Introduction

A phytochemical is a natural bioactive compound found in plant

foods that works with nutrients and dietary fiber to protect against
disease. Research suggests that phytochemicals, working together
with nutrients found in fruits, vegetables and nuts, may help slow the
aging process and reduce the risk of many diseases, including
cancer, heart disease, stroke, high blood pressure, cataracts,
osteoporosis, and urinary tract infections.
Pronounced "fight-o-chemicals," phytochemicals fight to
protect your health. They can have complementary and overlapping
mechanisms of action in the body, including antioxidant effects,
modulation of detoxification enzymes, stimulation of the immune
system, modulation of hormone metabolism, and antibacterial and
antiviral effect.
"Phyto" is a Greek word that means plant and
phytochemicals are usually related to plant pigments. So, fruits and
vegetables that are bright colors — yellow, orange, red, green, blue,
and purple — generally contain the most phytochemicals and the
most nutrients. You can benefit from all of the phytochemicals and
nutrients found in plant foods by eating 5-9 servings of fruits and
vegetables a day and eating more whole grains, soy and nuts. More
than 900 different phytochemicals have been found in plant foods
and more will be discovered. These protective plant compounds are
an emerging area of nutrition and health, with new research
reported everyday. Remember, to get your Phytos eat 5-9 servings
of colorful fruits and vegetables every day!
The following charts provide a description of the most
well researched phytochemicals and some of the fruits and
vegetables they are found in. Complete phytochemical analysis has
not been done on most fruits and vegetables. USDA will conduct
phytochemical analysis on approximately 100 of the most frequently
eaten fruits and vegetables during 2000-2001. Our charts will be
updated as more phytochemical research becomes available.
Current research suggests that most fruits and vegetables contain
phytochemicals and that many fruits and vegetables contain a wide
variety of Phytochemicals.

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History of phytochemical
Only a few years ago, the term "phytochemical" was barely known.
But doctors, nutritionists, and other health care practitioners have
long advocated a low-fat diet that includes a variety of fruits,
vegetables, legumes, and whole grains. Historically, cultures that
consume such a diet have lower rates of certain cancers and heart
disease.

Since the passage of the Dietary Supplement Health and Education

Act (DSHEA) in the United States in 1994, a large number of
phytochemicals are being sold as dietary supplements.

Evidence of phytochemical
It has become a widely accepted notion that a diet rich in fruits,
vegetables, legumes, and grains reduces the risk of cancer, heart
disease, and other illnesses. But only recently have researchers
begun to try to learn the effects of specific phytochemicals contain
those foods.

Much of the evidence so far has come from observations of

cultures whose diets consist mainly of plant sources, and which
seem to have lower rates of certain types of cancer and heart
disease. For instance, the relatively low rates of breast and
endometrial cancers in some Asian cultures are credited at least in
part to dietary habits. These cancers are much more common in
the United States, possibly because the typical American diet is
higher in fat and lower in fruits, vegetables, legumes, and grains.

Because of the number of phytochemicals and the complexity of

the chemical processes they are involved in, researchers face a
challenging task in trying to determine which phytochemicals in
foods may fight cancer and other diseases, which may have no
effect, and which may even be harmful.

Many studies have looked at the relationship between cancer risk

and eating fruits and vegetables, legumes, and whole grains. Most
of the evidence indicates that eating large proportions of these
foods seems to lower the risk of some cancers and other illnesses.

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Some of the links between individual phytochemicals and cancer
risk found in studies in the lab are very compelling and make a
very strong case for the need for further research. So far, however,
none of the findings is conclusive. It is still uncertain which of the
many phytochemicals in fruits and vegetables actively helps the
body fight disease.

Researchers have also shown much interest in phytochemical

supplements. Some lab studies in cell cultures and animals have
shown that certain phytochemicals have some activity against
cancer cells or tumors. But at this time there have been no strong
studies in humans showing that any phytochemical supplement
can prevent or treat cancer.

Until conclusive research findings emerge, health care

professionals advise a balanced diet with an emphasis on fruits,
vegetables, legumes, and whole grains. The interaction between
certain phytochemicals and the other compounds in foods is not
well understood, but it is unlikely that any single compound offers
the best protection against cancer. A balanced diet that includes 5
or more servings a day of fruits and vegetables along with foods
from a variety of other plant sources such as nuts, seeds, whole
grain cereals, and beans is likely to be more effective in reducing
cancer risk than eating one particular phytochemical in large
amounts.

Carotenoids are the pigments responsible for the colors of many

red, green, yellow and orange fruits and vegetables. Carotenoids
are a large family of phytochemicals which include alpha-carotene,
beta-carotene, lutein, lycopene, cryptoxanthin, canthaxanthin,
zeaxanthin, and others.
Carotenoids protect the body by decreasing risk of heart disease,
stroke, blindness, and certain types of cancer. They may also help
to slow the aging process, reduce complications associated with
diabetes, and improve lung function. Fruits and vegetables that are
dark green, yellow, orange or red contain carotenoids.The following
information describe four of the carotenoids.

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Source of phytochemical

Beta-Carotene
Beta-Carotene may help to slow the aging process, reduce the risk
of certain types of cancer, improve lung function, and reduce
complications associated with diabetes. Beta-carotene is found in
yellow-orange fruits and vegetables such as mangoes, cantaloupe,
apricots, papaya, kiwifruit, carrots, pumpkins, sweet potatoes, and
winter squash, and green vegetables, such as broccoli, spinach, and
kale.
Lutein
Lutein is essential for maintaining proper vision as we age. It has
been shown to reduce the risk of cataracts and macular
degeneration, the leading causes of blindness in older people and
may help reduce the risk of certain types of cancer. Kale, spinach
and collard greens contain the most lutein of any fruit or vegetable.
Other sources of lutein include kiwifruit, broccoli, collard greens,
brussels sprouts, swiss chard, and romaine lettuce.
Lycopene
Diets rich in lycopene have been shown to reduce the risk of
prostate cancer and heart disease. Lycopene is found in red fruits
and vegetables such as tomatoes and cooked tomato products, red
peppers, pink grapefruit, watermelon.
Zeaxanthin
Zeaxanthin may help to prevent macular degeneration, which is the
leading cause of visual impairment in people over 50. It may also
help to prevent certain types of cancer. Corn, spinach, winter
squash, and egg yolks contain zeaxanthin.
Flavonoids
Flavonoids are another large family of protective phytochemicals
found in fruits and vegetables. Flavonoids, also called bioflavonoids,
act as antioxidants. Antioxidants neutralize or inactivate highly
unstable and extremely reactive molecules, called free radicals that
attack the cells of our body every day. Free radical damage is
believed to contribute to a variety of health problems, including
cancer, heart disease and aging. There are many different types of
flavonoids and each appears to have protective health effects.
Some of the better known flavonoids include resveratrol,
anthocyanins, quercetin, hesperidin, tangeritin, kaempferol,
myricetin, and apigenin. Flavonoids are found in a variety of foods,
such as oranges, kiwifruit, grapefruit, tangerines, berries, apples,

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red grapes, red wine, broccoli, onions, and green tea. The five
primary flavonoids found in fruits and vegetables are:
Resveratrol
Resveratrol may reduce the risk of heart disease, cancer, blood
clots and stroke. Red grapes, red grape juice, and red wine contain
resveratrol.
Anthocyanins
Anthocyanins, which are particularly high in blueberries, have been
shown to protect against the signs of aging. In one study, elderly
rats that ate the equivalent of a half-cup of blueberries daily for eight
weeks improved balance, coordination, and short-term memory.
Scientists think these results may apply to humans as well.
Anthocyanins in blueberries and cranberries have also been shown
to help prevent urinary tract infections. Blueberries, cherries,
strawberries, kiwifruit, and plums contain anthocyanins.
Quercetins
Quercetins may reduce inflammation associated with allergies,
inhibit the growth of head and neck cancers, and protect the lungs
from the harmful effects of pollutants and cigarette smoke. Apples,
pears, cherries, grapes, onions, kale, broccoli, leaf lettuce, garlic,
green tea, and red wine contain quercetins.
Hesperidin
Hesperidin is a flavonoid that may protect against heart disease.
Hesperidin is found in citrus fruits and fruit juices, such as oranges
and orange juice, grapefruit and grapefruit juice, tangerines,
lemons, limes, mandarins, and tangelos.
Tangeritin
Tangeritin may help prevent cancers of the head and neck.
Tangeritin is found in citrus fruits and their juices.
Phenolic Compounds
Phenolic compounds may reduce the risk of heart disease and
certain types of cancer. Phenolic compounds may be found in
berries, prunes, red grapes and red grape juice, kiwifruit, currants,
apples and apple juice, and tomatoes.
Ellagic Acid
Ellagic acid is a phenolic compound that may reduce the risk of
certain types of cancer and decrease cholesterol levels. Ellagic acid
is found in red grapes, kiwifruit, blueberries, raspberries,
strawberries, blackberries, and currants.
Sulphoraphane
Sulphoraphane is in a class of phytochemicals called
isothiocyanates. Sulphoraphane may reduce the risk of colon
cancer. Cruciferous vegetables such as broccoli sprouts, broccoli,

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cauliflower, kale, Brussels sprouts, cabbage, bok choy, collard
greens, turnips and turnip greens contain sulphoraphane.
Limonene
Limonene is in a class of phytochemicals called mono-terpenes. It is
found in the rinds and the edible white membranes of citrus fruits,
such as oranges, grapefruit, tangerines, lemons and limes.
Limonene may help to protect the lungs and reduce the risk of
certain types of cancer.
Indoles
This family of phytochemicals may reduce the risk of certain types of
cancer, including breast cancer. Indoles are found in cruciferous
vegetables, such as broccoli, cauliflower, kale, brussels sprouts,
cabbage, bok choy, collard greens, watercress, and turnips and
turnip greens.
Allium Compounds
Allium compounds may reduce the risk of certain types of cancer
and lower cholesterol and blood pressure. Garlic, onions, chives,
leeks, and scallions contain allium compounds.

In fruit &vegetable
• Apples and apple juice contain phenolic compounds which may
protect against heart disease.
• Apricots (fresh and dried) contain beta-carotene which may help
slow the aging process, reduce the risk of certain types of cancer,
improve lung function, and reduce complications associated with
diabetes.
• Blackberries contain ellagic acid which may reduce the risk of
certain forms of cancer and decrease cholesterol levels.
• Blueberries contain anthocyanins which may protect against the
effects of aging. Blueberries also contain ellagic acid which may
reduce the risk of certain forms of cancer and decrease
cholesterol levels.
• Bok Choy contains a variety of phytochemicals including
sulphoraphane and indoles.

• Broccoli contains many different phytochemicals including

sulphoraphane, indoles, beta-carotene, lutein, and quercetins.
These phytochemicals may help slow the aging process, reduce
the risk of certain types of cancer, improve lung function, protect
against macular degeneration and cataracts, reduce inflammation
associated with allergies, and reduce complications associated
with diabetes.

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• Broccoli sprouts contain sulphoraphane which may reduce the
risk of certain types of cancer.
• Brussel sprouts contain a variety of phytochemicals including
sulphoraphane and indoles. These phytochemicals may reduce
the risk of certain types of cancer.
• Cabbage contains a variety of phytochemicals including
sulphoraphane and indoles. These phytochemicals may reduce
the risk of certain types of cancer.
• Cantaloupe contains beta-carotene which may help slow the
aging process, reduce the risk of certain types of cancer, improve
lung function, and reduce complications associated with diabetes.

• Carrots contain beta-carotene which may help slow the aging

process, reduce the risk of certain types of cancer, improve lung
function, and reduce complications associated with diabetes.
• Cauliflower contains a variety of phytochemicals including
sulphoraphane and indoles. These phytochemicals may reduce
the risk of certain types of cancer.
• Cherries contain anthocyanins which may protect against the
signs of aging. Cherries also contain quercetins which may
reduce inflammation associated with allergies, inhibit the growth
of head and neck tumors, and protect the lungs from the harmful
effects of pollutants and cigarette smoke.
• Chives contain allium compounds that may reduce the risk of
certain forms of cancer and lower cholesterol and blood pressure.
• Citrus fruits, such as oranges, grapefruits, and tangerines
contain hesperidin and tangeritin which act as antioxidants to
reduce the risk of heart disease and various types of cancer.
Citrus fruits also contain limonene which may protect the lungs.
• Collard greens contain lutein which may reduce the risk of
cataracts and macular degeneration. Collard greens also contain
indoles and sulphoraphane which may help decrease the risk of
certain types of cancer.
• Corn contains zeaxanthin which may help to prevent macular
degeneration, which is the leading cause of visual impairment in
people over 50.
• Currants contain ellagic acid which may reduce the risk of
certain forms of cancer and decrease cholesterol levels.
• Garlic contains allium compounds which may reduce the risk of
certain forms of cancers and lower cholesterol levels and blood
pressure. Garlic also contains quercetins which may reduce
inflammation associated with allergies, inhibit the growth of head
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 and neck tumors, and protect the lungs from the harmful effects
of pollutants and cigarette smoke.
• Kale contains a variety of phytochemicals including beta
carotene which may help slow the aging process, reduce the risk
of certain types of cancer, improve lung function, and reduce
complications associated with diabetes and lutein which may
reduce the risk of cataracts and macular degeneration. Kale also
contains indoles and sulphoraphane which may help decrease
cancer risk and quercetins which may reduce inflammation
associated with allergies, inhibit the growth of head and neck
tumors, and protect the lungs from the harmful effects of
pollutants and cigarette smoke.
• Kiwifruit contains a variety of phytochemicals, including beta-
carotene, lutein, anthocyanins, and ellagic acid. These
phytochemicals may reduce the risk of heart disease, certain
types of cancer, cataracts, and macular degeneration.
• Leaf Lettuce contains quercetins which may reduce
inflammation associated with allergies, inhibit the growth of head
and neck tumors, and protect the lungs from the harmful effects
of pollutants and cigarette smoke.
• Leeks contain allium compounds which reduce the risk of certain
forms of cancer and may lower cholesterol levels and blood
pressure.
• Mangoes contain beta-carotene which may help slow the aging
process, reduce the risk of certain forms of cancer, improve lung
function, and reduce complications associated with diabetes.
• Onions contain allium compounds which may reduce the risk of
certain forms of cancer and lower cholesterol levels and blood
pressure. Onions also contain quercetins which may reduce
inflammation associated with allergies, inhibit the growth of head
and neck tumors, and protect the lungs from the harmful effects
of pollutants and cigarette smoke.
• Papaya contain beta-carotene which may help slow the aging
process, reduce the risk of certain forms of cancer, improve lung
function, and reduce complications associated with diabetes.
• Pears contain quercetins which may reduce inflammation
associated with allergies, inhibit the growth of head and neck
tumors, and protect the lungs from the harmful effects of
pollutants and cigarette smoke.
• Pink grapefruit contains lycopene which may decrease risk for
prostate cancer and heart disease. Pink grapefruit also contains
hesperidin and tangeritin which act as antioxidants to reduce the

8
 risk of heart disease and various types of cancer as well as
limonene which may protect the lungs.
• Plums contain anthocyanins which may help protect against the
signs of aging.
• Prunes contain phenolic compounds which act as antioxidants
that may prevent the loss of long-term memory and learning
ability.
• Pumpkins contain beta-carotene which may help slow the aging
process, reduce the risk of certain types of cancer, improve lung
function, and reduce complications associated with diabetes.
• Raisins contain phenolic compounds that may act as powerful
antioxidants to help slow the aging process.
• Raspberries contain ellagic acid which may reduce the risk of
certain forms of cancer and decrease cholesterol levels.
• Red grapes and grape juice contain resveratrol and ellagic acid
which may lower the risk of heart disease and certain forms of
cancer.. Red grapes also contain quercetins which may reduce
inflammation associated with allergies, inhibit the growth of head
and neck tumors, and protect the lungs from the harmful effects
of pollutants and cigarette smoke.
• Red peppers contain lycopene which reduce the risk of prostate
cancer and heart disease.
• Romaine lettuce contains lutein which may reduce the risk of
cataracts and macular degeneration, the leading causes of visual
impairment in people over 50.
• Scallions contain allium compounds which may reduce the risk
of certain forms of cancer and lower cholesterol levels and blood
pressure.
• Spinach contains beta-carotene which may help slow the aging
process, reduce the risk of certain types of cancer, improve lung
function, and reduce complications associated with diabetes.
Spinach also contains lutein and zeaxanthin which may help
prevent blindness. People who eat lots of spinach have a
decreased risk of cataracts and macular degeneration, the
leading causes of visual impairment in people over 50.
• Strawberries contain anthocyanins which may protect against
the effects of aging. Strawberries also contain ellagic acid which
may reduce the risk of certain forms of cancer and decrease
cholesterol levels.
• Sweet potatoes contain beta-carotene which may help slow the
aging process, reduce the risk of certain types of cancer, improve
lung function, and reduce complications associated with diabetes.

9
• Swiss chard contains lutein which may reduce the risk of
cataracts and macular degeneration. Swiss chard also contains
indoles and sulphoraphane which may help decrease the risk of
certain types of cancer.
• Tomatoes and cooked tomato products contain lycopene which
may decrease risk for prostate cancer and heart disease. Tomato
products such as ketchup, tomato juice, and spaghetti sauce are
some excellent sources of lycopene.
• Turnips contain indoles and sulphoraphane which may help
decrease the risk of certain types of cancer.
• Watercress contains indoles and sulphoraphane which may help
decrease the risk of certain types of cancer.
• Watermeloncontains lycopene which may decrease risk for
prostate cancer and heart disease.
• Winter squash contains beta-carotene which may help slow the
aging process, reduce the risk of certain forms of cancer, improve
lung function, and reduce complications associated with diabetes.
Winter squash also contains zeaxanthin which may help to
prevent macular degeneration, which is the leading cause of
visual impairment in people over 50.

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Phytochemical

Definition & chemistry

Phytochemicals are non-nutritive plant chemicals that have
protective or disease preventive properties. There are more than
thousand known phytochemicals. It is well-known that plant
produces these chemicals to protect itself but recent research
demonstrates that they can protect humans against diseases. Some
of the well-known phytochemicals are lycopene in tomatoes,
Isoflavones in soy and Flavonoids in fruits. They are not essential
nutrients and are not required by the human body for sustaining life.

Phytochemicals are chemicals found in plants. Plant sterols,

flavonoids (FLAV'oh-noidz), and sulfur-containing compounds are
three classes of micronutrients found in fruits and vegetables.
These compounds may be important in reducing the risk of
atherosclerosis (ath"er-o-skleh-RO'sis), which is the buildup of fatty
deposits in artery walls. Within these categories are many possible
compounds, most of which aren't well described and whose modes
of action aren't established. Many other plant products may also be
linked to the atherosclerotic process, such as antioxidant vitamins,
phyto estrogens and trace minerals. These plant micronutrients will
clearly be the topic of future research. As work continues on all
these compounds, other unrecognized components in plants will be
identified that may have promise in reducing risk of cardiovascular
disease.

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Mechanism of action of Phytochemicals

There are many phytochemicals and each works differently. These

are some possible actions:

• Antioxidant - Most phytochemicals have antioxidant activity

and protect our cells against oxidative damage and reduce the
risk of developing certain types of cancer. Phytochemicals with
antioxidant activity: allyl sulfides (onions, leeks, garlic),
carotenoids (fruits, carrots), flavonoids (fruits, vegetables),
polyphenols (tea, grapes).

• Hormonal action – Isoflavones, found in soy, imitate human

estrogens and help to reduce menopausal symptoms and
osteoporosis.

• Interference with DNA replication - Saponins found in beans

interfere with the replication of cell DNA, thereby preventing the
multiplication of cancer cells. Capsaicin, found in hot peppers,
protects DNA from carcinogens.

• Anti-bacterial effect - The phytochemical allicin from garlic has

anti-bacterial properties.

Physical action - Some phytochemicals bind physically to cell walls

thereby preventing the adhesion of pathogens to human cell walls.
Proanthocyanidins are responsible for the anti-adhesion properties
of cranberry. Consumption of cranberries will reduce the risk of
urinary tract infections and will improve dental health.

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Phytochemical are useful in complication
These products are sold as a dietary supplement in the United
States. Unlike drugs (which must be tested before being allowed to
be sold), the companies that make supplements are not required
to prove to the Food and Drug Administration that their
supplements are safe or effective, as long as they don't claim the
supplements can prevent, treat, or cure any specific disease.

Some such products may not contain the amount of the herb or
substance that is written on the label, and some may include other
substances (contaminants). Actual amounts per dose may vary
between brands or even between different batches of the same
brand. Most such supplements have not been tested to find out if
they interact with medicines, foods, or other herbs and
supplements.

Even though some reports of interactions and harmful effects may

be published, full studies of interactions and effects are not often
available. Because of these limitations, any information on ill
effects an Phytochemicals in the amounts consumed in a healthy
diet are likely to be helpful and are unlikely to cause any major
problems. Some people assume that because phytochemical
supplements come from "natural" sources, they must be safe and
free from side effects, but this is not always true.

It is important to note that, especially when taken in large amounts,

many of them have side effects and possible interactions with
some drugs. Some of these interactions may be dangerous.
Before taking a phytochemical in supplement form, consider
talking to your doctor and pharmacist tobe sure it won’t interact
harmfully with other medicines or herbs you may be taking.

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LIST OF PHYTOCHEMICALS

Alkaloids
• Caffeine
• Theobromine
• Theophylline

Anthocyanins

Carotenoids
• Beta-Carotene
• Lycopene

Coumestans
Flavan-3-Oil

• Epicatechin
• Hesperidin
• Kaempferol
• Naringin
• Nobiletin
• Proanthocyanidins
• Quercetin
• Resveratrol
• Rutin
• Tangeretin

Hydroxycinnamic Acids

• Chicoric acid
• Coumarin
• Ferulic acid
• Scopoletin

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Isoflavones

• Genistein

Lignans

• Silymarin

Monoterpenes

• Limonene

Organosulfides

• Allicin
• Glutathione
• Indole-3-Carbinol
• Isothiocyanates
• Sulforaphane

Other Phytochemicals

• Damnacanthal
• Digoxin
• Phytic acid

Phenolic Acids

• Capsaicin
• Ellagic Acid
• Gallic acid
• Rosmarinic acid
• Tannic Acid

Phytosterols

• Beta-Sitosterol

Saponins

Triterpenoids

• Ursolic acid

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SCREENING
Definition
Screening is the process of separation and isolation of
active principle from plant sources.

Screening are helpful-

- To get lead for “Discovery of new therapeutic agents”.

- To find “New sources” for economic material.
- To help expand “CHEMOTAXONOMY”.
- To produce “Semisynthetic” derivatives.

For this purpose, following 3 essential steps are prescribed-

* Selection of plant.
* Phytochemicals screening.
* Phytopharmacological evaluation.

PHYTOCHEMICAL SCREENING APPROACHES

Ultimately, the goal in surveying plants for biologically active or
rnedicinally useful compounds should be to isolate the one or more
constituents responsible for a particular activity. Hence with the
selection of a specific plant for phytochemical investigation either on
the basis of one or more approaches set forth under
phytopharmacologic Approaches, or through some other avenue,
phytochemical screening techniques can be a valuable aid.

Certain investigators feel that an initial selection of investigational

plants should be made not on evidence that extracts elicit a
particular and interesting biological activity, but rather on the basis
that certain chemicals are present in the plant, relatives of which can
usually be associated with biological activity. Thus, some
investigators will select initially only alkaloid-containing plants for
study on the premises that (a) normally exert some type of
pharmacologic activity usually on the center nerves system but not
always so; m(b) the greatest
majority of natural products used in medicine today are alkaloidal in
16
nature;(c) tests for the presence of these compounds in plants are
simple, can be conducted rapidly, and are reasoanably reliable,
and(d)because of their chemical nature, alkaloids are more easily
manipulated making extraction and isolation less of a problem .
In addition, economics , as well as other factor associated with
biological testing, often force the investigation to pursue a
phytochemical group other then alkaloids be selected for
investigation say the flavonols , the diversity of expected biological
activities can be enormous. Willaman has surveyed the literature
and has found that at least natural flavonoids are known, occurring
in some families, genera, and species of plants . Also, some
different pharmacologic or biological activities have been reported
for one or more of flavonoids . More recently, horhammer and
wagner have reviewed the same area, and these numbers are
therefore to be increased . Also, orzechowski has considered the
role of flavonids as therapeutic agent. Along similar lines, the
coumarins have been repoterd to exert some 31 different biological
effert , and according to soine , their full range of farmacologic
activities is not apprericiated by most investigators. Other examples
pointing out the complexity of expected biological effects for any one
category of phytoconstituents could of course be made.In any event
publication representing the phytochemical screening approach for
out wigh those following phytopharmacologic avenues not only
numbers of report but in representation of total plant examined.

Since the number of chemical categories of plant constitutes is

great and each is capable of eliciting biological activity no attempt
will be made in this to be all enclusive. This section of the review will
be restricted to some consideration of phytochemical screening
methodology followed by discussions of those categories of
phytoconstituents

Which have been represented in major published surveys of

screening programs. These will include: alkaloids. Glycorides as a
general class (heteroside) sapgrams. (steroid and
triterpenoid).sterols. cardiac glycosides. Cyanogenetic glycosides.
Isothiocyanate glycosides. cyanogenetic glycosdes, isothiocyanate
glycosides, anthraquinones, flavonoids and related compounds.
Surveys which have decn condussed along with the general
metehodology involved. The examples to be coted are intended to
be representative of each class and are not recant to include all
available published data.

17
General considerations
A method for use in phytochemical screening should be

(a) Simple,

(b) Rapid,

(c) Designed for a minimum of equipment

(d) Reasonably selective for the class of compounds under study

(e)Quantitative in so far as having a knowledge of the lower limit of

detection is concered, and if possible

(f) Should give additional information as to the presence or absence

of specific members of the group being evaluated. Most published
procedures adhere to criteria.

(a) through (d) but few are designed to provide the information
included in (e) and (f).

In fact, certain procedure cannot be duplicated because of

insufficient details included in some report. For example,Arthur and
cheung in a phytochemical survey of Hong kong plants, screened
332 species for alkaloids. They equated the precipitates observed
following the additional of standard alkaloid precipitating reagents to
result obtained by addition the same reagents to standard solution
of 1:100, 1:500, 1:2500, and 1;10,000quinine sulfate. It is implied
that water was the solvent. However , the solubility of quinine sulfate
is stated to be 1gm.in 810 ml. of water. Along similar lines, will have
used the cyanidin test for the detection of the alfa-benzofurane
nucleus as indicative of the presence of flavanoids. They compare
a test result color with a similar color produced by a 0.1% solution of
rutin and equate it as a (+) reaction. Their extraction solvent is 95%
ethanol (but fresh plant inaterial was often extracted which would
decrease this pcrcentage considerably), and rutin is stated to be
only slightly soluble in ethanol and soluble about l Gm. in 8 L. of
water. We find that maximum solubility of rutin at room temperature
is about 0.02% for both 80 and 95% ethanol.

Webb, using a field method, estimated alkaloid precipitates with

reagents on a + to ++++ basis but used no reference for
comparison . IIe also states,.. on the other hand, while the method
may yield a percentage of ‘false positives; it has never failed to
18
detect species with’’ alkaloids . If the initial field test did indeed fail
to detect alkaloids, perhaps because of a low concentration in the
plant, how could it be determined that the test was infallible when
only field test positives species were colleted for more specific
laboratory examination .

Fundamental consideration-
One of the most important and fundamental consideration in
designing a phytochemical screening produce is the selection of
proper extraction solvent.It is often difficult to follow general or
expected solubility rule for a given class of phytoconsitutents scince
there are often substance of unknown Character present in crude
plant extracts that affect solubility. For example, woo has repoted
that effect of saponin in plant extracts on the solubility of certain
normally insoluble compounds using selected solvent. Apparently
saponin acts as a wetting agent to enhance the formation of
micelles ;thus an increase in solubility of certain constituents is
effected. This phenomena has been noted through the use of
synthetic detergents to enhance the solubility, and thus extract
ability of alkaloids from cinchona . Since saponins, or other similar
surface-active agents, do not occur universally in plants, prediction
of general solubilities for a class of phytoconstituents ppt. a major
problem. In our laboratory n-hexane-soluble extractives from
catharanthus lanceus were found to be rich in alkaloids. Subsequent
isolation of individual alkaloids from the crude mixture proved them
to be totally insoluble in n-hexane. Presumably the alkaloids occur in
the plant, at least in this instance dissolved in some lipid material,
the latter being soluble in n-hexane.

No solution is offered for these problemes involving solubility

except tosay that extract residues should always be examined with
a variety of solvents to determine whether solubility phenomena
have occurred.

Even though a great many problems are presented by the diverse

methodology utilized by investigatiors in phytochemical screening,
much useful information can be derived from published studies.
Positive test result are usually clear cut and on the other hand, must
be carefully weighed in terms of being due to real absence of the
test material in the sample being evaluated, or to the methodology
employed.

19
Pharmavegetables, their ethnomedicinal properties and
phytochemical screening of plant extracts.

20
Plant Species Common Ethnomedicinal Phytochemicals in the
Name Property Plant Extract
Abelmoschus Lagikway Emmenagogue; laxative Flavonoid, unsat. sterol &
manihot (L.) triterpene, steroid Medik
glycoside, tannin &
phenol
Amaranthus Kadyapa Antiinflammatory, Flavonoid, unsat. sterol &
spinosus L. antussive, diuretic, triterpene, cyanogenic
febrifuge, kidney, tonic, glycoside, tannin &
lactagogue, laxative phenol
Amaranthus Dalaura Antidysentery, antitussive Flavonoid, unsat. sterol &
tricolor L. Pula’t triterpene, tannin &
Amaranthus Dilaw phenol
tricolor L.
Amaranthus Kulitis Antiinflammatory, Flavonoid, unsat. sterol &
viridis L. antitumor, antitussive triterpene, cyanogenic
glycoside, tannin &
phenol
Broussonetia Salugim Alkaloid, flavonoid, unsat.
luzonica sterol & triterpene, steroid
(Blanco) var. glycoside, cyanogenic
luzonica glycoside, tannin &
phenol
Cayratia sp. (?) Yagini Flavonoid, unsat. sterol &
triterpene, tannin
& phenol
Centella Takip- Antiaging, antidysentery, Alkaloid, flavonoid, unsat.
asiatica Kuhol anti-inflammatory, sterol & triterpene, steroid
antitumor, diuretic, glycoside, cyanogenic
immunostimulant, liver glycoside,saponin,
tonic anthraquinone
Colubrina Kabatete Alkaloid, flavonoid, unsat. Alkaloid, flavonoid, unsat.
asiatica (L.) sterol & triterpene, sterol & triterpene, steroid
saponin, anthraquinone, glycoside, cyanogenic,
tannin & phenol glycoside, & saponin,
tannin
phenol
Corchorus Saluyot Demulcent
capsularis (L.) ligaw
Cordia Anonang Antidysentery, diuretic, Alkaloid, flavonoid, unsat.
dichotoma liver tonic sterol & triterpene, steroid
Forst. F. glycoside, cyanogenic
glycoside, saponin, tannin
& phenol
Eclipta alba (L.) Higis- Antianemia, Alkaloid, flavonoid, unsat.
Hassk. Manok immunostimulant,
21 sterol &
vermifuge triterpene, steroid
glycoside, cyanogenic
glycoside,saponin, tannin
Alkaloid screening
Prior to a consideration of screening plant material for alkaloids . it
would seem in order to define the term “alkaloids “ as used in this
review ;however the nature of the word . It self precludes any thing
more then a vague definition. Any one familiar with alkaloids surely
has a knowledge of their character but seldom can one give as
acceptable definition. Most authorities agree that chemical botanical
and pharmacologic implications most be reflected an one
acceptable definition. Hegnauers suggestion that;

Alkaloids are more or less toxic substances which act

primarily on the central nervous system have a basic character
contain heterocyclic nitrogen and are synthesized in plants from
amino acids or their immediate derivatives. In most eases they are
of limited distribution in the plant kingdom.

Seems as acceptable as any. For purposes of this

discussion we will utilize Hegnaures concept except. Of course, we
cannot be concerned with the site of mechanism of synthesis , thus
compounds such as aliphatic nitrogenous bases amides and the
amino acids themselves will not be considered as alkaloids,

Estimates for the distribution of alkaloids in vascular

plants have been placed as high as 15-20%, although this figure
appears some what high with respect to data derived from several
extensive phytochemical screening programs Will have screened
more then 4000 species of plants and report a distribution of about
10% alkaloids. Webb in his experience with some 1700 species
indicates alkaloids occurrence to be about 14% whereas the smith
kline & French survey found that about 10% of 25000 species
screened were positive for alkaloids . since a few of these
undoubtedly will be determined through future studies to be false
positive alkaloid containing species, 9-10%seems to be the more
logical estimate representing alkaloids yielding plant species.

Alkaloids are widely distributed in the plant kingdom although

certain groups have been shown to be characteristically devoid of
them excellent essays on this subject have been published by
willaman and Schubert and by Webb.

22
ALKALOID DETECTION

Since alkaloids usually occur in plants as their water-soluble salts,

some workers believe that extraction with acidulated water can
result in a crude extract which can be tested directly with one or
more standard alkaloid ppt. reagents. Other workers feel that the
presence in such an extract of materials that are capable of giving
false-positive alkaloids test necessitate a purification procedure
before valid result can be obtained. This is usually accomplished by
the addition of base and subsequent extraction with water-
immiscible organic solvent. The organic extract can then de tested
by application to filter paper, drying, and dipping or spraying with an
alkaloid detecting reagent that giver a chromo genic response with
alkaloids. If the latter method is not preferred, the organic solution
can be re-extracted with dilute acid and the usual alkaloid
precipitating reagents added to separate portions of this acid
extract.

Another method of removing impurities that are capable of giving

false-positive tests from an initial aqueous acidic extract is to “salt
out” these materials by the addition of powdered sodium chloride.
An additional procedure for alkaloid detection could be based on the
addition of alkali directly to the powdered plant sample, followed by
extraction with an appropriate organic solvent. This extract could
then be purified by partition as described above, or be tested
directly.

With respect to these general methods, certain anomalies have

been reported in the literature which should be pointed out. There is
no implication that these examples are frequently encountered in
alkaloid screening: however, one should be aware that they do exist.
Certain plants are known to contain labile non-basic constituents
and may yield nitrogenous materials on extraction with ammoniacal
solvents, while others contain alkaloids that are susceptible to
modification by acidic reagents. That proteins, which may be
present in aqueous or acidic aqueous plants extracts, can ppt. on
the addition of heavy metal alkaloid precipitating reagents and thus
yield false-positive tests, is well established. Such proteins can be
removed by treatment of the extract with sodium chloride prior to the
use of the heavy metal reagent, a procedure which usually salts out
the protein. However, alkaloids such as alstonine may be
quantitatively precipitated as hydrochloride under these conditions.
In the treatment of a crude plant extract to remove impurities by the
acid-base-organic solvent acid procedure, it is quite possible that
23
plants containing water-soluble alkaloid bases will go undetected.
Quaternary bases, amine oxides, betaines, and choline would full
into this category.

Variability of results in alkaloid testing of plant material

can, be induced by a number of factors such as age, climate habitat
plant part tested season time of harvest chemical races of plants
sensitivity of alkaloid type to reagents etc. A few examples regarding
these factors should serve to point out their importance Geijera
salicifolia was found by Webb to give consistently better alkaloid
tests as the broad leaf form than narrow leaf form even when the 2
were growing side by side in the field. In certain groups of plants
(i.e. composite) alkaloids often arc found only on or near the flower
tops and in the Apocynaceae. Alkaloids generally tend to
concentrate in the root or bark often to the exclusion of other parts
of the plant thus the proper selection of plant parts for testing is
quite important. To obtain equivalent results, quantitation of
precipitates obtained with alkaloid reagent is not always possible,
especially when comparing different genera or families. This is
exemplified through knowledge that galbulimima baccata was found
to be rated a ++++ in field tests and subsequent analysis resulted in
a yeald of 0.01%-0.05% of 4 alkaloids. A++++ rating for
Daphnandra aromatica was determined in the field and subscquent
analysis in the laboratory yielded 6+% of crude alkaloids. Anlireha
putaminosa loses 50% of its alkaloid decomposition rates have also
been noted for A. tennuifolia randia rubiaceous plants. Silica gel
drying of antirhea tennuifolia for 1 month resulted in material that
gave a ++++ alkaloid test, whereas this same plant dried in the
shade for 1month gave a negative alkaloid test. Acronychina baueri
on the other hand gave strong alkaloid positive tests when 124year
old herbarium specimens were evaluated Along similar lines ,
Raffauf and morris have reported that a plant sample identified as
Nicoliana attenuata and estimated to be some 1300 years old gave
positive alkaloid tests. Duboisia myoporoides yielded 3%of
hyoscyaminne when harvested in October but when harvested in
April of the same year 3% hyoscine was isolated. Example of
alkaloid decomposition as a result of milling dried plant matcrial
have also been cited. These examples should suffice to point out
just a few of the problems encountered by the natural product
investigator who is interested in the detection and isolation of
biologically active alkaloids.

24
SOME USEFUL ALKALOID PRECIPITATING REAGENTS

NAME COMPOSITION

Bouchardat Iodine-Potassium-Iodide

Dragendroff Bismuth potassium iodide

Ecolle Sillicotungstic acid

Hanger Picric acid

Kraut Iodine-zinc chloriodide

Marme Cadmium potassium iodide

Mayer Potassium mercuric iodide

Platinum chloride Chloroplatinic acid

Scheibler Phosphotungustic acid

Sonneschein Ammonium

hosphomolybdate

Valser Potassium mercuric iodide

Wagner Iodine potassium iodide

Bismuth antimony iodide

Bromauric acid

Bromoplatinic acid

Bromothalic acid

25
ALKALOID DETECTING REAGENTS
For detecting alkaloids in phytochemical screening, two
types of reagents are available, alkaloidal precipitants and spray or
dip reagents. In table-1 20 precipitating reagents commonly used for
the detection of alkaloids, whereas In table-2 present 25 reagents
that were used in 45 recent phytochemical surveys for alkaloids. At
least 2 reagents were used in 38 of the surveys, while 7 surveys
depended solely on 1 reagent to establish the presence of alkaloids.
Because of the variable sensitivities of these reagents and because
of their nonspecificity for alkaloids many investigators utilize 4 or 5
reagents in their screening of plant extracts, and only samples
yielding precipitates with all reagents are considered to contain
alkaloids. Fulton has tabulated some 200 of these reagents and
presents a great deal of information concerning their specilicity and
sensitivity. A series of papers by munch a al is concerned with the
effect of 17 different alkaloid detecting reagents on several classes
of nitrogenous bases. Travell has studied the sensitivity of mayers
and valsers reagents both solutions of potassium mercuric iodide
and potassium iodide and the letter from mercuric iodide and
potassium iodide.

The reagent used by most investigators for phytochemical

screening is essentionally the same formula that Mayer originally
introduced in 1862. Several investigalors have demonstrated. How
ever. That the original formula is perhaps the least sensitive for
alkaloid detection in comparison with many proposed modifications.
And travel has conclusively demonstrated the superiority of several
common alkaloid precipitating reagents using 40 different chemical
types. The reagents tested were mayers. Velsers wegners
Bouchardats hagers Schreibers. Silicotangstic acid. Dregendorll”s
Marme’s gold chloride.and sonnenschein’s. It was demonstrated in
this study that the various reagents exhibit wide differences in
sensitivity for structurally dissimilar alkaloids. None of the reagents
would detect ephedrine at a concentration of 0.1% but wagner’s
bouchardat’s. Dragendorff’s and scheibler’s each defected all of the
other alkaloids at concentrations ranging from 0.001 to
0.1%.Hager’s marme’s and gold chloride reagents were by far least
effective detecting reagent failing to react with 13 12 and 10
respectively of the 40 test alkaloids. All 3 of the Mayer’s formulations
were inferior to Valser’s reagent with respect to sensitivity and
specificity of alkaloid detection. It should be pointed out that the
majority of these precipitating reagents must be used to defect

26
alkaloids only in acid solution. And further more. That a large
number of naturally occurring mononitrogenous plant principles will
react to give false-positive tests. These will be discussed
subsequently,

ALKALOID DETECTING REAGENTS EMPLOYED IN

SCREENING PROGRAMS

REAGENT SURVEYS USED

Mayer reagent 39

Silicotungustic acid reagent 23

Dragondroff’s drop reagent 19

Wagner’s reagent 11

Dragendroff’s spray reagent 10

Sonnenschein’s reagent 09

Hanger’s reagent 07

Bouchardat’s reagent 03

Phophotungustic acid 02

Valser’s reagent 01

Chloroplatinic acid reagent 01

Chlorauric acid reagent 01

Sodium tetraphenylboron reagent 01

27
Investigators who prefer to use spot tests, or those who prefer to
chromatograph concentrated plant extracts for the detection of
alkaloids, have a variety of available reagents the most widely
utilized, however, are modifications of the original dragendorff drop
test reagent, which produce orange to red colors with most
alkaloids. Although a number of modified formula have been
proposed, each reported to have advantages over the others, the 2
most frequently utilized in phytochemical studies are the 1951
munier and macheboeuf and the these and rather modifications. A
literature search has revisited the availability of at least 15
modifications of the original Dragendorff drop test reagent which
produce orange to red colors with most alkaloids. Although a
number of modified formulas have been proposed each reported to
have advantages over the other the 2 most frequently utilized is
phytochemical studies are the 1951. munier and macheboeuf and
the these and rather modification. A literature search has revealed
the availability of at least 15 modification of the dragendroff spray
reagent .we are prompted to study the stability and sensitivity of
one of these modified reagents since a number of published reports
had commented on the need for their storage under refrigeration
with concomitant protection from light. It was determined that
prepared concentrates of the 1951 Munier machcbocuf
dragendorff’s reagent required a storage period of about I week prior
to its use in the preparation of the diluted reagent. Also the diluted
spray reagent should be stored for a minimum of 1 week prior to its
use for alkaloid detection in order to obtain maximum sensitivity. The
reagent maintained its stability and sensitivity for at least 6 months
and no special storage conditions were found necessary.

Some alkaloid detecting reagent are available which on reaction

with certain groups of alkaloids or with specific functional groups
produce characteristic chromogenic value in screening work but only
after alkaloids have been determined in the sample being evaluated.
A selected list of general as well as specific chromogenic reagents
has been prepared and is presented in table.

28
False-Positive Alkaloid Reaction –

Mechanism for the reaction between alkaloids and detecting

reagents are dependent chiefly on the chemical character of the
reagent. Fulton claddifies alkaloidal precipitates as

Those which react with basic compounds to form insoluble salts;

examples are silicotungstic phosphomolybdic and phosphotungstic
acids.

a) Those which react with alkaloids as loose complexes to form

precipitates; examples are Wagner’s and bouchardat reagents.

b) Those which react to form insoluble addition products through the

alkaloid nitrogen ; examples are the complex heavy metal salt
reagents Mayer’s valser’s marme’s and dragendorff’s arrd .

c) Those, which react through the attraction of organic acids with

basic alkaloids to form insoluble salts.

An example of such a reagent would be Hager’s (picric acid).

Obviously these are rather nonspecific reactions and a number of
nonalkaloidal plant constituents should be expected. These false
positive reactions are most liable to occur when testing an extract
that has not been treated by at least one acid base organic solvent
purification.

The most frequent false positive reactions have been attributed to

the presence of proteins which precipitate on the addition of heavy
metal containing reagents. Included in this category are ptomaine’s.
At least one textbook has indicated that amino acids will also
precipitate with general alkaloid reagent. However, a study by Winek
and Fitzgerald appears to disprove this allegation. Among other
substance reported in the literature as the cause of false-positive
alkaloid reaction are certain glycosides, and carbohydrate, betaine,
choline, purines, methylated amines, tannins and ammonium salts.
Recently, we were able to show that previous positive alkaloid tests
reported for extracts of piper methysticum were due to the alfa-
pyrones: kawain, dihydrokawain, methysticin, dihdromethysticin, and
yagonine. This prompted an investigation of the mechanism by
which nonalkaloidal compounds are able to elicit a positive reaction
with an alkaloid detecting reagent, in this case the modified
Dragendroff reagent. It was determined that any non-nitrogenous
29
organic compound having conjugated carbonyl or lactone functions
would react in a manner typical of alkaloids. These minimum
qualifications are quite prevalent in natural products and
undoubtedly many false-reactions are promulgated through this
mechanism. Fortunately, the majority of compound with these
functionalities can be separated from the alkaloids by treatment of
the extract with base, followed by extraction with organic solvent
which, in turn, is extracted with diluted aqueous acid. Another
interesting report that is by Bings and Locker, who in 1949isolated 3
compounds from Melicope ternate which give precipitates with the
usual alkaloid reagent and crystalline salts with acids. How ever
these compounds contained no nitrogen and proved to be the
completely alkylated hydroxylflavones meliternatin meliternin and
ternatin . It would appear that in the light of our work the false-
positive test for alkaloids was due to the presence of the conjugated
carbonyl in each molecule rather than as stated by the authors the
fact that each molecule was completely alkylated. Presumably other
flavones would react similarly as would most of the cardenolides
and bufadienolides.

Householder and camp have recently pointed out that treatment of

plant extracts with ammonium hydroxide and acetone can give rise
to artifacts which give positive reactions with the standard qualitative
alkaloid test reagents. These investigators were unable to identify
the condensation products formed in this reaction but presented
evidence to show that the rate of formation was affected by
exposure to light and the atmosphere.

A recent report by Russian works presented evidence for the

isolation of an alkaloid named rosmaricine from rosmarinus offcinalis
, This
anomaly of on alkaloid from a number of the mint family prompted
wankert and co-workers to investigate the validity of the Rassion
work. They found that rosmaricine was indeed not present in the
plant prior to the addition of ammonia {used by the Russian workers
in their isolation experiments}. And that this “alkaloid” was
undoubtedly formed as a result of the action of the base on the
precursor caruosic acid. Other anomalous alkaloid reactions have
been mentioned in the literature but as yet they cannot be
explained. For example. Samolus repens extracts give a black color
and precipitate with Dragendorff’s reagent. We have observed this
phenomenon frequently in the field testing of fresh plant material
and have assumed the reaction to be one of free iodine in the
reagent combining with starch to give a typical blue black cooler.
30
Extracts from Plagianthus divaricatus have been reported to give a
pink cooler. But no precipitate, with dragendorff’s reagent. Webb has
pointed out that about 9% of species of species tested in the filed for
alkaloid were found to be false positive reaction following
subsequent detailed laboratory analysis. On the bases of
experience resulting form test on the some 25000 plant species
Douglas has estimated that not more than 5% of initial positive
alkaline tests have been found to se done to nonalkaloidal entities.

False- negative alkaloid reaction – if one considers the

nonheterocyclic nitrogen bases as alkaloids it will be noted that the
greatest majority of these fail to react with the usual alkaloid
precipipatating reagent. Certain examples of this can be
documented. Also unless certain precautions are observed in the
test procedure quaternary alkaloids and amine oxides will not be
detected. That is if an acidic plant extract is treaterd with base and
extracted with an immiscible organic solvent both the aqueous
basic layer and the organic layer must be tested for alkaloids. In
most alkaloid screening procedures that have been reported the
basic layer has been neglected. Argument for this omission have
been based on the assumption that quaternary alkaloids or amine
oxide would not be expected to occur in plants to the exclusion of
tertiary bases which would be detected by this procedure. Raffauf
points out that this may be an erroneous assumption.

Alkaloid testing of herbarium specimens – the validity of

alkaloid test on plant material derived from herbarium sheets is
open the question. Often for many and varied reasons it is difficult
to find certain plants in their negative habitat at the time of
collection of indigenous flora and this alternative to collection has
been used by several investigators to survey a broad distribution of
plant task. An obvious disadvantage in the use of such material is
that usually only leaves and branches are available and instances
are known where in plants contain alkaloids in other organs but
their leaves and stems are relatively alkaloid free. Also, herbarium
material is often quite old and a number of examples can be cited
correlating alkaloid decomposition as a function of time. On the
other hand plants 1300 years old have been repoted still to give
alkaloid positive test. It is common practice in some herbaria to
treat specimens with formali could very well decompose many
alkaloids and mercuricwith certain alkaloidal precipitants to from
abnormal precipitates. With Mayer’s reagent this is evidence by a
bright orange precipitate this is evidence by a bright orange

31
precipitate with yellow streaks which eventually become red, and
with bochar dat’s reagent a pale purplish brown precipitate is
observed. In the some herbaria it is common to mark specimen in
a manner that treatment of this type can be easily ascertained
while in other that this practice in not carried out. Cainetal have
indicated that equivalent results were obtained in their studies with
fresh plant material dried herbarium specimen.

Field test for alkaloids in plants-- Investigator searching for new

alkaloids bearing species in remote or distant areas of the world
often find it difficult to return to make additional bulk plant
collection for laboratory study. Therefore, simple field tests for
alkaloids have been developed which are sufficiently reliable to
distinguish alkaloids, thus enableing bulk collections of these
species initially and eliminating the need for a return expedition.
These field tests can be classified in the following manner.

Organoleptic Evaluation- it has been suggested by wabb that at

least some of his collections of species for laboratory examination
were made on the basis of taste, in conjunction with some
knowledge of the botanical characteristics of the sample being
evaluated. That is he avoided tasting plants in such families as the
Anacardiaceae, Euphorbiaceae, etc, but bitterncss in a group such
as the lauraceae, particularly if a cryptocarya, would suggest
alkaloids and saponins can be made on the basis of taste, but only
after considerable experience. Also bitterness in the inner bark of
such groups as evodia, acoronychia and melicop in conjunction
with an observation of yellow pigmentation is suggestive of the
presence of alkaloids. While judgment such as approach.

Spot tests using alkaloid test paper

Kraft has development a simple device for alkaloid
detection in fresh plant material a process which consist of
impregnating filter paper with Dragendorff’s reagent fallowed by
drying. A plant part is incised with a razor blade and a small
amount of juice is applied to test paper which, if alkaloids are
present will give the characteristic orange color indicative of a
positive test. This method has been applied to the field testing of
about 1200 species of plant by nikonov and ban’kovskii who found
it to be acceptable with certain resertions. They found it unsuited
for plants containing pigment in the sap which masked positive
reaction and it was further determined that protoalkaloids such as
ephedrine were not determined. An important point that must be
32
emphasized is that workers stress that typical color of an alkaloid
positive reactive must be observed within 30 sec. from the time
that the sample was applied to the paper for a test to be
considered valid. We have used paper similar to this in our
laboratory for the detection of alkaloids in organic solvent during
chromo graphic separations and found that the after sample
solvent had evaporated in order for the reaction to take place.

Spot tests on paper using liquid reagents

The most extensive phytochemical survey for alkaloid in
the plant kingdom being conducted by scientists from the nature
product section smith kline & French laboratories Philadelphia.
This program was initiated in 1954 but extensive alkaloid testing
did not bening until 1958. briefly their approach consist of
semiconductor collection of plants all part s of the world, with tests
for alkaloid test for made in the field on all accessible parts of each
species. Those found to be promising as a source of alkaloid are
collected at the test site in sufficient quantity to enable laboratory
extraction of the alkaloid for subsequent pharmacologic study.
Extract from all plants show to contain alkaloids by this field test of
screening for several types of pharmacologic activity.

The field test for alkaloid used by this group has been
described and is essentially the same as that utilized by nikonov
and bankovskii with the exception of the special test paper. Instead
plants sap obtained by making an incision of the approate plant
part is applied to filter paper dried and a microdrop of specially
prepared dragendorff’s regent is added. Positive tests are
evaluated as previously described. All positive field tests are
confirmed by means of a laboratory alkaloid detection procedure.

Of 25000 species evaluated in this manner to date about 10%

have been recorded as alkaloid positive. About 5% of the plants
shown to contain alkaloids by the field test were not confirmed by
the laboratory procedure.

33
Abisch and reichstein have also utilized this spot test technique for
alkaloid detection in their study of plants of the apocynaceae A
sclepiadaceae and periplocaceae. However their extracts were
prepared from dry plant material.

Test Tube Spot Tests –

Culvenor and Fitzgerald have described a simple kit that
can be taken into the field for use in testing samples of plant
material for alkaloids. About 2-4 gm of fresh plants part is ground
in a small mortar with sand and sufficient chloroform to make
slurry. Ammoniacal chloroform is added and the mixture stirred for
1min prior to filtration into a small test tube. Extraction of the
alkaloids from the chloroform is accomplished by shaking the
solution with 0.5ml of 2N sulfuric acid and separation of the acid
layer by means of a medicine dropper. A few drop of this acid
extract are then tested with either Mayer’s reagent orsilicotungstic
acid to ascertain the presence of alkaloids. When samples were
analyzed by both the field method and a laboratory procedure it
was found that a number of weakly positive tests recorded through
use of the laboratory test were found to be negative in the field
test. The method of course fails to detect quaternary alkaloids and
this appears to be its major draw back.

Presumably many of the plantscollected for laboratory alkaloid

testing by Webb, Amarsingham and Arthur were field analyzed in a
similar manner to that describe above. However, their respective
reports failed to point out any consistency with regard to this
matter.

Alkaloid Surveys-
Although surveys for alkaloids, representing tests on
more than 15,000 species of plants, have been published, the data
that they present are often inconsistent because of variations in
testing methodology. That is, some of the procedure will detect
both quaternary and tertiary alkaloids, but the former group is
omitted from most surveys reports. Certain procedures involve
treatment of the alkaloid fraction to remove substances that often
give rise to false-positive alkaloid reaction, whereas others do not
include this extra step. Some methods are semiquantitative, while
others lack this desirable feature. A survey of the most extensive
34
and more frequently reported methods allows them to be classified
into 6 major categories. Perhaps the simplest method is that
represented group by group A in which either an acidic or aqueous
plant extract is prepared, with or without the use of heat, followed
by filtration and the addition of one or more alkaloidal reagent to
separate portions of the filtrate. Most investigatiors assess a rating
of o, or +1 to +4 on the lack of, or degree of precipitation following
use of the reagents. However, there is seldom any indication of the
alkaloids equivalent of these ratings. This undoubtedly could
present a problem to either a novice or one who is attempting to
duplicate results in a different laboratory. On the other hand, a
person experienced in alkaloid screening can usually assess this
+1to +4 rating system by a rule of thumb.The major drawback of
this method is that it results in the greatest number of false positive
reaction. An inspection of the compounds presented in fig 2 which
are representative of a great number of nonalkaloidal plant
eonsttuents capable of giving false-positive alkaloid reactions,
shows that for the most part they would. Be soluble either aqueous
or acidic media. Also although this method world not differentiate
between quaternary and tertiary alkaloids, nether would it fail to
detect one or the other.

Group Testing differs from group A only in that the filtrate is made
basic and extracted With an organic solvent {usually chloroform or
ether.} followed by extraction of the alkaloids from the organic
solution with dilute aqueous acid. The usual alkoloidal precipitants
are then added to separate portions of the acid extract, This
method had the advantage over the group. A procedure of
eliminating a great number of compounds from the final test extract
that are capable of eliciting false-positive alkaloid reactions;
however, any quaternary alkaloids present would also be
eliminated. Simple modifications in this method would allow one to
test for the latter group of alkaloids.

Since water will extract a number of non alkaloidal constituents

from plants and because there is a possibility of free alkaloid
bases existing in the plant as such and these would be water
insoluble most investigators utilize alcohol or alcohol water
mixtures as a primary extraction medium. Group c test methods
involve preparation of an alcohol extract followed by removal of
solvent and the addition of dilute acid to dissolve any alkaloids.
Some investigators test the resultant acid extract directly and stop
at this point. The advantages and disadvantages for this type of
testing are similar to those discussed for the group a methods.
35
Others will confirm initial positive reaction following a base organic
solvent acid extraction. These are the group C-2 methods which
are designed primarily to eliminate substances capable of eliciting
false positive alkaloids reactions. The group C-1 and c-2 methods.
Were designed and used most extensively by wall and co-workers
but there are 2 important features that should be discusscd
concerning these procedures first the test involves pre-cipitation of
free alkaloid bases from the initial acid extract with NaOH rather
than with NH,OH Thus. if a majority of the alkaloids in the sample
were phenolic in character (highly improbadle) the phenolates
formed on the addition of fixed alkali would be insoluble in the
immiscible organic solvent used for the extraction of the basic
alkaloid-containing solution. Subsequent extraction with dilute acid
would then result in a solution free from phenolic alkaloids and
would therefore not be representative of the true alkaloid content
of the sample A second problem associated with this method was
recognized by the workers themselves after screening the first
4000 accessions Because they were experiencing a lesser number
of positive results than would be expected from statistical averages
they increased the concentration of test solution so that l ml would
be equivalent to 4.0 gm. Of dry plant material previous test results
In the serics wcre rcported on solutions which represented only
0.2 gm of dry pample this of course made any ncgative alkaloid
test rcsults rcpored in the fist 4000 acccssions open to qucstion
Rccognizing this problem plants yiclding negative rcsults from the
latter group if availablc wcre rctestcd and the results included in
rcports on the final 2000 accessions the method does not include
specific provisions for the detection of quaternary bases but as
indicated previously modifications could be made so that this
procedure would detect these compounds.

36
Screening for Heterosides (Glycosides) -
Heterosides arc organic compounds in which a hemiacetal linkage
usually connects the anomeric carbon of a sugar (glycone) with an
alcohol or phenolic hydroxyl of a second nonsugar molecule
(aglycone) this type of linkage rise to the so-called o-heterosides
(e.g.,salicin) the most common type of Heterosides found in plants
If the anomeric carbon of the glycone is attached to an aglycone
through sulfur the S-heterosides are formcd (e.g.,sinigrin) A third
group are the N-hcterosides which involve attachment of the
glycone to an amino group of an aglycone (e.g.vicine,crotnoside)
Finally, the C-heterosides involve a carbon of carbon linkage of
glycone and aglycone (e.g.aloin).
As a general rule plant heterosides are easily
hydrolyzed with dilute acids or appropriate enzymes the C-
hcterosides arc a notable ex-ception as they are resistant to the
usual type of acid hydrolysis and require ferric chloride for this
purpose.
A number of different sugars are known to occur in
plants in combination with an equally large number of diverse
aglycones Paris has recently reviewed plant heterosides with
particular reference to the types and distribution in plats.
In most instances the biological activity of
heterosides can be attributed to the aglycone moiety the glycone is
mainly associated with the degree or modification of activity
primarily induced by the aglycone. However the cardiac
heterosides can be pointed out as a group that have no useful
biological activity unless the heteroside is intact thus we have the
economically important saponin heterosides and the medicinally
useful anthraquinone flavonid cyanogenetic isothioyanate and
cardiac groups.
From a chemical point of view there are 3 parts of
the heteroside molecule that can be used as a material of
detecting this group of compounds in plant material. First the
hemiacetal linkage between aglycone and glycol is usually not
associated with biological activity, nor can it be associated with any
specific aglycone. This part of the molecule does not appear
attractive as a means of detecting plant heterosides. Because of
the usual correlation of biological activity with the aglycone moiety
of heterosides and because this part of molecule often has
chemical properties amenable to ready detections, most
investigator have used it as a means of screening plant material
indirectly for heterosides.
37
Detection of Glycosides

If, however, heterosides must be intact to

exert their potential biological activity it would appear most fruitful
to detect the hemiacetal linkage in plant extracts as an identifying
feature of the presence of heterosides. Several investigator have
proposed application of those to the screening of plants for
hetersoidal attests to their complexity or inefficiency. Bourquelot
proposed a method for detecting and identifying hetersoidal based
on the determination of an “Enzymolytic index of reduction “obtain
by measuring the optical reaction of a heteroside-containing plant
extract before and after hydrolysiswith specific enzymes. Although
the method has some value it is time consuming and requires
large amounts of plant matcrial therclorc it would be difficult to
adopt to a large-scale screening program Bliss and Ramstad
devised a simple procedure that could be adapted for routine
screening. It consists of
(a) Separation of the heterosides in an extract by paper
chromatography
(b) hydrolysis of the heterosides on the chromatogram with proper
enzymes (i.e.a-glucoidase-invertin-Bglucosidase-emulsin) and
(c) Location of the reducing sugars formed on the chromatogram
by means of an appropriate reagent spray. Tahis method appears
to be least objectionable of many proposed. However it will detect
only those heterosides for which the selected enzymes have a
hydrolytic specificity Also optimal reaction conditions such as time
temperature and pH would have to be determined for a large
number of substrate heterosides to propose operating conditions
that would allow detection of the greatest number of compounds.
Janot et al and Paris have suggestcd chromatographic methods
for detecting heterosides similar to the method of Biss and
Ramstad but acid hydrolysis of the sample is induced to
supplement the action of enzymes. Other methods have been
proposed ,but ether they have not been applied successfully to
plant sample or certain limiting factor make them of doubtful value
for general screening.
Knapp and Beal have proposed a method involve the selective
extraction of heterosides from plant material using 80% ethanol
oxidation of the free sugar in the extract to their corresponding
carboxylic acids so that they will not be detected after hydrolysis of
the heterosides hydrolysis of the heterosides in the extract using
0.15 N sulfuric acid and hart (100) and (d) detection of hydrolyzed
glycones by means of paper chromatography. The major objection
38
to this procedure is that holosides, especially sucrose which is
wide spread in plants, are detected thus the method is of
decreased value.
Abich and Reichstein have utilized a rather simple procedure
which involves the preparation of an extract devoid of free sugars
hydrolysis of the extract with the Killani acid mixture and testing of
the hydrolysis products with Fehling’solution for evidence of
reduction. These investigators have pointed out the nonspecificity
of the test; however in a broad screening program false-positive
reactions must be accepted especially in the presence of a
completely acceptable and specific method of detection.

It does not appear that adequate methodology has been

developed to allow for an extensive screening of plants for
hetrosides based on the approaches described above. As
indicated previously, the majority of studies involving a search for
hetrosides in plant material concerned with tests designed to
detect specific aglycones. The more important of these will now be
considered.

Isolation of triterpinoid glycosides

This study reports the isolation and characterization of a new

triterpenoid glycoside extracted from the bark of Terminalia arjuna.
Theisolation of the organic compounds was done using simple
chromatographic technique. Compound characterization using
various spectroscopic technique identify the final isolated
compound as Olean-3 ,22 -diol-12-en-28 Dglucopyrano
-side-oic acid. The method of isolation is simple, cost effective
andefficient. The preliminary bioactivity of the compound was also
evaluated.

39
Extraction and isolation of glycosides

The sun-dried stem bark was crushed into fine powder.

Pulverized bark part 2.5 kg was exhaustively extracted with
ethanol (95%) at room temperature, 23 ± 2 oC, for 10 days. The
ethanolic extract was filtered, distilled and concentrated to obtain
the solid brownish residue (M.P = 158 oC). The yield was 7.1%
w/w. The final weight was noted and stored. The residue was
treated with water. The water soluble and insoluble portions were
separately collected by filtration (G4 crucibles). Initial study in
preparative TLC of the
water-soluble part does not give any spot in the chromatogram and
therefore, we did not take any further attempt to analyze the water-
soluble part.
The water insoluble alcohol extract was found to be
partially soluble in different organic solvents like ethyl acetate,
benzene, chloroform, carbon tetrachloride and methyl alcohol. So,
it was dissolved in ethyl alcohol and allowed to stand for nearly 4 h
then filtered and separated into ethyl alcohol soluble (A) and
insoluble (B) parts.The ethyl alcohol soluble (A) portion was
treated with equal volume of distilled water and ethanol mixture
(1:1) and then treated with ethyl acetate in separating funnel,
which separated into organic layer and the aqueous layers. The
process was repeated for at least
three times to ensure complete extraction From the organic layer
taken separately, the ethyl acetate was distilled out by and it was
further treated with dry petroleum benzene and the purity of the
compound was tested using thin layer chromatographic system
developed in a benzene and ethyl acetate (9:1)solvent system.
Three different spots were obtained when the chromatogram was
placed inside an Iodine chamber, indicating the presence of three
different compounds. All the three compounds were separated and
collected using preparative thin layer chromatography.

However, we failed to get a quantitative yield of the

materials and therefore, further analysis of the compounds was not
undertaken in the present investigation.The aqueous layer
obtained in the above process was treated with distilled water and
filtered. In this case, the thin layer chromatography developed in
benzene and ethyl acetate (9:1) solvent system does not yield any
spot thus confirming absence of any compounds. The residue
portion (B) was refluxed with petroleum benzene for 12 h using
40
reflux condenser and water bath. It was filtered and separated into
two parts, residue (C) and filtrate (D). The filtrate (D) was
subjected to preparative TLC and no spot was found, thus
confirming absence of any compound. The residue (C) was again
refluxed with dry benzenefor 12 h and filtered. No residue was
obtained in this case. So, the benzene soluble portion was
concentrated using a hot water bath to obtain a greenish-white
colored residue. This wasfurther treated with petroleum benzene
mixture (9:1) and recrystallized in benzene to give a white color
compound (D), (M.P. = 160 - 162 oC), with quantitative yield.

Screening of Anthraquinones

The largest groups of naturally occurring quinine substances

are the anthraquinones. Although they have a widespread use as
dyes, their chief medicinal value is dependent upon their cathartic
action. They are restricted distribution in the plant kingdom and are
found most frequently in members of the Rhamnaceae,
Polygonaceae, Rubiaceae, Leguminosae and Liliaceae. As found in
plant, they are usually carboxylated, methylated or hydroxylated
derivatives of the anthracines, anthrone, anthranol, anthraquinone,
or dianthrone. Hydroxylated anthracines often occur as hetrosides
linked with various sugars through one of the hydroxyl group. Other
types of anthracene hetrosides are represented as C-hetrosides in
which the sugar and aglycone are linked by a carbon to carbon
bond.

DETECTION OF ANTHRAQUINONES

For the qualitative detection of anthraquinones in plant

material, the Brontrager reaction, as modified by Kraus, appears to
be simplest to perform in the application to phytochemical
screening. The powdered sample (0.3gm) is boiled for a few minute
with 0.5 N KOIT (10ml) to which is added 1 ml. of dilute hydrogen
peroxide solution. After cooling the mixture is filtered and 5 ml.
acidified with 10 ml. of benzene in a separator and the benzene
layer takes on a yellow color . A 5-ml. sample of ammonium
hydroxide and a positive reactive for the presence of an
thraquinones is evidence by the formation of a red color in the
alkaline layer. normally if c-glycosides are present in a sample
41
being evaluated for anthra-quinones they will not be detected by the
usual Borntrager reaction as c-glycosides require special methods
for cleaving the sugar from the aglycone this can be done with ferric
chloride sodium dithionate or as described above with peroxide in
an alkaline medium It has been shown that this method results in a
mixture of products however this is not a disad vantage for a
general screening test other simple and rapid spot tests which
involve the direct addition of a reagent to the solid sample
(powdered drug) have been described they should be useful in
phytoehemical screening but to date have not been shown to be
ap-pliable for this type of work phytoehemcial surveys for
anthraquinones have been found only infrequently in the

Screening of Tannins

Two groups of phenolic constituents, hydrolysable and

condensed, comprise the tannins, substances which are important
economically as agents for the tanning of leather and for certain
medicinal purpose. More recently, evidence has been presented in
support of their potential value as cytotoxic neoplastic agents.

Properties of tannins
Hydrolysable tannins are yellow-brown amorphous
substances which dissolve in hot water to form colloidal dispersions.
They are astringent and have the ability to tan hide. Chemically
speaking, they are esters which can be hydrolyzed by boiling with
dilute acid to yield a phenolic compound, usually a derivative of
gallic acid, and a sugar. These are often referred to as pyrogallol
tannins.
Condensed tannins are polymers of phenolic compounds related to
the flavonoids and are similar in general properties to the hydrolysed
tannins but are not very soluble in water and following treatment
with boiling dilute acid red-brown insoluble polymers known as
phlabaphenes or tannins-red are formed.
Tannins are detected most simply in plant extract by the use
of the so called gelatin-block test which has been utilized
extensively in the phytochemical surveys. This test employs
aqueous extract prepared from 80% ethanol extracted plant
material. A sodium chloride solution is added to one portion of the
42
test extract, of 1% gelatine solution to a second portion, and the
gelatine salt reagent to a third portion. Precipitation with the latter
reagent or with both the gelatine and gelatine-salt reagents is
indicative of the presence of tannins. If precipition is observed only
with the salt solution a false-positive test is indicated. Positive test
are confirmed by the addition of ferric chloride solution to the extract
and should result in a blue, blue-black, green or blue-green color
and precipitate. Hoch has applied some 33 different classical tannin
detecting reagents to several tannin extract; however, the
nonspecificity of many of these would render them impractical for
use in general phytochemical screening work.

Extraction of tannins

The process used to extract tannins is the

hydrosolubilization Because this process operates with
temperaturesaround 100°C, the extraction process motives
ahydro cracking of sugar and others organic compounds with a
darkening of the final product. We studied the supercritical
extraction process as alternative procedure to obtain the natural
raw material to leather tannage . In which supercritical carbon
dioxide and polar ornon-polar co-solvents were used as solvents.
The advantages of supercritical extraction process withregard to
hydrosolubilization and solvent extraction are low extraction
temperature, shortextraction time and absence organic solvent
concentration in the extract. Another objective wasto compare the
different extraction technology.

MATERIAL AND METHODS

Natural Material
The raw material, black acacia bark, was provided by
EXTRATOS BRASIL. A part of bark was milled with cutting mill
(TECNAL - Willye TE 650) with 2.0 mm average particle diameter.
In the solvent extraction with Soxhlet apparatus, dry natural
material was employed. The dryer used Pansera, M. R. et al.
Brazilian Archives of Biology and Technology 996 was a BIOMATIC
Equipment. The tannin was dried for 10 days at 36°C.

43
Solvent Extraction Process

Samples (50 g of dark acacia bark) were extracted with

500 mL of solvent for 24 h. The extracted solution was
concentrated on a SAVANT Lyofilizator.

Supercritical Extraction Process

A Hewlett Packard 7680 extraction module was used to

perform all the experiments. This system consisted of a nozzle/trap
assembly that acted as a controllable variable restrictor, allowing
an instantaneous depressurization of the supercriticalfluid as well
as the decoupling of flow and pressure. The material to be
extracted was loaded into a self-sealing extraction cell of 7.0 mL
thickwalled stainless steel thimble. Supercritical fluid extracts were
deposited in an internal trap rinsed off into a vial with 1 mL of
hexane and methanol. Samples (0.4 g of bark) were extracted with
supercritical carbon dioxide according to the described procedure,
where different experiments to optimize the extraction conditions
were done.The operation temperature and pressure rangestested
were 40 to 80ºC and 150 to 200 bar,respectively. All other variables
were kept constant: CO2 flow, 2.0 mL/min; and extractiontime, 30
minutes..

44
Essential oils

Essential oils (volatile oils) in addition to their value as flavoring

agents and perfumes have been reported to have excellent
antibacterial and antifungal properties.
A few reports have been published which
inclued an evaluation of plant samples for the presence of
essential oils For maximum efficieney tests should be conducted
on fresh material since most of the volatile constituents of plants
are lost during drying. In most instances the methods that have
been employed since they of necessity had been conducted in the
field as organoleptie examina-tions. More elaborate laboratory
examinations have involved steam distillation followed by
measurement of the water immiscible oil and in some eases
followed by the application of chemical tests for terpenes Arthur
studied more than 700 species in North Borneo and Hong Kong
and simply chopped a small amount of fresh plant with a razor
introduced the into a test tube added hot water and boiled the
mixture Any characteristic odor of essential oils was then recorded.
Kohlmunzer evaluated some 59 species of plants from genera
known to have previously yielded economically important essential
oils (salvia Lavandula Mentha Rosmarinus Thymus etc). Following
steam distillation and subsequent measurement of the separation
oil, chemical tests for cineol were applied on the other hand Betts
has devised a method employing thin layer chromatography for the
evaluation of petroleum ether extracts from umbelliferous fruits in
which essential oils are universally soluble. Fluorescein-treatad
plates of his extracts were first viewed under ultraviolet light to
note the presence of dark quenching spots against a bright yellow
background Unsaturated compounds were then detected as spots
by exposure of the plates to bromine vapor which converted the
fluorescein to eosin and subsequent ultraviolet examination then
indicated unsaturated compounds against a dull background plates
were then sprayed with 2,4-dinitrophenylhydrazine which revealed
ketones and aldehydes as orange spots. A simple microcohobation
still for the estimation of quantities of essential oil ranging from 2-
50 al. in small (0.4 Gm.) samples of plant material has been shown
to screening large numbers of plant samples for essential oils on a
quantitative basis. At least this would be some improvement over
current organoleptie methodology..

Extraction: Hydro distillation method was used for the extraction

of the oil. The macerated plants were weighed and packed into the
45
round bottom flask. This was mixed with lots of water as the
extracting solvent. The distillates were then transferred into a
sample bottle.

Antibacterial assay: Pour plate method was used for the assay .
Nutrient agar was prepared by dissolving the appropriate quantity
in deionised water, homogenized and sterilized. The organism was
introduced into the sterile plate under aseptic conditions. 20ml of
the prepared nutrient was also poured into the petri dish, swirled
together and allowed to solidify. After solidifying, cork borer(9mm)
was used to bore two wells in the medium or culture and the oil
poured into the bored well to notice if there would be any zone of
inhibition or not. A positive control with Streptomycin was used.

Micro-organisms assay: The slants of nine organisms, six

bacteria and three fungi were collected from the laboratory stock of
the Department of Medical Microbiology, University College
Hospital UCH, Ibadan, Oyo state. The bacterial are Klebsiella
pneumoniae, Bacillus megaterium, Bacillus subtilis, Proteus
mirabilis, Pseudomonas aeruginosa and Eschrichia coli and the
fungi are Aspergillus favus, Trichoderma spp. And Aspergillus
niger.

SCREENING OF Flavonoids

46
Definition & Chemistry
The term favonoid refers to a class of plant secondary
metabolites based around a phenyl benzopyrone structure.
Flavonoids are most commonly known for their antioxidant activity.
Flavonoids are also commonly referred to as bioflavonoids in the
media –these terms are equivalent and interchangeable since all
Flavonoids are biological in origin.

The Flavonoid synthetic pathway begins with a product of

glycolysis phosphoenolpyruvate entering into the shikimate
pathway toyield phenylalanine. Phenylalanine is the starting
material of the phenylalanine metabolic pathway from which 4-
coumaryl- CoA is produced. This can be Combined with malonyl –
CoA to yield the true backbone of Flavonoids a group of
compounds called chalcones. Ring-closure of these compounds
results in the familiar form of Flavonoids a three ringed phenolic
structure (polyphenols).
Flavonoids have been referred to as “nature’s biological
response modifiers” because of strong experimental evidence of
their inherent ability to modify the body’s reaction to allergens
viruses and carcinogens. They show anti-allergic anti-inflammatory
anti- microbial and anti- cancer protectingagainst oxidative and
free radical damage.

Isolation of Flavonoids

It is estimated that about 8%of all carbon photosynthesized by

plant is converted into flavonoid closely related compound most
tanning are flavonoid. Flavonoid thus constitute one of the largely
occurring natural compound. In plant flavonoid aglycone occur in
variety of the structural form all contains 15carbon atom in main
basic nucleus and these are arranged in a C-C-C configuration.
That is two aromatic ring linked by 3corbon unit which may not
form a third ring. The flavonoid varied are all related by common
biosynthetic pathway which incorporate preculor form both the
shikmate and acetate mevalonate pathway. These compounds
occur in green plant with the exception of algae and horn weeds.
They are virtually in all plant parts including leaves, wood, bark,
pollen nector flowers brarriers and seeds in the few recoded cases
of the favonoids being found in animals.
The distribution of flavonoids is found in the largest way
in the higher plant Angiosperm. Important feature of the distribution

47
of Flavonoids in plants is the strong tendancy taxonomically
related plant to produce similar type Flavonoids.

ISOLATION-SOLUBILITY CHARACTERISTICS

Flavonoid aglycone polyphenols and as such passes the property

of phenotic I. e. they are slightly acidic and will thus dissolved in
alkali. However it is left in alkali in the presence of oxygen many
flavonoid will degrade flavonoid possess NO of unsubstituited –OH
sps or sugar or polar compound.
These are generally moderator dissolved in polar
solvent such as methanol ethanol acetone dimethyl sulfoxamide
dimethly furomide water etc presence of unattached sugar tends to
render the flavonoid more H2O soluble while polar aglycones such
as isoflavone flavonone and slightly methoxylated flavone and
flavonones tends to be more soluble in solvent such as ether and
propanol.

CONCLUSION

48
Thus the screening of the phytochemical constituent was studied
screening can be used in research & development for
specialization in field of pharmacy.

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49
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52