporting the teins(HDL); 8. endothelial dysfunction, increased arterial
extended benefits of nutraceuticals but it further needs more stiffness, increased vascular resistance, and hypercoagulabil-
investigations as described in following separate section of ity with coronary artery disease [55].
new literature evidences [39-41].
However the effect of bioactive foods is not known if
Clinical Trials bioactive food affects cardiovascular morbidity or mortality.
It might be beneficial to use bioactive food or nutraceuticals
Singh and coworkers used 400g/day of fruits, vegetables as supplements simultaneously with cardioprotective drug
and legumes in conjunction with mustered oil to decrease the therapy. Recently reported noninvasive imaging methods
risk of hypertension, diabetes and CAD in 1090s similar with such as Doppler echocardiography, carotid intima-media
DART, DART II, GISSI [42]. This diet was re-examined by thickness, pulsed tissue Doppler imaging, cardiac MRI and
DASH investigators and subsequently by other group to ob- radionuclide ventriculography to evaulate preejec-
serve the reduced risk of hypertension in USA [43, 44]. In tion/ejection ratio systolic dysfunction may be more useful to
further randomized, controlled intervention trials, Singh et establish the beneficial effect of nutraceuticals. Overall, tri-
al. 2002, 2008 administered 400 g/day of fruits, vegetables als evaluating cardiovascular mortality and mortality have
and nuts(almonds and walnuts) and another 400g/day of yielded conflicting results [56].
whole grains including legumes in conjunction with 25-
50g/day of mustered oil (ALA 2.9 g/day) in patients with Biochemical Basis of Nutraceuticals in Cardiac
high risk of vascular disease, which showed significant bene- Prevention
fit [23, 45-47]. Other workers also found a beneficial effect
Natural vegetables, herbs, plants, wild foods are complex
of fruit, vegetables, nuts and ω-3 fatty acids (EPA+DHA 1.8
in structural composition. The biochemical basis of individ-
g/day) rich foods to patients on risk of coronary artery dis-
ual source of these foods is not explored due to their com-
ease [46, 47]. A randomized, double blind placebo controlled
plex nature. Some of the evidences are in favor of the active
trial on 300 patients after MI supplemented with EPA+DHA food principles as nutraceuticals to show cardioprotective or
3.4-3.5 g/day or corn oil showed no change. Increased intake preventive supplements. Some of nutraceuticals are in the
of monounsaturated fatty acid and ω-3 fatty acids have been phase of clinical trial or already available as food supple-
suggested to be protective against diabetes and metabolic ment. Complementary and Alternative Medicine is emerging
syndrome whereas increased consumption of trans fatty ac- in prevention of chronic coronary and heart diseases as safe
ids, saturated fat and refined starches can predispose CVD. practice because of the high risk of mortality and long-term
India has a rapid economic development causing increased morbidity associated with surgical procedures of coronary
consumption of salt, tobacco, fat, sugar, and energy in the artery disease and high side effects of chemotherapy. Herbal
last four decades. There is increase in per capita income, medicines have shown reduced myocyte cell necrosis in cul-
gross domestic product, food production and automobile tured cells. The vitamins, minerals, dietary fat play a role in
production in the last four decades. relation to cardioprevention and control. The mechanisms of
This period from 1970 to 2008 has witnessed marked nutraceutical action can be discussed broadly in following
changes in nutraceutical rich diet and lifestyle, particularly in categories based on active metabolites present in nutraceuti-
the urban populations among Indians. New bioactive factors cals.
have came in light of cardiovascular mechanisms likely af- 1. Niacin-bound chromium is reported to enhance myocar-
fected by nutrients such as: 1. iodine induced T3 and nitric dial protection from ischemia-reperfusion injury [57].
oxide decreases SVR by dilation of the arterioles protein
kinase akt pathway via smooth muscle relaxation through 2. Mechanism of the antithrombotic effect was invented by
nuclear transcription mechanisms [48]; 2. fish consumption dietary diacylglycerol in atherogenic mice [58].
Bioactive Foods and Nutraceutical Supplementation Food, Diet and Disease 7

3. Protective effect of potassium against the hypertensive The possible reversal of increased total cholesterol, increased
cardiac dysfunction was associated with reactive oxygen LDL cholesterol, apolipoprotein B and decreased HDL con-
species reduction [59]. centrations in cardiovascular patients on bioactive foods and
nutraceuticals is controversial [65]. In several trials, total
4. The atherogenic process is reduced by regulation of co-
enzyme Q10 biosynthesis and breakdown. cholesterol levels, HDL, LDL-cholesterol, triglycerides,
apolipoprotein A and B and lipoprotein A were not signifi-
5. The n-3 fatty acids reduce the risk of cardiovascular dis- cantly improved with nutraceutical or vitamin-mineral treat-
ease. The evidence was explained and mechanisms was ment [65]. A trend was noted in favor of nutraceutical ther-
explored. apy with reduced total cholesterol TC level >240 mg/dL,
6. Mediterranean diet and optimal diets play role for pre- LDL > 155 mg/dL TC levels (significant only for >240
vention of coronary heart disease. mg/dL), and Body Mass Index > 25 kg/m2 was associated
with better improvements [66].
7. Alpha-tocopherol therapy was evidenced to reduce
oxidative stress and atherosclerosis. Control of lipid metabolism and cholesterol desaturation
in the blood has been cited as major factor in cardiovascular
8. Genetic deficiency of inducible nitric oxide synthase re- disease. The nutraceuticals have been reported as inhibitors
duces atherosclerosis and lowers plasma lipid peroxides of cholesterol synthesis and enhancing HDL lipoproteins in
in apolipoprotein E-knockout mice. the body. To explain the effect of nutraceuticals, two major
9. Glutathione is the liver's most abundant protective con- mechanisms play significant role in cholesterol saturation
stituent of antioxidant glutathione reductase enzyme. and lipoprotein synthesis. First, HMG CoA synthase enzyme
Glutathione functions as a substrate for the two key de- controls the mevalonate to HMG CoA formation that subse-
toxification processes in the liver: 1. transforming toxins quently used in cholesterol formation while cholesterol oxi-
into water soluble forms, 2. neutralizing and "conjugat- dase enzyme oxidizes cholesterol to desaturate it. Second,
ing" with toxins for elimination through the gut or the cholesterol esterification by LCAT and ACAT enzymes and
kidneys. If either of these processes is impaired for any subsequently apoprotein binding controls the lipoprotein
reason, toxins will accumulate in the body and lead to formation [67]. Mainly high density lipoprotein (HDL) plays
disease. The best nutrition with liver cardiovascular dis- significant role in scavenging cholesterol from blood as
ease focuses on improving the body's glutathione re- shown in Fig. (1). Low density lipoproteins (LDL) transport
serves [60]. is controlled by LDL receptors in the cells. LDL lipoproteins
10. The Soy isoflavone Haelan951 (genistein and genistin) get metabolized by lipo-oxygenase pathway as shown by
and garlic allicin were reported to have some role as a Fig. (2).
cardioprotective in humans [61]. Beta-glycoside conju- The anti-inflammatory effects and antithrombogenic ef-
gate, genistin is abundant in fermented soybeans, soy- fects of ω-3 fatty acids are eicosanoid–dependent process.
bean products such as soymilk and tofu. Beta-glycosyl More intake of EPA and DHA fatty acids increases these
bond of genistin is cleaved to produce genistein by mi- fatty acids in tissue, cellular and circulating lipids, along
crobes during fermentation to yield miso and natto. Soy with a simultaneous reduction in ω-6 fatty acids. EPA acts as
sauce has high isoflavone but low miso and natto con- a substrate for both cyclooxygenase (COX) and 5-
tents. How much soy isoflavones needed? 1.5-4.1 lipoxygenase (5-LOX) enzymes to make derivatives from
mg/person miso isoflavone and 6.3-8.3 mg/person natto arachidonic acid (ΑΑ) such as leucotriene B5 (LTB5) is only
respectively [62]. about 10% as potent as LTB4 as a chemotactic agent and in
11. Green tea has always been considered by the Chinese and promoting lysosomal enzyme release). The ω-3 fatty acids
Japanese peoples as a potent medicine for the mainte- also result with reduced formation of thomboxane-2
nance of health, endowed with the power to prolong life (TxA2)and prostacyclin I2 (PGI2), as AA is a TxA2 and PGI2
[63]. precursor and inhibiting platelet aggregation (a less throm-
bogenic state) as shown in Fig. 2).
12. The cardiovascular disease has been reported associated
with vascular endothelial growth factor [64]. The fatty acids display three major beneficial effects:
1.lipid lowering in blood; 2.antiarrhythmic effect in CHD; 3.
13. Some herbal plants act as cardioprotective medicine. The antithrombotic effects; 4. anti-atherosclerotic and anti-
herbal extracts are known to reduce the circulating mark- inflammatory effects; 4. improved endothelial function; and
ers of inflammation, including C-reactive protein (CRP), 5. lowering blood pressure.
interleukine-6 (IL-6), tumor necrosis factor-α (TNF-α),
serum amyloid A (SAA). From biochemistry standpoint, the beneficial effect of ω-
3 fatty acids on blood lipids is by the stimulation of the gene
14. Combination of garlic, ginko biloba, herbs with rever- expression of lipoprotein lipase (LPL) enzyme in human
astrol inhibited a full 92 percent of age-related gene adipose tissue with increase in the LPL mRNA. It results
changes in the heart [2, 64]. with post-heparin LPL activity, in conjunction with the low-
ering effect of these fatty acids on the triglyceride levels,
Lipid Metabolism and Fatty Acid Modifiers as Basis of
postprandial lipaemia and the levels of the highly athero-
CVD and role of Nutraceuticals
genic, small and dense LDL particles [24]. These fatty acids
Lipid metabolism is established a major factor in cardio- increase the expression of genes encoding enzymes critical
vascular protection by supplementing omega fatty acids as to hepatic and skeletal muscle fatty acid β-oxidation while
described with recent developments for interested readers. repressing genes encoding glycolytic, lipogenic and choles-
8 Innovations And Solutions, Inc. Sharma,Shrinivas

Fig. (1). Proposed role of LDL oxidation in the thrombogenesis and endothelial dysfunction. LDL crosses the endothelium in a concentra-
tion-dependent manner and can become trapped in the extracellular matrix (1). The subendothelium is an oxidizing environment, and if the
LDL remains trapped for a sufficiently long period of time, it undergoes oxidative changes (2). Mildly oxidized forms of LDL contain bio-
logically active phospholipid oxidation products that affect the pattern of gene expression in endothelial cells (ECs), leading to, among other
things, changes in the expression of monocyte binding molecules (designated X-CAM), monocyte chemoattractant protein (MCP-1), and
macrophage colony stimulating factors (CSFs) (3). These factors in turn promote the recruitment of monocytes (4) and drive their phenotypic
differentiation to macrophages (5). Further oxidation leads to alterations in apolipoprotein B such that LDL particles are recognized and in-
ternalized by macrophages (6), progenitors of the lipid-laden foam cells. Marked increases in lipid and cholesterol oxidation products render
the LDL particles cytotoxic, leading to further endothelial injury (7) and favoring further entry of LDL and circulating monocytes and thus a
continuation of the disease process.

Fig. (2). The omega 3 and omega 6 fatty acids synthesize ecosanoids in the myocardial cells.

terolgenic enzymes. This twofold action results in the de- erators- activated receptors (PPARs) α, β and γ – and by
crease in lipid synthesis and a subsequent increase in lipid regulating the transcription factor sterol regulatory element
oxidation favorable for nutraceutical intervention. Despite binding proteins (SREBPs) 1 and 2[25]. Ω-3 fatty acids also
the fact that the exact mode of action of ω-3 fatty acids is not decrease excitability and cytosolic calcium fluctuations of
fully understood, it is speculated that ω-3 fatty acids interact ventricular myocytes via inhibition of Na+ and L-type Ca+2
with three nuclear receptors–hepatic nuclear factor (HNF)- channels. The mechanisms of action of ω-3 fatty acids have
4α, liver X receptors (LXR) α and β and peroxisome prolif- not been fully elucidated.
Bioactive Foods and Nutraceutical Supplementation Food, Diet and Disease 9

TREATMENT RECOMMENDATIONS FOR Government Policy: Criteria of Suggested Practice of


NEUTRACEUTICALS IN CARDIOVASCULAR Nutraceuticals in Cardiovascular Prevention
PREVENTION
The awareness of complementary and alternative medi-
Who Need the Alternative Approaches of Nutraceuticals cine (CAM) is increasing rapidly among common public in
in CVD developed countries [69]. Government agencies are actively
participating in safe delivery of bioactive foods and dog-
Children below 18 years probably do not need nutraceu-
watch if any side effect. Several government reports have
ticals. Adults over 20-40 years need nutraceuticals and moni-
showed positive role to introduce new functional foods &
toring CVD. Persons over sixty years in age, need CVD/
nutraceuticals in CVD/CHD prevention in favor of guava,
CHD watch and nutraceuticals as mandatory daily dietary
dietary fibers, soy, phystoesterogens, herbs, cruciferous
supplements in practice. These senior persons may show
vegetables [70]. Both bioactive food and nutraceuticals in
the following major symptoms as causes of cardiovascular
diets were suggested as preventive in cardiovascular disease.
disorders and CVD development [68].
Main causative factors of cardiovascular disease were free
• Poor cytokines, inflammatory proteins gradually lead to radicals, vitamin C,D,E deficiency, selenium deficiency and
apoptosis, loss of immunity loss of cellular immunity in patients on daily diet [71].
• Arteries and veins (and other tissues) become less elastic, Recently, National Heart and Lung Institute put forth the
as evidenced by our skin. Blood pressure may rise, as efforts on alternative ways of cardiovascular disease preven-
arteries lose their elasticity. (The amino acid taurine, tion as public awareness to main focus on life style, preven-
found in fish, softens arteries and veins, as well as other tion and control care measures, eating habits, hazardous con-
connective tissue.) taminants with several successful attempts of antioxidants,
garlic, vitamins [72]. Under supervision and dogwatch, most
• Inflammation and cholesterol-filled growths (plaques) in of the bioactive foods on counters and nutraceuticals are
our blood vessels reduce their rates of flow. The loss of marketed as some of them listed in Table 2.
elasticity causes the heart to pump with less power and
force. Bioactive Foods and Nutraceuticals in CVD/CHD: A
Survey
• Insulin levels begin to rise as old cells become less re-
sponsive to insulin, and the pancreas increases its output In recent years during 2002-2008, the major focus was on
to compensate. This eventually leads to Type II diabetes more evidence based wider use of omega 3 fatty acids com-
and pancreatic cardiovascular disease in which old cells bined with multivitamin-multimineral and isolated bioactive
no longer respond to insulin and end up with heavy components from plants and functional foods in various car-
cardiovascular damage and cardiovascular disease. diovascular disease types. In last 4 years maximum efforts
were devoted on reviews and compilation of evidenced ex-
• Kidneys lose reserve capacity, gradually fail to do normal perimental results on vegetarianism in reducing cardiovascu-
function and develop cardiovascular disease. lar disease progress and identification of associations of ac-
• Reduced cell mediated immunity and humoral immunity tive food components in diet with reduced lipids, myocardial
leads to immune deficiency and cardiovascular disorders. necrosis and apoptosis. However, NHLI views that sequen-
tial events during the nutraceutical treated cell growth or
Present State of Art on Nutraceutical Medicine in arrest cardiovascular disease are controversial [73]. The use
Cardiovascular Prevention of fish oils in elderly patients was revisited if any relation
with arrhythmia and contractility. The literature during years
FDA requires appropriate scientific evidence regarding
2002-2008 suggested major information for following: 1.
safety of nutraceutical use as daily prescription. However,
direct link of vitamins, minerals in cardiovascular disease
new recommendations suggested that daily diet must contain
prevention; 2. new bioactive food components with new
6.25 grams of soy protein per serving, micro-compound al-
mechanism of lipid lowering; 3. more controlled trials and
licin (a small component of garlic) ad libitum amount, ecos-
regulated studies under federal support; 4. new awareness of
apentanoic acid/docosaheaxanoic acid as polyunsaturated
unpopular foods and common shelf food supplements in car-
fatty acids (PUFAs) from fish or fish oils. The complemen-
diovascular disease prevention; 5. new federal and statuary
tary medicine and alternative medicine approach is emerging
guidelines on nutraceutical recommended allowances and
as regulated tool to prescribe the norms of nutraceuticals as
marketing.
daily supplements in cardiovascular disease and other
diseases [69]. The following information is grouped based on literature
on nutraceuticals and nutraceuticals in cardiovascular disease
Insurance and Prescription management with major focus on controlled randomized
National and federal agencies such as NCI and FDA need trials in experimental cardiovascular diseases and clinical
evidences and established data in large trials to approve nu- cardiovascular disease subjects. The description is divided
traceuticals in clinical practice. In lack of such evidences and into three sections.
database, still nutraceutical practice remains at the door steps Bioactive foods and nutraceuticals in cardiovascular pre-
as nonprescription self-prescription available on counter. As vention during years 2000-2008: Nutraceuticals and local
a result, insurance companies still shy to accept nutraceuti- foods were suggested as readily available and their use with
cals as prescription. possibility of alternative pharmacotherapy to prevent cardio-
10 Innovations And Solutions, Inc. Sharma,Shrinivas

Table 2. The Table Represents the FDA Approved Nutraceuticals with Recommended Quantity and Sources of Nutraceuticals on
Shelf in Super Markets

Nutraceuticals Quantity Needed Common American Sources

Vitamin D 400 IU a day(2000 IU) Walmart's "OneSource" multivitamins

Multivitamin-minerals As 1 pill daily Centrum Silver A-Z with minerals

Natural Vitamin E (4 tocopherols + 4 tocotrienols) Two 400 IU capsules a week (800 mg) GNC natural Vitamin E

Selenium 200 mcgs. a day Walmart's "OneSource" multivitamins

Aspirin or ibuprofen* Baby aspirin a day nonprescription counter

Chocolate (best if fat-free) ?, 3 servings home made, Food emporium

Green tea ?, 3 servings home made, Foof emporium

Lycopene Cooked tomato sauces Domino's Pizza

Fish (tuna, salmon, mackerel)** or EHA+DHA Two servings a week Fresh phytosterols at Publix

Publix, at the edge of the produce section.


Soy "meat", cheese, milk Ad libitum
mozzarrela, sausage, burgers

Broccoli, cabbage, cauliflower Sulfhydrals ad libitum Piccadilly's tastes pretty good.

Blueberries A few tablespoons a day Publix' frozen foods (N. side, S. aisle)

Strawberries 4 or 5 large a day Publix' frozen foods (N. side, S. aisle)

Old-fashioned oatmeal One ounce? Publix, all supermarkets

Legumes (beans) Two servings a week Publix, all supermarkets

Low-fat blueberry yogurt 2 or 3 times a week Publix, all supermarkets

Yellow vegetables Ad libitum Publix(Piccadilly's tastes pretty good).

Purple grape juice, or red wine A glass a day Publix, for Welsh's grape juice

Turmeric roots Two capsules daily GNC natural body products

Herbs Two pills daily St John Warts natural source

Garlic, Soy products Ad libitum Walmart's "OneSource" ampoules

*aspirin and ibuprofen primarily act as anti inflammation. (Other agents such as fish also have anti-inflammatory properties.); **Tuna and mackerel contain mercury, dioxin, and
PCB's, . The salmon fish is safe. Winn Dixie farm-raised salmon. canned salmon provides omega-3 fatty acids and , taurine which are vital to the nervous and cardiovascular systems
(modified from the Source[Sharma 2009]).

vascular diseases [73-75]. Less known bioactive foods con- The possible reasons of cardioprotection by ω-3 fatty
taining ephedra and caffeine were reported to improve elec- acids in bioactive foods were:
trocardiograhic and hemodynamic effects [75]. Clear cardio-
• Lipid lowering (reduction of fasting triglycerides,
protective role of vitamin E and antioxidant supplements was
attenuation of postprandial triglyceride response)
reviewed in prevention of cardiovascular diseases [76-81].
Homocysteine, taurine, vitamins and omega 3-fatty acids • Antiarrhythmic effects
were reinvestigated and confirmed their value in cardiovas- • Antithrombotic and other effects on the haemostatic sys-
cular prevention [82-86]. tems (i.e. reduced platelet reactivity, moderately longer
Mechanism of cardiovascular prevention by nutraceuti- bleeding times, reduced plasma viscosity)
cals: Mainly cholesterol rich dietary fats enhances the risk of • Inhibition of atherosclerosis and inflammation via inhibi-
coronary heart disease while omega 3/omega 6 fatty acids tion of smooth muscle cell proliferation, altered eicosa-
reduce the risk of cardiovascular diseases and play cardio- noid synthesis, reduced expression of cell adhesion
protective role in primary, secondary and late- onset diseases molecules and suppression of inflammatory cytokines
[87-89]. Interestingly, author described the excessive linoleic production (IL’s, TNF-α) and mitogens
acid is manifested as ‘linoleic acid syndrome’ in coronary
heart disease [89]. Conjugated lineleic acid was reported as • Improvement of the endothelial function [through en-
protective against cardiac hypertrophy [90]. Omega 3 fatty hancement of nitric oxide – dependent and nitric oxide
acids mainly lower the blood lipids. independent vasodilatation]
Bioactive Foods and Nutraceutical Supplementation Food, Diet and Disease 11

• Improvement in blood pressure. [6] Rishi RK. Nutraceuticals: borderline between food and drug?
Pharm Rev 2006; 2: 51-53.
III. CVD/CHD in the human body and nutraceutical pro- [7] Pawlus AD, Kinghorn DA. Review of the ethnobotany, chemistry,
tection: Supplementation of fish oils dominate the scenario biological activity and safety of the botanical dietary supplement
of lipid lowering in cardiovascular diseases [90]. New can- Morinda citrifolia (noni). J Pharm Pharmacol 2007; 59(12): 1587-
609.
didates such as cinnamon, ginko biloba, bioactive peptides [8] Visioli F, Bogani P, Grande S, Galli C. Mediterranean food and
have been introduced in the list of nutraceuticals with health: building human evidence. J Physiol Pharmacol 2005; 56
cardioprotective properties [85, 91, 92] (1): 37-49.
[9] Dujardin KS, Dumotier B, David M, Guizy M, Valenzuela C, Hon-
Challenges, Hypes, Hopes and Futuristic role of deghem LM. Ultrafast sodium channel block by dietary fish oil
prevents dofetilide-induced ventricular arrhythmias in rabbit hearts.
Nutraceuticals in Cardioprotection Am J Physiol Heart Circ Physiol 2008; 295(4): H1414-21.
[10] Boots AW, Haenen GR, Bast A. Health effects of quercetin: from
Most of the success of nutraceuticals is based on self- antioxidant to nutraceutical. Eur J Pharmacol 2008; 585(2-3): 325-
prescription and own individual experiences. Still it is far to 37.
realize the miraculous benefits of nutraceuticals unless con- [11] Bo S, Pisu E. Role of dietary magnesium in cardiovascular disease
trolled clinical trials support the evidences and facts of nu- prevention, insulin sensitivity and diabetes. Curr Opin Lipidol
traceutical preventive therapeutic efficacy. Major challenge 2008; 19(1): 50-6.
[12] Navas-Acien A, Bleys J, Guallar E. Selenium intake and cardiovas-
is early detection of cardiovascular disease and timely effec- cular risk: what is new? Curr Opin Lipidol 2008; 19(1): 43-9.
tive treatment. In spite of all tools available, cardiovascular [13] Juan YS, Hydery T, Mannikarottu A, et al. Coenzyme Q10 protect
disease is major health hazard. The major available data on against ischemia/reperfusion induced biochemical and functional
nutraceutical benefits in cardiovascular disease comes from changes in rabbit urinary bladder. Mol Cell Biochem 2008; 311(1-
2): 73-80.
epidemiological health and population statistics. The reduced [14] Spitberg VL. Bovine milk fat globule membrane as a potent nu-
cardiovascular disease incidence due to nutraceuticals seems traceutical. J Dairy Sci 2005; 88: 2289-94.
hype but greater hopes are anticipated with advancements in [15] Hamblin M, Smith HM, Hill MF. Dietary supplementation with
food science. However, still cardiovascular disease remains a vitamin E ameliorates cardiac failure in type I diabetic cardiomy-
major threat because of high mortality compounded with opathy by suppressing myocardial generation of 8-iso-
prostaglandin F2alpha and oxidized glutathione. J Card Fail 2007;
incomplete success of chemotherapy and surgery interven- 13(10): 884-92.
tion. In future, bioengineered nutraceuticals will play signifi- [16] Novgorodtseva TP, Vitkina TI, Karaman IuK, Aminina VF. Appli-
cant role in CVD prevention as alternative therapeutics. cation of food supplement on the base of potassium and magnesium
at experimental cardiopathology. Vopr Pitan 2007; 76(5): 55-9.
CONCLUSION [17] Cybulska I. N-3 polyunsaturated fatty acid in cardiovascular dis-
ease. Przegl Lek 2006; 63(8): 685-7.
Bioactive foods with rich nutraceuticals still are growing [18] Ismail HM. The role of omega-3 fatty acids in cardiac protection:
an overview. Front Biosci 2005; 10: 1079-88.
in number as healthy food products introduced by companies [19] Rupp H, Wagner D, Rupp T, Schulte LM, Maisch B. Risk stratifi-
and investigations suggest high hopes of nutraceuticals in cation by the "EPA+DHA level" and the "EPA/AA ratio" focus on
cardiovascular disease prevention. The primary focus still anti-inflammatory and antiarrhythmogenic effects of long-chain
remains on dyslipidemia and lipid lowering by fish oils and omega-3 fatty acids. Herz 2004; 29(7): 673-85.
bioactive foods. The role of governments and globalization [20] Mesa MD, Buckley R, Minihane AM, Yaqoob P. Effects of oils
rich in eicosapentaenoic and docosahexaenoic acids on the oxidi-
will certainly support the health risks and clinical trials on zability and thrombogenicity of low-density lipoprotein. Athero-
new bioactive foods and nutraceuticals. The nutraceuticals sclerosis 2004; 175(2): 333-43.
are becoming popular as they are harmless and natural food [21] Tikkanen MJ, Wähälä K, Ojala S, Vihma V, Adlercreutz H. Effect
constituents. The nutraceuticals are still food supplements of soybean phytoestrogen intake on low density lipoprotein oxida-
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Received: July 29, 2009 Revised: September 3, 2009 Accepted: September 5, 2009

© Rakesh Sharma, Bharati D Shrinivas; Licensee.


This is an open access article licensed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
by-nc/3.0/) which permits unrestricted, non-commercial use, distribution and reproduction in any medium, provided the work is properly cited.
Foods,diets and disease (C)2009 Innovations And Solutions,Inc
Editors:Rakesh Sharma,Bharati D Shrinivas
_____________________________________________________________

Comparison of Cholesterol Lowering Diets: Apple, Casein


Cytochrome P450 protein and Cholesterol 7α Hydroxylase Activities in Hamsters

Rakesh Sharma1, Rakesh K Tandon2

Department of Gastroenterology,
All India Institute of Medical Sciences, New Delhi 110029 India

Abstract

Lithogenic diet, casein and apple fiber diets were fed to hamsters for 3-5 weeks. For
control group, animals were fed on normal Purina chow without any supplement. The
cholesterol lowering effect of lithogenic diet, casein and apple diets were compared.
After dietary regimen, animals were screened for any gall stone formation. The isolated
liver microsomes were separated from animals and tested for the cholesterol-7α
Hydroxylase (CH) enzyme activity measurement in all three groups. The control animals
did not show any gall stone formation and their CH enzyme activities were normal. The
lithogenic diet showed significantly enhanced CH enzyme activities while animals fed on
casein and apple diet regimen showed moderate increase in microsomal CH enzyme
activity indicated cholesterol lowering in liver. In conclusion, cholesterol 7α hydroxylase
may be a biomarker of cholesterol status in the body and microsomal CH enzyme may be
lowered down after treatment of casein and apple diets.
Key words: cholesterol 7α hydroxylase, apple, casein, hamster, gall stone, cholesterol
lowering

Introduction

Five million Americans suffer from some type of diagnosed symptomatic cholesterol
saturation disorder while an even larger number are believed to be suffering from an
undiagnosed cholelithiasis related disease. The cholesterol saturation in bile and blood is
a common problem as hypercholesterolemia and it leads to gall stones mainly
complicated with atherosclerosis, obesity and fatty liver sooner or later [1,2,3]. Dietary
fibers, bran, pectins as bioactive foods have proven effective in serum cholesterol
lowering or hypocholesterolemic effect. Apple dietary fiber and rice bran, a
predominantly insoluble fiber source was reported to show hypocholesterolic effect in
hamsters [4]. Apple fiber is among top five high fiber diets rich in cellulose,
hemicellulose, lignin, pectin contents with potentials of hycholesterolemic effect[5,6].
Having evidences of their hypocholesterolemic effects, the mechanism of these dietary

1
Present corresponding address: EB-5, Saket colony, New Delhi 110023 India
2
Present corresponding address: Department of Food and Nutrition, Florida State University, Tallahassee,
Florida 32304 USA
fibers still is not clearly defined how cholesterol concentration in bile and liver is
regulated by enzymes. Cholesterol is ultimate saturated lipid synthesized from precursor
acetyl CoA (formed as end product of glycolysis) to mevalonate, sequalene and farnesyl
phosphate intermediates through several biochemical reactions. Cholesterol serves as
precursor of mobile lipids in the blood and bile emulsion made up of very low density
(VLDL) low density (LDL), and high density (HDL) lipoproteins. In bile, cholesterol is
synthesized from its precursor acetyl CoA to hydroxyethyl CoA and further to
mevalonate intermediate precursor to synthesize cholesterol as shown in Figure 1. In
catabolism, cholesterol breakdown takes place by cholesterol 7α hydroxylase enzyme[7].
The hydroxyl group on 7th carbon in cholesterol plays a major metabolic regulatory role
in the action of cholesterol 7α hydroxylase enzyme activity. Dietary cholesterol controls
the biosynthesis of cholesterol by inactivating the existing 3-hydroxymethylglutaryl CoA
reductase (HMG-CoA Reductase) and suppressing the synthesis of additional reductase
[8,9]. We showed the dietary fiber binds and removes the extra bile acids from the system
and thereby causes cholesterol to be converted into replacement bile acids. However,
postcholesystectomy develops symptoms despite of function gall bladder [10]

In previous experiments, dietary supplemented bile acids and salts in combination


showed cholesterol desaturation and reduced gallstone formation in animals and human
both [11-17]. The major factor of cholesterol desaturation was the physical phase
transformation of cholesterol into bile cholesterol esters and metabolic desaturation of
cholesterol because of cholesterol conversion into different lipoproteins [18-23]. In
present study, role of hepatic cholesterol 7α hydroxylase in cholesterol lowering and its
regulation or reduced HMG-CoA: Cholesterol 7 α hydroxylase ratio is explored by apple
diet supplementation to hamsters.

Materials and Methods

Animals: Hamsters weighing 150-225 gm were locally raised at animal laboratory at All
India Institute of Medical Sciences, and placed in plastic cages fed on cholesterol rich
diet for the period of one-four weeks under constant supervision for any change in weight
as per Institute Animal Care and Use Committee. Animals were grouped in four groups:
control group, lithogenic or high cholesterol group, apple supplemented group, and casein
supplemented group fed on normal Purina (Rodent Ralston Purina), high cholesterol diet,
apple supplemented diet, casein supplemented diet respectively, with ad libitum water
[24]. After different intervals, cytochrome P450 protein and CH enzyme were estimated
as biomarker of time dependent cholesterol changes and cholesterol regulating CH
enzyme in liver.

The lithogenic high cholesterol dietary additive supplements* were used to fed animals
and to develop cholesterol gall stones in animals as positive control animal group. Other
two experimental animal groups included apple diet** and casein diet*** groups. The
Purina diet contained sucrose (550 g/kg wt), vitamin mix (40 g/kg wt)
*Cholesterol pure form 0.25% added in diet (w/w)
**Apple pulp fiber was added in Purina 5% in diet (w/w)
***Casein powder (250 g/kg animal weight) was added in Purina 5% in diet (w/w)
At the end of each experiment, animals were killed by decapitation within 2-h span in an
order determined to distribute time of death among different dietary treatments
throughout that period. Livers were stored at -80 °C [24].

Isolation of hepatic microsomes: The hepatic microsomal preparations from hamster


livers were done by liver cell isolation, density gradient ultrafast centrifuge at x10 5 g
centrifugal force to isolate the microsomal fraction as source of cytochrome P450 and
cholesterol 7α hydroxylase enzyme activity. The microsomal isolation was done by our
modified method as described elsewhere [25].

Cholesterol enzyme estimation and enzyme activity: Cholesterol was delivered in


Tween 80 as previously described and modified by our group [22]. Briefly, in a total
volume of 500 μl, each assay tube contained 75 mmol/l phosphate buffer, pH 7.4, 1
mmol/l EDTA, 2.5 mmol/l DTT, 5 mmol/l MgCl2, 3 mmol/l NADPH, [4 14C]cholesterol
(1.1×106 dpm, 100 000 pmol=0.2 mmol/l), 0.78 mg Tween 80 and 500 μg of liver
microsomal protein. The preincubation time during which NADPH was omitted was 2
min at 37°C. The incubation time was 20 min.

The improved assay for cholesterol 7α-hydroxylase activity in hamster liver microsomes
was performed as following [22]. In a total volume of 500 μl, each assay tube contained
75 mmol/l phosphate buffer, pH 7.4, 1 mmol/l EDTA, 0.5 mmol/l DTT, 5 mmol/l MgCl2,
1 mmol/l NADPH, [14C]cholesterol (1.1×106, 26 000 pmol, 52 μmol/l), 4.5 mg HPBCD,
10 mmol/l glucose-6-phosphate, 2 U glucose-6-phosphate dehydrogenase and 250 μg of
liver microsomal protein. Assay tubes were incubated at 37°C in a shaking water bath
(Maxi-shake, Heto, OSI, France). The preincubation time during which only NADPH
was omitted was 5 min and the incubation time following initiation of the assay with
NADPH was 6 min. The reaction was terminated by adding 40 μl of 5 mol/l NaOH. Zero-
controls were always run in parallel by the addition of 5 mol/l NaOH at the beginning of
the preincubation. The sterols were extracted (after neutralization) with 4.3 ml of
dichloromethane–ethanol (5:1, v/v, plus 1.2 ml of H2O). A mixture of unlabeled 7α- and
7β-hydroxycholesterol was added and [14C]7α-hydroxycholesterol was separated from
7β-hydroxycholesterol by thin-layer chromatography (TLC) on silica gel G using a
double migration technique with ethyl acetate–hexane (1:1, v/v). In all cases, activity was
linear with respect to the amount of microsomal protein added and the incubation time.
The amount of endogenous cholesterol present in assays ranged from 4.75–5.5 μg for 250
μg of microsomal protein.

Enzyme cholesterol 7α hydroxylase reaction and effect of cytochrome P450 and


cholesterol additives: In control and apple diet supplemented animal liver microsomal
preparations, cholesterol 7α hydroxylase enzyme activity were mesaured at different
concentrations of cytochrome P450 protein. In other set of experiment, cholesterol 7α
hydroxylase activity was measured at different cholesterol concentrations added in the
reaction mixture.
Results

The hamsters were active healthy and responsive throughout the span of study. The
weight of animals varied in the range of 20%-40% after lithogenic diet. The animals did
not show any symptoms of indigestion after dietary treatment of casein and apple fiber.

The treatment of casein and apple fiber showed measurable difference in the CH
enzyme activity in microsome. However, control animal CH activity remained constant
through out study. The CH enzyme activity was linearly changed with the hydroxylation
of cytochrome P450 protein. The cytochrome P450 hydroxylation was proportional to
the cholesterol added in the enzyme reaction medium. The treatment of high casein and
apple fiber contents in hamsters did not affect the enzyme activity in reaction medium
and medium conditions.

Table 1: Cholesterol 7α hydroxylase specific activities in animals fed on different diets.

Diet Micrososomal Cytochrome P450/mg Cytochrome P450/mg protein


Cholesterol 7α protein (total) (fraction A)
Hydroxylase* _________________________ _________________________
Content 7α hydroxylase content 7α hydroxylase
Control 2.20 + 0.25 18.4 + 0.2 0.70 + 0.1 18.5 + 2.2 0.85 + 0.15
Lithogenic 1.79 + 0.20 10.2 + 0.9 0.65 + 0.1 16.5 + 2.0 0.75 + 0.20
Apple diet 3.45 + 0.50 24.6 + 2.0 1.2 + 0.15 20.5 + 2.5 1.8 + 0.15
Casein diet 2.81 + 0.30 12.2 + 0.2 0.48 + 0.5 -- --
Specific activity of CH enzyme* is expressed as pmoles cytochrome P450 catalyzed per
minute.
Figure 1: The cholesterol 7α hydroxylase enzyme activities are shown as histogram
graphs.

The cholesterol 7 hydroxylase enzyme activity and cytochrome 450 concentration in


liver cell microsomes: The purity of microsomes was 96% based on microscopy. In
isolated microsomes the indicator enzymes showed the active state of microsomes
without enzyme deactivation throughout the experiments. The enzyme activity was
substrate concentration dependent and substrate specific. The end product of cholesterol
conversion to hydroxycholesterol by CH enzyme indicated the nature of enzyme reaction
as dependent on cytochrome P450 and NADP reduction at optimum pH 7.4 in Tris buffer
medium. The addition of different cholesterol concentrations in enzyme reaction medium
containing control microsome showed the linear change in CH enzyme specific activity.
The cholesterol lowering effect of diets in different animal groups was in the increasing
order of control group I > lithogenic diet II > casein group III > apple diet group IV as
shown in Table 1 and Figure 1. The effect of each individual diet was distinct and CH
activity was decreased in animals after diet treatment while enzyme reaction medium did
not show any change in cholesterol and cytochrome P 450 contents. It indicated further
that enzyme activity changes were due to diet treatment.

Table 2: Effect of cytochrome P450 and cholesterol concentration on purified


cholesterol 7α hydroxylase system (microsomal).

Diet (group) Concentration of Cholesterol 7α hydroxylase


Cytochrome P450 concentration activity
(in nanomoles)** added (picomoles/min)
Control 0.250 25 n mol 36.5
Apple diet 0.250 25 n mol 35.0
Control 0.50 25 n mol 55.0
Apple diet 0.50 25 n mol 60.25
Control 1.0 25 n mol 57.0
Apple diet 1.0 25 n mol 65.5

Control + cholesterol 0.5 50 µM* 10.5


Control + cholesterol 0.5 100 µM* 28.5
Control + cholesterol 0.5 200 µM* 34.2
Control + cholesterol 0.5 400 µM* 55.0
Control: Group I; Apple diet: Group IV
*Group I animal liver microsomes were added with different cholesterol concentrations.

Animals on apple supplemented diets showed dependence of cholesterol 7α hydroxylase


on cytochrome P450 protein content and cholesterol in the reaction mixture. With
increasing cytochrome P450 protein in microsomes, enzyme elevated and apple diet
enhanced further the enzyme activity. It indicated the apple diet effect independent from
cytochrom P450 protein. The cytochrome P450 protein stimulated enzyme activity
behavior was close to sigmoid pattern at constant cholesterol concentrations. Enzyme
activity enhancement showed dependence with both cytochrome P450 protein
concentrations and cholesterol concentrations. The cholesterol concentrations in reaction
mixture and enzyme activities showed linear relationship as shown in Table 2.

Discussion

The cholesterol plays a major role in body as precursor of several vitamins, steroids, bile
salts and synthesis of esters. The increased amount of cholesterol in the blood and tissues
was discovered in the middle of past century and present time it is serious health hazard
in stroke, cardiovascular and renal occlusion as cholesterol deposits around the walls with
time [1,3,7,9,12,15]. However, in liver its role is more aggressive as cholesterol
saturation during bile salt synthesis in bile formation. The balance between two enzymes
for: mevalonate conversion in to HMG CoA by hydroxymethylglutaryl CoA reductase
enzyme and cholesterol conversion to hydroxycholesterol by cholesterol 7α hydroxylase
enzyme, is the key of cholesterol saturation or cholesterol desaturation in the body[8,9,
22]. The initial cholesterol saturation leads to effect bile saturation and slowly occludes
vascular walls in renal, cardiovascular and cerebrovascular system[1,17,24].
An apple a day provides great phytonutrients (phyto=plant) and a good dose of fiber.
One medium apple contains about 23 grams of carbs and 4 grams of fiber. Apple pulp
processing and juice manufacturing is now a booming industry and recently apple
products are identified with cholesterol lowering property [12,14,15,16]. The dietary
fiber content in apple further adds up the double benefit of cholesterol lowering with
renal dysfunction and enhanced fecal steroid excretion [12,14,15,16].

Table 3: Apple dietary composition is shown in whole apple and apple pulp in fiber content

Apple Pulp fiber

Source Serving Content % energy


raw 1 small 55-60* 3.0
raw 1 med 70 4.0
raw 1 large 80-100* 4.5
baked 1 large 100 5.0
apple sauce 2/3 cup 182 3.6
Whole Apple
raw 1 whole 17 0.8
dried 2 halves 36 1.7
canned in syrup 3 halves 86 2.5

*Important as dietary fiber is, laboratory technicians have not yet been able to ascertain
the exact total content in many foods, especially vegetables and fruits, because of its
complexity. Consequently, estimates vary from one source to another. Where differing
estimates have been found, an approximation is given in the chart, as indicated by an
asterisk. The same symbol following calorie content means the number of calories has
been estimated, varying according to other added ingredients, especially fats and sugars,
and to the size of the "average" fruit or vegetable unit. Reference: Bowel Function and
Dietary Fiber: Warren Enker http://www.wehealny.org/healthinfo/dietaryfiber/index.html)

Lithogenic diets are advocated as inducing cholesterol saturation due to their action on
cholesterol sterol to make cholesterol free as one of the major lithogenic effect. These
lithogenic diets are considered to have negative effect on omega 3 and omega 6 fatty
acids in the body and remain a major focus of their characteristic in plaque formation or
atherosclerosis. The cholesterol lowering diets rich in omega 3 and omega 6 fatty acids
have been investigated and reported to show major benefit in lipid lowering including
cholesterol lowering [25]. The diets rich in fibers have been a major attention since last
decade due to their double benefit to reduce lipids with cholesterol desaturation in the
body and maintaining intestinal satiety free from any microvillus membrane damage
[11,12,13,14,17].
The apple fiber was recently reported as major fruit content in the bulk of apple. The
apple fiber was rich in cellulose, hemicellulose, lignin, and pectin contents and very
compatible to the human digestive system free from any tropical sprue, pain or
constipation[15,16,17]. The apple fiber has significant activity to lower down the
cholesterol in the liver. The study focused on unique possibility of cholesterol lowering.
The cause of cholesterol desaturation or cholesterol lowering could be either rapid
cholesterol conversion to its hydroxylated product or it could be slowing down of
cholesterol precursor HMG CoA formation to make less available cholesterol or to
deprive the cholesterol formation. Here are several issues remain unanswered. First, the
proposed cholesterol 7 hydroxylase activity is not solely cholesterol specific but includes
other cholesterol derivatives or its analogues. Second, dependence of cytochrome P450
and NADP reduction is not solely represents hydroxylation reaction but other redox
reactions active in the medium at optimal pH 7.4 and temperature 37 °C. Third, liver
microsomes are very specific to intracellular conditions such as state of substrate,
reaction medium composition, physiological variables etc. During liver cell fractionation
and microsomal isolation there is every possibility of deactivation of enzyme and loss of
enzyme protein. Fourth, the dietary effect on cholesterol lowering is not single
biochemical or metabolic disorder but diet may also effect the other lipids and bile salts
made from cholesterol. So, the dietary effect is a compound effect on group of lipids in
both gall bladder and blood participating in lipid disorder. Fifth, cholesterol is not single
lipid compound to represent the lipid disorder or bile disorder in the body as other lipid
compounds also compete with cholesterol during bile formation or lipid lowering. Sixth,
hypocholesterolemic effect is really increased by activation of fiber or pulp binding with
alkaline earth metals, hydroxides, carbonates and phosphates or not.

Recently, bioactive foods were highlighted to play major role in cholesterol


desatutaion and lipid lowering such as guggulipid, garlic, fish fatty acids. Still fiber rich
foods remain less known and there is paucity of information on the role fibers in lipid and
cholesterol management. The study throws a possibility of the cholesteremic action of
naturally available apple fibers and casein proteins in the body applicable to human.
Casein is known to play a major role in cholesterol lowering and increasing HDL
cholesterol in the body [27]. Still it remains to investigate if the biochemical action of
both casein and apple diet is similar or analogous. Other available fiber rich foods do
have potential of lowering lipids in the body such as soy, pectins, brans of cellulose, rice,
oat [13,14,16,27]. Still main problem of isolated principle or fraction from complex
fibers, diets and bran remains to solve while evaluation of cholesterol lowering based on
enzyme activity is specific to cholesterol.

How to enhance the hypocholesterolemic effect by apple diets?


The method includes the steps of apple pulp processing by: (i) disrupting (rupturing)
the cell structure of the pulp material, (ii) reacting the disrupted pulp material with a first
reactant(s) capable of chemically modifying at least a portion of the pendant hydroxyl
groups on the fiber material contained in the pulp to pendant groups capable of
chemically coupling with alkaline earth metal ions, and then (ii) reacting the modified
fiber material with a second reactant(s) capable of chemically coupling an alkaline earth
metal ion to the modified pendant groups. An exemplary process includes the steps of
(aa) preconditioning the pulp by reacting the pulp with an aqueous solution of NaOH,
(bb) reacting the preconditioned pulp with an aqueous solution of CH2 Cl-COOH to
carboxylate the pendant hydroxyl groups on the fiber material contained in the
preconditioned pulp, and then (cc) reacting the carboxylated fiber material with Ca(OH)2
so as to bond Ca.supplementation to the pendant carboxyl groups [13,14,16,27].

What present study highlights?


Present study is a preliminary report indicating an evidence of apple diet benefit in
cholesterol lowering and a possible enzyme mechanism responsible of cholesterol
lowering while cytochrome P450 protein may act as biomarker in serum to evaluate the
cholesterol lowering.

Current state of art is in the direction of investigation on dietary modification and


effect on active or inactive forms of enzyme, better tracer techniques of enzyme
estimation, better understanding of enzyme heterogeneity and physiological effects
among species, apple processing advanced methods, and effective cholesterol lowering
by dietary servings. The present study has limitation in predicting the effect of apple
dietary fiber on cholesterol desaturation or lipid lowering as the study showed only
evidence as preliminary data to support the possibility of cholesterol lowering by apple
fiber supplementation to animals. It needs more investigations at different amounts of
apple pulp supplementation in diet and estimation of cholesterol 7α hydroxylase in
optimized reaction mixture. Other issue is cytochrome P450 protein may not represent as
true cholesterol 7α hydroxylase enzyme in liver. It needs additional experiments of
isolation of apple dietary fiber principle(s) responsible of cholesterol lowering in blood or
liver and additional benefits in renal dysfunction, fecal excretion etc. Present time, apple
punch is FDA approved safe table drink with hope of cholesterol lowering effect but
unconfirmed.

Conclusion

Apple diet is rich in pulp and apple fiber may have the cholesterol lowering effect on
the lipid metabolism in body. Cholesterol 7 Hydroxylase is rate limiting enzyme of
cholesterol degradation. Apple pulp fiber may have stimulatory effect on cholesterol 7α
hydroxylase enzyme while cholesterol also showed enhanced enzyme activities.

Acknowledgements

The financial grant from Indian Council of Medical Research to first author under
supervision of Professor RK Tandon for this study is highly appreciated. The assistance
of Mr Kishan Lal is highly appreciated to carry out daily lab work.

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APPENDIX:
EXTRA MATERIAL TO READ FROM LITERATURE
1. HUGH B. MATHESON, IVETTE S. COLON AHD JON A. STORY: Cholesterol 7
Hydroxylase Activity Is Increased by Dietary Modification with Psyllium Hydrocolloid,
Pectin, Cholesterol and Cholestyramine in Rats.

2. LIEN B. NGUYEN, SARAH SHEFER, GERALD SALEN, JOHN Y. L. CHIANG, AND


MUKUL PATEL: Cholesterol 7a-Hydroxylase Activities From Human and Rat Liver
Are Modulated In Vitro Posttranslationally by Phosphorylation/Dephosphorylation
Biochemical and Molecular Roles of Nutrients

Cholesterol 7«-Hydroxylase Activity Is Increased by


Dietary Modification with Psyllium Hydrocolloid,
Pectin, Cholesterol and Cholestyramine in Rats1«2«3
HUGH B. MATHESON,4 IVETTE S. COLON5 AHD JON A. STORY6

Department of Foods and Nutrition, Purdue University, West Lafayette, IN 47907

cholesterol concentrations in rats fed normal and


ABSTRACT Sources of dietary fiber known to alter cholesterol-supplemented diets (Anderson and Chen
cholesterol metabolism and/or bile acid pool size were 1979, Tsai et al. 1976). There is also evidence that oat
fed to rats, and activity of the rate-limiting step in bile
acid synthesis, cholesterol 7«-hydroxylase, was bran, a fiber source with both soluble and insoluble
components, has a hypocholesterolemic effect in rats

Downloaded from jn.nutrition.org at Florida State Univ on July 26, 2009


measured. In the first experiment, semipurified diets
containing 5% cellulose, psyllium hydrocolloid, pectin or and humans (Anderson et al. 1984, Arjmandi et al.
oat bran as dietary fiber sources or 2% Cholestyramine 1992, Chen et al. 1981, Shinnick et al. 1988). Rice
were fed to groups of 10 male Wistar rats for 4 wk. In bran, a predominantly insoluble fiber source, has been
the second experiment, groups of six rats were fed diets
containing 5% cellulose, rice bran, oat bran or psyllium
reported to reduce serum and liver cholesterol in
with and without 0.25% cholesterol. In the first ex hamsters (Kahlon et al. 1992). Despite considerable
periment, the activity of cholesterol 7«-hydroxylase effort, the hypocholesterolemic mechanism of these
(prnol-mhr^mg protein"1) was highest in the
fibers has not yet been clearly defined.
cholestyramine-treated group (95.6 ±3.6), followed by We have previously observed increases in the pool
groups fed psyllium (35.5 ±3.5) or pectin (36.0 ±4.5), sizes of bile acids in rats fed pectin and psyllium
which exhibited more than twice the enzyme activity of
groups fed cellulose (16.9 ±1.9) or oat bran (12.3 ± (Matheson and Story 1994), suggesting an increase in
2.0). In the second experiment, feeding cholesterol the catabolism of cholesterol through conversion to
resulted in significantly higher enzyme activity when cel bile acids. The rate-controlling enzyme in the for
lulose (65%), oat bran (118%) and rice bran (60%) mation of bile acids is cholesterol 7a-hydroxylase (EC
were fed, but no difference in activity was observed when 1.14.13.17) (Danielsson et al. 1967, Myant and
cholesterol was added to the psyllium-containing diet.
Higher activity of cholesterol 7a-hydroxylase when Mitropoulos 1977), which is expressed exclusively in
pectin or psyllium rather than cellulose was fed may the liver and whose mRNA levels can be increased by
explain the almost twofold higher bile acid pool sizes dietary bile acid séquestrants, cholesterol or
previously reported in response to feeding either of these mevalonate and decreased by dietary bile acids
fibers. These data support the hypothesis that the (Jelinek et al. 1990, Noshiro et al. 1990).
hypocholesterolemic effect of soluble fibers is modulated
through increased synthesis and therefore pool size of
bile acids. J. Nutr. 125: 454-458, 1995.

INDEXING KEY WORDS: 'These data were presented in part at Experimental Biology 93,
April 1, 1993, New Orleans, LA [Matheson, H. B. & Story, J. A.
•cholesterol-7a-hydroxylase •rats (1993) Changes in the activity of cholesterol 7a-hydroxylase by
•dietary fiber •cholesterol dietary modification using cellulose, psyllium, pectin, oat bran and
Cholestyramine. FASEB. J. 7: A722 (abs.|].
Supported in part by the Indiana Agricultural Research Pro
grams (paper no. 14,339|, American Institute for Cancer Research
(86A-25), and the Procter and Gamble Company.
There has been a great deal of interest in the 3The costs of publication of this article were defrayed in part by
hypocholesterolemic properties exhibited by some di the payment of page charges. This article must therefore be hereby
etary fibers. Fibers with predominantly water-soluble marked "advertisement" in accordance with 18 USC section 1734
components have proved to be more effective in solely to indicate this fact.
4Current address: Department of Medical Nutrition, Huddinge
reducing human serum cholesterol than have in
University Hospital F60, NOVUM, S-141 86 Huddinge, Sweden.
soluble types of fiber (Anderson et al. 1988, Behall 5Current address: Nutrition Department, General Mills, Ine,
1990, Everson et al. 1992, Jenkins et al. 1993). It has P.O. Box 1113, Minneapolis, MN 55440.
also been reported that soluble fibers such as pectin 6To whom correspondence and reprint requests should be ad
and psyllium hydrocolloid significantly reduce serum dressed.

0022-3166/95 $3.00 ©1995 American Institute of Nutrition.


Manuscript received 23 May 1994. Initial review completed 11 July 1994. Revision accepted 29 August 1994.
454
FIBER MODIFIES CHOLESTEROL 7a-HYDROXYLASE 455

This study was designed to investigate whether cholesterol (Rudel and Morris 1973) and insulin.
increases in the enterohepatic circulation of bile acids Serum insulin was measured by RIA (Rat Insulin RIA
in rats, induced by dietary fiber, resulted from alter Kit, Lineo Research, St. Louis, MO). The ex
ation in the activity of cholesterol 7a-hydroxylase and perimental protocol was reviewed and approved by
whether there was any modulation of this effect by the Purdue University Animal Care and Use Com
the addition of low levels of dietary cholesterol. mittee.
Cholesterol 7a-hydroxylase assay. Liver micro-
somes were isolated by ultracentrifugation (Nord
MATERIALS AMD METHODS strom et al. 1977) and stored in liquid nitrogen. Ac
tivity of cholesterol 7a-hydroxylase was measured
Animals and diets. Male Wistar rats (HarÃ-an using incorporation of liposome solubilized
Sprague Dawley, Indianapolis, IN), initially weighing cholesterol isotope into microsomal preparations
62-88 g, were individually housed and adapted to a (Junker and Story 1985). The results are expressed as
reverse light cycle (dark 0400-1600 h), which op picomoles of 7a-hydroxycholesterol produced per
timized cholesterol 7a-hydroxylase activity at 1000 h minute per milligram of microsomal protein (Bio-Rad
(6 h after the beginning of the dark cycle) and Protein Assay, Bio-Rad Laboratories, Hercules, CA)
minimized variation due to diurnal changes (Myant (Bradford 1976).
and Mitropoulos 1977). During a 7-d stabilization Statistical analysis. Experimental values were ana

Downloaded from jn.nutrition.org at Florida State Univ on July 26, 2009


period rats were given free access to ground nonpu- lyzed using the Statistical Analysis System, version
rified diet (Rodent Laboratory Chow®, Ralston 6.07 (SAS Institute, Gary, NC). Experiment 1 em
Purina, St. Louis, MO). In both experiments the basal ployed a one-way ANOVA and Experiment 2 a two-
diet contained sucrose (550 g/kg), casein (250 g/kg), way ANOVA, with dietary cholesterol and dietary
corn oil (100 g/kg), AIN-76 mineral mix (40 g/kg) and fiber as main effects. In both experiments, ANOVA
AIN-76A vitamin mix (10 g/kg) (Story et al. 1981). was followed by Fisher's least significant difference
In the first experiment, 50 rats were randomly test (Snedecor and Cochran 1989) to compare mean
assigned to one of five treatment groups and fed for 28 values where appropriate. Data are presented as the
d the basal diet with cellulose, pectin, oat bran or mean of 10 or 6 rats in Experiments 1 and 2, respec
psyllium hydrocolloid added at 5% or cholestyramine tively, with pooled SEM.
added at 2%; in each case fiber was added at the
expense of sucrose. In the second experiment, 48 rats
were randomly assigned to one of eight treatment
groups and fed for 28 d a semipurified diet with 5% RESULTS
cellulose, oat bran, rice bran or psyllium, with and
without addition of 0.25% cholesterol, added at the Final body weights and average weight gain were
expense of sucrose. Body weight and food intake were not significantly different among any of the groups, in
monitored each week. either experiment.
At the end of each experiment animals were killed In the first experiment, when cellulose, oat bran,
by decapitation, beginning at 1000 h. Animals were pectin, psyllium or cholestyramine was fed, serum
killed within a 2-h span, in an order determined to and liver cholesterol concentrations were comparable
distribute time of death among the different dietary among the groups. Liver weight in animals fed pectin,
treatments throughout that time. Serum and liver psyllium or cholestyramine was significantly lower
samples were stored at -80°C until analysis for than in those fed cellulose or oat bran (Table 1). The

TABLE 1

Experiment 1: Serum and liver cholesterol concentrations in rats fed diets containing cellulose, psyllium,
pectin, oat bran or cholestyramine1

liver
GroupCellulose-fedOat weightg16.2a16.2a14.4b14.9b14.9b1.2Serum
cholesterolmmol/L2.252.511.912.172.690.17Liver
cholesterol\im cholesterol\imol119a116a119a109a96b0.65Serum
insulinnmol/L0.155ab0.179
/g7.507.248.287.246.470.54Total
ol

bran-fedPectin-fedPsyllium
14b0.128ab0.148ab0.020
-fedCholestyramine-fedPooled

errorLiver
Values are means, n = 10. Within a column, values with different superscripts are significantly different (P < 0.05].
456 MATHESON ET AL.

lower total liver cholesterol level in the psyllium consumption resulted in the lowest liver
cholestyramine-fed group would explain some of the cholesterol concentration in rats fed cholesterol
difference but does not account for differences in (Table 2). Total liver cholesterol level responded in a
groups fed pectin or psyllium. similar fashion (Table 2).
Compared with cellulose-fed controls, rats fed 2% There was no difference in the activity of
cholestyramine had a much higher activity of cholesterol 7a-hydroxylase in the cellulose-, oat bran-
cholesterol 7a-hydroxylase, which was of a mag or rice bran-fed groups in the absence of dietary
nitude similar to that previously reported (Hylemon cholesterol. When cholesterol was added to each of
et al. 1989). There was no difference in enzyme ac these diets, activity of the enzyme was significantly
tivity when oat bran was fed compared with cellulose, greater but the values in the three groups did not
but both psyllium and pectin consumption resulted in differ (Fig. 2). This was not reflected in the psyllium-
activities that were twice that of rats fed cellulose, a fed group, in which inclusion of cholesterol in the
difference that was significant for both groups (Fig. 1). diet had no effect and enzyme activity was signifi
In the second experiment, which was designed to cantly higher than that observed in all other groups
test the modulation of the effects of cholesterol with and without dietary cholesterol.
feeding by cellulose, rice bran, oat bran and psyllium,
serum cholesterol was significantly lower in rats fed
the diet containing psyllium without cholesterol than
DISCUSSION

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any other dietary group. There was no difference in
liver cholesterol concentration or total liver
cholesterol when the diets were fed without added There have been several hypotheses proposed to
cholesterol, as was observed in Experiment 1. When explain observed reductions in serum cholesterol con
cholesterol was added to the cellulose diet, there was centrations in response to soluble components of di
approximately a fivefold higher concentration of liver etary fiber. One of the most widely discussed is in
cholesterol. When cholesterol was added to the other creased excretion of neutral and acidic biliary sterols
diets, the extent of liver cholesterol accumulation and diet-derived cholesterol, due to reduced intestinal
was modulated by the dietary fiber sources. Oat bran absorption and/or increased luminal binding of bile
resulted in a cholesterol concentration that was sig acids. This would lead to an increased conversion of
nificantly lower than that observed in rats fed cel exogenous and endogenous cholesterol to bile acids in
lulose with added cholesterol, whereas rice bran and the liver, causing an elevation in LDL receptor ex
pression, thereby reducing serum cholesterol concen
tration. However, there have been conflicting reports
-7 100
±bTbTCTC
40 -,
-'5 90
:2

40 -
o.0)E

-•CE
30

20 -
~51.
TO
10
-0

-a
t>C
«-= C
§ '?C C
w
._ y *- U
3E
= ^ » 2

5"50
=!" ÕT o-
°
ra

FIGURE 2 Activity of hepatic cholesterol 7a-hydroxylase


FIGURE 1 Hepatic cholesterol 7a-hydroxylase activity in in rats fed semipurified diets containing 5% cellulose, oat
rats fed semipurified diets containing 5% psyllium bran, rice bran or psyllium hydrocolloid with (filled bars)
hydrocolloid, pectin, oat bran or cellulose or 2% and without (open bars) 0.25% dietary cholesterol. Values
cholestyramine. Values are means ±SEM (n = 10). Bars with are means ±SEM (n = 6). Bars with different letters are
different letters are significantly different (P < 0.05). significantly different (P < 0.05).
FIBER MODIFIES CHOLESTEROL 7a-HYDROXYLASE 457

TABLE 2

Experiment 2: Serum and liver cholesterol levels in rats fed cellulose, rice bran, oat bran or psyHium with
and without added dietary cholesterol1

Liver liver
GroupCellulose-fedRice cholesterol
weightS16.9cd+ cholesterolmmol/L2.09a2.15a2.08a2.26a2.24a2.30a1.77b2.07a3.72Liver
cholesterolÃ-imol/g7.83d38.0a7.66d22.6C6.96d30.0
cholesterol\imol129d742a132d

19.4a17.1cd+
bran-fedOat
20.0a172bcd+
bran-fedPsyllium-fedPooled
19.0a15.5d+

18.4abc0.56Serum
SEMDietary

Downloaded from jn.nutrition.org at Florida State Univ on July 26, 2009


^Values are means, n ==6. Within a column, values with different superscripts are significantly different (P < 0.05].

concerning fecal steroid excretion during dietary fiber 7a-hydroxylase increased in response to cholesterol
intervention (Kritchevsky and Story 1988). Although feeding to approximately the same extent in the
soluble fibers generally result in higher fecal bile acid groups fed insoluble fiber (cellulose or rice bran) and
excretion in humans (Jenkins et al. 1993, Story 1985), in the group fed the partially soluble oat bran. In the
the evidence is not consistent in rats, with both case of psyllium, the high activity of the enzyme was
higher and lower levels of daily fecal acidic steroid not further increased by addition of cholesterol to the
excretion having been reported for several soluble diet.
fibers (Ide et al. 1990). The above hypothesis requires It has been shown that there are separate hepatic
an increased flux of cholesterol conversion to bile pools of cholesterol depending on its exogenous or
acids and therefore an increase in the biosynthetic endogenous origin (Balasubramaniam et al. 1973,
capacity of the bile acid pathway. Liscum and Dahl 1992) and that there is some stimu
There is evidence that insulin stimulates activity lation of activity of cholesterol 7a-hydroxylase by
of cholesterol 7a-hydroxylase at concentrations as increased microsomal cholesterol availability (Straka
low as 1 nmol/L and that this response is dose de et al. 1990). Results from our experiments do not
pendent, occurring within 30 min (Skett 1990). The agree with the concept that the primary controlling
factor influencing cholesterol 7a-hydroxylase activity
small differences in serum insulin, which were sig
nificant only between rats fed oat bran and those fed is total hepatic cholesterol concentration. These data
pectin (difference between the two being 0.065 nmol/ suggest that, in spite of differences in liver cholesterol
L) (Table 1), probably did not affect enzyme activity. concentration and content in rats fed cellulose, rice
This difference in insulin concentration may have had bran or oat bran with added dietary cholesterol,
enzyme activity is comparable for all three groups of
more effect on glycogen accumulation and, as a
rats. The ability of rice bran and oat bran to prevent
result, liver weight.
cholesterol accumulation when cholesterol is added
Groups fed the soluble fibers pectin and psyllium
to the diet does not seem to be mediated by changes
had greater than twice the activity of cholesterol
7a-hydroxylase than those fed cellulose or oat bran in bile acid synthesis, as indicated by their similar
cholesterol 7a-hydroxylase activities. Other compo
(Fig. 1 and 2). This difference in enzyme activity is of nents of these substances evidently are involved in
a similar magnitude to the previously observed bile their hypocholesterolemic potential.
acid pool size (2.7 to 3.1 times) in rats fed pectin or These results are consistent with the hypothesis
psyllium compared with those fed cellulose suggested by our earlier work (Matheson and Story
(Matheson and Story 1994). This may have been suffi 1994), indicating that soluble dietary fiber influences
cient to partially prevent liver cholesterol accumu cholesterol metabolism by causing changes in the
lation when dietary cholesterol was added because, spectrum of circulating bile acids. It was suggested
although cholesterol feeding resulted in higher that these changes in bile acid composition and the
hepatic concentration of cholesterol in the group fed concomitant changes in hydrophobicity of bile would
psyllium, the increase was not as great as that alleviate the bile acid feedback inhibition on
resulting from cholesterol feeding when cellulose or cholesterol 7a-hydroxylase. These experiments sub
oat bran were fed (Table 2). Activity of cholesterol stantiate this change in bile acid synthesis, further
458 MATHESON ET AL.

supporting our hypothesis that the hypocholestero- Ide, T., Horii, M., Yamamoto, T. & Kawashima, K. (1990) Con
trasting effects of water-soluble and water-insoluble dietary
lemic effect of soluble fibers is modulated through
fibers on bile acid conjugation and taurine metabolism in the
increased synthesis and therefore pool size of bile rat. Lipids 25: 335^340.
acids. Jelinek, D. F., Andersson, S., Slaughter, C. A. & Russell, D. W.
(19901 Cloning and regulation of cholesterol 7a-hydroxylase, the
rate-limiting enzyme in bile acid synthesis. J. Biol. Chem. 265:
8190-8197.
ACKNOWLEDGMENTS Jenkins, D.J.A., Wolever, T.M.S., Rao, V., Hegele, R. A., Mitchell, S.
J., Ransom, T.P.P., Boctor, D. L., Spadafora, P. J., Jenkins, A. L.,
The authors would like to express their thanks to Mehling, C., Relie, L. K., Connelly, P. W., Story, J. A., Furumoto,
Emily Furumoto for her excellent technical assistance E. J., Corey, P. & Wiirsch, P. (1993) Effect on blood lipids of very
high intakes of fiber in diets low in saturated fat and
and to Penny Keller for help in preparation of the cholesterol. N. Eng. J. Med. 329: 21-26.
manuscript. Junker, L. H. & Story, J. A. (1985) An improved assay for cholesterol
7a-hydroxylase activity using phospholipid liposome solubilized
substrate. Lipids 20: 712-718.
Kahlon, T. S., Choq, F. L, Sayre, R. N. & Betschart, A. A. (1992)
LITERATURE CITED Cholesterol lowering in hamsters fed rice bran at various levels,
defatted rice bran and rice bran oil. J. Nutr. 122: 513-519.
Anderson, J. W. & Chen, W.-J.L. (1979) Plant fiber carbohydrate and Kritchevsky, D. & Story, J. A. (1988) The influence of dietary fiber
lipid metabolism. Am. J. Clin. Nutr. 32: 346-363.

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on cholesterol metabolism in experimental animals. In: CRC
Anderson, J. W., Story, L., Sieling, B., Chen, W.-J.L., Petro, M. S. & Handbook of Dietary Fiber in Human Nutrition (Spiller, G. A.,
Story, J. (1984) Hypocholesterolemic effects of oat bran or bean ed.), pp. 129-142. CRC Press, Boca Raton, FL.
intake for hypocholesterolemic men. Am. J. Clin. Nutr. 40: Liscum, L. & Dahl, N. K. (1992) Intracellular cholesterol transport.
1146-1155. J. Lipid Res. 33: 1239-1254.
Anderson, J. W., Zettwoch, N., Tietyen-Clark, J., Oeltgen, P. & Matheson, H. B. & Story, J. A. (1994) Dietary psyllium hydrocolloid
Bichop, C. W. (1988) Cholesterol-lowering effects of psyllium and pectin increase bile acid pool size and change bile acid
hydrophilic mucilloid for hypercholesterolemic men. Arch. composition in rats. J. Nutr. 124: 1161-1165.
Intern. Med. 148: 292-296. Myant, N. B. & Mitropoulos, K. A. (1977) Cholesterol 7a-hydroxy-
Arjmandi, B. H., Ahn, J., Nathani, S. & Reeves, R. D. (1992) Dietary lase J. Lipid Res. 18: 135-153.
soluble fiber and cholesterol affect serum cholesterol concen Nordstrom, J. L., Rodwell, V. W. & Mitschelen, J. J. (1977) Intercon
tration, hepatic portal venous short-chain fatty acid concen version of active and inactive forms of rat liver hydroxymethyl-
tration and fecal sterol excretion in rats. J. Nutr. 122: 246-253. glutaryl-CoA reducÃ-ase. J. Biol. Chem. 252: 8924-8934.
Balasubramaniam, S., Mitropoulos, K. A. & Myant, N. B. (1973) Noshiro, M., Nishimoto, M. & Okuda, K. (1990) Rat liver
Evidence for the compartmentation of cholesterol in rat-liver cholesterol 7a-hydroxylase pretranslational regulation for cir-
microsomes. Eur. J. Biochem. 34: 77-83. cadian rhythm. J. Biol. Chem. 265: 10036-10041.
Behall, K. M. (1990) Effect of soluble fibers on plasma lipids, Rudel, L. L. & Morris, M. D. (1973) Determination of cholesterol
glucose tolerance and mineral balance. Adv. Exp. Med. Biol. 270: using o-phthaldialdehyde. J. Lipid Res. 14: 364-366.
7-16. Shinnick, F. L., Longacre, M. J., Ink, S. L. &.Marlett, J. A. (1988) Oat
Bradford, M. M. (1976) A rapid and sensitive method for quanti - fiber: composition versus physiological function in rats. J. Nutr.
tation of microgram quantities of protein utilizing the principle 118: 144-151.
of protein-dye binding. Anal. Biochem. 72: 248-254. Skett, P. (1990) Role of hormones in mixed-function oxidases.
Chen, W.-J.L., Anderson, J. W. &. Gould, M. R. (1981) Effects of oat Biochem. Soc. Trans. 18: 24-26.
bran, oat gum and pectin on lipid metabolism of cholesterol-fed Snedecor, G. W. & Cochran, W. G. (1989) Statistical Methods. Iowa
rats. Nutr. Rep. Int. 24: 1093-1098. State University Press, Ames, IA.
Danielsson, H., Einarsson, K. & Johansson, G. (1967) Effect of Story, J. A. (1985) Modification of steroid excretion in response to
biliary drainage on individual reactions in the conversion of dietary fiber. In: Dietary Fiber (Vahouny, G. V. & Kritchevsky,
cholesterol to taurocholic acid. Eur. J. Biochem. 2: 44—49. D., eds.), pp. 253-264. Plenum Publishing, New York, NY.
Everson, G. T., Daggy, B. P., McKinley, C. &. Story, J. A. (1992) Story, f. A., Czarnecki, S. K., Tepper, S. A. & Kritchevsky, D. (1981)
Effects of psyllium hydiophilic mucilloid on LDL-cholesterol Dose response to dietary cholesterol in the rat. Nutr. Rep. Int.
and bile acid synthesis in hypercholesterolemic men. f. Lipid 24: 465-470.
Res. 33: 1183-1192. Straka, M. S., Junker, L. H., Zacarro, L., Zogg, D. L., Dueland, S.,
Hylemon, P. B., Struder, E. J., Pandak, W. M., Heuman, D. M., Everson, G. T. &. Davis, R. A. (1990) Substrate stimulation of
Vlahcevic, Z. R. & Chiang, J.Y.L. (1989) Simultaneous meas 7a-hydroxylase, an enzyme located in the cholesterol-poor en-
urement of cholesterol 7a-hydroxylase activity by reverse-phase doplasmic reticulum. J. Biol. Chem. 265: 7145-7149.
high performance liquid chromatography using both endogenous Tsai, A. C., Elias, J., Kelly, J., Lin, R.-S. C. & Robson, J.R.K. (1976)
and exogenous [4-14C)cholesterol as substrate. Anal. Biochem. Influence of certain dietary fibers on serum and tissue
182: 212-216. cholesterol levels in rats. J. Nutr. 106: 118-123.
Cholesterol 7a-Hydroxylase Activities From Human and Rat
Liver Are Modulated In Vitro Posttranslationally by
Phosphorylation/Dephosphorylation
LIEN B. NGUYEN,1 SARAH SHEFER,1 GERALD SALEN,2 JOHN Y. L. CHIANG,3 AND MUKUL PATEL1

Purified cholesterol 7a-hydroxylases (C7aH) from hu- ciency of C7aH (activity per protein mass unit) is modu-
man and rat liver microsomes, and from transformed lated, in vitro, posttranslationally by a phosphorylation/
Escherichia coli expression systems, were incubated dephosphorylation mechanism in both the human and
with 0.3 mmol/L [g-32P] adenosine triphosphate (ATP) in the rat enzymes. (HEPATOLOGY 1996;24:1468-1474.)
the presence and absence of bacterial alkaline phospha-
tase (AP) or rabbit muscle adenosine 3*,5*-cyclic mono-
Cholesterol 7a-hydroxylase (C7aH) (EC 1.14.13.17) is the
phosphate (cAMP)-dependent protein kinase. The
first step and rate-limiting enzyme in the conversion of cho-
amounts of 32P incorporation after separation of human
lesterol to bile acids in the liver.1,2 This enzyme has been
and rat C7aH proteins by sodium dodecyl sulfate–poly-
purified by various laboratories,3-6 its gene cloned, sequenced,
acrylamide gel electrophoresis (SDS-PAGE) were re-
localized to chromosome 8q11-q12, and partially character-
lated to C7aH catalytic activities (determined by a radio-
ized.7-11 The catalytic activity of C7aH is regulated by a diur-
isotope incorporation method) and enzyme protein mass
nal rhythm and various dietary, drug, and hormonal fac-
(determined by Western blotting and laser densitome-
tors.12-17 The preferred substrate pool for C7aH is newly
try). Both human and rat C7aH activities significantly
synthesized cholesterol.18 Therefore, bile acid synthesis is
decreased after dephosphorylation by AP (057%–072%)
regulated by the activity of both C7aH and 3-hydroxy-3-
and increased up to twofold with phosphorylation by
methylglutaryl coenzyme A reductase, the rate-limiting en-
rabbit muscle cAMP-dependent protein kinase. The in-
zyme that controls the formation of endogenous choles-
creases in C7aH activities were proportional to the
terol.15-17 The supply of cholesterol up-regulates C7aH activ-
amounts of cAMP-dependent protein kinase used, and
ity in the rat,15,19 but inhibits it in the rabbit,20 the hamster,19
were coupled to 32P incorporation into the purified en-
and the African Green monkey.21 The reasons for such species
zymes. Both the activation of C7aH and the amounts of
32 differences have not been established. There is still a contro-
P incorporation were time-dependent and reached a
versy about mechanisms by which C7aH activity is controlled
maximum after 1 hour of incubation with 5 U of cAMP-
by the enterohepatic flux of bile acids.15,16 Earlier studies
dependent protein kinase. In a second set of experi-
have suggested that C7aH activity could be regulated post-
ments, purified human and rat liver C7aH were dephos-
translationally by mechanisms involving cytosolic factors,22
phorylated by 30-minute incubation with AP, followed
disulfide bonds in the enzyme structure,23 and phosphoryla-
by inactivation of the phosphatase by the inhibitor NaF,
tion/dephosphorylation of the enzyme protein.24-28 In contrast
and rephosphorylation of C7aH by 30-minute incubation
to 3-hydroxy-3-methylglutaryl coenzyme A reductase, which
with rabbit muscle cAMP-dependent protein kinase or
is deactivated by phosphorylation and stimulated by dephos-
bovine heart cAMP-independent protein kinase. Re-
phorylation,29 it was earlier suggested that C7aH was deacti-
phosphorylation of the dephosphorylated C7aH pro-
vated by phosphatase-mediated dephosphorylation and stim-
teins by cAMP-dependent protein kinase increased
ulated by a number of protein kinases.24-26 However,
C7aH catalytic activities up to fourfold, and the stimula-
phosphorylation/dephosphorylation as a mechanism for post-
tion in catalytic activities paralleled the increases in 32P
translational regulation of C7aH activity is still disputed by
incorporation into the purified enzymes. Bovine heart
some investigators.29-31
protein kinase was as potent as rabbit muscle cAMP-
The objectives of this study are to (1) examine the effects
dependent protein kinase in stimulating catalytic activ-
of phosphorylation/dephosphorylation on catalytic activities
ity and 32P incorporation into the human C7aH protein.
of various preparations of purified C7aH from human and
Because the protein mass of these purified enzymes did
rat liver; and (2) relate changes in catalytic activities under
not change, the short-term regulation or catalytic effi-
varying phosphorylation/dephosphorylation conditions to the
amounts of 32P incorporated into the purified enzyme pro-
teins.
Abbreviations: C7aH, cholesterol 7a-hydroxylase; AP, alkaline phosphatase; cAMP,
adenosine 3*,5*-cyclic monophosphate; SDS-PAGE, sodium dodecyl sulfate–polyacrylamide MATERIALS AND METHODS
gel electrophoresis; ATP, adenosine triphosphate.
From the 1Department of Medicine and the Liver Center, University of Medicine and Purification of Human and Rat C7aH. Four preparations of puri-
Dentistry of New Jersey–New Jersey Medical School, Newark, NJ; 2Gastroenterology Sec- fied rat C7aH were used: two were isolated as previously described5
tion, Veterans Administration Medical Center, East Orange, NJ; and 3Department of Bio- from cholestyramine-fed rats and two were obtained from Esche-
chemistry and Molecular Pathology, Northeastern Ohio Universities College of Medicine, richia coli that had been transformed with an expression vector con-
Rootstown, OH. taining a C7aH complimentary DNA (cDNA).9 Human C7aH protein
Received June 28, 1996; accepted July 25, 1996. was expressed similarly and purified from transformed E. coli.10 The
Supported in part by U.S. Public Health Service Grants DK 26756, HL 17818, DK 44442,
and the Research Service Veterans Administration Medical Center.
C7aH proteins isolated from E. coli expression systems were trun-
Address reprint requests to: Lien B. Nguyen, Ph.D., UMDNJ-NJ Medical School, Depart-
cated enzymes that lacked the N-terminal 24-amino acid residues
ment of Medicine, 185 South Orange Ave., MSB H-532, Newark, NJ 07103. but were catalytically similar to the full-length enzymes purified
Copyright q 1996 by the American Association for the Study of Liver Diseases. from human and rat liver microsomes.9,10 All purified enzyme prepa-
0270-9139/96/2406-0028$3.00/0 rations showed single bands upon sodium dodecyl sulfate–polyacryl-
1468

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HEPATOLOGY Vol. 24, No. 6, 1996 NGUYEN ET AL. 1469

amide gel electrophoresis (SDS-PAGE)32 and had a molecular weight prestained molecular-weight standards (Bio-Rad Laboratories, Mel-
of 51,000. Polyclonal antibodies against rat and human enzymes ville, NY) and purified C7aH proteins as markers, and separated
were raised in rabbits as described previously.5 These specific anti- by electrophoresis32 using a Hoeffer system (Model SE600, Hoeffer
bodies could cross-react with heterologous antigens of both full- Scientific Instruments, San Francisco, CA). The gels were stained
length and truncated enzymes.5 with Coomassie Brilliant Blue, and 32P radioactivity in the gel slices
Phosphorylation/Dephosphorylation of C7aH. Aliquots (0.5-4 mg) of containing the C7aH bands were determined by liquid scintillation
human- and rat-purified C7aH proteins were incubated at 377C for spectroscopy.
30 minutes with varying amounts of E. coli–type III alkaline phos- Assay of C7aH Activity, Mass, and Catalytic Activity. The phosphor-
phatase (AP) or rabbit muscle adenosine 3*,5*-cyclic monophosphate ylation/dephosphorylation of the microsomal and purified C7aH pro-
(cAMP)-dependent protein kinase (Sigma Chemical Co., St. Louis, teins, or rephosphorylation of AP-treated C7aH, were performed as
MO) in buffer (50 mmol/L Tris, 100 mmol/L NaCl, 5 mmol/L MgCl2 , described above, except that unlabeled ATP was used. After inactiva-
pH 7.4) containing phospholipids (5 mg dilauryl phosphatidylcholine tion of the AP/protein kinase with 50 mmol/L NaF and 10 mmol/L
dissolved in 5 mg deoxycholic acid) in a final volume of 50 mL. The disodium ethylenediamine tetraacetate, cofactors needed for the
AP was solubilized in 20 mmol/L imidazole buffer, pH 7.4, diluted assay for C7aH catalytic activity (5 mmol/L dithiothreitol, 30 mmol/L
to 100 U/mL, and added in increasing amounts (0.2, 0.5, and 1 U) nicotinamide, and 2 U of reduced nicotinamide adenine dinucleotide
as active or heat-inactivated phosphatase (boiled for 5 minutes for phosphate cytochrome P450 reductase in 100 mmol/L K2HPO4 , pH
control samples). The cAMP-dependent protein kinase was dissolved 7.4) were added with the labeled substrate (200 nmol [4-14C]cholest-
at a concentration of 800 U/mL in 100 mmol/L K2HPO4 , pH 7.4, and erol, from Amersham Life Science, Arlington Heights, IL, dissolved
added in aliquots containing 1, 2, and 5 U, together with 5 mCi [g- in 10 mL of 50% b-cyclodextrin,33 from Cyclodextrin Technologies,
32
P] adenosine triphosphate (ATP) (Amersham Life Science, Arling- Inc., Gainesville, FL) in a total volume of 0.48 mL. The mixtures
ton, IL; diluted with unlabeled ATP for a final concentration of 0.3 were preincubated for 2 minutes at 377C, and the 7a-hydroxylation
mmol/L) and 50 mmol/L cAMP. In a time-course study of the phos- reactions were started with the addition of 20 mL of reduced nicotin-
phorylation reaction, 5 U of cAMP-dependent protein kinase was amide adenine dinucleotide phosphate (1.2 mmol/L final concentra-
added to 2 mg C7aH with [g-32P]ATP and cAMP (as above) and the tion). The products were extracted, separated by thin-layer chroma-
mixtures were incubated for various intervals (15, 30, 60, and 120 tography, and quantitated by liquid scintillation counting as
minutes). previously reported.34
In a second set of experiments, the purified human and rat C7aH The C7aH protein mass was measured by immunoblotting and
proteins (2-4 mg) were incubated with AP (0.5 U) for 30 minutes at densitometry after SDS-PAGE separation. The proteins and
377C in a total volume of 30 mL (50 mmol/L Tris, 100 mmol/L NaCl, prestained markers were transferred electrophoretically from the
5 mmol/L MgCl2 , 5 mg dilauryl phosphatidylcholine/deoxycholic acid, SDS-PAGE gels to nitrocellulose membranes (Amersham Life Sci-
pH 7.4). The phosphatase was then inactivated with 5 mL of 500 ence) at 100 V for 2 hours in a Trans-Blot Cell, equipped with a
mmol/L NaF, then increasing amounts (0, 1, 2, 5 U) of rabbit muscle Model 200/2.0 power supply (Bio-Rad Laboratories) and a refriger-
cAMP-dependent protein kinase or bovine heart cAMP-independent ated circulating bath (Haake, Inc., Saddle Brook, NJ). The mem-
protein kinase (Sigma Chemical Co., St. Louis, MO) were added with branes were blocked at room temperature for 2 hours (20 mmol/L
5 mCi [g-32P]ATP with or without cAMP, to bring the total volume Tris, 150 mmol/L NaCl, 0.02% NaN3 , 5% nonfat dry milk, 0.2%
to 50 mL. The mixtures were incubated for an additional 30 minutes Tween-20, pH 7.6), and incubated overnight in heat-sealed bags at
to rephosphorylate the dephosphorylated (AP-treated) C7aH pro- 47C with polyclonal rabbit anti-human C7aH IgG at a concentration
teins, before the measurements of 32P incorporation, C7aH catalytic of 5 mg/mL in Tris-buffered saline (20 mmol/L Tris, 500 mmol/L NaCl,
activity, and protein mass. pH 7.5) containing 5% nonfat dry milk. The membranes were washed
To investigate the role of endogenous hepatic protein kinase/phos- three times (10 minutes each) with Tris-buffered saline containing
phatase in the activation/deactivation of C7aH, livers from adult 0.05% Tween-20 and incubated with biotinylated anti-rabbit IgG
Sprague Dawley rats were used. Two rat liver microsomes (P and (Vector Laboratories, Inc., Burlingame, CA) at a concentration of 5
dP) were prepared with different buffers. The homogenizing buffer mg/mL in Tween-20 Tris-buffered saline for 1 hour at room tempera-
for microsomes P is a phosphate buffer (50 mmol/L K2HPO4 , 154 ture. After washing in Tween-20/Tris-buffered saline the membranes
mmol/L KCl, 1 mmol/L disodium ethylenediamine tetraacetate, 1 were incubated for 1 hour at room temperature with horseradish
mmol/L dithiotreitol, 75 mmol/L nicotinamide, pH 7.4) that contains peroxidase conjugated to strepavidin (Amersham Life Science). The
50 mmol/L of the phosphatase inhibitor NaF; the homogenizing conjugated C7aH protein bands were detected by color development
using 4-chloro-1-naphthol–containing reagents (Bio-Rad Labora-
buffer for microsomes dP is the same, except 50 mmol/L imidazole
tories) according the the manufacturer’s instructions. The protein
and 50 mmol/L KCl replace K2HPO4 and NaF, respectively. The mi-
mass of C7aH under various experimental conditions was quanti-
crosomal fractions, separated by ultracentrifugation between
tated by densitometric scanning of the immunoblots with a LKB
10,000g and 100,000g, were resuspended in buffers (microsomes P:
2202 laser densitometer equipped with a LKB 2220 recording inte-
100 mmol/L K2HPO4 , 50 mmol/L NaF, 1 mmol/L disodium ethylene-
grator, and C7aH relative peak area used as protein mass units.
diamine tetraacetate, 5 mmol/L dithiotreitol, 75 mmol/L nicotin-
The catalytic efficiency (catalytic activity per protein mass unit)
amide, 20% glycerol, pH 7.4; microsomes dP: same buffer with imid-
of phosphorylated and dephosphorylated C7aH was calculated by
azole and KCL replacing K2HPO4 , and NaF) and stored at 0807C
dividing the C7aH catalytic activity (pmol/mg protein/min) by the
until used for enzymatic assays. Dephosphorylation of microsome P
enzyme protein mass (relative peak area/mg protein).
was carried out by incubating 100 mg microsomal proteins with 5 U Statistical Analysis. Data were analyzed statistically by the one-
of AP at 377C for 30 minutes in a buffer (50 mmol/L Tris, 100 mmol/ way ANOVA, comparison of confidence intervals of the means, or
L NaCl, 5 mmol/L MgCl2 , pH 7.4). Phosphorylation of microsomal the unpaired t test.35
protein dP was carried out by incubating 100-mg microsomal proteins
with 100 U of rabbit muscle cAMP-dependent protein kinase, 2 mmol/ RESULTS
L ATP, and 50 mmol/L cAMP at 377C for 30 minutes in the same
Tris buffer. After the phosphorylation/dephosphorylation reactions, Table 1 shows C7aH catalytic activities in rat liver micro-
50 mmol/L NaF and 10 mmol/L disodium ethylenediamine tetraace- somes P (prepared from liver homogenized with a phosphate
tate were added to deactivate phosphatase/protein kinase prior to the buffer in the presence of the phosphatase inhibitor NaF) and
measurements of C7aH catalytic activities. Preliminary experiments microsomes dP (prepared in the absence of phosphate and
with the same microsomal preparations showed that varying the NaF), as well as the effects of AP and cAMP-dependent pro-
composition of the C7aH assay buffer (phosphate, imidazole, or Tris) tein kinase on microsomal C7aH activities. Microsomes dP
used during the incubation with the [14C]cholesterol substrate did showed significantly lower C7aH catalytic activities (060%,
not change C7aH catalytic activities. P õ .01) than microsomes P, where the phosphorylated state
Measurements of 32P Incorporation. At the end of the phosphoryla-
tion, dephosphorylation or rephosphorylation reactions as described
of C7aH was protected. Dephosphorylation of microsomes P
above, the incubation mixtures were ultrafiltrated (Centricon-30; with AP significantly reduced C7aH catalytic activities,
Amicon, Beverly, MA) to remove unreacted [32P]ATP, and dissolved whereas phosphorylation of microsomes dP with cAMP-de-
in 50 mL of gel-loading buffer (100 mmol/L Tris, 200 mmol/L dithio- pendent protein kinase raised C7aH catalytic activities to
threitol, 4% SDS, 0.2% bromophenol blue, 20% glycerol, pH 6.8). The the high level found in microsomes P.
samples were loaded on 1.5-mm-thick 10% polyacrylamide gel with When human and rat purified C7aH proteins were incu-

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1470 NGUYEN ET AL. HEPATOLOGY December 1996

TABLE 1. Effects of AP and cAMP-Dependent Protein Kinase on


C7aH Activities in Rat Liver Microsomes
C7aH Activities†
Treatment* pmol/mg/min

Microsomes P 107.2 { 6.3 (100)


Microsomes dP 43.1 { 8.6‡ (40)
Microsomes P { AP 36.2 { 7.6‡ (34)
Microsomes dP { cAMP-dependent protein kinase 108.4 { 26.8 (101)

* Microsomes were prepared from liver homogenized with either a phos-


phate buffer containing 50 mmol/L NaF (microsomes P) or an imidazole buffer
with 50 mmol/L KCl replacing NaF (microsomes dP). Treatment with 5 U of
AP or 100 U of cAMP-dependent protein kinase, 2 mmol/L ATP, and 50 mmol/
L cAMP was for 30 minutes, followed by deactivation of the phosphatase and
protein kinase, before cofactors and substrate were added for the assay of
C7aH activities.
† Means { SEM from three to six animals, with percentage in parentheses.
‡ Significantly lower than activities in microsomes P, P õ .01.

bated for 30 minutes with increasing amounts of AP, C7aH


catalytic activities significantly decreased and reached maxi-
mum inhibition with 0.5 U AP (P õ .01, Table 2). In contrast,
rabbit muscle cAMP-dependent protein kinase increased
C7aH catalytic activities with the increasing amounts of pro-
tein kinase (Table 2). C7aH proteins purified from livers of
cholestyramine-fed rats and isolated from E. coli expression
systems gave similar results, and have been combined in the
presentation of data in all tables and figures. When human
and rat purified C7aH proteins were incubated with 5 U
of rabbit muscle cAMP-dependent protein kinase for longer
periods (up to 2 hours), C7aH catalytic activities increased
linearly with incubation times and reached a maximum (a
twofold increase relative to baseline) after 1 hour of incuba-
tion (Fig. 1). Thus, C7aH catalytic activities significantly
decreased with AP-mediated dephosphorylation and substan-
tially increased with protein kinase–mediated phosphoryla-
tion (Fig. 1). The increases in catalytic activities of both hu- FIG. 1. Effects of AP and rabbit muscle cAMP-dependent protein kinase on
man and rat purified C7aH after phosphorylation were human and rat C7aH activities. Purified C7aH proteins (2 mg) were incubated
without (C, control), and with AP (0.5 U) or cAMP-dependent protein kinase
(KN, 5 U) for 1 hour before the assay for C7aH catalytic activities. Means {
SEM from three measurements of E. coli-expressed human C7aH, and multiple
TABLE 2. Effects of AP and cAMP-Dependent Protein Kinase on measurements of four purified rat C7aH proteins (two from livers of cholestyra-
mine-fed rats and two isolated from E. coli expression systems, which gave
Purified C7aH Activities
similar results). Catalytic activities representing 100% for human and rat
C7aH Activities† C7aH were 1.96 { 0.31 and 2.13 { 0.31 nmol/mg/min, respectively. *Signifi-
Source Treatment* nmol/mg/min cantly different from control, P õ .01.

Human Control, 0 U 1.96 { 0.31 (100)


AP
coupled to increases in amounts of 32P incorporated into the
0.2 U 1.04 { 0.39 (53)
0.5 U 0.55 { 0.21‡ (28)
C7aH proteins. As with C7aH catalytic activities, amounts
1.0 U 0.77 { 0.22§ (39) of 32P incorporation increased linearly with C7aH protein
cAMP-dependent protein kinase concentrations (Fig. 2A) and incubation times (Fig. 2B), and
32
1U 1.85 (94) P incorporation/mg C7aH protein reached a maximum after
2U 2.29 (117) incubation with [g-32P]ATP and 5 U of cAMP-dependent pro-
5U 2.94 { 0.09\ (150) tein kinase for 1 hour (Fig. 2B).
Rat Control, 0 U 2.13 { 0.31 (100) Figure 3 presents the catalytic activities of human and rat
AP C7aH proteins after dephosphorylation by 30-minute incuba-
0.2 U 1.18 { 0.68 (55)
tion with 0.5 U of AP, inactivation of the phosphatase with
0.5 U 0.91 { 0.19‡ (43)
1.0 U 0.92 { 0.19‡ (43)
NaF, then rephosphorylation of the C7aH proteins by 30-
cAMP-dependent protein kinase minute incubation with increasing amounts of two different
1U 2.20 { 0.20 (103) protein kinases. Both human and rat C7aH catalytic activi-
2U 2.60 { 0.32 (122) ties significantly increased with rephosphorylation by cAMP-
5U 3.15 { 0.45 (148) dependent protein kinase, and these increases were protein
kinase concentration-dependent. With 5 U of cAMP-depen-
* Incubation for 30 minutes with 2-4 mg C7aH. dent protein kinase, human and rat C7aH catalytic activities
† Means { SEM from two to four measurements of purified human C7aH
were stimulated fourfold and twofold, respectively, relative
and at least two measurements of each of four different preparations of purified
rat C7aH (two from livers of cholestyramine-fed rats and two from E. coli
to baseline activities of the dephosphorylated enzymes. The
expression systems). Values in parentheses are percentages. bovine heart cAMP-independent protein kinase was as potent
‡ Significantly lower than control, P õ .01. as the rabbit muscle cAMP-dependent protein kinase in up-
§ Significantly lower than control, P õ .05. regulating the human C7aH catalytic activities. It should be
\ Significantly higher than control, P õ .05. noted that the rephosphorylation reactions and up-regulation

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HEPATOLOGY Vol. 24, No. 6, 1996 NGUYEN ET AL. 1471

of catalytic activities shown in Fig. 3 were not complete be-


cause the rephosphorylation was performed for only 30 min-
utes; yet, the up-regulation of enzyme catalytic activities was
already significant. The stimulation of human and rat C7aH
catalytic activities by rephosphorylation with increasing
amounts of rabbit muscle cAMP-dependent protein kinase
and bovine heart protein kinase paralleled the increases in
32
P incorporation into the C7aH proteins (Fig. 4). Both C7aH
catalytic activities and 32P incorporation into the human and
rat C7aH proteins increased linearly with the increments
in amounts of cAMP-dependent protein kinase. The human
C7aH protein was rephosphorylated (32P incorporated into
the enzyme protein) and its catalytic activity was stimulated
FIG. 2. Effects of C7aH concentrations and incubation times on the as efficiently by the bovine heart protein kinase as by the
amounts of 32P incorporated into the purified C7aH proteins. (A) 0.2 to 4 mg cAMP-dependent protein kinase (Fig. 4).
C7aH was incubated in a total volume of 50 mL with 0.3 mmol/L [g-32P]ATP, Figure 5 shows the SDS-PAGE separation of human and
50 mmol/L cAMP, and 5 U of cAMP-dependent protein kinase for 30 minutes.
(B) 2 mg C7aH was incubated with 0.3 mmol/L [g-32P]ATP, 50 mmol/L cAMP, rat C7aH proteins after incubation with cAMP-dependent
and 5 U of cAMP-dependent protein kinase for 15-120 minutes. Means { SEM protein kinase without (Fig. 5A) and with (Fig. 5B) prior
from three to four measurements of E. coli–expressed human C7aH, and dephosphorylation by bacterial AP. In the absence of protein
multiple measurements of three purified rat C7aH proteins (one from livers kinase and AP (Fig. 5A lanes 4 and 9, Fig. 5B lane 7), one
of cholestyramine-fed rats and two isolated from E. coli expression systems).
major band was detected slightly above the 50,000 molecular-
weight marker. Neither the addition of varying amounts of
the rabbit muscle cAMP-dependent protein kinase (Fig. 5A),
nor the incubation with AP before addition of cAMP-depen-
dent protein kinase (Fig. 5B) changed the apparent mass of
the purified C7aH proteins. The constant relative protein
mass of human and rat C7aH proteins under various experi-
mental conditions was substantiated by densitometric mea-
surements of the C7aH protein bands in the immunoblots,
which remained unchanged. The catalytic efficiencies (nmol/
min/protein mass unit) of both human and rat C7aH proteins
(Table 3), markedly decreased after dephosphorylation by AP
and increased after rephosphorylation by cAMP-dependent
protein kinase.
DISCUSSION
Both human and rat purified C7aH activities significantly
decreased with AP-mediated dephosphorylation and in-
creased two fold to fourfold by cAMP-dependent protein ki-
nase–mediated phosphorylation. When purified C7aH pro-
teins were phosphorylated by cAMP-dependent protein
kinase, the amounts of 32P incorporated into both human
and rat C7aH proteins increased linearly with C7aH protein

FIG. 3. Effects of rephosphorylation by rabbit muscle cAMP-dependent FIG. 4. The amounts of 32P incorporation (solid and open circles) and C7aH
protein kinase (solid bars) and bovine heart protein kinase (hatched bars) on catalytic activities (solid and open triangles) after rephosphorylation of C7aH
the catalytic activities of dephosphorylated C7aH. The broken lines represent with rabbit muscle cAMP-dependent protein kinase and bovine heart protein
activities of C7aH after dephosphorylation by 30-minute exposure to 0.5 U kinase. After dephosphorylation of C7aH by 30-minute exposure to AP, then
of AP (Human Å 550 { 207 pmol/mg/min; Rat Å 907 { 187 pmol/mg/min). inactivation of the phosphatase with NaF, the C7aH proteins were rephosphor-
Rephosphorylation was performed by incubation for 30 minutes with 0.3 mmol/ ylated for 30 minutes with 0.3 mmol/L ATP and 5 U of rabbit muscle cAMP-
L ATP, 5 U of protein kinase with or without cAMP, after inactivation of AP dependent protein kinase (solid lines and filled symbols) or bovine heart cAMP-
with NaF. Averages from two measurements of the E. coli–expressed human independent protein kinase (broken lines and open symbols). Averages of two
C7aH protein and means { SEM from measurements of four purified rat C7aH measurements of the E. coli–expressed human C7aH protein and means {
proteins (two from livers of cholestyramine-fed rats and two isolated from E. SEM from measurements of four purified rat C7aH proteins (two from livers
coli expression systems). of cholestyramine-fed rats and two isolated from E. coli expression systems).

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1472 NGUYEN ET AL. HEPATOLOGY December 1996

concentrations and incubation times, and occurred in parallel TABLE 3. Effects of Dephosphorylation by AP and
to the stimulation in catalytic activities. With 5 U of cAMP- Rephosphorylation by cAMP-Dependent Protein Kinase on Catalytic
dependent protein kinase, both the amounts of 32P incorpo- Efficiency of C7aH
rated into human and rat C7aH proteins and C7aH catalytic Source Treatment* Catalytic Efficiency†
activities reached maximum levels after 1 hour of incubation.
These observations demonstrated that, in in vitro experi- nmol/min/protein mass unit
ments, both human and rat C7aH are regulated posttransla- Human Control, 0 U 3.9 { 0.6
AP
tionally by a phosphorylation/dephosphorylation mechanism.
0.2 U 1.9 { 0.3‡
In contrast to 3-hydroxy-3-methylglutaryl coenzyme A reduc- 0.5 U 1.1 { 0.2§
tase, which is inactivated with phosphorylation, C7aH is 1.0 U 1.6 { 0.1‡
stimulated by the incorporation of phosphate groups and de- AP (0.5 U) / cAMP-dependent
activated with their removal. protein kinase
The C7aH cDNA used to obtain the E. coli–expressed rat 0U 1.1 { 0.2
C7aH protein was isolated with antibodies against C7aH 1U 3.3 { 1.0
purified from liver of rats that were fed the bile acid seques- 2U 4.3 { 1.4
trant cholestyramine to enhance C7aH formation.7,9 C7aH 5U 5.0 { 0.2\
Rat Control, 0 U 4.9 { 0.8
purified from cholestyramine-fed rats and preparations from
AP
E. coli were similar in catalytic activities, responses to AP 0.2 U 2.7 { 1.0
and protein kinase, as well as 32P incorporation. The similar- 0.5 U 1.6 { 0.6‡
ity in the data from the two sources of rat enzymes is evi- 1.0 U 1.7 { 0.6‡
denced by the coefficients of variation, which were less than AP (0.5 U) / cAMP-dependent
14% when C7aH activities in untreated or protein kinase– protein kinase
treated rat enzymes from both sources were pooled and ana- 0U 1.6 { 0.6
lyzed. The coefficient of variation of 32P incorporation mea- 1U 2.8 { 0.8
surements in both the liver and the E. coli-expressed enzymes 2U 3.4 { 0.5
5U 4.5 { 0.8Ø
is also small (less than 8%).
Diven et al.24,25 showed that the inactivation of C7aH by * Incubation of 2-4 mg C7aH with or without AP for 30 minutes, inactivation
dephosphorylation could be effected by phosphatases of he- of AP with 50 mmol/L NaF, then incubation for an additional 30 minutes with
patic source. Our results with microsomal C7aH, which or without rabbit muscle cAMP-dependent protein kinase.
showed high C7aH activity in microsomes P, where the phos- † Means { SEM from four measurements of purified human C7aH and two
phorylated form of enzyme was protected during preparation, to four measurements of each of three different preparations of purified rat
and loss of activity when this microsomal fraction was ex- C7aH (one from cholestyramine-fed rat liver and two from E. coli expression
posed to AP, suggest a role of hepatic phosphatase/protein systems).
‡ Significantly lower than control, P õ .05.
kinase in the regulation of microsomal C7aH in the liver. It
§ Significantly lower than control, P õ .01.
is interesting to find that the E. coli–expressed C7aH protein \ Significantly higher than samples incubated with only 0.5 U of AP, P õ
is phosphorylated partially, because it could be deactivated .01.
by AP-mediated dephosphorylation and its activity restored Ø Significantly higher than samples incubated with only 0.5 U of AP, P õ
and enhanced above baseline values with protein kinase– .05.
mediated phosphorylation. Apparently, C7aH can be phos-
phorylated/dephosphorylated by protein kinase/phosphatase
with broad substrate specificity. and possibly other endogenous kinases, could effectively up-
In our in vitro experiments, the stimulation of C7aH cata- regulate C7aH in humans. The rabbit muscle cAMP-depen-
lytic activities by phosphorylation is not limited to cAMP- dent kinase used in this study is commercially prepared ac-
dependent protein kinase. The bovine heart protein kinase, cording to a published method.36 One unit is defined as the

FIG. 5. SDS-PAGE separation of human and rat C7aH after phosphorylation/dephosphorylation and rephosphorylation reactions. (A) Lanes 5 and 10,
molecular weight markers (top to bottom: b-galactosidase, 121,000; bovine serum albumin, 86,000; ovalbumin, 50,700; carbonic anhydrase, 33,600; soybean
trypsin inhibitor, 27,800; lysosyme, 19,400), lanes 1-4, 4 mg purified C7aH from rat liver with 5, 2, 1, 0 U cAMP-dependent protein kinase (KN); lanes 6-9, 4
mg purified human C7aH protein with 5, 2, 1, 0 U cAMP-dependent protein kinase. (B) Lane 2, molecular-weight markers as in (A); lanes 1, and 3-5, 4 mg
purified human C7aH dephosphorylated by AP prior to rephosphorylation by 0, 5, 2, 1 U cAMP-dependent protein kinase; lanes 6 and 7, incubations of human
C7aH in the absence of AP and absence of both AP and protein kinase, respectively, in the dephosphorylation/rephosphorylation experiment.

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HEPATOLOGY Vol. 24, No. 6, 1996 NGUYEN ET AL. 1473

amount that will transfer 1 pmol of phosphate from [g-32P]- Acknowledgment: The dedicated technical assistance of
ATP to hydrolyzed and partially dephosphorylated casein per Eva Paroulek is acknowledged.
minute at pH 6.5 at 307C. It should be noted that the specific
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