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Plant Secondary Metabolites: Biosynthesis, Classification, Function and

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Journal of Pharmacy and
Pharmacology
Volume 2, Number 7, July 2014 (Serial Number 8)

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 Journal of
Pharmacy and Pharmacology

Volume 2, Number 7, July 2014 (Serial Number 8)

Contents
Reviews

377 Plant Secondary Metabolites: Biosynthesis, Classification, Function and Pharmacological

Properties
Justin N. Kabera, Edmond Semana, Ally R. Mussa and Xin He

Original articles

393 Evaluation of the Anticonvulsant Activity of the Leaf Methanol Extract of Crassula arborescens
(Mill.) Willd. (Crassulaceae) in Mice
George Jimboyeka Amabeoku, Oluchi Nneka Mbamalu, Tasneem Davids, Samukelisiwe Fakude, Anda
Gqwaka, Fiona Mbai, Reighman Pieterse and Aneesa Shaik

404 Can Dietary Pomegranate Peels Reduce Stress Responses Associated with Group Mixing of
Holstein Beef Calves?
Sarah Weyl-Feinstein, Alla Orlov, Moran Yishay, Rotem Agmon, Machteld Steensels, Vered Sibony,
Ilan Halachmi, Ido Izhaki and Ariel Shabtay

416 Immune System and Body Defence Enhancement Effects of Oral Dihydroartemisinin in Wistar
Albino Rats
A. Utoh-Nedosa Uchechukwu, A. Akah Peter, Nedosa S. Kenechi, Anowi F. Chinedu, Adeyanju N.
Oluwafemi, Nedosa V. Ikenna, Onyekwelu N.A. and Ojemudia Thiophilus

422 Neuropathology of Schizophrenia

Villeda-Hernández Juana, Sánchez Martínez Rodrigo, Totxo-Guerrero Sebastián, Manzanarez-Colin
Mariel Carolina, Palacios-Escalona Sergio, Franco-Del Toro Perla Guadalupe, Cuevas-Nuñez Flora
Minerva, Alonso-Zuñiga Rosa Emma, Peralta-Rodríguez Brenda and Rembao-Bojórquez Daniel
Tristán-Agundis Ma. Francisca

432 The US Payor Landscape: Results from a Survey of Medical Directors and Pharmacy Directors
on Comparative-Effectiveness Research
Richard A. Brook, Jeff A. Carlisle, Stanton R. Mehr and James E. Smeeding
Journal of Pharmacy and Pharmacology 2 (2014) 377-392
D DAVID PUBLISHING

Plant Secondary Metabolites: Biosynthesis,

Classification, Function and Pharmacological Properties

Justin N. Kabera1, Edmond Semana1, Ally R. Mussa1 and Xin He1, 2

1. Department of Pharmacology, School of Chinese Materia Medica, Tianjin University of Traditional Chinese Medicine, Tianjin
300193, China
2. Tianjin State Key Laboratory of Modern Chinese Medicine, Tianjin 300193, China

Abstract: Secondary metabolites, also known as phytochemicals, natural products or plant constituents are responsible for medicinal
properties of plants to which they belong. The role they play in the plant is not, to date, well known or understood, but it may be beyond
the protection. Their classification is based on chemical structure, composition, their solubility in various solvents, or the pathway by
which they are synthesized. The main classification system includes three major groups: terpenoids, alkaloids and phenolics. For each
one, we find subclasses with complexity in structure. In this review, we deal with the description of second metabolites, their
biosynthesis, function, and the current pharmacological findings. Natural products are an important source of drug candidates in
pharmaceutical industry, more deeply we understand them, the easier it is for scientists to intervene in alleviating different kind of
diseases. The recent references have been consulted for presenting updated information, but also showing the new potentialities of plant
second metabolites in drug research and development.

Key words: Secondary metabolites, biosynthesis, terpenoids, alkaloids, phenolics, pharmacological activities.

1. Introduction extremely diverse; thousands of them have been

identified in several classes. Each plant family, genus,
Humans have used medicinal plants throughout their
and species produce a characteristic mix of these
history life, and long time before, good records were
chemicals, and they can sometimes be used as
kept about herbs use [1]. Texts from ancient China and
taxonomic characters in classifying plants [8].
India provide a good example of very early use of
Many scientific sources state that their role is not
medicinal herbs. They contain prescriptions of
crucial for living cells in normal growth, development,
countless plant-derived medicines [2, 3]. In modern
and reproduction [9], but they act in defense purposes
times, natural products from plants have been isolated
to protect a plant from any possible harm in the
for drug discovery and development. During the last 20
ecological environment [10] and other interspecies
to 30 years, the analysis of secondary plant products
protection [11]. Therefore, they are usually synthesized
has progressed a lot [4]. The use of modern analytical
in plants for particular needs, while the primary
techniques like chromatography, electrophoresis,
metabolites have generally the shared biological
isotope techniques and enzymology have succeeded in
purposes across all species. Secondary metabolites
the elucidation of exact structural formulas and the
may often be created by modified synthetic pathways
most important biosynthetic pathways [5, 6].
from primary metabolite, or share substrates of primary
The secondary metabolites from plants, which are
metabolite origin. Plants have been evolving to adapt
distinguished from primary metabolites such as nucleic
the environment with genetic encoding of useful and
acids, amino acids, carbohydrate, fat, etc. [7] are
diverse synthases for secondary metabolites [12]. In
Corresponding author: Xin He, Ph.D., professor, research human life, these compounds are used as medicines,
field: pharmacology (pharmacokinetic and pharmacodynamic).
flavorings, or relaxing drugs, especially essential oils.
E-mail: hexintn@163.com.
378 Plant Secondary Metabolites: Biosynthesis, Classification, Function and Pharmacological Properties
In most references, it is stated that the secondary
metabolites extracted from plants are subdivided in
three major classes: terpenoids, alkaloids and phenolics.
They contain numerous natural products with
interesting pharmacology activities [13, 14].
In this review, we highlight the secondary
metabolites classified in three main groups mentioned
above, showing pharmacological activities leading to
drug discovery and development. Plant constituent
should be regarded as a complex mixture of several
unwanted chemicals, refined of the objective of
identifying and isolating an active principle. In order to
study the activity of a given medicinal plant, it is often
necessary to purify it or to isolate a specific compound [15].
However, some drug resistance cases observed in
infectious diseases or cancer, the approach of synergy
therapy is applied to overcome the challenge and
improve the treatment [16].
2. Biosynthesis
The pathways of biosynthesis are responsible for the
occurrence of both primary and secondary metabolites
[17, 18]. Biosynthetic reactions are energy consuming,
fuelled by the energy released by glycolysis of
carbohydrates and through the citric acid cycle.
Oxidation of glucose, fatty acids and amino acids
results in ATP (adenosene triphosaphate) formation,
which is a high-energy molecule formed by catabolism
of primary compounds. ATP is recycled in fuel
anabolic reactions involving intermediate molecules on
the pathways. Whereas, catabolism involves oxidation
of starting molecules, biosynthesis or anabolism
involves reduction reaction. Hence, the need of
reducing agent or hydrogen donor, which is usually the
NADP (nicotinamide adenine dunucleotide phosphate).
These catalysts are known as coenzymes and the most
widely occurring is CoA (coenzyme A) made up of
ADP (adenosine diphosphate) and pantetheine
phosphate [19].
The most common pathways taken for biosynthesis
are performed through the pentose for glycosides,
polysaccharides; shikimic acid for phenols, tannins,
aromatic alkaloids; acetate-malonate for phenols and
alkaloids and mevalonic acid for terpenes, steroids and
alkaloids [20]. As showed in the Fig. 1, the scheme
outlines how metabolites from the process of
photosynthesis, glycolysis and Krebs cycle are tapped
off from energy-generating process to provide
biosynthetic intermediates. By far, the important
building blocks employed in the biosynthesis of
secondary metabolites are derived from acetyl-CoA
(acetyl coenzyme A), shikimic acid, mevalonic acid
and 1-deoxylulose 5-phosphate [21].
3. Structure and Classification
As said previously, the classification of secondary
metabolites consists of terpenoids, alkaloids and
phenolics [13]. Glycosides, tannins and saponins are
part of them according their specific structure.
3.1 Terpenoids
Terpenoids constitute a large family of
phytoconstituents of little functional and structural
common ground. Steroids, carotenoids, and gibberelic
acid are just some of its members. They are composed
by the most important group of active compounds in
plants with over than 23,000 known structures. They
are polymeric isoprene derivatives and synthesized
from acetate via the mevalonic acid pathway. During
their formation, the isoprene units are linked in head
and tail fashion. The number of units incorporated into
a particular terpene serves as a basis for their
classification. Many of them have pharmacological
activity and are used for diseases treatment both in
humans and animals. According to Tada et al. cited by
Ref. [22], diterpenes tend to be most abundant in
Lamiaceae family and have antimicrobial and antiviral
properties.
Some interesting compounds are extensively used in
the industry sector as flavors, fragrance, spices [23, 24].
Several thousand different types of molecules
from very different plant groups have been isolated and
 Plant Secondary Metabolites: Biosynthesis, Classification, Function and Pharmacological Properties
Fig. 1 Biosynthesis scheme of plants secondary metabolites [21].
characterized. Despite their varied structures, all of
them are synthesized by only a few pathways as shown
below in Fig. 2.
3.1.1 Functions
Plants tissues are related to adaptation to both abiotic
and biotic stressors such herbivores and pathogens.
However, the high volatility and reactivity of some
terponoids may also affect the atmosphere composition [25].
Volatility of terpenoids provides, for sessile plants, a
tool for communication with other organisms such as
379
neighboring plants, pollinators and foes of herbivores,
via air-bone infochemicals. From a physiological
standpoint, briefly, plant volatiles are involved in three
critical processes, namely plant—plant interaction, the
signaling between symbiotic organisms, and the
attraction of pollinating insects. Their role in these
“housekeeping” activities underlies agricultural
applications that range from the search for sustainable
methods for pest control to the production of flavors
and fragrances [26].
380 Plant Secondary
S M
Metabolites: B
Biosynthesis, Classificatio
on, Function and
a Pharmac
cological Properties

Fig. 2 Exam
mple of sterol syynthesis (sourcce: weekpedia,, February 13, 2014).

3.1.2 Pharrmacologicall Activities esseential oils haas revived inn recent decaades with thee
Extensivee biological investigatioons have been b poppularity of arromatherapy,, a branch of o alternativee
carried out within the group
g and thhese studies have
h med dicine that claims
c that essential oills and otherr
revealed a broad spectrrum of pharrmacological and arommatic compoounds have cuurative effectts. Accordingg
physiologicaal properties. Some of theem have led to a several findings of research, they are rep puted to havee
number of teerpenoids gainning medicinall applications [26]. variious pharmaccological effeects such as antibacterial,,
The recent findings demonstrate that cerrtain antiifungal and anntiviral [29].
nitrogenous terpene derrivatives posssess the pootent They
T are knnown to bee characterizzed by bothh
anti-hypertensive activityy and may inndicate a new w era beaautifully and powerfully
p fraagrant. Thus, they providee
in medicine through the synthetic
s terppenoids path. The prottection to thee plants against predatorss and diseasee
antimicrobiaal and inseccticidal propperties of other o andd play a role inn plant pollinnation [30]. Although
A theyy
terpenoids have
h led to theeir utilization as pesticidess and are fat soluble, they
t do not innclude fatty liipids or acidss
fungicides inn agriculture and horticultture [27, 28]. founnd in vegeetables and animal oilss. Single orr
commbined extraact stimulatte the olfacctory nerve,,
3.2 Essentiaal oils
sending messagees to the brainn’s limbic sysstem (the seatt
Essential oils are naatural aromaatic and vollatile of memory,
m leaarning, and eemotion) thatt are said too
compounds found in the parts of plants. Interesst in trig
gger physiollogical respoonses (e.g., eucalyptuss
 Plant Secondary Metabolites: Biosynthesis, Classification, Function and Pharmacological Properties 381

relieves congestion, lavender promotes relaxation, and
menthe piperita promote exercise performance) [31, 32].
The use of oils and their solutions have been shown to
have certain effects but are not standardized. The few
risks involved include allergic reactions [33, 34].
3.3 Alkaloids
The alkaloids present the group of secondary
metabolites that contain basic nitrogen atoms. Some
related compounds with neutral and weakly acid
properties are also included in the alkaloids. In addition
to carbone, hydrogen and nitrogen, this group may also
contain oxygen, sulfur and rarely other element such as
chlorine, bromine and phosphorus [18]. Alkaloids are
produced by a large variety of organisms, such as
bacteria, fungi, animals but mostly by plants as
secondary metabolites. Most of them are toxic to other
organisms and can be extracted by acid-base. They
have diverse pharmacological effects [48], and have a
long history in medication [49].
The boundary between alkaloids and other
nitrogen-containing natural compounds is not
clear-cut [21]. Compounds like amino acids, proteins,
peptides, nucleotides, nucleic acid, and amines are not
usually called alkaloids.
Compared with most other classes of secondary
metabolites, alkaloids are characterized by a great
structural diversity and there is no uniform classification
of them [13]. First classification was based on the
common source because no information about chemical
structure was yet available. Recent classification is
based on similarity of the carbon skeleton [14].
Alkaloids are biosynthesized from amino acids such as
tyrosine [50]. The typical example is the biosynthesis of
morphine that includes a phenol coupling reaction
involving a benzylisoquinoline alkaloid.

Table 1 Important molecules of terpenoids [35].

Number of carbone Name Example
C5 Hemiterpene Isoprene, prenol, isovaleric acid
C10 Monoterpene Limonene, eucalyptol, pinene
C15 Sesquiterpene ABA (abscisic acid)
C20 Diterpene Gibberellin
C25 Sesterterpenes
C30 Triterpene Brassinosteroids, squalen, lanosterol
C40 Tetraterpene Carotenoids, lycopen
C> 40 Polyterpenes Ubuquinones, rubber, cytokonines, vitamine E

Table 2 Summary of some terpenoids properties.

Name of terpenoid Location Pharmacological activity References
Isoprenol Synthesis Act as alcohol [36]
Anticonvulsant agent in valeria, used largely in
Isovaleric acid Essential oils [37, 38]
perfumery
Fragrant, botanical insecticide, anti
Limonene Essential oils [39, 40]
carcinogenic, anti bacterial
Antibacterial, exert effect on CNS (centre
Linalool Essential oils [41, 42]
nervous system)
ABA Anti transpirant [43]
Synthesized plants at areas of Anti oxidative effects, antimicrobial, anti tumor
Phytoalexins
pathogen infection and neurotrophic effects
Gibberlin Gibberella fujikuroi Promoting growth and elongation of plant cells [44, 45]
Brassinosteroids Lychnis viscaria, Brassica napus Involved in plant protection [46, 47]
sterols Natural in plants, animals and fungi Nutritional supplements and medicinal
Carrots, chloroplasts and Antioxidants, sunlight protection, immune
Carotenoids
chromoplasts of plants function
382 Plant Secondary Metabolites: Biosynthesis, Classification, Function and Pharmacological Properties

Fig. 3 The Biosynthesis of morphine [51, 52].

Table 3 Pharmacological effects of some well known alkaloids.
Alkaloid name Source Pharmacological activity References
Atropa belladonna, Darura stramonium, Competitive antagonist of muscarinic acetylcholine
Atropine [53, 54]
Mandragora officinarum receptors, anti cholinergic, anti myopia effects
Berberis species, Hydrastis, Canadensis,
Anti inflammatory, anti bacterial/viral, recently
Xanthorhiza simplicissima, Phellodendron
experiments showed anti diabetic and beneficial
Berberine amurense, Coptis chinensis, Tinospora [55-58]
effects on cardiovascular system and anti cancer and
cordifolia, Argemone mexicana and
others disorders such as intestinal
Eschscholzia californica
Analgesic, antitussive, anti diarrheal, antidepressant,
Codeine Papaver somniferum [59-62]
sedative and hypnotic properties
Coniine Conium macularum, Sarracenia flave Neurotoxin, poisonous [63, 64]
Cytisine
Labum and cytisus of Fabaceae family, most
(baptitoxine, Acetylcholine agonist, smoking cessation drug [65, 66]
extracted from seeds of Cytisus laborinum
sophorine)
Act on CNS (central nervous system), on myenteric
Morphine Papaver somniferum and poppy derivatives plexus, acute pulmonary edema and reduce the [67-69]
shortness of breath
Stimulant, antiherbivore, insectide, anti
Nicotine Solanaceae plants family [70-73]
inflammatory
Cinchona succirubra, C. calisya, c. ledgeriana, Antimalarial, antpyretic, analgesic,
Quinine [74-77]
plants of Rubiaceae family. anti-inflammatory, antiarrhythmic, bacteriostatic
Solanum tuberosum, s. lycopersiam, s. igrum, Antifungal, antipesticide, sedative, anticonvulsant,
Solanine [78-81]
plants of Solanaceae family anticarcinogenic, anti inflammatory
Strychnos nux-vomica, loganiaceae plants
Strychnine Persticide, strong poisonous, Convulsant [82-85]
family
Thebaine
Papaver bracteatum Analgesic, not therapeutically used. [86-89]
(paramorphine)
Tomatine Green parts of tomato plants Immune effects, anticancer, antifungal, poisonous [90-92]
 Plant Secondary Metabolites: Biosynthesis, Classification, Function and Pharmacological Properties
3.4 Phenolics
Phenolic compounds from plants are one of largest
group of secondary plants constituents synthesized by
fruits, vegetables, teas, cocoa and other plants that
possess certain health benefits. They are characterized
by the antioxidant, anti-inflammatory,
anti-carcinogenic and other biological properties, and
may protect from oxidative stress and some diseases
[93]. Simple phenolics are bactericidal, antiseptic and
anthlemintic. Phenol itself is a standard for other
antimicrobial agents [94]. They are distributed in
almost all plants and subject to a great number of
chemical, biological, agricultural, and medical
studies [95, 96].
They are diverse in structure, and present in common
the hydroxylated aromatic rings (e.g., flavan-3-ols).
Most of phenolic compounds are polymerized into
larger molecules such as the PA (proanthocyanidins;
condensed tannins) and lignans. Furthermore, phenolic
acids may occur in food plants as esters or glycosides
conjugated with other natural compounds such as
flavonoids, alcohols, hydroxyfatty acids, sterols, and
glucosides [95]. Hydroxybenzoic and
hydroxycinnamic acids present two main phenolic
compounds found in plants. In tea, coffee, berries and
fruits, the total phenolic comounds could reach up to
103 mg/100 g fresh weigh [97].
3.3.1 Classification and Structure
The approach to classifying plant phenolics are
based on: (1) a number of hydroxylic groups. So, they
may be divided on 1-, 2- and polyatomic phenols.
OH
Phenol
Fig. 4 Examples of simple phenolics, C6 (phenol, hydroquinone and pyrogallol acid).
383
Phenolic compounds containing more than one
OH-group in aromatic ring are polyphenols; (2)
chemical composition: mono-, di, oligo- and
polyphenols; (3) substitutes in carbon skeleton, a
number of aromatic rings and carbon atoms in the side
chain. According to the latter principle, phenolic
compounds are divided into four main groups:
phenolics with one aromatic ring, with two aromatic
rings, quinones and polymers.
Phenolic compounds with one aromatic ring: a large
number of compounds, among them are simple phenols
(C6), phenol with attached one (C6-C1), two (C6-C2)
and three (C6-C3) carbon atoms.
Phenolic compounds with two aromatic rings: this
group includes benzoquinones and xanthones (C6-C1-C6)
containing two aromatic rings which are linked by one
carbon atom; stylbenes (C6-C2-C6) which are linked by
two carbon atoms; and flavonoids, containing three
carbon atoms (C6-C3-C6). Flavonoids, depending on the
structure of propane unit and an attaching place of side
chain B, are divided into flavonoids in strict sense,
which are derived from chromane or chromone,
isoflavonoids and neoflavonoids.
Polyphenolics are more than 8,000 different
compounds identified to date. That is why the
terminology and classification of polyphenols is
complex and confusing. Although all polyphenols have
similar chemical structures, there are some distinctive
differences. Based on these differences, polyphenols
can be subdivided into two classes: flavoinoids and non
flavonoids, like tannins [98].
OH OH
HO OH
OH
Pyrogallol acid
Hydroquinone
384 Plant Secondary Metabolites: Biosynthesis, Classification, Function and Pharmacological Properties

OH O OH
HO OH
C
OH

OH COOH

Salycilic acid Gallic acid

Fig. 5 Examples of C6-C1 phenolics: gallic acid and salicylic acid.

O
C CH3
COCH3

R R

O CH3
R
OH
R
Acetophenones Apocynin
Fig. 6 Examples of C6-C2: acetophenones, apocynin.

HC COOH
HO COOH
R
COOH
HO
R
R Caffeic acid HO
Hyroxycinnamic acids
P-coumaric acid
Fig. 7 Phenolics with C6-C3 (phenylpropanoids): hydroxycinnamic acid, ferulic acid and sinapic acid.

R
R O R R R R R

R CH C R
H
R
R O R Stylbenes (C6-C2-C6)
Xanthones( C6-C1-C6)
R
O B
R R
A C
R R
R R Flavonoids (C6-C3-C6)
Fig. 8 Some phenolics with two aromatic rings (xanthones: C6-C1-C6; stylbenes: C6-C2-C6 and flavonoids: C6-C3-C6).
 Plant Secondary
S M
Metabolites: B
Biosynthesis, Classificatio
on, Function and
a Pharmac
cological Properties 3855

O O R O R
R
R R

R
R
O R O O
na
aphtaquinones(C10) antra
aquinones((C14)
benzoquinone
es(C6)
Fig. 9 Main n groups of qu
uinines: benzooquinones, nap
phtoquinones, and anthraqu
uinones polyph
henolics (flavo
onoids, tanins,,
glycosides, saaponins).

O
O O
O

Struc
cture of the flavone
f
Isofla
avone struc
cture
Neo
oflavonoids structure
Fig. 10 Basiic structures off flavonoids.

Fig. 11 Tannic acid, type of

o tannins.

bluee pigmentattion in petaals designed d to attractt

3.4 Flavonoids
Flavonoidds stand as thhe first classs of polyphennols.
They are waater-soluble pigments founnd in the vacuuoles
of plant cellls. They cann also be diivided into three
t alsoo act as chemical
groups: anthhocyanins, flaavones and flaavonols. Theyy are
widely distrributed in plaants, fulfillingg many functtions
such as flow wer coloratioon, producingg yellow, redd or
polllinator animaals. In higheer plants, flaavonoids aree
invo olved in UVU (ultraviollet) filtration n, symbioticc
nitrrogen fixationn and floral pigmentation n. They mayy
c messengers, physiological
p l
regu ulators, and cell cycle innhibitors. Flaavonoids aree
secrreted by the root
r of their hhost plant Rhiizobia help inn
the infection stage of their syymbiotic relattionship withh
386 Plant Secondary Metabolites: Biosynthesis, Classification, Function and Pharmacological Properties
legumes like peas, beans, clover, and soy [99].
Rhizobia living in soil are able to sense the flavonoids
and this triggers the secretion of nod factors, which in
turn are recognized by the host plant and can lead to
root hair deformation and several cellular responses
such as ion fluxes and the formation of a root nodule.
Some flavonoids have inhibitory activity against
organisms that cause plant disease, for example,
Fusarium oxysporum [100].
They have become very popular because their health
benefits. Some of the activities attributed to them
include: anti-allergic, anti-cancer, antioxidant,
anti-inflammatory and anti-viral [101, 102]. The
flavonoids quercetin is known for its ability to relieve
high fever, eczema, asthma and sinusitis.
Epidemiological studies have illustrated that heart
diseases are inversely related to flavonoid intake.
Studies have shown that flavonoids prevent the
oxidation of low-density lipoprotein thereby reducing
the risk for the development of atherosclerosis [103,
104]. The contribution of flavonoids to the total
antioxidant activity of components in food can be very
high; for instance red wine contains high levels of
flavonoids, mainly quercetin and rutin. The high intake
of it by the French might explain why they suffer less
from coronary heart disease than other Europeans,
although their consumption of cholesterol rich foods is
higher (French paradox) [105]. Many studies have
confirmed that one or two glasses of red wine daily can
protect against heart disease [106]. Their antioxidant
activities in chemical and biological assays are
undisputed, and many are associated with the
health-promoting effects of fruits and vegetables.
However, extending these effects to entire organisms,
and clinical outcomes in human disease in particular,
remains a controversially discussed topic in nutrition
science and disease prevention [107]. Recently, it has
been mentioned that growing consensus for the
hypothesis that the specific intake of food and drink
containing relatively high concentrations of flavonoids
may play a meaningful role in reducing the risk of
CVD (cardiovascular disease). The reviewers stated
that research to date has been of poor quality. The large
and rigorous trials are needed to better investigate the
possible adverse effects associated with excessive
polyphenol intake. Currently, a lack of knowledge
about safety suggests that polyphenol levels should not
exceed that which occurs in a normal diet [108].
3.5 Tannins
Tannin is the name derived from French “Tanin”
(tanning substance) and used for a range of natural
polyphenols. The tannins are the phenolic compounds
that precipitate proteins. They are composed by a very
diverse group of oligomers and polymers. They can
form the complex with proteins, starch, cellulose and
minerals. They are synthesized via shikimic acid
pathway, also known as the phenylpropanoid pathway.
The same pathway leads to the formation of other
phenolics such as isoflavones, coumarins, lignins and
aromatic aminoacids. Tannins are water soluble
compounds with exception of some high molecular
weight structures. They are usually subdivided in two
groups: HT (hydrolysable tannins) that include
gallotannins, elligatannins, complex tannins, and PA,
also known as condensed tannins [16].
The tannins also constitute the active principles of
plant-based medicines. According the literature, the
tannins containing plants are used as astringents
against diarrhea [109], adiuretic against stomach and
duodenal tumors [110], and anti-inflammatory [111].
3.6 Glycosides
Glycosides may be phenol, alcohol or sulfur
compounds. They are characterized by a sugar portion
or moiety attached by a special bond to one or
non-sugar portions. Many plants store chemicals in the
form of inactive glycosides, which can be activated by
enzyme hydrolysis [112]. For this reason, most
glycosides can be classified as prodrugs since they
remain inactive until they are hydrolyzed in the large
bowel leading to the release of the aglycone, the right
 Plant Secondary
S M
Metabolites: B
Biosynthesis, Classificatio
on, Function and
a Pharmac
cological Properties 3877

effeect on membrranes and skinn [119].

H OH They
T are largeely distributedd in plant kin
ngdom, whichh
hav
ve many physsicochemical (foaming, em mulsification,,
H O
soluubilization, swweetness andd bitterness) an nd biologicall
HO
H O O
OH prop perties (haeemolytic, anntimicrobial, antioxidant,,
H OH molluscacide, inssecticide and ichthyocide), exploited inn
OH OH man ny applications in food, ccosmetics, ph harmaceuticall
Fig. 12 Saliccin, a glycosidee type related to
t aspirin. induustries and soil bioremediation. Among thee
saponins properties, CMC C (critical mi cellarr
con
ncentration), maximum surface density d andd
agg
gregation num mber (numberr of monomerrs in a micellee)
are of great impportance for application as a surfactantss
and
d foaming ageents. These arre influenced d by variabless
such h as temperatture, salt concentration, aq queous phasee
pH,, solvent conccentration and type, such as ethanol orr
metthanol.

Fig. 13 Type of saponin: solanine

s chemiical structure. 4. Conclusion
C ns
active constiituent. In
nvestigating the secondaryy metabolitess remains thee
The classsification of glycosides is based on the larg
gest area of reesearch wherre a lot of wo ork related too
nature of agllycone, whichh can be any of
o a wide rangge of theiir functions, pharmacologgical propertties must bee
molecular tyypes includinng phenols, quinines,
q terpenes andd will still be carried out. Skills,, knowledgee
and steroidss. They are heterogeneoous in structture, emb bedded in thhe natural sciences such h as botany,,
therefore, thhey are not eaasy to learn as
a specific grroup cheemistry or biochemistry
b y and pharm macology iss
and are deescribed herre in this review for the requuired. The knowledgee of indiv vidual classs
conveniencee. Glycosiddic bonds are of great g connstituents is essential foor developin ng assurancee
significance, since they link monosacccarides togeether quaality methods, extraction pprocedures, understanding
u g
to form oliggosaccharidees and polysaaccharides [113, of pharmacologgical activityy, pharmacokinetics andd
114]. Conceerning the thherapeutic acttions in diffeerent mosst importantlyy the potentiaal toxicity andd interactionss
studies, it has been shown that glycosides are withh pharmaceuttical drugs. A As this review
w summarizedd
anticancer [115], expecctorant [1166], sedative and the complexity of second m metabolites, eaach group orr
digestives prroperties [1177, 118]. classs, may have a specific stuudy for moree informationn
in order
o to preeview the peerspectives in n new drugss
3.7 Saponinss
reseearch and devvelopment. T Therefore, forr some, suchh
Saponins are compounnds whose active portions formf as glycosides, they seem tto present difficulties
d inn
colloidal sollutions in waater, which produce
p latheer on learrning them specifically
s bbecause of heterogeneity
h y
shaking andd precipitate cholesterol. They occuur as andd complexityy in their strructure. In addition,
a thee
glycosides whose
w aglycone is tripennoid or sterooidal functions of secoond metaboliites on plantss are not welll
structures. The
T combinattion of lipophhilic sugars att the undderstood, but at least peopple such as scientists
s andd
end gives thhem the abillity to lower surface tenssion, herbbalists still apppreciate how
w much they contribute too
producing the
t detergennt characterisstic or soap--like relief and heal huuman and animal ailmentss.
388 Plant Secondary Metabolites: Biosynthesis, Classification, Function and Pharmacological Properties
Acknowledgments
This work was supported by the National Natural
Science Foundation of China (81373890); the Research
Fund for the Doctoral Program of Higher Education
(20121210110011), and the Natural Science
Foundation of Tianjin (No. 12JCZDJC26100).
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Journal of Pharmacy and Pharmacology 2 (2014) 393-403
D DAVID PUBLISHING

Evaluation of the Anticonvulsant Activity of the Leaf

Methanol Extract of Crassula arborescens (Mill.) Willd.
(Crassulaceae) in Mice

George Jimboyeka Amabeoku, Oluchi Nneka Mbamalu, Tasneem Davids, Samukelisiwe Fakude, Anda Gqwaka,
Fiona Mbai, Reighman Pieterse and Aneesa Shaik
Discipline of Pharmacology, University of the Western Cape, Cape Town 7535, Western Cape, South Africa

Abstract: Crassula arborescens (Mill.) Willd. subsp. Arborescens is widely used for the treatment of various ailments including
diarrhoea, corns, epilepsy and as a purgative. However, no information exists in any literature to verify the acclaimed effectiveness of
C. arborescens in the treatment of the various ailments. The study, therefore, intended to investigate the anticonvulsant activity of the
leaf methanol extract of C. arborescens in mice. Acute toxicity study and phytochemical qualitative analysis of the plant extracts
were also carried out. Chemically-induced convulsion methods were used to assess the anticonvulsant activity of C. arborescens.
Standard methods were used for the acute toxicity study and phytochemical analysis of the chemical components of the plant extract.
PTZ (pentylenetetrazole), bicuculline, picrotoxin, NMDLA (N-methyl-DL-aspartic acid) or strychnine produced tonic convulsions in
all the mice used. Leaf methanol extract of Crassula arborescens, muscimol, phenobarbitone or diazepam significantly antagonised
PTZ, bicuculline or picrotoxin-induced convulsion. C. arborescens or LY233053 significantly antagonised NMDLA-induced tonic
convulsion. C. arborescens or phenobarbitone significantly antagonised strychnine-elicited tonic convulsion. Phenytoin or DMSO
(dimethylsulfoxide) did not significantly affect the tonic convulsion produced by PTZ, bicuculline, picrotoxin, NMDLA or
strychnine. The LD50 value obtained from intraperitoneal administration of C. arborescens was 781.6 mg/kg while that following oral
administration of the plant extract was over 4,000 mg/kg. The phytochemical qualitative analysis done showed the presence of
flavonoids, tannins, reducing sugar, saponins and triterpene steroids. The data obtained in the study show that the leaf methanol
extract of Crassula arborescens has anticonvulsant activity which may be underpinned by GABAergic, glutaminergic and
glycinergic mechanisms. The high LD50 value obtained following the oral administration of the plant extract shows that the leaf
methanol extract is non-toxic to animals.

Key words: Crassula arborescens, Crassulaceae, anticonvulsant activity, GABAergic, glutamatergic and glycinergic mechanisms,
mice.

1. Introduction wood. The plant grows up to a height of between 1.2

m to 4 m. The leaves are round to broadly ovate and
The use of medicinal plants in South Africa in the
are approximately 3 × 3 cm in size. They are thick and
treatment and/or management of diseases is an age
fleshy with a grey waxy bloom. The apex is round,
long tradition. Crassula arborescens (Mill.) Willd.
obscure and sharp, and the margins are reddish
subsp. Arborescens (Crassulaceae) is one of the
rimmed. The flowers are whitish-pink in colour [1].
medicinal plants used in the country to treat certain
Crassula arborescens is indigenous to South Africa
ailments. The plant species belongs to the family of
and Western Cape Province in particular. It is locally
Crassulaceae. It is a succulent plant with a soft bark,
known in Afrikaans as “beestebal”. It is distributed in
which is smooth and grey-green in colour, and soft
the Western Cape Province from the Little Karroo to
the Hex River Valley. It is also found in KwaZulu
Corresponding author: George Jimboyeka Amabeoku,
Ph.D., professor, research fields: pharmacology and Natal and Swaziland where it is known as
ethnopharmacology. E-mail: gamabeoku@uwc.ac.za.
394 Evaluation of the Anticonvulsant Activity of the Leaf Methanol Extract of Crassula arborescens (Mill.)
Willd. (Crassulaceae) in Mice
“umchobozovithi” [1]. Infusion of the leaves has been
used in the cape to treat epilepsy and corns [1, 2].
Despite the claim of therapeutic success of the plant
species by traditional medicine practitioners in the
treatment and management of epilepsy, no scientific
evidence has been found in any literature to verify the
claim. Aim of the present study was to evaluate the
anticonvulsant activity of Crassula arborescens in
vivo, and analyse the chemical composition by
colorimetric assays and HPLC analysis. Acute toxicity
study of the plant species was also carried out.
2. Materials and Methods
2.1 Plant Material
Fresh leaves of Crassula arborescens were
collected from Kirstenbosch National Botanical
Gardens, Cape Town, South Africa. They were
authenticated by Mr F. Weitz, a taxonomist in the
Department of Biodiversity and Conservation Biology,
UWC (University of the Western Cape). A voucher
specimen (CR 22) was then deposited in the
Herbarium at the University of the Western Cape.
2.2 Preparation of the Methanol Extract and Stock
Solutions
Fresh leaves were separated from the branches,
weighed (1 kg), and washed with distilled water. After,
the leaves were air dried for an hour and dried in an
oven at 35 °C for 2 days. Leaves dried were ground
using a commercial blender affording a fine powder
(442.5 g), and was extracted (60 g) in a Soxhlet
extractor with 500 mL of methanol for 5 h. The filtrate
was evaporated to dryness using a Buchi RE11
rotavapor and Buchi 461 water bath. A yield of 18.2 g
crude methanol extract of C. arborescens was
obtained. This was preserved in a refrigerator for
further use. Fresh solution of the methanol extract was
prepared on each day of the experiment by dissolving
a weighed quantity of the methanol extract in a small
volume of DMSO (dimethylsulfoxide) and made up to
the appropriate volume with physiological saline. The
solutions were administered intraperitoneally (i.p.) to
mice in a volume of 1 mL/100 g of body weight of
animals.
2.3 Animals
Male albino mice bred in the Animal House of the
Discipline of Pharmacology, School of Pharmacy,
University of the Western Cape, South Africa were
used throughout the experiments. Animals weighing
between 18-30 g were used in groups of eight per dose
of plant extract or drug. They had access to food and
water ad libitum. All animals were fasted for 16 h
during which they had access to water before the
experiments commenced. A 12 h light/12 h dark cycle
was maintained during the experiment. An ambient
room temperature of 22 ± 1 °C was also maintained.
All animals were used for one experiment only.
2.4 Drugs and Chemicals
PTZ (pentylenetetrazole, Sigma Chemical Co.),
picrotoxin (Sigma Chemical Co.), NMDLA
(N-methyl-DL-aspartic acid, Sigma Chemical Co.),
strychnine (Sigma Chemical Co.), phenobarbitone
sodium (BDH Chemicals Ltd), 5,5 diphenylhydantoin
sodium salt (phenytoin, Sigma Chemical Co.),
muscimol (Sigma Chemical Co.) and LY233053
(Sigma Chemical Co.) were all dissolved in
physiological saline to appropriate volumes. (+)
Bicuculline (Sigma Chemical Co.) was suspended in a
small volume of Tween 80 and adjusted to the
appropriate volume with physiological saline.
Diazepam (Valium, Roche, South Africa) was
dissolved in a minimum amount of propylene glycol
and made up to the appropriate volume with
physiological saline. DMSO (dimethylsulfoxide,
Sigma Chemical Co.) solution was prepared by
dissolving equal volume, used to dilute the plant
extract, in an appropriate volume of physiological
saline. All drugs were injected intraperitoneally (i.p.)
in a volume of 1 mL/100 g of animal. Control animals
received equal volume injections of the appropriate
 Evaluation of the Anticonvulsant Activity of the Leaf Methanol Extract of Crassula arborescens (Mill.)
Willd. (Crassulaceae) in Mice
vehicles which include physiological saline and
DMSO. Fresh plant extract and drug solutions were
prepared on each day of the experiment. The doses
and pre-treatment times of the leaf methanol extract of
C. arborescens and the standard antiepileptic drugs
used were obtained from preliminary studies in our
laboratory. The pre-treatment times following the
administration of PTZ, bicuculline, picrotoxin,
NMDLA or strychnine were 15 min (plant extract), 10
min (phenobarbitone), 20 min (diazepam), 20 min
(phenytoin), 30 min (LY233053), 1 h (muscimol) and
15 min (DMSO solution).
2.5 Phytochemical Qualitative Analysis
The methods of Ikhiri et al. [3] and Harborne [4]
were used for the phytochemical qualitative analysis
of the dried powdered leaf of C. arborescens for
various chemical compounds (Table 1).
2.6 Assessment of Anticonvulsant Activity
Modified method of Vellucci and Webster [5] by
Amabeoku and Chikuni [6] was used to assess the
anticonvulsant effect of the leaf methanol extract of
Crassula arborescens. Each mouse was housed in a
transparent perspex mouse cage 30 min before the
commencement of the experiment to acclimatize to
their new environment. PTZ (95 mg/kg), bicuculline
(40 mg/kg), picrotoxin (15 mg/kg), NMDLA (400
mg/kg) and strychnine (2 mg/kg), all standard
convulsant drugs, were each administered
intraperitoneally to induce tonic administration of
each convulsant drug for tonic convulsions which
manifested as tonic hind-limb extensions. The animals
were observed for 30 min following the administration
of each convulsant drug for tonic convulsions which
manifested as tonic hind-limb extensions. The time of
the onset of tonic convulsions and the number of
animals convulsing or not convulsing were obtained
during the 30 min period of observation. Experiments
were repeated with other groups of animals pre-treated
with either the leaf methanol extract of the plant
species (25-200 mg/kg, i.p.), phenobarbitone (12
395
mg/kg, i.p.), diazepam (0.5 mg/kg, i.p.), phenytoin (30
mg/kg, i.p.), muscimol (2 mg/kg, i.p), LY233053 (5
mg/kg, i.p.) or DMSO (0.25 mL, i.p.) before the
administration of any of the convulsant agents. The
ability of the plant extract to prevent or delay the onset
of the tonic hind limb extensions was taken as an
indication of anticonvulsant activity [6]. Animals that
did not convulse during the 30 min period of
observation were considered as not having convulsed.
2.7 HPLC Analysis
The leaf methanol extract of Crassula arborescens
was analysed using HPLC in order to characterise the
plant species. Chromatographic system: Agilent 1200
system consisting of degassing system, quaternary
pump, auto loading sampler, thermostatted column
compartment, diode array detector, fluorescence
detector, analyte fraction collector and Agilent
ChemStation software; column: Phenomenex Luna
(C18) 5 μm and dimensions (250 cm × 4.6 mm).
Chromatographic conditions: mobile phase degassed
with helium; solvent: water containing 0.01% formic
acid; solvent B: acetonitrile containing 0.01% formic
acid; mode: flow rate, 0.8 mL/min; injection volume:
50 μL, detector, UV at 370 nm. The analysis was
monitored at several wavelengths ranging from 210
nm to 370 nm, the specific wavelength of interest
being 350 nm. The HPLC operating conditions were
programmed to give the following: 0 min, solvent B:
18%; 15 min, solvent B: 25%; 20 min, solvent B: 35%;
30 min, solvent B: 90%. The run rate was 30 min.
Table 1 Phytochemical screening of dry powder of C.
arborescens leaf.
Compounds Tests
Saponins Frothing test
Tannins Ferric chloride test
Reducing sugar Fehling’s test
Flavonoids Shinoda’s test
Triterpene steroids Salkowski test
Alkaloids Dragendorff’s test
Cardiac glycosides Kedde’s test
Quinones Hydrochloric acid/ether test
396 Evaluation of the Anticonvulsant Activity of the Leaf Methanol Extract of Crassula arborescens (Mill.)
Willd. (Crassulaceae) in Mice
2.8 Acute Toxicity Study
The method described by Lorke [7] and modified
by Ojewole [8] and that of Hilaly et al. [9] were used
to determine the median lethal dose (LD50) of the leaf
methanol extract of Crassula arborescens. Mice were
fasted for 16 h and then randomly divided into groups
of eight mice per cage. Graded doses of the plant
extract (100, 200, 400, 800, 1,200, 1,600, 2,000, 2,400,
2,800, 3,200, 3,600 and 4,000 mg/kg) were separately
administered orally by means of a bulbed steel needle
to mice in each test group. The control group received
0.25 mL (p.o.) of physiological saline by means of a
bulbed steel needle. The acute toxicity experiment was
repeated by administering the graded doses of the leaf
methanol extract of the plant species or control vehicle
to other groups of animals intraperitoneally. The mice
in both the test and control groups were then allowed
free access to food and water, and observed for over 5
days for signs of acute toxicity including death. Log
dose-response curves were then constructed for the
plant extract from which the median lethal dose was
calculated where applicable.
2.9 Statistical Analysis
The data on the onset of tonic convulsions were
analysed using one-way ANOVA (analysis of
variance) followed by Dunnett’s multiple comparison
test (GraphPad Prism, version 5.0, Graph Pad software,
Inc., San Diego Cap 2130, USA). The number of
animals convulsing was analysed using the
Chi-squared test [10]. Data obtained were expressed
as mean (± S.E.M). P-values less than 5% (P < 0.05)
were considered statistically significant.
2.10 Ethical Consideration
The experimental protocol used in this study was
approved (07/04/31) by the University of the Western
Cape Ethics Committee, Bellville 7535, South Africa
and conforms to the University’s Regulations Act
concerning animal experiments.
3. Results
3.1 Phytochemical Analysis
The phytochemical qualitative analysis of the dried
powdered leaf of Crassula arborescens revealed the
presence of the following chemical constituents:
triterpene steroids, saponins, tannins, reducing sugar
and flavonoids.
3.2 Anticonvulsant Activity Assessment
3.2.1 Effect of Leaf Methanol Extract of Crassula
arborescens on PTZ (Pentylenetetrazole)-Induced
Tonic Convulsion
PTZ (pentylenetetrazole, 95 mg/kg, i.p.) induced
tonic convulsion in the eight mice used. Leaf
methanol extract of Crassula arborescens (25 mg/kg,
i.p.) did not significantly affect the onset or incidence
of the tonic convulsion elicited by PTZ (95 mg/kg,
i.p.). The plant extract (50 mg/kg, i.p.) did not
significantly affect the incidence but significantly
delayed the onset of the tonic convulsion produced by
PTZ (95 mg/kg, i.p.). Crassula arborescens (25 mg/kg
and 50 mg/kg, i.p.) protected 12.5% and 37.5% of the
mice against PTZ (95 mg/kg, i.p.)-induced tonic
convulsion, respectively. Leaf methanol extract of
Crassula arborescens (100-200 mg/kg, i.p.)
significantly delayed the onset and also significantly
decreased the incidence of PTZ (95 mg/kg,
i.p.)-elicited tonic convulsion in a dose dependent
manner. The doses of the leaf methanol extract of
Crassula arborescens (100 mg/kg and 200 mg/kg, i.p.)
protected 62.5% and 75% of the animals respectively
against the tonic convulsion produced by PTZ (95
mg/kg, i.p.). Phenobarbitone (12 mg/kg, i.p.) and
muscimol (2 mg/kg, i.p.) significantly delayed the
onset of PTZ (95 mg/kg, i.p.)-induced tonic
convulsion and also significantly decreased the
incidence of the tonic convulsion. Phenobarbitone (12
mg/kg, i.p.) and muscimol (2 mg/kg, i.p.) protected
87.5% of the animals against the tonic convulsion
respectively. Diazepam (0.5 mg/kg, i.p.) profoundly
 Evaluation of the Anticonvulsant Activity of the Leaf Methanol Extract of Crassula arborescens (Mill.) 397
Willd. (Crassulaceae) in Mice

antagonised PTZ (95 mg/kg, i.p.)-induced tonic convulsion by protecting 87.5% of the animals each,
convulsion by protecting 100% of mice against the against the convulsion. Phenytoin (30 mg/kg, i.p.) or
convulsion. DMSO (0.25 mL, i.p.) or phenytoin DMSO (0.25 mL, i.p.) did not affect the tonic
(30 mg/kg, i.p.) did not significantly affect the onset convulsion produced by bicuculline (40 mg/kg, i.p.)
or incidence of PTZ (95 mg/kg, i.p.)-induced tonic (Table 3).
convulsion (Table 2). 3.2.3 Effect of Leaf Methanol Extract of Crassula
3.2.2 Effect of Leaf Methanol Extract of Crassula arborescens on Picrotoxin-Induced Convulsion
arborescens on Bicuculline-Induced Convulsion Picrotoxin (15 mg/kg, i.p.) produced tonic seizures
All the control mice treated with 40 mg/kg (i.p.) of in all the mice used. C. arborescens (50-200 mg/kg,
bicuculline exhibited tonic convulsion. Leaf methanol i.p.) significantly antagonised picrotoxin (15 mg/kg,
extract of C. arborescens (25 mg/kg, i.p.) did not i.p.)-induced tonic convulsion by delaying the onset of
affect the onset or incidence of the tonic convulsion the convulsion and reducing the number of animals
produced by bicuculline (40 mg/kg, i.p.). The dose of convulsing significantly. These doses of
50 mg/kg (i.p.) of the plant extract significantly C. arborescens protected between 62.5% and 87.5%
delayed the onset but did not significantly affect the of mice against the convulsion induced by picrotoxin.
incidence of the tonic convulsion. It protected 50% of C. arborescens (25 mg/kg, i.p.) did not affect the
mice against bicuculline (40 mg/kg, i.p.)-induced onset or incidence of pictotoxin produced tonic
tonic convulsion. Leaf methanol extract of convulsion. Phenobarbitone (12 mg/kg, i.p.),
C. arborescens (100-200 mg/kg, i.p.) dose diazepam (0.5 mg/kg, i.p.) and muscimol (2 mg/kg,
dependently and significantly delayed the onset of i.p.) all significantly delayed the onset and
bicuculline (40 mg/kg, i.p.)-induced tonic convulsion significantly decreased the incidence of the tonic
and also significantly reduced the number of animals convulsion elicited by picrotoxin (15 mg/kg, i.p.).
convulsing. The doses of 100 mg/kg and 200 mg/kg Phenobarbitone or diazepam protected 100% of the
(i.p.) protected 62.5% and 75% of mice respectively animals while muscimol protected 87.5% of the
against the tonic convulsion. Phenobarbitone (12 animals against picrotoxin-induced tonic convulsion.
mg/kg, i.p.), diazepam (0.5 mg/kg, i.p.) or muscimol DMSO (0.25 mL, i.p.) or phenytoin (30 mg/kg, i.p.)
(2 mg/kg, i.p.) significantly delayed the onset and the neither affected the onset nor the incidence of the
incidence of bicuculline (40 mg/kg, i.p.)-induced tonic tonic convulsion produced by picrotoxin (Table 4).
Table 2 Effect of leaf methanol extract of CA (Crassula arborescens) on PTZ (pentylenetetrazole)-induced seizures in mice.
Percentage Onset of tonic
PTZ CA PB DZ PN MS DMSO No. con/
protection convulsion (min)
(mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mL) No. used
(%) Mean ± SEM
95 - - - - - - 8/8 4.30 ± 0.62
95 25 - - - - - 7/8 12.5 4.55 ± 0.85
95 50 - - - - - 5/8 37.5 18.24* ± 3.63
95 100 - - - - - 3/8+ 62.5 21.42* ± 5.10
++
95 200 - - - - - 2/8 75 28.15* ± 5.32
+++
95 - 12 - - - - 1/8 87.5 23.76* ± 1.58
++++
95 - - 0.5 - - - 0/8 100 0*
95 - - - 30 - - 8/8 0 4.31 ± 0.44
+++
95 - - - - 2 - 1/8 87.5 28.56* ± 3.16
95 - - - - - 0.25 8/8 0 4.49 ± 0.30
*
P < 0.001 compared to PTZ (95 mg/kg, i.p.) control, ANOVA (n = 8). +P < 0.05, ++P < 0.01, +++P < 0.005, ++++P < 0.001 compared
to PTZ (95 mg/kg. i.p.) control, Chi-squared test (n = 8). PB: phenobarbitone; DZ: diazepam; PN: phenytoin; MS: muscimol; DMSO:
dimethylsulfoxide; No. con/No. used: number convulsed/number used.
398 Evaluation of the Anticonvulsant Activity of the Leaf Methanol Extract of Crassula arborescens (Mill.)
Willd. (Crassulaceae) in Mice

Table 3 Effect of leaf methanol extract of CA (Crassula arborescens) on BIC (bicuculline)-induced seizures in mice.
Percentage Onset of tonic
BIC CA PB DZ PN MS DMSO No. con/
protection convulsion (min)
(mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mL) No. used
(%) Mean ± SEM
40 - - - - - - 8/8 3.25 ± 0.74
40 25 - - - - - 8/8 0 5.93 ± 0.63
40 50 - - - - - 4/8 50 18.25* ± 2.87
+
40 100 - - - - - 3/8 62.5 27.46** ± 1.48
++
40 200 - - - - - 2/8 75 27.72** ± 1.31
+++
40 - 12 - - - - 1/8 87.5 26.23** ± 1.06
+++
40 - - 0.5 - - - 1/8 87.5 27.15** ± 1.11
40 - - - 30 - - 8/8 0 3.31 ± 0.49
40 - - - - 2 - 1/8+++ 87.5 28.28** ± 1.33
40 - - - - - 0.25 8/8 0 3.29 ± 0.54
*
P < 0.005, **P < 0.001 compared to bicuculline (40 mg/kg, i.p.) control, ANOVA (n = 8). +P < 0.05, ++P < 0.01, +++P < 0.005
compared to bicuculline (40 mg/kg, i.p.) control, Chi-squared test (n = 8). PB: phenobarbitone; DZ: diazepam; PN: phenytoin; MS:
muscimol; DMSO: dimethylsulfoxide; No. con/No. used: number convulsed/number used.

Table 4 Effect of leaf methanol extract of CA (Crassula arborescens) on PIC (picrotoxin)-induced seizures in mice.
Percentage Onset of tonic
PIC CA PB DZ PN MS DMSO No. con/
protection convulsion (min)
(mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mL) No. used
(%) Mean ± SEM
15 - - - - - - 8/8 10.75 ± 0.75
15 25 - - - - - 8/8 0 10.88 ± 0.44
15 50 - - - - - 3/8+ 62.5 23.50* ± 3.17
++
15 100 - - - - - 2/8 75 26.88** ± 2.29
+++
15 200 - - - - - 1/8 87.5 27.48** ± 1.98
15 - 12 - - - - 0/8++++ 100 0**
++++
15 - - 0.5 - - - 0/8 100 0**
15 - - - 30 - - 8/8 0 11.00 ± 0.65
15 - - - - 2 - 1/8+++ 87.5 29.13** ± 0.88
15 - - - - - 0.25 8/8 0 11.13 ± 0.88
*
P < 0.005, P < 0.001 compared to picrotoxin (15 mg/kg, i.p.) control, ANOVA (n = 8). P < 0.05, P < 0.01, +++P < 0.005, ++++P
** + ++

< 0.001 compared to picrotoxin (15 mg/kg, i.p.) control, Chi-squared test (n = 8). PB: phenobarbitone; DZ: diazepam; PN: phenytoin;
MS: muscimol; No. con/No. used: number convulsed/number used; DMSO: dimethylsulfoxide.

3.2.4 Effect of Leaf Methanol Extract of Crassula
arborescens on N-methyl-DL Aspartic Acid-Induced
Tonic Convulsion
The dose of 400 mg/kg (i.p.) of N-methyl-DL
aspartic acid produced tonic convulsion in the eight
mice used for the assessment. LY233053 (5 mg/kg,
i.p.) significantly delayed the onset of N-methyl-DL
aspartic acid (400 mg/kg, i.p.)-induced tonic
convulsion and significantly reduced the number of
animals convulsing. Crassula arborescens (100-200
mg/kg, i.p.) significantly delayed the onset of
N-methyl-DL aspartic acid (400 mg/kg,
i.p.)-produced tonic convulsion but did not
significantly affect the incidence of the convulsion.
The dose of 200 mg/kg (i.p.) of C. arborescens
protected only 12.5% of the animals. C. arborescens
(25-50 mg/kg, i.p.), phenobarbitone (12 mg/kg, i.p.),
diazepam (0.5 mg/kg, i.p.), phenytoin (30 mg/kg, i.p.)
or DMSO (0.25 mL, i.p.) did not affect the onset of
the tonic convulsion induced by N-methyl-DL
aspartic acid (400 mg/kg, i.p.) or the number of mice
convulsing (Table 5).
3.2.5 Effect of Leaf Methanol Extract of Crassula
arborescens on Strychnine-Induced Tonic Convulsion
All the mice given strychnine (2 mg/kg, i.p.)
exhibited tonic convulsion. Leaf methanol extract of C.
 Evaluation of the Anticonvulsant Activity of the Leaf Methanol Extract of Crassula arborescens (Mill.) 399
Willd. (Crassulaceae) in Mice

Table 5 Effect of leaf methanol extract of CA (Crassula arborescens) on NMDLA (N-methyl-DL aspartic acid)-induced
seizures in mice.
Percentage Onset of tonic
NMDLA CA PB DZ PN LY DMSO No. con/
protection convulsion (min)
(mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mL) No. used
(%) Mean ± SEM
400 - - - - - - 8/8 2.18 ± 0.52
400 25 - - - - - 8/8 0 2.39 ± 0.40
400 50 - - - - - 8/8 0 2.44 ±0.28
400 100 - - - - - 8/8 0 5.81* ± 0.15
400 200 - - - - - 7/8 12.5 6.32* ± 0.11
400 - 12 - - - - 8/8 0 2.20 ± 0.41
400 - - 0.5 - - - 8/8 0 2.30 ± 0.73
400 - - - 30 - - 8/8 0 2.19 ± 0.44
400 - - - - 5 - 1/8+ 87.5 24.91** ± 1.63
400 - - - - - 0.25 8/8 0 2.15 ± 0.52
*
P < 0.05, **P <0.001 compared to NMDLA (400 mg/kg, i.p.) control, ANOVA (n = 8). +P < 0.005 compared to NMDLA (400
mg/kg, i.p.) control, Chi-squared test (n = 8). PB: phenobarbitone; DZ: diazepam; PN: phenytoin; LY: LY233053; No. con/No. used:
number convulsed/number used; DMSO: dimethylsulfoxide.

Table 6 Effect of leaf methanol extract of CA (Crassula arborescens) on SCN (strychnine)-induced seizures in mice.
Percentage Onset of tonic
SCN CA PB DZ PN DMSO No. con/
protection convulsion (min)
(mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mL) No. used
(%) Mean ± SEM
2 - - - - - 8/8 3.18 ± 0.22
2 25 - - - - 8/8 0 3.33 ± 0.31
2 50 - - - - 7/8 12.5 8.78* ± 0.17
+
2 100 - - - - 3/8 62.5 12.06* ± 0.27
2 200 - - - - 3/8+ 62.5 16.59* ± 0.18
++
2 - 12 - - - 2/8 75 21.43* ± 0.28
2 - - 0.5 - - 8/8 0 3.81 ± 0.60
2 - - - 30 - 8/8 0 3.57 ± 0.94
2 - - - - 0.25 8/8 0 3.25 ± 0.75
*
P < 0.001 compared to strychnine (2 mg/kg, i.p.) control, ANOVA (n = 8). +p < 0.05, ++P < 0.01 compared to strychnine (2 mg/kg,
i.p.) control, Chi-squared test (n = 8). PB: phenobarbitone; DZ: diazepam; PN: phenytoin; No con/No. used: number
convulsed/number used; DMSO: dimethylsulfoxide.

arborescens (100-200 mg/kg, i.p.) significantly
antagonised strychnine (2 mg/kg, i.p.)-induced tonic
convulsion by significantly delaying the onset of the
convulsion and significantly reducing the number of
animals convulsing. Both doses of 100 mg/kg (i.p.)
and 200 mg/kg (i.p.) of C. arborescens protected
62.5% of mice against strychnine (2 mg/kg,
i.p.)-induced tonic convulsion. C. arborescens (25
mg/kg, i.p.) did not significantly affect the tonic
convulsion elicited by strychnine (2 mg/kg, i.p.).
However, the dose of 50 mg/kg (i.p.) significantly
delayed the onset of the tonic convulsion but did not
affect the number of animals convulsing. C.
arborescens (50 mg/kg, i.p.) protected only 12.5% of
mice against strychnine (2 mg/kg, i.p.)-induced tonic
convulsion. Phenobarbitone (12 mg/kg, i.p.)
significantly antagonised the tonic convulsion
produced by strychnine (2 mg/kg, i.p.).
Phenobarbitone (12 mg/kg, i.p.) protected 75% of
mice against strychnine (2 mg/kg, i.p.)-induced tonic
convulsion. Diazepam (0.5 mg/kg, i.p.), phenytoin (30
mg/kg, i.p.) or DMSO (0.25 mL, i.p.) did not affect
strychnine (2 mg/kg, i.p.)-induced tonic convulsion in
any significant manner (Table 6).
400 Evaluation of the Anticonvulsant Activity of the Leaf Methanol Extract of Crassula arborescens (Mill.)
Willd. (Crassulaceae) in Mice

Fig. 1 HPLC fingerprint of methanol extract of Crassula arborescens.

Volume of injection: 50 μL; sample concentration: 22 mg/mL; column: Phenomenex Luna (C18); system of elution: A: water (0.01%
formic acid), B: acetonitrile (0.01% formic acid), flow rate: 0.8 mL/min, 0 min, solvent B: 18%; 15 min, solvent B: 25%; 20 min,
solvent B: 35%; 30 min, solvent B: 90%.

dose of 800 mg/kg and from the doses of 1,200-4,000

3.3 HPLC Analysis
The HPLC fingerprint of C. arborescens obtained
showed characteristic peaks at the following retention
times (minutes): 8.834, 11.776, 17.485 and 26.075
(Fig. 1).
3.4 Acute Toxicity Study
Leaf methanol extract of Crassula arborescens
(100-4,000 mg/kg), given orally, did not produce any
signs of acute toxicity in or deaths of the mice used.
The NOAEL (no-adverse-effect-level) was observed
at 4,000 mg/kg (p.o.) which was the highest dose
administered during the acute toxicity testing. The
LD50 of the plant extract when given orally, was
therefore, taken to be greater than 4,000 mg/kg.
However, when the same dose range (100-4,000
mg/kg) of Crassula arborescens was used and the
plant extract administered to mice intraperitoneally,
signs of acute toxicity, such as piloerection, salivation
and hypoactivity were observed at the dose of 400
mg/kg. Two mice also died at this dose. Other signs
such as diarrhoea and tremor were observed in higher
doses in addition, before death. Five mice died at the
mg/kg, all the animals died. The LD50 of the plant
extract when given intraperitoneally, was found to be
781.6 mg/kg.
4. Discussion
According to Rang et al. [11], Waller et al. [12] and
Burnham [13], the imbalance between GABA (gamma
aminobutyric acid)-mediated inhibition and
glutamate-mediated excitation in the brain underpins
epilepsy. It has been postulated that the inhibition or
enhancement of GABAergic neurotransmission at
GABAA receptors may cause or antagonise convulsion.
On the other hand, the inhibition or enhancement of
glutamatergic neurotransmission at NMDLA receptors
may antagonise or cause convulsion. The data
obtained in this study indicate that the leaf methanol
extract of Crassula arborescens antagonised the tonic
convulsion elicited by PTZ, bicuculline, picrotoxin,
NMDLA or strychnine. The study also shows that
diazepam, phentobarbitone or muscimol antagonised
PTZ-, bicuculline- or picrotoxin-induced tonic
convulsion but did not affect NMDLA-induced tonic
convulsion. Phenytoin did not affect PTZ-,
 Evaluation of the Anticonvulsant Activity of the Leaf Methanol Extract of Crassula arborescens (Mill.)
Willd. (Crassulaceae) in Mice
bicuculline-, picrotoxin-, NMDLA- or
strychnine-induced tonic convulsion. LY233053
antagonised NMDLA-induced tonic convulsion.
Strychnine-induced tonic convulsion was antagonised
by phenobarbitone. Rang et al. [11], De Sarro et al.
[14] and Meldrum [15] reported that PTZ may be
producing convulsion by inhibiting GABA-mediated
inhibition at the GABAA receptors in the brain.
Phenobarbitone and diazepam, standard
anticonvulsants, are thought to produce their
anticonvulsant effects by acting at their respective
receptors on the GABAA receptor-chloride ionophore
complex to enhance GABAergic neurotransmission
[11, 15, 16]. Phenobarbitone and diazepam shown in
this study to antagonise PTZ-induced tonic convulsion
may be doing so by enhancing GABAergic
neurotransmission. Phenytoin, another standard
antiepileptic drug, known to produce its
anticonvulsant effect by blocking sodium ion entry
into the brain cells [10], did not affect PTZ-induced
tonic convulsion in this study. According to Rang et al.
[11] and Lança [17], muscimol, a selective GABAA
receptor agonist, stimulates GABAA receptors in the
brain to produce effects similar to that of naturally
occurring GABA. Muscimol was shown in this study
to antagonise PTZ-induced tonic convulsion. Leaf
methanol extract of C. arborescens antagonised
PTZ-induced tonic convulsion which may probably
implicate GABAergic mechanism in its anticonvulsant
activity. Phenobarbitone, diazepam and muscimol, a
selective GABAA receptor agonist, known to enhance
or mimic GABA neurotransmission [11, 17], were
shown in this study to antagonise tonic convulsion
produced by either bicuculline or picrotoxin.
Bicuculline, a selective GABAA receptor antagonist, is
thought to produce its convulsant activity by blocking
GABAergic neurotransmission at GABAA receptor in
the brain [11]. According to Rang et al. [11],
picrotoxin produces its convulsant activity by
blocking GABAA receptor-linked chloride ion
channels and preventing chloride ion entry into the
401
brain cells thus inhibiting GABA neurotransmission.
Phenytoin, known to produce convulsion by blocking
sodium ion entry into the brain cells, did not alter
bicuculline- or picrotoxin-induced tonic convulsion.
In this study, C. arborescens antagonised the tonic
convulsion produced by either bicuculline or
picrotoxin thus supporting the earlier suggestion that
GABAergic mechanism may be involved in the
anticonvulsant activity of the plant extract. Rang et al.
[11], Besancon et al. [18] and Chapman and Meldrum
[19] reported that NMDLA, a specific agonist at the
NMDA receptors, produces convulsion by stimulating
NMDA receptors in the brain to mimic the effect of
the naturally occurring glutamate which is an
excitatory neurotransmitter. LY233053, a competitive
NMDA receptor antagonist, which acts by blocking
the effect of glutamate at NMDA receptors [20, 21],
was shown in this study, to antagonise
NMDLA-induced tonic convulsion. Phenobarbitone
and diazepam, known as enhancers of GABAergic
neurotransmission and Phenytoin, known as blocker
of sodium ion entry into the brain cells [11], did not
affect NMDLA-tonic tonic convulsion. Leaf methanol
extract of C. arborescens significantly delayed the
onset of NMDLA-tonic tonic convulsion indicating
that glutamatergic mechanism may be involved in its
anticonvulsant activity. According to Rang et al. [11],
strychnine produces convulsion by blocking receptors
for glycine in the brain. In this study, phenobarbitone
and leaf methanol extract of C. arborescens
antagonised strychnine-induced tonic convulsion. The
possibility, therefore, exists that glycinergic
mechanism may also be involved in the anticonvulsant
activity of C. arborescens. It has been reported that
different mechanisms may be involved in the
anticonvulsant activity of phenobarbitone [11]. The
enhancement of glycinergic neurotransmission may
probably be one of such mechanisms involved in the
anticonvulsant activity of phenobarbitone. Rang et al.
[11] reported that benzodiazepines do not alter
strychnine-induced convulsion in experimental
402 Evaluation of the Anticonvulsant Activity of the Leaf Methanol Extract of Crassula arborescens (Mill.)
Willd. (Crassulaceae) in Mice
animals. It is pertinent to note that Lambert et al. [22]
and Boyd et al. [23] reported the use of moderate to
high doses of IV phenobarbitone, IV diazepam and IV
phenytoin to prevent strychnine convulsion in
strychnine poisoning in humans. Phytochemical
qualitative analysis of C. arborescens carried out in
this study revealed the presence of saponins, triterpene
steroids, flavonoids, tannins and reducing sugar. It is
important to mention that studies by Singh et al. [24],
Ibrahim et al. [25], Van Heerden et al. [26] and
Chauhan et al. [27] have shown that flavonoids,
saponins and triterpene steroids have anticonvulsant
activities suggesting that these compounds, also being
present in the plant extract, may probably be
contributing to the anticonvulsant activity of C.
arborescens. In this study, a fairly high LD50 of
probably over 4,000 mg/kg was obtained when leaf
methanol extract of C. arborescens was given orally
suggesting that it is safe in mice at this dose level. On
the other hand, the LD50 obtained when the plant
extract was given intraperitoneally was 781.6 mg/kg.
However, only the oral route has been used by
traditional medicine practitioners in South Africa to
administer the plant extract in the form of infusion or
decoction in the treatment of epilepsy and other
ailments.
5. Conclusion
The data obtained in this study indicate that C.
arborescens has anticonvulsant activity which may be
underpinned by GABAergic, glutamatergic and
glycinergic mechanisms. The HPLC fingerprint of C.
arborescens obtained showed characteristic peaks at
the following retention times (minutes): 8.834, 11.776,
17.485 and 26.075. The results obtained also indicate
that the plant extract may be safe in mice when given
orally.
Acknowledgments
This study was financially supported by the
National Research Foundation (67983), South Africa.
The authors wish to thank Mr Franz Weitz and Mr
Simthembile Pambuka for their valuable technical
assistance.
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Journal of Pharmacy and Pharmacology 2 (2014) 404-415
D DAVID PUBLISHING

Can Dietary Pomegranate Peels Reduce Stress

Responses Associated with Group Mixing of Holstein
Beef Calves?

Sarah Weyl-Feinstein1, 2, Alla Orlov1, Moran Yishay1, Rotem Agmon1, Machteld Steensels3, Vered Sibony1, Ilan
Halachmi3, Ido Izhaki2 and Ariel Shabtay1
1. Department of Ruminant Science, Institute of Animal Science, Newe Ya’ar Research Center, Agricultural Research Organization,
Ramat Yishay 30095, Israel
2. Department of Evolutionary and Environmental Biology, Faculty of Sciences, University of Haifa, Haifa 3498838, Israel
3. Institute of Agricultural Engineering-Agricultural Research Organization (ARO)-The Volcani Center, Bet-Dagan 50250, Israel

Abstract: The study investigated whether dietary CPE (concentrated pomegranate peel extract) may mitigate the negative effects
associated with group mixing prior to marketing and enhance beef meat quality. Twenty-two bull calves were reared in triplets and
divided to control (n = 9) and CPE-treated (n = 13) groups. CPE was supplemented during eight months of rearing, and calves were
mixed 34 d prior to marketing to control and treatment groups. Calves were monitored for weight gain, rumination, activity, metabolic
and oxidative stress responses. Finally, meat quality traits were examined. The results find that pre-mixing activity (P < 0.0001),
non-esterifies fatty acids (P = 0.02) and plasma testosterone (P = 0.005) levels were higher in the treatment group. Following mixing,
activity (P < 0.0001) and plasma anti-oxidative capacity (P = 0.05) increased in the treatment compared to the control group. In spite
of the above, dietary supplementation of CPE didn’t reveal improvement of meat quality parameters by means of meat pH and shelf life.
It indicated that improved serum anti-oxidant capacity in the CPE calves was not sufficient to prevent the mixing effects on meat
quality. A combined CPE concentration and mixing management should be examined in order to reduce mixing related effects on meat
quality.

Key words: Bull calves, group-mixing, CPE, anti-oxidative capacity, non-esterified fatty acid, testosterone.

1. Introduction Pre-slaughter handling can affect both carcass and

meat quality. The major influence of pre-slaughter
Animal handling practices prior to marketing are a
handling on lean meat quality is through the potential
growing concern in many countries around the world,
effect on muscle glycogen stores. If these stores are
as they have impact on animal welfare [1]. At the
depleted by chronic stress, the extent of postmortem
course of handling, animals are exposed to physical
acidification is reduced, leading to the production of
and psychological stresses, such as the breakdown of
DCB (dark cutting beef), which is prone to spoilage
social groupings and mixing with unfamiliar animals [2].
and has poor organoleptic qualities. The major cause of
Mixing of unfamiliar bulls causes injuries, bruises
DCB is mixing unfamiliar animals, thus promoting
and body weight losses [3], leading to significant
agonistic behavior, particularly in young bulls.
economical outcome [4].
Therefore pre-slaughter handling practices which
Lawrie and Ledward (2006) [5] reported that with
encourage mixing increase the incidence of DCB [6],
higher levels of stress poorer meat quality is eminent.
leading to harmful economic implications.
However, the mixing policy is an integral part of
Corresponding author: Sarah Weyl-Feinstein, Ph.D.,
research fields: cattle health and nutrition. E-mail: beef cattle production. So, one should seek solutions to
sarah@volcani.agri.gov.il.
 Can Dietary Pomegranate Peels Reduce Stress Responses Associated with Group Mixing of Holstein 405
beef Calves?

reduce the deleterious impact of mixing unfamiliar 2. Materials and Methods

individuals. Since oxidative stress is the outcome of a
2.1. Calves’ Husbandry and Nutrition
line of behavioral and physiological responses, a notion
we intended to explore was whether long-term Twenty-two young Holstein bull horned calves, not
supplementation of dietary antioxidants might reduce castrated, were included in this experiment. They were
the oxidative stress effects induced by dramatic event randomly divided into two groups: control (C) (n = 9,
such as group mixing. BW = 281.5 ± 93, age 7.2 ± 0.1 months) and treatment
Natural antioxidants can protect the cellular (T) (n = 13, BW = 295.5 ± 82, age 7.5 ± 0.07 months)
components from oxidation processes caused by (BW and age; P > 0.05). The calves were housed in
reactive oxygen species [7], including those which groups of three in 9 m2 pens.
occur during and after the slaughter. Lipid oxidation in Both groups were reared in the same paddock and
the meat results in the formation of free radicals, which fed TMR (total mixed ration) (Table 1). Weight
may lead to the oxidation of meat pigments and to the measurements were performed monthly, 1 DBM (day
generation of rancid odors and flavors [8]. Dietary before mixing), 3 and 33 DPM (days post mixing).
supplementation of vitamin E, a potent lypophilic Calves in the T group received the CPE
anti-oxidant, may for instance, slows down lipid supplementation between the ages of 7.3 ± 0.07 and
oxidation, and hence expand the shelf-life of the meat 15.7 ± 0.07 months. The CPE was initially poured over
[9] and improve lipid stability and meat color [10]. the TMR at a concentration of 3%, on DM (dry matter)
Several phenolic compounds such as gallic acid, Table 1 Ingredient and chemical composition of the TMR
cyanidin, quercetin and catechin were also found to (total mixed ration) fed to experiment calves.
have a high antioxidant activity [11]. These Components (g/kg of DM) Amount
phytochemicals, together with punicalagins, ellagic Ingredients composition
Barley grain 402
acid and others, are the principal anti-oxidants in the
Ground maize grain 264
pomegranate fruit [12]. The antioxidant characteristics Soybean meal (solvent-extracted) 27.06
of all parts of the pomegranate fruit, including the peel, Wheat barn 51.6
were proven in several studies [13, 14]. Pomegranate Gluten feed 35.8
was already tested as a potential beneficial additive for Vetch hay (Vicia) 81
Wheat silage 89
enhancing meat quality. It was found that adding
Broiler liter silage 33
pomegranate components to goat [15] and chicken Minerals ans vitamins 2.56
patties [16] had some positive effects of reducing meat NaCL 3.8
lipid peroxidation. Limestone 10.15
Chemical components
Based on pioneer studies on pomegranate peel
ME, MJ/kg of DM 2.83
dietary supplementation [17], a commercial by-product, CP, g/kg of DM 132
namely, CPE (concentrated pomegranate peel extract) Organic matter, g/kg of DM 950
has recently been developed. This additive was already NDF, g/kg of DM 240
found to induce various productive and Ether extract, g/kg of DM 28
Soluble carbohydrates (g/kg) 461
health-promoting effects [17-19]. However, to the best
Roughage (g/kg) 170
of our knowledge, the potential of CPE to attenuate Ash (g/kg) 50
oxidative stress damages related to mixing of beef Ca (g/kg) 8
cattle, including effects on meat quality traits, haven’t P (g/kg) 4.68
Vitamin A (IU) 7000
been tested yet.
406 Can Dietary Pomegranate Peels Reduce Stress Responses Associated with Group Mixing of Holstein
beef Calves?
basis, for three months, thereafter concentration was
increased to 4%, as shown in Fig. 1. The CPE of the
“wonderful” cultivar was supplied by Gan Shmuel
Food Ltd. (Gan-Shmuel, Israel). It was made by
chopping up the pomegranate parts remaining after
pomegranate juice production, including peels and
residual arils, followed by extraction in water, filtering,
evaporation procedures and pasteurization [18]. The
final extract was standardized to ensure uniform and
constant DM content [17, 18].
The CPE composition used in this experiment (in
g/kg of DM) was: soluble sugars (288), crude protein
(20.8), ash (50.2), total soluble phenolics (63),
punicalagins (26.5), ellagic acid (2.5); the pH of the
extract was 2.65 [19].
Thirty four days before marketing the calves were
moved to two paddocks, as requested by the abattoir,
one for the C and the other for the T group. The calves
were finally weighed on the marketing date, at a mean
age of 523 ± 0.34 days and sent to a commercial
abattoir.
2.2. Rumination and Activity Measurements
In order to examine the behavioral changes
associated with group mixing, all calves were equipped
with rumination tags (Hi-Tag TM, SCR Engineers,
Netanya, Israel) that measured the duration of daily
rumination in minutes (min/day). The rumination tags
Fig. 1 Experiment schedule.
(1) Growth period of calves since parturition; (2) Separation of
22 calves into control and treatment calves, reared in triplets; (3)
CPE was poured on the ration up to the end of experiment; (4)
Calves were mixed (from triplets in a pen) into control (n = 9)
and treatment (n = 13) groups; (5) All calves were sent to
slaughter in organic groups. TMR—total mixed ration in first,
second and third periods of experiment fed to all calves, TMR
1, 2, and 3 slightly differ in feed composition.
were hung on the neck of each calf during the entire
trial period. The rumination data were logged in 2h
blocks transferred to the computer and analyzed using
Matlab software (Matlab 6.1, MathWorks, Natick,
Massachusetts, USA). Activity evaluation included the
total number of steps, number of rest-bouts (number of
times a calf lies down and stands up again) and
rest-time (duration of rest in minutes). These data were
recorded by a behavior sensor tag (Pedometer PlusTM,
AfiFarm® Dairy Herd Management software) that was
fitted to the forelimb of each calf. Daily activity is
presented for 24 h period, day-time activity was
considered as the activity during light hours, hence
from 04:00 am until 18:00 pm, and night-time activity
was defined as the period between 18:00 pm and 04:00
am. The variables that were collected were analyzed in
the following form: sum of daily (24 h), day-time and
night-time number of steps, sum of daily, day-time and
night-time lying down time in minutes, and sum of the
number of daily, day-time and night-time rest-bouts.
Average values for every parameter were obtained for
each group for the different periods: 20 DBM (days
before mixing), 24 h PM (post-mixing), 2, 3-14 and
15-25 DPM.
2.3 Blood Sampling
Blood was sampled 1 DBM, 1 and 3 DPM from the
caudal vein, using EDTA-containing tubes,
heparinized tubes and serum clot activator tubes. The
blood was centrifuged at 1,500 g at 4 °C to separate the
cells from plasma and to collect serum. These samples
were frozen in liquid nitrogen and kept at -20 °C until
use.
2.4 Determination of Serum Anti-oxidant Capacity
Two methods were used to evaluate total
antioxidant capacity, the FRAP (ferric reducing
antioxidant power) assay [20] and the
Chemiluminescence-inducing cocktail method [21,
22]. The FRAP assay uses antioxidants as reluctant in
a redox-linked colorimetric method, employing an
 Can Dietary Pomegranate Peels Reduce Stress Responses Associated with Group Mixing of Holstein
beef Calves?
easily reduced oxidant system. Analyses were
performed in duplicates on 1 DBM, 1 and 3 DPM
heparinized plasma samples, and data were presented
as µM ascorbic acid equivalents.
For the Chemiluminescence-inducing cocktail
method we used serum samples. This method is based
on generation of light-conjugated free radicals
production. The anti-oxidant capacity of a sample is
evaluated by its potential to quench the light generated
by the system. Thus, lower LDCL (luminol-dependent
Chemiluminescence) values reflect samples with
higher anti-oxidant capacity. The reaction cocktail was
comprised of Hank’s buffer (cat #02-016-1A, BI),
H2O2 and 1 mM of cobalt chlorine, sodium selenite,
and luminol. A volume of 20 µL serum was added to
the reaction mixture and analyzed immediately in a
Lumac type 2500 M luminometer for LDCL generation.
The samples were measured during 6.5 min and values
were calculated as the sum counts of the entire
measurement period.
2.5 Analysis of Plasma NEFA (Non-esterified Fatty
Acids)
We used a two reaction, enzymatic-based assay that
was adapted and validated to quantify NEFA in bovine
heparinized plasma, using micro-titer plates [23]. The
analysis was performed in duplicates samples on 1
DBM, 1 and 3 DPM using the commercial kit
NEFA-HR (2) R1 set, (cat #. 434-91795 Wako
chemicals GmbH).
2.6 Determination of Plasma Testosterone
Concentrations
In order to discover whether the dietary CPE
influenced the testosterone levels, and hence the calves’
behavior, we sampled blood from the caudal vein 3
months and 1 day before mixing into EDTA-containing
tubes. Testosterone levels were measured in duplicates
using a DRG Elisa kit (ELISA EIA-1559, DRG
Instruments, GmbH, Marburg, Germany), according to
the manufacturer’s instructions.
407
2.7 Meat Quality Analyses
The calves were slaughtered at a commercial
abattoir one day after arrival. The pH of m. LTL
(longissimus thoracis et lumborum) was measured
using a glass electrode 24 h after the carcasses were
chilled.
At slaughter, the pelvic, kidney, testicles and heart
fat depots were weighed. The quarters of the carcasses
were chilled overnight in a refrigerated room, followed
by dismantling of the carcasses. Slices of about 250 g
from the M. longissimus dorsi between the 12th and
13th ribs were sampled from the carcass of each calf.
These samples were kept in vacuum bags at -20 ºC.
These slices were used for analyses of DM, ash, water
content, crude protein, ether extract, fatty acid profile
and lipid oxidation. To this end, 5 g ground meat
samples were dried at 105 ºC for 24 h [24].
Determination of ether extract was carried out by the
Soxhlett method [25], and FAME (fatty acid methyl
esters) was performed as previously described [26]
with some modifications [27, 28]. The extent of meat
lipid oxidation was determined in duplicates by the
formation of TBARS (thiobarbituric acid-reactive
substances) as previously described [29, 30] with some
modifications.
Meat samples were tested for TBARS values one
day (D1), 4 days (D4), 9 days (D9), 16 days (D16) and
31 days (D31) after thawing. The results are calculated
[31] and expressed as milligrams malondialdehyde/kg
meat.
2.8 Statistical Analysis
All means are presented ±SEM and analyzed with
the use of SPSS for Windows (version 17.0). The
differences of weight gain and meat analyses were
analyzed using the 2-way ANOVA test. For plasma
NEFA levels, testosterone levels, fatty acid profile and
fat percentages in meat; we used an independent
one-tailed student t-test. The differences in activity,
rumination, FRAP, and anti-oxidative activity analysis
among groups was tested using a repeated
408 Can Dietary Pomegranate Peels Reduce Stress Responses Associated with Group Mixing of Holstein
beef Calves?

measurement ANOVA (analysis of variance). The

meat analyses differences were tested using the
one-way ANOVA test. Comparison of meat pH
distribution was tested using Chi-square test. We used
the Spearman correlation coefficient (r) to test the
one-tailed non-linear correlation between variables.
Statistical significance was declared at a probability
level of P ≤ 0.05.
Fig. 2 The effect of group mixing on weight gain of Holstein
3. Results bull calves.
1 DBM—1 day before mixing, 3 and 33 DPM—days post
3.1 Mixing Effect on Weight Gain, Plasma Levels of mixing. The mixing had a significant effect on pooled groups
NEFA and Testosterone Concentrations (P = 0.04), however, no mixing x treatment interactions were
revealed (P = 0.9). Data are presented as means ± SEM.
The group mixing had a significant impact on the
animals weight gain with no difference between the C
and T groups (pooled groups effect; P = 0.04, group x
mixing days; P = 0.9; Fig. 2). Average weight loss of
calves was 6.1 ± 3 and 6.3 ± 2 kg for the C and T groups,
respectively. Moreover, even 33 DPM, the average
weight gain did not return to the values obtained before
mixing. Additionally, one calf suffered from a broken
leg on the day following mixing and needed to be
Fig. 3 The effect of dietary CPE on plasma NEFA levels
excluded from the experiment.
following mixing.
In response to mixing, plasma NEFA levels Samples were taken 1 DBM (one day before mixing), one day
increased significantly for pooled groups (P < 0.0001). and three DPM (days post mixing). Data are presented as
However, NEFA concentrations were statistically means ± SEM. Significance refers to the difference between
groups on each tested day. CPE—concentrated pomegranate
higher for T over C group in all sampled days (Fig. 3).
peel extract. NEFA—non esterifies fatty acids.
Additionally, plasma testosterone concentrations were
significantly higher in the T group, 3 months and 1 day
before mixing (Fig. 4). A positive correlation was
found between testosterone levels three months DBM
and weight gain 1 DBM (rs = 49, P = 0.02, n = 18).
Additionally, testosterone levels three months before
mixing was positively correlated to testosterone
concentration 1 DBM (rs = 54, P = 0.01, n = 18),
NEFA levels 1 DPM (rs = 45, P = 0.02, n = 19),
number of steps 20 DBM (rs = 44, P = 0.04, n = 17),
steps 1 (rs = 44, P = 0.03, n = 18) and 3 DPM (rs = 43, Fig. 4 The effect of CPE on plasma testosterone
P = 0.03, n = 18). The testosterone levels 1 DBM were concentrations in Holstein bull calves.
also positively correlated to testosterone levels three 3 months BM—three months before mixing, 1 DBM—one day
post mixing. Data are presented as means ± SEM and
months earlier (rs = 54, P = 0.01, n = 18), NEFA levels significance refers to t-test comparison between control and
1 DPM (rs = 62, P = 0.003, n = 18), and number of treatment groups. CPE—concentrated pomegranate peel extract.
 Can Dietary Pomegranate Peels Reduce Stress Responses Associated with Group Mixing of Holstein 409
beef Calves?

steps 1 (rs = 66, P = 0.002, n = 17) and 3 DPM (rs = 45,
P = 0.03, n = 17). In addition, testosterone levels 3
months before mixing negatively correlated to the
rest-time 3 DPM (rs = -56, P = 0.009, n = 17). Finally,
the number of steps 20 DBM and NEFA levels 1 DPM
were positively correlated (rs = 0.59, P = 0.006, n =
17).
3.2 The Effect of Mixing on Plasma Antioxidant
Capacity
The total antioxidant capacity evaluated by the
FRAP method was not affected by mixing. The values
were 181.6 ± 15 and 189.5 ± 13 on 1 DBM, 173.6 ± 19
and 178.1 ± 21 on 1 DPM, and 210.7 ± 24 and 198.3 ±
20 M ascorbic acid equivalent on 3 DPM, for the C
and T groups, respectively (P > 0.05). However, the
anti-oxidative capacity of the serum measured by the
chemiluminesce method revealed an improved
antioxidant capacity for the T group (Fig. 5). The
anti-oxidant capacity of the T group was significantly
superior relative to the C group only 1 DPM, as judged
by the lower LDCL values in the different days.
However, when we tested only the effect of the
treatment on LDCL, without considering the day factor,
the results were significant (P = 0.04). Furthermore,
there was no interaction between treatment and days in
relation to mixing (P = 0.8).
3.3 Activity and Rumination Following Group Mixing
The mixing had a significant effect on the calves’
activity pooled groups: the daily steps in the 20 DBM,
24 h, 2, 3-14 and 15-25 DPM was 3,508 ± 445, 10,305
± 783, 4,062 ± 638, 4,913 ± 657 and 6,391 ± 1,167
steps/d, respectively (P < 0.0001). Also the daily
number of rest-bouts was affected by the group mixing,
being 19.1 ± 0.8, 10.4 ± 0.7, 16.4 ± 1.1, 17 ± 0.5 and
19.7 ± 0.9 rest-bouts/d, respectively (P < 0.0001), for
20 DBM, 24 h, 2, 3-14 and 15-25 DPM. Daily rest-time
differed significantly between the periods around
mixing: the calves rested on 20 DBM, 24 h, 2, 3-14 and
15-25 DPM a sum of 702 ± 12, 384 ± 17, 609 ± 20, 580
± 12 and 701 ± 19 min/d, respectively (P < 0.0001).
The differences in activity and resting parameters
between C and T groups are elaborated in Table 2. It is
noticeable that both the daily steps and the day-time
steps of the T group are significantly higher than those
of the C group, however, during the night-time, the
differences in number of steps between the groups were
not significant.
The daily sum of rest-time (m/d) in the two groups
was not different during the pre-mixing period (P =
0.6), nor 24 h post mixing (P = 0.8). 24 h-2 DPM, the
C group rested longer as compared to the T group (P =
0.05), but no differences among the groups were shown

Table 2 Daily and day-time activity (steps) and rest time in C (control) and T (treatment) groups in relation to mixing.
Period T/C Steps Rest time
Daily DT Daily DT
Pre-mixing C 2,109 ± 167 1,328 ± 199 703 ± 19 388 ± 17
T 3,169 ± 249*** 1,944 ± 216*** 701 ± 15ⁿ 369 ± 13ⁿ
24h PM C 7,851 ± 413 5,996 ± 310 377 ± 23 152 ± 11
T 13,395 ± 702*** 9,269 ± 477*** 391 ± 28ⁿ 182 ± 17ⁿ
24h-2 DPM C 1,968 ± 153 2,128 ± 178 657 ± 17 352 ± 16
T 6,129 ± 574*** 3,635 ± 369*** 561 ± 30* 299 ± 22ⁿ
3-14 DPM C 2,700 ± 145 2,623 ± 344 585.5 ± 10 330 ± 9
T 7,232 ± 513*** 5,039 ± 971*** 574 ± 23ⁿ 315 ± 13ⁿ
15-25 DPM C 4,862 ± 338 1,837 ± 281 723 ± 20 450 ± 15
T 6,891 ± 2,048ⁿ 3,734 ± 940*** 678 ± 27ⁿ 384 ± 16**
Values are presents as means ± SEM. C—control, T—treatment, Daily—24 h period, DT—day time (daily-night time)—between
04:00 am and 18:00 pm. Rest time is measured in minutes per day and day time. Pre-mixing mean—average of the 20 days prior to
mixing, PM—post mixing. Numbers indication for two-tail P value is between treatment pairs in the same categories: ⁿ—not significant,
*P ≤ 0.05, **0.001 < P < 0.01, *** P < 0.0001.
410 Can Dietary Pomegranate Peels Reduce Stress Responses Associated with Group Mixing of Holstein
beef Calves?
Fig. 5 The effect of dietary CPE on plasma anti-oxidative
capacity of Holstein bull calves.
1 DBM—one day before mixing, 1 and 3 DPM—one and three
days post mixing. T-test result of ANOVA repeated
measurement revealed an effect of treatment alone (P = 0.04),
but no interaction between treatment and days in relation to
mixing (P = 0.8). CPE—concentrated pomegranate peel extract.
LDCL—luminol dependent chemiluminesce. Please note that
lower LDCL counts represent higher anti-oxidative capacity.
in the 3-14 and 15-25 DPM (Table 2). In addition, the
rest at night-time was longer for all calves, with a
non-significant tendency of the C group to lie down
longer than the T group.
Rest-bouts, which are positively correlated with
rest-time [32] decreased 24 h PM and then calves
gradually restored their pre-mixing values 15-25 DPM.
Daily, day-time and night-time number of rest-bouts
were similar in the two groups in all periods. However,
the impact of mixing regardless of treatment was
clearly noticeable (P < 0.0001).
The average daily rumination duration was affected
by mixing (the difference between 20 DBM and 24 h, 2,
3-14 and 15-25 DPM were significant regardless of
group identity; P < 0.0001), however, it did not differ
between groups. Prior to mixing, the C and T groups
ruminated 306.6 ± 24 and 330.5 ± 18 min/d,
respectively (P = 0.5). 24 h PM, rumination activity
took place for only 175.8 ± 17 and 157.5 ± 17.8 min/d
for the C and T groups, respectively (P = 0.3);
rumination increased back to its initial pre-mixing
values 24 h-2 DPM and 3-14 DPM (290.8 ± 12 and
295.8 ± 19 min/d, P = 0.4), for the C and T groups,
respectively. Interestingly, during the of 15-25 DPM
period, rumination differed significantly among groups
(333.6 ± 24 m/d and 349.8 ± 18 m/d for the C and T
groups, respectively (P = 0.04).
3.4 Results of Meat Quality Analyses
There were no differences between groups in the
following parameters: DM, ash, water content, protein
and fat in meat (P > 0.05). The average pH values for
all meat samples regardless of treatment were 6.5 ±
0.02. While meat pH values of the T group were 6.55 ±
0.03, pH of the C group was 6.60 ± 0.03. These values
are higher in comparison with LTL of other Holstein
calves (where 60% of carcasses had pH < 6 as oppose
to 21% of carcasses in the current trial) reared, handled
and slaughtered under the same conditions (P = 0.009).
The overall fat percentages dressed out of the net
carcass weight did not differ between the groups (P >
0.05).
The composition of FAME detected in the meat did
not differ among groups. The TBARS values of the C
and T groups on D1, D4, D9, D16 and D31 post
thawing were similar. The results of the repeated
measurements test showed that the rise with time after
thawing was significant (P < 0.0001); however, there
was no effect of the treatment (P = 0.8) nor was there
an interaction between time and treatment (P = 0.9).
4. Discussion
4.1 Effect of CPE Supplementation on Behavior and
Metabolism
NEFA (non-esterified fatty acids) are released from
lipid stores and oxidized in the liver as an alternative
energy source. Thus, its concentration in the plasma is
an indication for lipid mobilization to overcome
current demand for energy [33]. The T group had
higher circulating levels of NEFA compared to the
control group. This could be explained by either direct
CPE effects on fat metabolism, androgenic effect of
CPE on NEFA concentration, or a combination of both.
Dietary pomegranate peels were shown to modulate
carbohydrate and fat metabolism [34]. Indeed,
decreased levels of blood triglycerides, cholesterol and
 Can Dietary Pomegranate Peels Reduce Stress Responses Associated with Group Mixing of Holstein 411
beef Calves?

glucose were demonstrated following pomegranate 4.2 Effect of Mixing on Behavioral and Physiological
consumption [34, 35]. Various mechanisms of action Measures
might explain these effects. Among them one can
Group mixing was accompanied by a high degree of
include metabolic effects such as: (1) suppression of
physical activity, which gradually decreased during the
energy intake and inhibition of pancreatic lipase
post-mixing period. The social and environmental
activity, leading to decreased absorption of dietary fat
changes which are associated with the establishment of
[36], (2) changing the interplay between the metabolic
group social hierarchy [46], were behaviorally
hormones leptin, insulin (decrease of both) and
characterized by aggressive butting, pushing, frequent
adiponectin (increase) [37], and (3) activation of
mounting and increased vocalization (data not shown).
PPARα, PPARβ/δ and PPARγ [38, 39], the central
Warriss et al. [47] reported that behavioral interactions
modulators of lipid and carbohydrate metabolism.
and associated physical activity occurring during group
However, dietary CPE could have also influenced
mixing had led to a considerable rise in plasma creatine
calves lipid metabolism through regulation of the
phosphokinase activity and free fatty acid
androgen hormone testosterone. In the current study,
concentration. In the present study we observed a
plasma testosterone levels of the T calves were higher
noticeable effect of mixing on activity as judged by the
than those of the C group. This, in turn, was also
number of increased steps along with a tendency of
reflected in the significantly higher activity (already
decreased rest-time. This stressful stimulus was further
pre-mixing), and the positive correlation between the
pronounced by increased plasma NEFA levels and
testosterone levels 3 months before mixing and activity
decreased rumination and weight gain that we assume
1 and 3 DPM, suggest together that CPE might have
were a result of decreased feed intake. These factors
potentially altered calves temperament and may be
similarly affected both groups with no apparent
used in order to predict stress response to mixing
advantage for the CPE calves. Interestingly, however,
months before it occurs. Also in previous studies
the significantly higher activity levels of the T group
pomegranate supplementation was associated with
was not further reflected by an attenuation of their daily
increased plasma testosterone levels [40] and activity
rumination time, nor by decreased weight gain,
[41]. Testosterone administration to non-obese aging
compared with the control. It is thus hypothesized that
men prevented visceral fat accumulation [42].
CPE might have contributed to higher efficient
An androgenic effect attributed by testosterone was
utilization of the diet by the T group.
previously suggested to cause higher frequency of
interactions, aggression and movement [43]. 4.3 Effect of CPE on the Antioxidant Capacity of Serum
Rebuffé-Scrive et al. [44] demonstrated that and Meat
administration of testosterone led to decrease of Several studies demonstrated a higher anti-oxidant
abdominal adipose tissue LDL, as well as increased activity of the pomegranate peels in relation to the juice
lipolytic responsiveness to norepinephrine. [48], mainly due to water-soluble polyphenols,
Interestingly, a reciprocal effect of free fatty acids on anthocyanins and hydrolyzable tannins [49, 50].
testosterone metabolism was reported in vivo [45]. Phenolic compounds attain their active anti-oxidant
However, weight gain, fat depots, as well as fat content activity through free-radical scavenging activity [51],
and composition in the meat did not differ among transition-metal-chelating activity [52] and/or
groups, perhaps since pomegranate effect on fat singlet-oxygen quenching capacity [53]. These
metabolism is localized to specific fatty tissue such as mechanisms may explain the results obtained herein
sub-dermal or abdominal fat [44]. for the T calves which serum was characterized by
412 Can Dietary Pomegranate Peels Reduce Stress Responses Associated with Group Mixing of Holstein
beef Calves?
higher anti-oxidative activity, in comparison with
control. Recent studies from our research team in dairy
cows [18] demonstrated that milk anti-oxidative
potential can be improved in response to dietary CPE
supplementation. Thus, we hypothesized that similar
effects might have also been attained in meat. However,
the results shown herein raise the possibility that
dietary CPE components are not accumulated in meat,
as in the case of milk. It cannot be excluded, however,
that the timing of group mixing might have obscured
the beneficial contribution of edible CPE on meat
anti-oxidative parameters. Indeed, the potential of
pomegranate was uncovered in a study which tested the
in vitro effect of pomegranate on goat meat patties. It
was shown that the average TBARS values during
refrigerated storage were lower in these tested samples
[15]. Similar effects were reported also in the case of
chicken patties [16], however, other dietary
anti-oxidant did not affect the total anti-oxidant status
of the plasma and meat pH [53]. These discrepancies
may arise from the different application methods used.
The dietary concentration of CPE (4% of DM of TMR)
we used during the experiment may have not been
sufficient for proper anti-oxidative effect on meat lipid
oxidation. Generally, tannins that are abundant in
pomegranate peels are known to bypass the rumen and
are absorbed in the small intestine [54]. Punicalagin,
which is responsible for about half of the total
anti-oxidant capacity of the juice [50], is highly
bioavailabile, since it can be completely absorbed as an
intact molecule in the plasma [55]. However, it can also
be hydrolyzed in cells to yield ellagic acid [56] and
only traces of punicalagin metabolites can be detected
in tissues [57, 58]. It was suggested that the poor
absorption of ellagic acid may prevent tissues from
attaining sufficient concentrations for further effects
[59]. Again, it cannot be excluded that the timing of
group mixing might have obscured the beneficial
contribution of edible CPE. Hence, further research is
necessary to ascertain whether feeding CPE at higher
levels or perhaps in different forms, may potentially
enhance the quality of fresh meat. This should be
considered within a broader management practice
which would also include timing of groups mixing.
4.4 Effect of Mixing on Meat pH
DCB (dark cutting in beef) is a state in which the
ultimate pH post mortem measured after 12-48 hours is
≥ 6. This can occur when animals are exposed to
chronic or long term stress before slaughtering [60].
The meat pH measurements in the current study are
considered higher than recommended (mean ± SEM;
6.5 ± 0.02), and we assume that this might be related to
the mixing that had occurred 34 days pre-slaughter.
Mixing unfamiliar beef bulls shortly or immediately
before loading led to difficulties in unloading them
from the truck [47] and reduction of meat quality [6,
61]. Evidence in the literature as for the recommended
period between group mixing and slaughter are not
unequivocal; looking for methods to prevent DCB,
Warriss et al. [47] found that, two days were required
for unfamiliar young bulls to recover from mixing
stress, by means of sufficient replenishment of muscle
glycogen stores and pH < 6. Similarly, Tennessen et al.
[62] observed that 10 days post-mixing, bulls showed
very little aggressive behavior. Yet, the process of
restoring a new social hierarchy for stranger bulls can
take at least 12 weeks [63].
Based on the precise activity data monitored in the
present study, calves do not seem to restore their
pre-mixing behavior even 25 d post mixing and their
pH levels after slaughter were higher than 6, even when
slaughter occurred 34 days post mixing. These
discrepancies might be explained by differences in
breeds, rearing and slaughter practices, however,
further investigations are required to explore this issue.
5. Conclusions
Mixing of unfamiliar calves has strong behavioral
and physiological implications. This was demonstrated
by the increased steps, NEFA levels, anti-oxidative
activity and decreased rest, rumination and weight gain.
 Can Dietary Pomegranate Peels Reduce Stress Responses Associated with Group Mixing of Holstein
beef Calves?
Speaking of meat quality traits, the consequences of
mixing may have further led to improper pH levels of
the meat after slaughter. From this point of view, the
activity monitoring systems utilized hereby and
testosterone levels, may serve as predictive tool for the
recovery period needed from mixing to marketing.
Dietary supplementation of 4% CPE during 8
months, before marketing, did not prevent reduction of
meat quality traits as judged, at least, by its pH values.
On the other hand, CPE was found to increase the
calves steps, plasma testosterone levels and
anti-oxidative capacity. Apparently, CPE
supplementation likely benefits calves coping with a
stressful event, however, these beneficial effects might
have gone obscured due to improper timing of group
mixing, or relative to the distance from slaughter. Yet,
further investigations are required in order to find out if
a different combination of CPE concentration and
mixing management would be expressed in
enhancement of meat quality.
Acknowledgements
We would like to express our appreciation to “Tnuva”
abbatoir staff, especially to head veterinarian, Dr.
Wassim. We would also like to express our gratitude to
Gan Shmuel Food Ltd. for their cooperation on this
study.
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Journal of Pharmacy and Pharmacology 2 (2014) 416-421
D DAVID PUBLISHING

Immune System and Body Defence Enhancement

Effects of Oral Dihydroartemisinin in Wistar Albino Rats

A. Utoh-Nedosa Uchechukwu1, A. Akah Peter2, Nedosa S. Kenechi3, Anowi F. Chinedu4, Adeyanju N.

Oluwafemi5, Nedosa V. Ikenna6, Onyekwelu N.A. 7 and Ojemudia Thiophilus5
1. Department of Pharmacology and Toxicology, Nnamdi Azikiwe University, Awka, Anambra State, Nigeria.
2. Department of Pharmacology and Toxicology, University of Nigeria, Nsukka, Enugu State, Nigeria
3. Evangelical Churches of West Africa Hospital (ECWA), Egbe, Kogi State, Nigeria
4. Department of Pharmacognosy and Traditional Medicine, Nnamdi Azikiwe University, Awka, Anambra State, Nigeria.
5. Department of Parasitology, Veterinary Research Institute, Vom, Plateau State, Nigeria
6. Department of Industrial Microbiology, Federal University of Technology, Owerri, Imo State, Nigeria
7. Department of Biochemistry, Nigerian Institute of Trypanosome Research, Vom, Plateau State, Nigeria

Abstract: Some drugs like clozapine, interferons and cyclosporine affect the number and function of white blood cells. This study
examined the effect of oral dihydroartemisinin on the white blood cells; the lymph and intestinal glands of the intestinal wall and the
open circulation of the spleen of Wistar albino rats. Five dosages of oral DHA (dihydroartemisinin), 1, 2, 60 and 80 mg/kg were
administered for 5 days or 7 days to 10 sets of 5 test rats weighing 104-106 grams. Equivalent doses of distilled water were given to 4
rats of similar weight and age to serve as controls in each of these tests. A group of five test and four control young adult albino rats
which weighed 75-90 grams were given a repeated dose of the 1 mg/kg oral DHA with a rest period of 1 week between the two dosage
regimens. The results of the study showed that oral dihydroartemisinin treatment produced highly statistically significant increases in
the percentage neutrophil count (P < 0.01); the percentage lymphocyte count (P < 0.01, P < 0.03); the percentage monocyte count; the
population of the cells of the intestinal glands and intestinal solitary and aggregated lymph glands; the number of the cells of the slow
and open circulation of the spleen of the dihyrdroartemisinin-treated rats in comparism with the controls. These increases were dose,
dose repetition and time dependent. The results suggest that oral dihydroartemisinin treatment had immune defence enhancement
effects in the treated rats.

Key words: Immune system enhancement, body defence, oral dihydroartemisinin, rats.

1. Introduction which is referred to as the possession of innate or

natural immunity. Cytotoxic T lymphocytes, for
The body of animals is continually invaded by
example, are the body’s main defence against
micro-organisms which grow on its skin and mucous
intracellular viruses and the blood filtering action of the
membranes from which they can spread to the interior
spleen removes spherocytes and other abnormal red
of the organism. The blood and tissue fluid of animals
cells [1-4]. The skin and the mucous membranes form
usually contain nutrients sufficient to sustain profuse
the first line of defence of the body of animals against
growth of micro-organisms, fungi, yeasts and parasitic
injury and infection. The lymph nodes of the intestinal
infestations. The healthy non-immunized animal body
wall also play important roles in maintenance of the
usually has a number of defence mechanisms that
immune system. The neutrophils of the white blood
enable it to defend itself against microbial invasion
cell population are the major fighters of the body
involved in chemo tactic defense of the body.
Corresponding author: A. Utoh-Nedosa Uchechukwu,
Ph.D., research fields: phamacology and toxicology. E-mail: Lymphocytes are the key components of the immune
unedosa@yahoo.com.
 Immune System and Body Defence Enhancement Effects of Oral Dihydroartemisenin in Wisara Albino
Rats
system as they have the remarkable ability to produce
antibodies against millions of foreign agents. These
antibodies not only kill the invading agents but also
memorize the structure of the invading organisms and
use this information to eliminate the invaders next
time they invade the host organism. The slow
component of the spleen circulation in which blood
leaves arterioles and percolates through large
numbers of phagocytes and lymphocytes before
entering the sinuses of the spleen to pass back into the
general circulation is a significant component of the
immune system.
Some drugs can impact on the number and function
of white blood cells. For example, clozapine, an
antipsychotic drug has a rare adverse effect of causing
total absence of all granulocytes (neutrophils,
basophils and eosinophils). Interferons used to treat
multiple sclerosis like rebif, avonex and betaseron as
well as immunosuppressive drugs like sirolimus,
mycophenolate mofetil, tachrolimus and cyclosporine,
can cause leucopenia [3-5].
We examined the effects of oral dihydroartemisinin
on some tissues of the intestinal wall, the white blood
cells and the slow open circulation of the spleen that
are involved in the body’s defence.
This study examined the effect of oral
dihydroartemisinin on the monocytes, neutrophils and
lymphocytes of the blood; on the intestinal and lymph
glands of the intestinal wall and on the lymphoid
tissues of the non-enclosed circulation of the spleen.
This study examined the effects of oral
dihydroartemisinin on some tissues of the intestinal
wall, the white blood cells and the slow open
circulation of the spleen that are involved in the body’s
defence.
2. Materials and Methods
The effects of four dosages of oral
dihydroartemisinin on the monocytes, neutrophils and
lymphocytes of the leukocyte population; the intestinal
exocrine/lymph glands and on the cells of the open
417
circulation of the spleen; were evaluated with 5
dosages of DHA on five adults test and 4 control rats
which weighed 104-106 g.
The dosages of oral DHA used in the test were 1, 2,
60 and 80 mg/kg and they were administered for 5 days
or 7 days to a group of the test rats. A group of 5 test
and 4 control young adult albino rats which weighed
75-90 g were given a repeat dose of the 1 mg/kg oral
DHA with a rest period of one week between the
administration of the two dosage regimens. The control
rats were given orally administered distilled water in
each experiment. The drug and distilled water were
administered to the test and control rats by oral
intubation.
Blood samples from the test and control rats were
collected from the subclavian artery 24 h after the
administration of the last dose of each dosage regimen
of DHA tested and put into blood collection bottles
which contained EDTA anti-coagulant.
The spleen and strips of the intestine of the sacrificed
test and control rats were collected and put into after
their gross anatomical examinations.
Photomicrographs of the tissues of these organs were
prepared and the differential white blood cell count of
the test and control rats were determined using
conventional histological photomicrograph preparation
and haematological methods.
3. Results
The results of the study showed that oral
dihydroartemisinin treatment produced a highly
statistically significant increase in the percentage
neutrophil count (P < 0.01) as well as in the percentage
lymphocyte count (P < 0.01, P < 0.03) (Figs. 1 and 2)
in treated rats which were absent in controls. It also
caused increases in the percentage monocytes count,
but their numbers in the sample were too small for their
level of significance to be computed.
The enhancement effect of DHA on the percentage
neutrophil and percentage lymphocyte counts were
dose, dose repetition and time dependent. The 2 mg/kg
418 Immun
ne System an
nd Body Defen
nce Enhance
ement Effects
s of Oral Dihy
ydroartemisen
nin in Wisara
a Albino
Rats

dose was thee maximal response dose of the populaation
increase effeects of DHA on the percenntage neutropphils
count and thhe percentage lymphocyte count.
Oral dihhydroartemisinin treatment also cauused
noticeable inncreases in thhe population of the cells of the
open circulaation part of the spleen which
w are staained
yellow in Fig. 4a in compparism with that of the conntrol
(Fig. 4b).
4. Discussiions
White bloood cells aree cells of the immune sysstem
thatt defend the body againsst infectious diseases andd
antiigenic foreignn substances [6]. Several diverse
d typess
of leukocytes
l exxist in the bloood and otherr parts of thee
boddy but they arre all produceed from the haemopoeitic
h c
stem
m cell. There are two broadd groups of leeukocytes thee
phaagocytes and the immunocytes [6]. Granulocytes
G s
whiich include thhe neutrophills, basophils, eosinophilss
andd monocytes, make up thhe phagocytees while thee
lym
mphocytes, thheir precursorr cells and th he mast cellss
mak ke up the imm munocytes [6].
The
T neutrophiils are multi-llobed; make up u 54%-62% %

35
30
25 5
5 days DHA tre
eatment
20
15
10 Control Exp. O
C Of 5 days 
5 D
DHA treatmen nt
0
7
7 days DHA tre
eatment.

Control Exp, off 7 days 
C
D
DHA treatmen nt.
C
Column1

Fig. 1 The effect

e of dihyd
droartemisinin treatment on the mean perrcentage neutroophil count off dihydroartem
misinin-treated
d
and control albino
a rats.

100
900
800
700 5
5 days DHA tre
eatment
600
500
40
0 Control Exp. Of 5 days 
C
300
200 D
DHA treatmen t
100
0 7
7 days DHA tre
eatment.

Control Exp, off 7 days 
C
D
DHA treatmen t.
C
Column1

Fig. 2 The effect

e of dihydrroartemisinin treatment on the mean perccentage lymphocyte count off dihydroartem
misinin-treated
d
and control albino
a rats.
 Immune System and Body Defence Enhancement Effects of Oral Dihydroartemisenin in Wisara Albino 419
Rats

(a) (b)
Fig. 3 (a): there was no multiplication of the cells of the intestinal wall and in the cells of node glands of the intestinal walls of
control albino rats; (b) the effect of dihydroartemisinin treatment caused an increase of the population of the cells of the
intestinal (middle of photomicrograph) and node glands (upper left corner of photomicrograph) of the intestinal walls of
dihydroartemisinin treated albino rats.

(a) (b)
Fig. 4 (a): there was no proliferation of the cells of the slow and open circulation of the spleen of control albino rats, there
was also no expansion of the white blood cell population of the control rats. (b) the effect of dihydroartemisinin treatment
caused an increase in the number of the cells of the slow and open circulation of the spleen in the orange coloured areas of the
photomicrograph of the spleen of dihydroartemisinin-treated albino rats. The treatment also expanded the white blood cell
population in the areas coloured purple (pointed to by two arrows).

of leukocytes in an adult human; have a life span of 6 h Monocytes constitute 2%-8% of leukocytes; have
to few days in spleen or in other tissues and their main kidney shaped nucleus; have a life span of hours to
targets are bacteria and fungi [5, 7, 8]. days; target various infections and foreign antigens and
420 Immune System and Body Defence Enhancement Effects of Oral Dihydroartemisenin in Wisara Albino
Rats
leave the blood stream to become scavenging
macrophages [6-8].
Lymphocytes are the immunologically competent
cells that assist the phagocytes in the defence of the
body against infective organisms and foreign invasion [6].
Lymphocytes contain a large, round deeply staining
nucleus; have a life span of weeks to years; as B cells
target pathogens; as CD4+ (helper) T cells,
intracellular bacteria; as CD8+ cytotoxic T cells, target
virus-infected and tumor cells and as γ δ T Natural
Killer cells, target virus-infected and tumor cells [6-9].
The action of the innate defence system against
micro-organisms that have penetrated into interstitial
tissues and the vascular compartment is largely
phargocytosis and activation of the complement
pathway. The actions of the innate system when
exposed to microbial infection are also critical to the
recruitment and activation of cells of the adaptive
immune response.
The main cells that mediate phagocytosis are
mononuclear phargocytic and granulocyte cell
populations (especially, the neutrophils) [3]. The cells
that mediate innate immunity include neutrophils,
macrophages, natural killer cells, cytotoxic T-cells and
non-proffessional immunocytes like
epithelial/endothelial cells [3].
Lymphocytes transform to B lymphocytes in the
foetal liver and after birth, in the bone marrow. T and B
lymphocytes are morphologically similar but can be
identified by markers on their membrane [1].There are
three types of T cells, cytotoxic T cells, helper T cells
and memory T cekks while B cells differentiate into
plasma cells and memory B cells [1].
Thymocytes are cells that develop in the thymus and
develop as T-cell precursors. In immunossupression,
the antibodies developed against T-lymphocytes bind
to the surface of circulating T-lymphocytes which then
undergo various reactions like complement-mediated
destruction; antibody-dependent cytotoxicity;
apoptosis or opsonization [2- 4, 6].
There are several lymphoid tissues in the body. They
include the spleen, the thymus, the lymph nodes, the
payer’s patches, the tonsils, the adenoids and the
appendix [6, 9, 10].
Some leukocytes migrate into tissues of the body to
take up permanent residence in these tissues examples
of which are the Kupffer cells of the liver, histocytes,
dendritic cells, microglia and mast cells. Such migrated
leukocytes still serve a role in the immune system, in
their new residence although some like dendritic cells
re-migrate to local lymph nodes on ingesting antigens.
Lymphoid tissues act as obstruction to disease
infective organisms; foreign particles and chemical
poisons. In diseases like small pox, typhoid fever and
scarlet fever, many of the lymph nodes scattered all
over the body, are inflamed, swollen, painful and
tender [9, 10].
Acquired immunity responses which are
antigen-specific responses are mediated primarily by
lymphocytes. The MHC (major histo-compatibility
complex) protein-peptide on the surface of the
antigen-presenting cells bind to appropriate T cells,
hence T cells must recognize a very wide variety of
complexes.
Immunosuppressant drugs like calcineurin inhibitors,
anti-proliferative drugs, glucocorticoids and
immunosuppressive anti-bodies inhibit cellular or
humoral or both immune responses. Cyclosporine, for
example, selectively suppresses cell-mediated
immunity, prevents graft rejection and yet leaves the
recipient with enough immunity to fight bacterial
infections [2]. The major adverse side effect of
cyclosporine is nephrotoxicity [4]. Many of the adverse
effects of cyclosporine are dose dependent [4].
Corticosteroids have potent immunosuppressant and
anti-inflammatory effects as they inhibit several
aspects of the immune response especially the MHC
expression and proliferation of T lymphocytes and
depression of production of adhesion molecules [1-4].
Corticosteroids are widely used as companion drugs
to cyclosporine in various organ transplants.
Immunosuppressant anti-metabolite agents are
 Immune System and Body Defence Enhancement Effects of Oral Dihydroartemisenin in Wisara Albino
Rats
generally used in combination with corticosteroids and
the calcineurin inhibitors, cyclosporine and tacrolimus
[1-4].
The immunosuppressant antimetabolite (nucleotide
analog) is rapidly proliferated in the immune response
and their dependence on the de novo synthesis of
purines required for cell division, predominantly halts
the production of lymphocytes required for the immune
response [1-4].
The proliferative effect of oral dihydroartemisini
obtained in this study shows that it can counter many of
the adverse effects of immunosuppressant agents.
5. Conclusions
Comparism of the results in test and control rats in
this study enable us to conclude that the proliferative
effects of oral dihydroartemisinin on the neutrophil,
monocyte (Fig. 1) and lymphocyte populations (Fig. 2)
of the white blood cells; on lymphoid tissue cells of the
spleen (Fig. 4); on intestinal mucus gland cells (Fig. 3)
and on the cells of the intestinal lymph glands (Fig. 3)
of albino rats demonstrated that dihydroartemisinin
produced body defence and immune defence
enhancement properties in dihydroartemisinin
421
treated rats.
References
[1] Barrett Kim, E., Barman, M. S., Boitano, S., and Brooks H.
L. 2010. Ganong’s Review of Medical Physiology, 23rd
ed., Tata McGraw Hill, New Delhi, New York.
[2] Tripathi, K.D. 2006. Essentials of Medical Pharmacology,
St Louis: Jaypee Brothers Medical Publishers (P) LTD.
[3] Denyer, P. S., Hodges, A. N., and Gormmon, Sean. P.
2004. Hugo and Russell’s Pharmaceutical Microbiology.
7th ed., Blackwell Science Ltd and Blackwell Publishing
Company.
[4] Richard, A. H., Clark A. M., Finkel R., Rey, A. J., and
Whalen, K., eds. 2012. Lippincott’s Illustrated
Pharmacology Reviews, 5th ed., Baltimore: Wolters
Kluwer and Lippincott Williams and Wilkins.
[5] Albert, B. 2005. Molecular Biology of the Cell. NCBI
Bookshelf. Accessed April 14, 2007.
[6] Leif, J. 2000. Semester 4 Medical lectures at Uppsala
University.
[7] Gartner, L. P., and Hiatt, J. L. 2006. Colour Textbook of
Histology, 3rd ed., Saunders, 225.
[8] Victor D., Paul W., Burkitt, R., and George, H. 1979.
Functional Histology: A Text and Colour Atlas. Edinburgh:
Churchill Livingstone.
[9] Tripathi, K. D. 2006. Essentials of Medical Pharmacology,
St Louis: Jaypee Brothers Medical Publishers (P) LTD.
[10] Hoffbrand, A. V., and Pettit, J. E. 1993. Essential
Haematology. 3rd ed., Oxford: Blackwell Science Ltd.
Journal of Pharmacy and Pharmacology 2 (2014) 422-431
D DAVID PUBLISHING

Neuropathology of Schizophrenia

Villeda-Hernández Juana1, Sánchez Martínez Rodrigo2, Totxo-Guerrero Sebastián3, Manzanarez-Colin Mariel

Carolina3, Palacios-Escalona Sergio3, Franco-Del Toro Perla Guadalupe4, Cuevas-Nuñez Flora Minerva4,
Alonso-Zuñiga Rosa Emma5, Peralta-Rodríguez Brenda1 and Rembao-Bojórquez Daniel1 Tristán-Agundis Ma.
Francisca2
1. Department of Neuropathology, National Institute of Neurology and Neurosurgery, Distrito Federal 14269, Mexico
2. Faculty of Medicine, National Autonomous University of México, Distrito Federal 03000, Mexico
3. School of Medicine, Guadalajara University, Guadalajara 44100, México
4. School of Medicine, San Luis Potosí University, San Luis Potosí 78000, México
5. Department of Pathology, Fray Bernardino Álvarez Psychiatric Hospital, Distrito Federal 14000, Mexico

Abstract: The paper is to study the structural changes in cortex and cerebellum of autopsies of subjects with schizophrenia. In the
Pathology Department of the Psychiatric Hospital “Fray Bernardino Álvarez”, there are 410 autopsy cases with diagnosis of mental
disease, from which 52 match schizophrenia. The aim of this work is to describe structural changes in the autopsy material and make
markers in order to confirm neurodevelopment alterations. The tissues were preserved in formol, paraffin-embedded; cut into 5-7 µm
slices; stained with H-E (Hematoxilin and Eosin), Bielschowsky, crystal fast of violet. The observed changes were brain asymmetry,
cortical dysplasia, neuronal distortion, axon bifurcation, neuronophagia and generalized demyelination. Schizophrenia is the result of
several happenings, it is important to devise new treatment strategies for prevention and genetic counseling for population.

Key words: Schizophrenia, cortical dysplasia, heterotopias, migration failure.

1. Introduction on some toxic action or anatomical process [6].

According to epidemiologic, clinical, genetic and
Schizophrenia is a severe and chronic mental illness
pathological researches, the application of such
(or group of illnesses) associated with high prevalence
techniques to populations of relatives that are at
(about 0.5% to 1% population worldwide). Symptoms
increased risk of developing schizophrenia because of
of schizophrenia typically emerge during adolescence
their genetic and shared environmental relationship to
or early adulthood [1], is characterized by profound
someone with the illness has not yet been reported [7].
disruptions in the thinking, language, perception,
Studies of neuroanatomical asymmetries in the
represents the 14th leading cause of years of life lost
relatives of patients with schizophrenia would provide
due to disability at global level [2, 3]. Kraepelin in
critical information about whether genetic influences
1919 [4] defined the nature of dementia praecox as
are a likely contributor to asymmetry disturbances in
almost certainly involving heredity and as a “tangible
schizophrenia. In-vivo brain mapping studies tend to
affection of the brain, probably damage or destruction
group brain structures into two broad categories: (1)
of cortical cells and chemical disturbances”. Bleuler,
cortical mantle; (2) deep nuclei or structures
in 1911 [5] wrote about the etiology of schizophrenia:
represented by a surface with globular shape at a level
one must acknowledge that, at least the great majority
of magnetic resonance image analysis. Previous
of clinical pictures, the name of dementia praecox, rest
volumetric studies have revealed volume reduction in
the cortical area, especially the temporal lobe [8, 9].
Corresponding author: Tristán-Agundis Ma. Francisca,
profesor, research feild: pathology. E-mail:
As novel evidence emerge of the schizophrenia is
hipocrates07@yahoo.com.
 Neuropathology of Schizophrenia
the early neurodevelopment disturbances [3] new
genes involved with neural proliferation, migration or
synapse formation are discovered.
The most robust and important histological findings
in schizophrenia are both negative. The
neuropathology of schizophrenia is not degenerative.
There are no discrete lesions such as neurofibrillary
tangles amyloidal plaques, Lewy bodies or other such
features visible using a range of routine and
immunocytochemical stains, which would indicate the
presence of any known neurodegenerative process.
Importantly, this conclusion applies even to patients
with schizophrenia who had sufficiently severe
cognitive impairment to warrant the label of dementia [7].
There is no gliosis (the proliferation and hypertrophy
of astrocytes, the supporting cells of the brain) in
patients with schizophrenia. Gliosis is a sign of
inflammation, injury or other ongoing pathological
processes. Hence, the lack of gliosis is taken as a sign
that the disorder is likely to be neurodevelopmental in
origin, affecting mechanisms involved in the normal
maturation of the brain [7].
Also, neuropathological changes have been
observed and studied by several authors. Brain bulk
decrease was described since the decade of the 1920s
and, since the developing of magnetic resonance,
dimension decrease of temporal lobe, hippocampus
and amygdala were more specifically described
[10,11]. Gray matter bulk reduction and ventricle
dimension increase have been described, too.
The histological findings in the two limbic regions
consisted mainly of poorly developed structure in the
upper layers, with a heterotopic displacement of single
groups of nerve cells in the entorhinal region.
Particularly, the disturbed structure of the second layer
Pre-alpha in medial and central fields of the entorhinal
region, situated in the parahippocampal gyrus (group
2a), suggests a disturbance of neuronal migration in a
later phase of cortical development [12], were
reported heterotopias and irregular arrangement or
neurons, invaginations in the entorhinal cortex
423
surface, and heterotopias of layer II neurons. Nieto in
1972 [13] found fibrillary gliosis (reactive astrocytosis)
in 70% of their cases of schizophrenia and Tristán et
al. [11] published the first article in Mexico about
histopathological changes in schizophrenia.
The aim of this paper is to study of structural
changes in cortex and cerebellum of autopsies of
subjects with schizophrenia.
2. Materials and Methods
In the Pathology Department of the Psychiatric
Hospital “Fray Bernardino Álvarez”, there are 410
autopsy cases with diagnosis of mental disease, from
which 52 match schizophrenia; for this work, we
present 12 cases. There is a subtle number
predominance of the female cases in the study group
and the ages range between 14 and 69 years old, being
the first psychotic episode the most common between
the ages of 15 to 20. The cortex, the hippocampus and
the cerebellum were studied.
The brain of regions (cortex, cerebellum) was
paraffin embedded and cut on a rotary microtome.
Paraffin sections were cut at a thickness of 5-7 µm
prior to staining; sections were mounted from distilled
water onto gelled slides. Gelatin coated slides were
prepared by immersion in a 35 °C solution of 1% pig
skin gelatin (Sigma; type A, 300 Bloom) and then
oven dried overnight at the same temperature. The
sections were mounted onto the slides from distilled
water and then air dried for at least 30 min on a slide
warmer at 35 °C. The sections are first deparaffinized
trough two 10 min changes of xylene and then the
sections are rehydrated through a graduated alcohol
series, omitting the basic alcohol solution, once in
distilled water, Harris hematoxylin will stain
everything on the slide and hold fast to the tissue
when rinsed, therefore, after staining and rinsing with
water, the next step is to differentiate or take out the
excess hematoxylin from everything except the
nucleus, the slides are agitated in a mild acid alcohol
solution that slowly removes the excess hematoxylin,
424 Neuropathology of Schizophrenia
but this is generally addressed by standardizing a set
procedure that will remove the optimal level of dye,
after differentiating the slides are rinsed and placed in
a bluing solution (ammonia water), which will cause
the nucleus to turn a deep purplish blue color. Sigma
has Harris hematoxylin in their product line for
laboratories that prefer regressive staining, after
hematoxylin staining the slides is rinsed, stained in
eosin, dehydrated with graded strengths of alcohols,
cleared in xylene and mounted.
For Bielschowsky technique, the sections are
washed in 4 changes of distilled water for 30 minutes
and placed in a coupling covered with 20% silver
nitrate solution at 37 °C for 15 min, washed in
distilled water; then placed in freshly prepared
ammoniacal silver solution for 10 minutes or until the
sections ceases to become darker, washed quickly in
distilled water, reduced in 10% neutral formalin in
tap water for about 10 minutes to 37 °C, and placed in
5% sodium thiosulfate solution to remove any
unreduced silver wash well in distilled water,
dehydrated clear and mount. For PAS technique: the
section is placed in 0.5% periodic acid solution for
15 minutes, washed in distilled water; then placed in
Schiff¨s reagent about 15 minutes, washed in hot
distilled water (40 °C) for 20 minutes; and then
counterstained with hematoxilin about 15 seconds,
dehydrate, clear, and mount. Analyzed by Leica
microscope tree channel system (red, green, blue)
color Sony video camera (SSC-DC14 Japan)
connected to the microscope through and adapter with
color neutral optics. A true-color image analyzer,
IM1000 (Leica, Cambridge, UK), was used.
3. Results
3.1 ANP-79
A 28 years old woman, she was no family history of
mental illness. She was the youngest of three brothers.
Her psychobiology development was normal until the
onset of her symptoms. Her first psychotic episode
was at the age of 28 years; she began with soliloquies,
and catatonia. She was referred to this institution at
admission that she was with mutism, catatonia, and
hallucinations.
The patient died five days after her admission.
3.1.1 Macroscopic Examination
The fixed brain weighed 1,450 g. There were focal
areas of lissencephaly in the temporal lobes, and in the
occipital region pachygyria was found.
3.1.2 Microscopic Examination
Cortical dyslamination, hypertrophic and
dysmorphic neurons with enlarged cell bodies as well
as nuclei and aggregates were visible, cytomegaly,
loss and acute neuronal necrosis with shrunken,
hyperchromatic and ballooned cells were identified in
affected areas. The cerebellar examination revealed
Purkinje’s cell depopulation (Fig. 1).
3.2 ANP-195
A 42-year old woman, here was family history of
mental illness by his mother; two of her aunts and her
sister suffered seizures. Little was known about her
birth or her early development. Along life she was
quiet and shy. She developed as clerk for 17 years.
She rarely consumed alcoholic beverages.
Her first psychotic episode was at the age of 40; she
began with visual, auditory hallucinations,
megalomaniac, delusional thought, and negativism. She
received antipsychotic medication for one year. At
admission she was excited, hyperkinetic, disoriented in
time and place, and referred visual and auditory
hallucinations. The neurologic examination just revealed
reflex exacerbated. During her hospitalization, the patient
was treated with antipsychotic medications and hospital
discharged after three months. The patient returned to
hospital few days later in comatose state and died.
3.2.1 Macroscopic Examination
The fixed brain weighed 1,500 g. There were areas
of subarachnoid hemorrhage at the parietal lobes and
the left hemisphere was larger in its antero-posterior
axis than the right hemisphere. There were focal areas
of lissencephaly in frontal, temporal and parietal lobes.
At coronal section, the left hemisphere was larger with
 Neuropathology of Schizophrenia 425

Fig. 1 ANP-79 (A) Brain with of lissencephaly; (B) hemispheric asymmetry; Cortex (C) and (D) dysmorphic neurons and
balloon cells (red arrows) (C) Hematoxilin-Eosin and D) violet cresil ) 40X.

Fig. 2 ANP-195, (A) Brain with lissencephaly; (B) hemispheric asymmetric; (C) and (D) cx, dysmorphic and balloon cells
were observed (red arrows); (C) Bielschowsky and (D) H-E 40X.
426 Neuropathology of Schizophrenia

asymmetrical thickening of the gray matter, and
hemorrhagic areas.
3.2.2 Microscopic Examination
Cortical dyslamination, neural loss, bipolar neurons,
dysmorphic neurons, neuromegaly, axons in different
directions and heterotopias were described. The
cerebellar examination revealed Purkinje’s cell
depopulation (Fig. 2).
3.3 ANP-227
A 44-year old woman, there was family history of
rheumatic cardiopathy, in her father and her mother,
and a nephew with seizures during the infancy. Her
birth and psychobiological development were
apparently normal. She had good grades at school, at
the age of 33-year old began with physical and verbal
aggressiveness, hypersexuality, soliloquies and
isolation.
Place, indifferent and suspicious. Her answers were
inappropriate during the examination. Neither
delusions nor hallucinations were referred. The
clinical examination reported right central facial palsy,
pupillary reflexes diminished, soft palate asymmetry
and slight edema in both legs. During the
hospitalization, the patient referred visual and auditory
hallucinations. For the next three months, the patient
deteriorated in her medical status and died.
3.3.1 Macroscopic and Microscopic Examination
The brain weighed 1.125g. The left hemisphere was
asymmetric, histopathologic examination revealed a
Cortical dysplasia type IIb characterized by abnormal
cortical lamination, neuromegaly, balloon dysmorphic
cells, axons with different direction, Purkinje cell
heterotopias were also found in the granular layer of
the cerebellum. Neither tumor nor vascular
malformations were found (Fig. 3).
3.4 ANP-303
A 58-year-old man—he was the first in a sib ship of
six. His mother died at 76-years old; apparently she

Fig. 3 ANP-227 (A) The Brain showed asymmetry and lissencephaly; (B) and (C) is observed in the parietal cortex section
dysmorphic and pycnotic neurons and neuromegaly; (D) cerebellum showed Purkinje´s cell depopulation and migration
failure of these cells (red arrows), H-E, 40X.
 Neuropathology of Schizophrenia 427

suffered from psychotic disorders, no psychomotor
development disturbances were reported, during 27
years worked as tourist guide.
The patient had lived an apparently normal life until
the onset of the psychotic episodes at age of 23 years
old, since then he was under medical treatment, until
one month before the hospitalization, when he refused
to take his medication. At admission he was oriented
in time, place and person. The attention and
compression were inadequate, also incoherent and
inconsistent language was reported. No delusions or
hallucinations were found at first. The clinical
examination was normal. While staying at hospital,
the patient seemed to Have hallucinations, during the
clinical interviews he answered with inappropriate
answers and his language continued to be incoherent
and inconsistent. The cognitive functions were
diminished. The patient died after three months.
3.4.1 Macroscopic Examination
The brain weighed 1,500 g. Both hemispheres
presented asymmetry. The left hemisphere was shorter
primarily in the frontal and temporal lobes, the
coronal section showed volume of the left hemisphere
decreased and smaller gyri
3.4.2 Microscopic Examination
There was abnormal cortical lamination,
dysmorphic and neural loss, with axons in different
direction, between the second and the third layer
(Fig. 4).
3.5 ANP-336
A 59-year old men—there was no family story of
mental disorder, the birth and the psychomotor
development were normal, at the age of 8-years old he
suffered head trauma with loss of consciousness for
five minutes, the subject was alcoholic and consume
cannabis, at the age of 25 started with visual and
auditory hallucinations, negativism, isolation, and
disorientation in time and place. His cognitive
function was decreased. The patient received
antipsychotic drugs for 17 years. At the moment
of admission in this hospital the patient presented

Fig. 4 ANP-303 (A) and (B) Cortex, dysmorphic and nodular Neurons (circle) with axons in different direction, shrunken
and picnotic neurons (red arrows); (C) cerebellar examination revealed Purkinje cell depopulation, Heterotopias and
hypoplasias cerebellar, dysplasias, and migration disorders 20×; (D) Magnification of “C”, H-E, 40X.
428 Neuropathology of Schizophrenia

hypovolemic shock, secondary to upper
gastrointestinal bleeding, and fluid resuscitation was
started, and during the intervention the patient was
excited and took off the catheter, the subject died four
days after his admission.
3.5.1 Macroscopic Examination
The autopsy revealed pachygyria and lissencephaly
in coronal section, and asymmetrical thickening of
gray matter was observed.
3.5.2 Microscopic Examination
We observed cortical dyslamination, neural loss,
dysmorphic neurons, neuromegaly, demyelination,
with heterotopias in white matter, the cerebellum
showed Purkinje’s cell depopulation and migration
failure of these cells, the folia are short and show a
narrow molecular layer above a crowded row of
Purkinje cells, these cells are often misplaced into the
molecular layer, there are few distinct pericellular
baskets, ectopic granule cell somata can be found at
any level within the molecular layer, fibrillary gliosis
extends through the cortex and white matter (Figs. 5
and 6).
4. Discussion
Neuropathological findings from clinical and
postmortem studies converge in support of the
hypothesis that schizophrenia is associated with
disturbed architecture, abnormal cell arrangement and
heterotopias in cortex and cerebellum, the researchers
should be trained in the morphological study of the
brain and not only in the molecular approach. Our
study confirms the findings of ventricular enlargement
and changes in selected structures in this with
schizophrenia patients.
Schizophrenia is the result of a series of events that
begin with a defective neurodevelopment that, at the
present time, have been demonstrated through
neuroimaging studies [12]. Therefore, it is important

Fig. 5 ANP-336 (A) Brain with lissencephaly; (B) asymmetry; (C) CX Neuromegaly, dysmorphics with shrunken and
hyperchromatic neurons with axons in different direction; (D). Dysmorphic Purkinje cells (red arrows), H-E, 40X.
 Neuropathology of Schizophrenia
Fig. 6 ANP-336, cerebral cortex, astrocytes with augmented activity and size were observed, glia fibrillar acidic protein
40X.
not only to devise new treatment strategies, but also
the prevention then doing genetic counseling in order
to control this disease (considering at least three
generations). Structural alterations are observed in all
brain lobes predominantly in frontal lobe, temporal
and cerebellum in this study. There is asymmetry of
the left hemisphere and hippocampus is reduced in
size. While the presence of an abnormality in the
normative pattern of structural asymmetry in the
siblings of schizophrenia patients may be due to either
genetic or shared environmental factors, prior twin
and adoption studies of subjects with schizophrenia
have indicated that shared environmental factors have
a negligible contribution to overall etiology of
schizophrenia [14]. In the cases studied, there is
enlargement of the ventricles with reduced cortical
volume structural alterations are observed in all brain
lobes predominantly in frontal.
Several works are suggestive of changes in the
microstructure of white matter in schizophrenia.
Myelinated structures are an attractive candidate for
an anatomical substrate for disconnectivity, and may
429
act synergistically with synaptic changes to result in
functional disconnectivity although the relationship
between white and grey matter changes in the illness
remains unclear [15].
The histopathological findings confirm the
diagnosis reported by other researchers [3, 10, 12]. In
most of the cases, there is hemispherical asymmetry
(Fig. 1). All the cases show cortical dysplasia (Fig. 2)
generalized demyelination and neuronal migration
failure was also found. T he histopathological changes
in front temporal areas predominated (Fig. 5).
However, it must be emphasized that there is as yet no
unequivocal neuropathological evidence for the
neurodevelopment hypothesis as would be provided
by, for example, clear and consistent evidence of focal
dysplasias [7]. In the frontal cortex and the temporal
lobe neurons of different size with undulating axons
are seen in different directions. In our study, we
observed neuronal depopulation in the frontal lobe and
Purkinje. These abnormalities suggest a disorder of
connectivity in brain regions arising during embryonic
development.
430 Neuropathology of Schizophrenia
One of the preliminary findings that suggested a
role for astrocytes in schizophrenia was altered cell
density in several brain regions thought to be involved
in the pathophysiology of the illness. Thus, it was
hypothesized that a loss of astrocytes, or compromised
astrocytic functioning in those particular brain regions,
could contribute to brain dysfunction in schizophrenia.
Although, subsequent research has failed to show
prominent changes in astrocyte density in select brain
regions in schizophrenia, an alternate hypothesis
suggests that a global astrocytic lesion may give rise
to brain abnormalities observed in the illness [16] we
find gliosis in all areas studied in patients with
schizophrenia. In direct contrast to gliosis, early
studies of astrocyte-related pathology in schizophrenia
reported the loss of astrocytes in several cortical areas
already known to contain neuronal abnormalities,
including the pole frontal cortical and motor cortex
[17, 18].
Reports of cerebellar pathology in childhood show
remarkable recovery of function with development.
Indeed, cerebellar dysfunction most likely dates to
childhood, and he may have, therefore, compensated
for these deficits over time, although with some
manifestations still measurable. In summary, case is
consistent with a neurodevelopmental syndrome,
representing a unique neurologic variation of
schizophrenia and psychotic illness [19]. In our study,
we found very similar clinical characteristics to those
reported inn others studies. It has been hypothesized
that the disorder originates from brain
neurodevelopmental neuropathology with symptoms
and neuropsychological deficits arising from
alterations in precisely defined brain regions or
functional neuronal circuits [20].
Our study found heterotopias, hypoplasias
cerebellar, dysplasia, and migration disorders existed
in all patients with schizophrenia.
There are several reports that associated this disease
with ventricular enlargement and decreased cortical
volume. The pathology is neither focal nor uniform,
being most convincingly demonstrated in the
hippocampus, prefrontal cortex and dorsal thalamus
[7]. There are several reports of the findings reported
in cortex, thalamus, and hippocampus; however, there
are very few histopathological descriptions of the
cerebellum, so we consider that in this study our
findings in cerebellum are of great importance.
5. Conclusions
The onset of schizophrenia is related to structural
alterations due to a defective neurodevelopment,
which is supported by the findings of this work. With
these data, we believe that we can say the
neuropathology of schizophrenia as a disorder in brain
development.
The authors declare that they have no conflicts of
interest to disclose.
Acknowledgments
The authors thank Alonso-Zuñiga Rosa Emma
Weinberg for comments on the manuscript; Fray
Bernardino Hospital ease by obtaining the autopsy.
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[4] Kraepelin, E. 1919. Dementia –Praecox and Paraphrenia.
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[5] Bleuler, E. 1911. Dementia Praecox or the Group of
Schizophrenias or the Group of Schizophrenias.
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[6] Lehmann, H. E., and Ban, T. A. 1997. “The History of
the Psychopharmacology of Schizophrenia.” Can. J.
Psychiatry 42 (2): 152-62.
[7] Harrison, P. J. 2008. “Neuropathology of Schizophrenia.”
Psychiatry 7 (10): 421-4.
[8] Honea, R., Crow, T. J., Passingham, D., and Mackay, C.
E. 2005. “Regional Deficits in Brain Volume in
Schizophrenia: A Meta-analysis of Voxel-Based
Morphometry Studies.” Am. J. Psychiatry 162 (12):
2233-45.
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Li, T., and Gong, Q. 2009. “Association of Cerebral
Deficits with Clinical Symptoms in Antipsychotic-Naive
First-Episode Schizophrenia: An Optimized Voxel-Based
Morphometry and Resting State Functional Connectivity
Study.” Am. J. Psychiatry. 166 (2): 196-205.
[10] Harrison, P. J. 1999. “The Neuropathology of
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Interpretation.” Brain 122 (4): 593-624.
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Guevara, J. 2005. “Histopathology Changes in the
Central Nervous System in the Patients with
Schizophrenia.” Psiquis.14 (4):98-102.
[12] Jakob, H., and Beckmann, H. 1986. “Prenatal
Developmental Disturbances in the Limbic Allocortex in
Schizophrenics.” J. Neural. Transm. 65 (3-4): 303-26.
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Pathology of the Nervous System, edited by Minckler, J.,
New York: McGraw Hill.
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Individuals with Schizophrenia and Their Non-psychotic
Siblings.” Neuroimag. 47 (4): 1221-9.
[15] Walterfang, M., Wood, S. J., Velakoulis, D., and Pantelis,
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918-48.
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[17] Benes, F., Davidson, J., and Bird, E. 1986. “Quantitative
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Journal of Pharmacy and Pharmacology 2 (2014) 432-438 D DAVID PUBLISHING

The US Payor Landscape: Results from a Survey of

Medical Directors and Pharmacy Directors on
Comparative-Effectiveness Research

Richard A. Brook1, 2, Jeff A. Carlisle3, Stanton R. Mehr4 and James E. Smeeding1, 5

1. TPG National Payor Roundtable, Glastonbury, Connecticut 06033, USA
2. The JeSTARx Group, New Jersey 07435, USA
3. The Pharmacy Group, Glastonbury, Connecticut 06033, USA
4. SM Health Communications, Valley Cottage, New York 10989, USA
5. The JeSTARx Group, Dallas, Texas 75219, USA

Abstract: The paper is to determine the types of approaches preferred by medical and pharmacy directors of US payer organizations
to enhance the Pharmacy & Therapeutic decision-making process and how medications accepted onto the formulary should be
covered. An online interactive survey of US medical and pharmacy directors was conducted in 2012. In addition to a 10-point Likert
scale (10 = agree completely, 1 = disagree completely), qualitative responses and interpretive analysis were used to explore beliefs
about certain statements. The results showed that the 30 respondents (20 medical directors and 10 pharmacy directors) rated current
progress in obtaining usable CER (comparative-effectiveness research) at only an average of 4.17 on the 10-point scale. They hoped
to regularly utilize CER information in formulary decision making by 2015 (average rating, 6.03). The rating of evidence-based
medicine use in coverage decision making today was somewhat higher, at an average of 7.08 (medical directors, 7.38; pharmacy
directors, 6.40). The survey participants believe that emerging CER results will greatly affect the following areas:
optimization/improvement of clinical guidelines, medical/pharmacy benefit management, assessments of the value and
appropriateness of interventions, and pharmaceutical research and development. Therefore, payers expect CER to play an increasing
role in helping them determine the value of new therapies.

Key words: Payer, survey, P&T Committees, comparative-effectiveness research, managed care formularies.

1. Introduction with appropriate cost sharing. Bodies that determine

whether to cover new technologies for their members,
To control the growth of healthcare costs and
such as P&T (Pharmacy & Therapeutic) Committees
ensure appropriate utilization of pharmaceutical
and technology assessment entities, have relatively
products, payers (health plans, insurers, pharmacy
little information on the value of these technologies
benefit managers, and third-party administrators) use a
when the coverage decision is made. Questions of
variety of management tools, including prior
coverage must be answered on both an individual and
authorization, generic substitution, step therapy,
population-based level. They include: Which is the
adherence management, and innovative pharmacy
appropriate population(s) for treatment? In whom does
benefit designs. The introduction of new
the intervention work best? Do the marginal benefits
pharmaceutical and biotechnology agents significantly
outweigh the expected risks [1]?
affect how payers balance access to these products
The limitations of relying the placebo-controlled
Corresponding author: Richard A. Brook, M.S., research study gold standard for evidence-based decision
fields: health economics, outcome research and payor making have been well described [2]. One trend that is
marketing. E-mail: rich.brook@tpg-nprt.com.
 The US Payer Landscape: Results from a Survey of Medical Directors and Pharmacy Directors on
Comparative-Effectiveness Research
gaining visibility is the generation and use of CER
(comparative effectiveness research) for medical
interventions [3, 4]. This effort seeks to obtain and
evaluate not only clinical evidence regarding the use
of one product versus an active comparator (i.e., not
placebo), but also use RWE (real-world evidence) in
the comparison [5]. Much of the ground work for
incorporating CER was laid by the Institute of
Medicine and by the ISPOR (International Society of
Pharmacoeconomics and Outcomes Research) [6].
Current efforts by governments,
quasi-governmental entities, and private-public
collaborations to introduce better comparative study
information to the formulary decision-making
process are underway [3, 7]. The case has also been
made that the pharmaceutical industry should
embrace the growing need for CER as an opportunity
[8], and some organizations (e.g., AstraZeneca) have
actively sought partnerships to develop more CER [9,
10]. Once CER research becomes more readily
available, some of the major challenges in this area
will include weighing and interpreting the data for
use in the coverage decision-making process, better
understanding the place pharmacogenomics and its
implications for clinical evidence, as well as
recalibrating the place of placebo-controlled clinical
research in this hierarchy.
The literature is unclear as to the readiness of
payers to begin utilizing these data and the types of
real-world evidence available today. The objective of
this study was to determine the types of approaches
preferred by medical and pharmacy directors of US
health plans, insurers, and PBMs (pharmacy benefit
managers) to enhance the P&T decision-making
process. The authors sought to better understand how
CER is viewed in the context of the coverage
decisions and how emerging CER evidence can be
better incorporated into it. A further goal of the study
was to learn more about payers’ current and expected
use of CER data in coverage decisions.
433
2. Methods
2.1 Survey Tool
To obtain these perspectives, an online, interactive
survey tool was employed. The 26-question survey
instrument was developed by the researchers to obtain
payers’ perceptions on the shifting landscape in
benefit management and coverage, anticipated trends
in healthcare costs, and the implications of healthcare
reform. In addition to recording participants’
demographic information, the survey included
questions that solicited ratings based on a 10-point
Likert scale (10 = agree completely, 1 = disagree
completely) and qualitative responses and interpretive
analysis to explore beliefs about certain statements.
The survey was designed to be completed within 20
minutes. Material or financial incentives were not
offered for completion of the survey.
2.2 Study Participants
In April 2012, invitations to participate in the
research project were sent to 235 medical directors
and pharmacy directors from a proprietary database
(developed by the authors). Invitations were sent via
Email. The survey was fielded over 3 weeks.
2.3 Data Analysis
Responses were collated using Microsoft Excel and
analyzed in aggregate and by respondent title (i.e.,
pharmacy director or medical director). Responses
from medical directors and pharmacy directors who
were not currently employed by US health plans and
insurers at the time of the survey were excluded from
analysis. Correlations were sought in terms of relative
weights given to specific concepts as well as
frequently mentioned phrases and concepts provided
as open-ended responses.
3. Results
3.1 Respondents
A total of 35 payers responded to the survey
434 The US Payer Landscape: Results from a Survey of Medical Directors and Pharmacy Directors on
Comparative-Effectiveness Research

invitation (12.8% response rate). Five respondents indicated that current progress in obtaining usable
were excluded from the analysis, because they were CER information was slow overall (Fig. 1). However,
not currently employed by US health plans or insurers they anticipated regularly utilizing CER information
at the time of the survey. The eligible group of in formulary decision making by 2015 (overall mean
respondents (N = 30) included 20 medical directors rating, 6.03 (on the 10-point Likert scale)). Their
and 10 pharmacy directors. A significant portion of mean rating of the use of EBM (evidence-based
these executives represented more than one plan type medicine) in coverage decision making today was
in their organization (Table 1). The majority somewhat higher, at an average of 7.08, but there was
represented commercial plans (51%), followed by less agreement between medical directors and
medicare plans (22%) and medicaid plans (27%). pharmacy directors (mean ratings, medical directors =
Furthermore, 44% of the payers represented multistate 7.38; pharmacy directors = 6.40).
or regional plans, 24% represented national plans, and
22% were part of organizations covering state-wide or Table 1 Demographics.
Plans represented Percentage (%)
local areas only.
Commercial plans 51
3.2 Views on CER Results and Utilization Medicaid HMO/PPO 16
Medicaid FFS 6
Study participants were asked about their Medicare HMO/PPO 18
expectations for the release of CER data. They Medicare FFS 9

We are making great progress in obtaining 4.1

usable information on the comparative
effectiveness of therapies 4.4

I expect to regularly utilize comparative- 6.0

effectiveness information in formulary decision
making by 2015 6.1

Managed care commonly uses evidence- 7.4

based medicine today in coverage decision
making 6.4

Medical directors (n = 20)

Pharmacy directors (n = 10) 0 2 4 6 8 10
I disagree I agree
completely completely

Fig. 1 Respondents expectations and anticipated use of CER data: mean ratings.
Respondents were asked about the degree to which they concurred with the following statements (1 = disagree completely; 10 =
agree completely).
 The US Payer Landscape: Results from a Survey of Medical Directors and Pharmacy Directors on 435
Comparative-Effectiveness Research

The survey participants were asked about the areas The survey participants were also queried about the
for which they believe the increased volume of CER one change they would like to make in their medical
results would have the greatest implications. They benefit management program. None of their
specified a number of areas, and the most common open-ended comments were mentioned significantly
responses were optimization/improvement of clinical more frequently than others, but such areas as
guidelines (22.6%), medical and pharmacy benefit focusing on value-based insurance design, improving
management (19.4%), evaluation of the value of adherence, better management of infusible products in
interventions (16.1%), appropriateness of care (16.1%), the physician office, and including pharmacists in
and pharmaceutical research and development (6.5%). medical benefit management were often cited (Table 3).
When asked to comment on the one change they
would make to improve their plan’s or PBM’s P&T 4. Discussion
Committee decision-making process, 27.6% offered
4.1 Challenges to Managing the Costs of Specialty
that they would use more CER results and emphasize
Drugs
value in the decision-making process. Interestingly,
they also related concerns regarding the composition The coverage decision-making process is becoming
of the P&T committee and obtaining adequate increasingly important and complex [2, 11],
physician input (17.2%) (Table 2). A total of 13.3% particularly with the forecast introduction of
would enhance the physician/specialist presence on ever-greater numbers of relatively high-cost specialty
the review committee, and 6.6% mentioned that they pharmaceuticals [2, 12]. These late-stage
would increase the time allotted to allow for a more investigational medications will likely be subject to
in-depth evaluation during the P&T Committee increased scrutiny to determine appropriate coverage
review. on the medical benefit or the pharmacy benefit, because
Table 2 Responses grouped by category to the question: if you could make one change to your Plan’s/PBM’s P&T Process,
what would it be?
CER
 Better use of CER data
 Better quality of CER data (including current and new therapies in head-to-head trials)
P&T Committee composition
 More members on the P&T committee
 Increased buy-in of clinicians
 Greater presence of clinical key opinion leader on committee
 Greater subspecialist utilization on committee
Review and analysis
 Greater automation in analysis
 Faster process
 More time to conducted more detailed review
Miscellaneous
 Better integration of medical and pharmacy
 More clarity in category classifications (e.g., Medicare part B vs. part D)
 Demand improved transparency
 Focus more on the therapy’s value proposition (i.e., similar to the evaluation of the UK National Institute for Clinical
Excellence) and designate expected outcomes prior to acceptance
 Begin to relax historical “two alternative therapy rule”, and move toward therapeutic substitution
 Move towards 4-tier benefit designs
436 The US Payer Landscape: Results from a Survey of Medical Directors and Pharmacy Directors on
Comparative-Effectiveness Research

Table 3 Responses to the question: If you could make one change to strengthen your organization’s medical benefit
management program, what would it be?
 Disallowing or limiting coverage (e.g., coinsurance) for interventions of little value or nonessential care
 Staggered system of member awards and penalties for noncompliance
 Management of drugs administered in doctors’ offices/hospitals/infusion centers
 Prior authorization for medical benefit drugs
 Stronger database to link medical to pharmacy benefit for patient out-of-pocket spending
 Addressing coverage limits
 Additional medical director staffing (and including pharmacists in medical benefit management)

they are anticipated to be relatively high cost and
some may be subject to inappropriate utilization.
Newer modalities to optimize the management of
these products may be necessary, such as covering the
product as part of the medical benefit but managing
utilization through pharmacy benefit tools.
The anticipated budgetary effects of these
medications weigh on the minds of managed care
executives [13]. When asked in the present study,
“What do you believe are the most significant
challenges to the future of managed care?” they
provided a litany of responses, from the workings of
health reform legislation to individual conditions. A
total of 23.7% responded with issues related to the
cost of care, and 15.6% referenced specialty
pharmaceuticals specifically. The question of what
value these agents bring to health plans and their
members can only be answered with more CER data
and appropriate interpretation of this information.
4.2 P&T Committee’s Concern
Indeed, P&T Committees will face numerous
additional challenges in the future. These will no
doubt include the evaluation of biosimilar products
[14], the incorporation of genetic testing into
medication access process, and the weighing of
various types of outcomes evidence (e.g., patient-
reported outcomes, secondary clinical outcome
measures, and proxies for other quality indicators)
into coverage decision making. Yet, each shines a
light on the assessment of a product’s value.
4.3 Slow Uptake of CER
The use and incorporation of CER in the past has
been mixed. Although it has greatly altered the
utilization of breast mastectomy and lumpectomy in
breast cancer [15] and radically curtailed the use of
autologous bone-marrow transplantation after high-
dose chemotherapy in patients with advanced breast
cancer [16], it did not change practices in other
important therapeutic areas like hypertension and the use
of first- versus second-generation antipsychotics to treat
schizophrenia [17].
It is clear that payers and policymakers are focused
on using EBM in coverage decision making, both in
the US and abroad [18-20]. Yet, this is not the only or
most important aspect of the coverage decision. For
example, the availability of an unusual volume of
CER data favoring the type 2 diabetes injectable
liraglutide[20, 21] did not yield an advantage in the
determination of coverage: Based on pricing, at least
one major pharmacy benefit manager (ExpressScripts)
decided not to cover liraglutide [22, 23]. In general,
managed care executives want to see more CER data
and to incorporate this evidence into clinical pathways
and coverage decision making. Intuitively, the ability
to evaluate and compare the value proposition of new
technologies seems to be linked with the need for
better-quality evidence, specifically that provided by
CER. Comparative-effectiveness research results are
anticipated to play a significant role in the calculation
of the value for future coverage decisions. This
research demonstrates a level of impatience with the
pace of CER and we hope that it will be more readily
available in the near future to assist in coverage
decision making.
Based on the results of this survey, additional
research should be conducted to determine whether
 The US Payer Landscape: Results from a Survey of Medical Directors and Pharmacy Directors on
Comparative-Effectiveness Research
US payers have preferences as to whether CER is
generated through public, private, or private/public
partnerships and the actual impact of CER on value
determinations.
4.4 Study Limitations
The study sample size was limited; as a result, the
study was not powered to compare responses between
medical and pharmacy executives. The database used
to recruit respondents was proprietary and may have
introduced biases based on relationships. Financial
compensation may have prompted greater
participation from the target population.
Although compensation was not provided for
participation, statistical bias related to respondent
motivation (by subject matter), or relationship to the
authors, cannot be ruled out.
The survey instrument was developed to elicit
responses that could be the subject for future, focused
research. Its intended purpose was not for in-depth
subject exploration. The responses, which highlight
participants’ interest in CER and the present and
future roles of CER, shaped the initial findings of this
“landscape” picture. As a result, these data should be
interpreted with caution, pending the completion of
more in-depth investigation.
5. Conclusions
The environment for P&T Committee decision
making in managed care is undergoing a series of
changes, and payer medical directors and pharmacy
directors, who commonly serve as P&T Committee
members, have distinct opinions as to how to alter the
process to adapt to these influences. Comparative-
effectiveness research will play an enhanced role in
helping formulary decision makers determine the
value of new therapies and where these agents will fit
into clinical practice guidelines, drug formularies, and
the medical benefit.
Additional research is necessary to determine the
types of CER preferred by coverage decision makers,
437
which can have implications for its credibility (e.g.,
the sponsors or source), how it is interpreted (e.g.,
health economic or clinical outcomes), and how it can
contribute to better understanding of the practical
value of an intervention.
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