PHYTOCHEMICAL

SCREENING AND
ANTIOXIDANT PROPERTY
OF CHILI PEPPER
(Capsicum annuum)
LEAF EXTRACT

Background of the study

Plants can provide remedies that are more suitable for the
human body than the commercially available synthetic
chemical products, and are the best agents for treating
diseases.
As time passed by, people discovered that plants can be used
for different purposes. They also acknowledged different
varieties of plants with various medicinal properties.
Some people who cannot afford to buy medicines resort to
the use of herbal medicines as an alternative to treat minor
ailments.
WHO estimated that 80% of people worldwide rely on
herbal medicines for some part of their primary healthcare.

 The practice on the utilization of medicinal plants dates back

to the very earliest periods of known history. There is
evidence of medicinal plants having been used in the
treatment of diseases, revitalizing the body system in almost
all civilizations - India, the Philippines, the Egyptians, the
Chinese, and even the Greek and the Romans.
The wide spread use of herbal medicine remedies and
preparations, as those described in ancient texts such at the
Vedas and the Bible, and obtained from commonly used
traditional herbs and medicinal plants, has been traced to the
occurrence of natural products with medicinal properties.
The use of traditional medicine and medicinal plants in most
developing countries is a normative basis for the maintenance
of good health.

PHYTOCHEMICALS
These are the chemical compounds that occur naturally
in plants (phyto means plant in Greek).
Some are responsible for color and other organoleptic
properties, such as the deep purple of blueberries and
the smell of garlic.
Phytochemicals may have biological significance, for
example carotenoids or flavonoids, but are not
established as essential nutrients.
There may be as many as 4,000 different
phytochemicals.
Have the potential to affect diseases such as cancer,
stroke or metabolic syndrome.

PHYTOCHEMICAL SCREENING
It is a process where important phytochemical constituents
that exhibit biological activity is determined. Among these
phytochemicals are the alkaloids, saponins, steroids,
flavonoids, tannins and compounds having polyphenol,
antraquinones and cyanoglycosides.
1. ALKALOIDS- They are a structurally diverse group of over
12,000 cyclic nitrogen-containing compounds that are found in
over 20% of plant species.
This group also provides the cholinesterase inhibiting
treatments routinely prescribed for the cholinergic
deregulation of Alzheimers disease (AD), such as
galantamine, huperzine, physostigmine, and rivastigmine.

 A number of alkaloids are used in drugs. Quinine,

derived from the bark of the tropical cinchona tree,
which helps in curing malaria. Cinchona bark also
produces quinidine, which is used primarily to control
abnormalities of heart like fibrillations and heart block.
Alkaloids like vincaleukoblastine and vincristine,
derived from periwinkle plant, are useful for the
treatment of lymphoma and childhood leukemia
respectively.
Many alkaloids are still used in medicine, usually in the
form of salts, including the following : antitumor,
antihypertensive, anesthetic, cough medicine, analgesic,
antipyretic, and antimalarial.

2. TANNIS- Tannins are complex chemical substances derived

from phenolic acids. They occur in many species of coniferous

trees as well as a number of flowering plant families.
Medical research has shown that tannin found in cranberries
is highly effective in preventing urinary tract infections by
preventing E.coli bacteria from adhering to walls of the
urinary tract. Similarly, the anti-adhesive property may
reduce the ability of H. pylori to cause stomach ulcers.
Various experiments and studies show that they also have
anti-carcinogenic, anti-mutagenic, anti-oxidative and antimicrobial properties.
They have been reported to exert other physiological effects,
such as to accelerate blood clotting, reduce blood pressure,
decrease the serum lipid level, produce liver necrosis, and
modulate immune responses.

3. ANTHRAQUINONES- These are aromatic organic compounds that

occur naturally in certain plants, fungi and insects. Since, they contribute
to the coloring pigment of such organisms, the compound is used
commercially to manufacture different dyes. Other commercial
applications of anthraquinones are a catalyst used in the production of
wood pulp and paper and a derivative, called 2-ethylanthraquinone, is
used to manufacture hydrogen peroxide.
Huang et al. (2007) determined the anti-cancer properties of
anthraquinones from Rhenium palmatum. Rhubarb has been used as a
traditional Chinese medicine since ancient times and today it is still
present in various herbal preparations. The most abundant
anthraquinone of rhubarb, emodin, was capable of inhibiting cellular
proliferation, induction of apoptosis, and prevention of metastasis.
Aloe-emodin is another major component in rhubarb found to have
anti-tumor property.
Rhein is the other major rhubarb anthraquinone, although less well
studied. This compound could effectively inhibit the uptake of glucose
in tumor cells, cause changes in the membrane associated functions and
lead to cell death.

4. FLAVONOIDS- It is the collective term for a large group of

natural dyes found in the outer layers of almost all fruits and
vegetables, but also in some nuts, cereals, tea and cocoa.
Chemically, they belong to the polyphenols.
Many of the studies on flavonoids have been done on
materials in test tube or animals, so it is not entirely clear
how effective they are in humans, but they may lower the
risk of a variety of health problems, including cardiovascular
diseases, age-related degenerative diseases, and cancers.
They may also help prevent tooth decay and reduce the
occurrence of common illnesses like the flu.
Preliminary research indicates that flavonoids may modify
allergens, viruses, and carcinogens, and so may be biological
response modifiers. In vitro studies show that flavonoids
also have anti-allergic, anti-inflammatory, anti-microbial,
anti-carcinogenic, and anti-diarrheal activities.

5. STEROIDS- Plant steroids are types of natural organic compounds

found in plants. Many types of plant steroids exist and play important
roles in the biological processes of plants, such as growth and
development, cell division, and resistance to damage from
environmental stresses like cold weather. Some plant steroids are
also useful for their effects when consumed by human beings
because their presence decreases the amount of cholesterol in the
bloodstream.
6. CYANOGENIC GLYCOSIDES- They are widely distributed over
80 different plant families, often legumes and grasses. They are
usually found in plants together with hydrolytic enzymes. Hence,
most of the cyanogenic glycosides are lost after plant harvest due to
spontaneous hydrolysis.

7. SAPONINS- Saponins are naturally occurring compounds

that are widely distributed in all cells of legume plants. Clinical
studies have suggested that these health-promoting components,
affect the immune system in ways that help to protect the human
body against cancers, and also lower cholesterol levels.
Saponins also decrease blood lipids, lower cancer risks, and
lower blood glucose response. A high saponin diet can be used
in the inhibition of dental caries and platelet aggregation in the
treatment of hypercalciuria in humans, and as an antidote
against acute lead poisoning.

ANTIOXIDANTS

They are substances that protect cells against the effects of

free radicals. Free radicals are molecules produced when the
body breaks down food or by environmental exposures like
tobacco smoke and radiation Free radicals damage cells, may
play a role in heart disease, cancer and other diseases.
Antioxidant substances include Beta-carotene, Lutein,
Lycopene, Slelenium, Vitamin A, Vitamin C, and Vitamin E.
Antioxidants are found in many foods. These include fruits
and vegetables, nuts, grains, and some meat, poultry and fish.
Studies have shown that most of the antioxidant compounds
posses anti-inflammatory, anti-atherosclerotic, anti-tumour,
anti-mutagenic, anti-carcinogenic, anti-bacterial, and anti-viral
activities .The ingestion of natural antioxidants has been
associated with reduced risks of cancer, cardiovascular
disease, diabetes, and other diseases associated with ageing.

DPPH Assay Method

Anti-radical activity assay is based on the reduction of 1,1diphenyl-2-picrylhydrazyl (DPPH). Due to the presence of an
odd electron it gives a strong absorption maximum at 517 nm.
Briefly, the reaction mixture contained 0.08ml of 50 mm TrisHCL buffer (pH 7.4) and 0.1ml of 100M DPPH dissolved in
ethanol and various concentrations of different antioxidants in
0.02 ml solution. The mixture is shaken and the absorbance at
517 nm is read at various time intervals.

CHILI PEPPER(Capsicum annuum)

Capsicum annuum is a species of the plant genus Capsicum, ordersolanales. Family- solanaceae.
Native to southern North America and northern South America,
this species is the most common and extensively cultivated of the
five domesticated capsicums. Despite being a single species, it has
many forms, with a variety of names even in same language.
The plant is perennial, with a densely branched stem. While the
species can tolerate most climates, it is especially productive in
warm and dry climates.
It is rich in Vitamins A, C, Iron and Calcium. It contains VitaminG, Magnesium, Phosphorus, Sulphur; it also has some B-complex,
and is rich in Potassium.

SIGNIFICANCE OF THE STUDY

Ways to cure diseases and other ailments
using herbal medicines.
To learn about the various phytochemicals
present in chili pepper.
To determine if chili pepper adds nutritional
value to food it is added to.
To promote the use of herbs and other plants
in making medicines and curing diseases.

OBJECTIVE OF THE STUDY

The study generally aims to conduct a qualitative
phytochemical screening and antioxidant property of chili
pepper (Capsicum annuum) leaf extract.
It seeks to answer questions likes1) Which of the following secondary metabolites are present
in the sample?
a. Alkaloids
b. Tannins
c. Anthraquinones
d. Flavonoids
e. Saponins
f. Phenolics
g. Phytosterols

2. What will be mean absorbance and percent (%) inhibition

of free radical using DPPH (Diphenyl Picryl Hydrazyl) in
evaluation of the antioxidant property of chili pepper at the
following functions?
a. 25 mcg/ml
b. 50 mcg/ml
c. 100 mcg/ml
d. 150 mcg/ml
e. 200 mcg/ml
3. What is the difference between the antioxidant property of
chili pepper (Capsicum annuum) leaf extract and the
standard drug ascorbic acid?

SCOPE AND DELIMITATION

The study will be delimited to the determination of the phytochemical
constituents and the antioxidant property of chili pepper (Capsicum
annuum) leaf extract.
The phytochemical constituents are limited to plant secondary
metabolites. Only the fresh and healthy leaves of Capsicum annuum
randomly picked from propagated plants in Bulala Norte, Vigan City,
Tamag and Cagayan in Ilocos Sur, will be used in study.
The extraction process of the leaves of Capsicum annum for the
phytochemical screening and the antioxidant property will be
conducted by the analyst at the University of Santo Tomas, Manila
City from November - December 2015.
For the antioxidant property testing, fresh leaves of Capsicum annum,
also collected from propagated plants in Bulala Norte, Vigan City and
Tamag, Vigan City, Ilocos Sur, will be analyzed at the University of
Santo Tomas, Manila City from November - December 2015. The
Diphenyl Picryl Hydrazyl (DPPH) method will be used.

Requisites for Phytochemical

Screening

Selection of promising plant materials

Proper collection of selected plants.
Authentication of plant materials
Drying of plant materials
Grinding of the dried plants
Garbling of the dried plants
Packing, storing and preserving
Extraction and fractionation of constituents
Methods of separation and purification
Methods of identification of isolated compounds

METHODOLOGY
The different procedures that will be employed in the study are
adopted from various authors, hence these are standard methods. The
first part discusses the procedure for phytochemical screening,
employing the presence of the secondary metabolites described by
Guevara (2005). The second part discusses the procedure for
antioxidant property testing employing the DPPH assay method by
Bozin et al.,(2006) and Nyaa et al.,(2009)
Phytochemical Screening
Plant extractiona. About 500 grams of the ground fresh plant material will be weighed
in an Erlenmeyer flask and will be treated with sufficient 95% ethyl
alcohol to completely submerge the material.
b. Stopper the flask and keep the material soaked for 48 hours.
c. The mixture will be filtered through a Buchner funnel with gentle
suction.

d. The flask and plant material will be rinsed with fresh

portions of alcohol. Washings and plant material will be
transferred to the funnel, combining the washings with the
first filtrate.
e. Gentle suction will be applied to complete collection of
the plant extract. Then, the plant residue will be discarded.
f. The filtrate will be concentrated under vacuous
temperature below 50 degree Celsius to about 20 ml.
g. The extract will be stored tightly stoppered, preferably in
the cold (0-5 degree Celsius) until used.

Test for the Alkaloids (Dragendroffs and Meyers Tests)

a. An equivalent of 20g plant material will be taken from the stock
plant extract and will be evaporated to incipient dryness over a
water bath. It will be evaporated to a syrupy consistency over a
steam bath, to which will be added 5 ml of 2M HCl, will be heated
for about 5 minutes and then cooled;
b. About 0.5 g NaCl will be added, then stirred and filtered;
c. The residue will be washed with enough 2M HCl to bring the
filtrate to a volume of about 5 ml;
d. 1 ml of the filtrate will be tested with 2-3 drops of Dragendroffs
reagent;
e. Another 1 ml of the filtrate will be tested with 2-3 drops of
Mayers reagent;
Positive results for alkaloids: with Dragendroffs reagent, a
positive test is indicated by an orange precipitate, while with
Mayers reagent, a positive test is indicated by a white precipitate.

Test of tannins (Spot test)

a. Ten grams of finely cut fresh leaves of C. annuum will be
macerated with 10 ml of 0.1M HCl, and filter off the residue;
b. The filtrate will be evaporated to 1 ml volume;
c. A drop of the prepared acid plant filtrate will be placed on the
yellow reagent paper, heated on top of a hot plate enough to
hasten the reaction, then will dry the paper in about three
mintues;
d. The colour of the spot will be observed, and subsequently the
resulting strip of paper will be dipped into a container of water
for few minutes to leach out the yellow nitroso compound;
e. Any changes in colour of the spot was observed.
Positive results for tannins: The formation of a red-brown stain
on the yellow reagent paper is indicative of the presence of
tannins.

Test for Anthraquinones (Borntragers test)

a. An equivalent of 1g plant material will be taken from the
stock plant extract and evaporated to incipient dryness over a
steam bath.
b. The residue will be taken with 10 ml diluted water and
filtered; discarding the residue;
c. The aqueous filtrate will be extracted twice with 5 ml
portions of benzene, combining the benzene extracts, then
treated with 5ml ammonia solution;
Positive result: A red coloration in the lower ammoniacal layer
indicated the presence of anthroquinones.

Test for Flavonoids (Wilstatter Test)

a. An equivalent of 10g plant material will be taken from the
stock plant and evaporated to incipient dryness over a steam
bath.
b. It will be cooled to room temperature; defatted by taking up
the residue with petroleum ether, discarding the petroleum
ether extract;
c. The aqueous layer will then be defatted with 10ml of 80%
ethyl alcohol, filtered and treated with 0.5 ml conc. HCl.
d. Four pieces of magnesium turnings will be added to the
filtrate and observed for any color changes for 10 minutes.
Positive result: Colors ranging from orange to red, to crimson
and magenta, and occasionally to green or blue may be
observed.

Test for Steroids (Liebermann-Burchard Test)

a. An equivalent of 10 g plant material will be taken from the plant stock and
evaporated to incipient dryness over a water bath, and cooled to room
temperature;
b. It will be defatted by taking up the residue with 6 ml of hexane and water
(2:1), partitioned by gently shaking the mixture in a test tube; the upper hexane
layer will be pipetted out. The treatment will be repeated with hexane until most
of the colored pigments have been removed, and discarded all the hexane
extracts properly;
c. The defatted aqueous layer will be heated over a water bath to remove the
residual hexane then cooled to room temperature;
d. One third of the defatted aqueous layer free from hexane, will be added with
10 ml dichloromethane and the mixture will be stirred for a few minutes and
allowed to stand;
e. The lower dichloromethane extract will be pipetted off and dried by passing it
through about 100 mg anhydrous sodium sulphate placed over dry filter in a
funnel; it will be treated with 3 drops of acetic anhydride, then one drop of conc.
sulphuric acid;

f. Any immediate color change will be observed.

g. It will be allowed to stand for an hour and further color
changes will be observed.
Positive result gives colors ranging from blue to green, red, pink,
purple or violet.
Test for Saponins (Froth Test)
a. A volume of the plant extract equivalent to 2g plant material
will be transferred to a test tube;
b. 10 ml of distilled water will be added to the test tube,
stoppered and shaken vigorously for 30 seconds, and allowed to
stand for 10 minutes to observe for honeycomb froth from the
surface of the liquid.
Positive result: If the honeycomb froth greater than 2 cm
height from the surface of the liquid persists after 10 minutes, the
sample was considered positive for saponins.

Test for Cyanogenic Glycosides (Grignard Test)

a. Five grams of the crushed plant material will be placed in a test
tube, moistened with enough water, to which a few drops of
chloroform will be added to enhance enzyme activity. 1 ml 1%
emulsion solution will be added to insure hydrolysis of the
glycoside;
b. The test tube will be stoppered with a cork from which will be
suspended a strip of yellow picrate paper, taking note that it
should not touch the inner side of the test tube;
c. The tube will be kept at room temperature for 3 hours, and any
color change in the picrate paper will be observed.
Positive result: Appearance of various shades of red within 15
minutes when the tube is warmed, gives the relative measure of
the concentration of cyanogenic glycosides.

Antioxidant Property Testing

Extraction of Plant Material
a. 500 grams of the ground fresh plant material will be weighed
in an Erlenmeyer flask and treated with sufficient 95% methyl
alcohol to completely submerge the material, stoppered and
allowed to stand for 48 hours;
b. The mixture will then be filtered through a Buchner funnel
with gentle suction to complete collection of plant extract;
c. The filtrate will be concentrated under vacuum at temperature
below 50 degree Celsius to about 20 ml, and submitted to the
laboratory for the antioxidant property testing.

Antioxidant Property Testing Proper

The leaves extract at various fractions (25 mcg/ml, 50
mcg/ml, 100 mcg/ml, 150 mcg/ml, 200 mcg/ml) will be
mixed with 2 ml of 30 M DPPH solution. The same amount
of concentration will be prepared for the positive control,
ascorbic acid. After 30 minutes incubation period at 37
degree Celsius, the absorbance will recorded at 517 nm using
a spectrophotometer. Inhibition of free radical by DPPH in
percent will be calculated using the following formula:
(Abscontrol Abssample)
Inhibition (%) = x 100%
Abscontrol
where Abscontrol is the absorbance of the control (containing all
reagents except the test samples) and Abssample is the
absorbance of test samples.

 In the present study, DPPH radical will be used as substrate

to evaluate the free radical scavenging activity of the
Capsicum annuum leaf extract. The reduction capability of
the DPPH radicals will be determined by the decrease in its
absorbance at 517 nm, which is induced by antioxidants.
The spectrophotometric method of determining the
antioxidant property of the Capsicum annuum leaf extract is
based on the principle that the lower the absorbance reading
of the extract, the greater is the antioxidant property in
comparison with the controls used.