PHYTOCHEMICAL SCREENINGS AND ANTIOXIDANT

PROPERTY EVALUATION FOR THE CRUDE EXTRACTS

OF THE STEMS, LEAVES, AND FRUITS OF Averrhoa
bilimbi Linn.
Berdin, N. M; Carreon, J. B. and Monte, J.
Natural Research Group, Department of Chemistry, University of San Carlos, Talamban, Cebu
City, Philippines.
Summer Science Internship Program

ABSTRACT
In the Philippines, Averrhoa bilimbi, or locally known as kamias, is a
common plant. It is an easy access to the locals as an alternative medication
for several diseases such as skin eruptions, inflammations, coughs, fever,
and likes. However, the use of the plant as medications is only a common
belief and isnt scientifically studied to be safe and effective. The study is
done to determine the phytochemicals present in the plant that could prove
the effectiveness of A. bilimbi as medicine to several diseases. Also, this
study is done to evaluate the antioxidant property of the plant. However,
only the leaves, stems, and fruits were separately experimented. Previous
studies on the plant suggested the presence of flavonoids, saponins, and
alkaloids in the bark extracts of A. bilimbi. Phytochemical screening and
DPPH Radical Scavenging Assay were done to the separate crude extracts of
the stems, leaves, and fruits of A. bilimbi. The result of the experiments
showed that: (1)the stems of A. bilimbi contain tannins, saponins, and
terpenoids; (2)the leaves of A. bilimbi contain tannins and saponins; (3)the
fruits of A. bilimbi contain saponins and cardiac glycosides; (4)and very slight
antioxidant property of A. bilimbi.
CHAPTER 1: INTRODUCTION
1.1. Rationale of the Study
From ancient times plants
have
provided
a
source
of
inspiration
for
novel
drug
compounds, as plant derived
medicines
have
made
large
contributions to human health and
well-being. Many Indian Plants are

used therapeutically for their antidiabetic effect and antibacterial

activities. Averrhoa bilimbi is
medicinally used as a folk remedy
for many symptoms. It is used for
the treatment of fever, mumps,
pimples, inflammation
of
the
rectum and diabetes, itches, boils,
rheumatism, syphilis, bilious colic,
whooping cough, hypertension,

stomachache, ulcer, and as a

cooling drink.
Averrhoa bilimbi is a multipurpose, long-lived tropical plant
commonly known as Bilimbi,
Cucumber Tree belonging to
family Oxalidaceae. The plant has
an enormous fiscal value since
most of the parts like leaves, bark,
flowers, fruits, seeds, roots or the
whole plant are used as alternative
medicine to treat a variety of
disease especially diabetes. In the
present review, we tried to give the
existing
information
on
photochemical
constituents,
conventional medicinal uses and
anti-microbial, anti-inflammatory,
cytotoxic activities, anti-oxidant
activity,
antifertility,
and
antibacterial activities and other
biological activities of Averrhoa
bilimbi. The extract of various parts
of Averrhoa bilimbi is medicinally
used as a folk remedy for many
symptoms and showed significant
pharmacological activities so it is
necessary to perform further
investigation
to
isolate
such
pharmacological active compounds
which can be used in production of
novel drugs for various diseases.
Overall, this paper gives an
overview on covering the biology,
and
various
commercial
and
therapeutic applications.
As a part of the study, crude
extracts from the fruits, stems, and
leaves of Averrhoa bilimbi were
obtained
for
phytochemical

screenings and evaluation for antioxidant property.

1.2. Statement of the Problem

The study aimed to:
1. determine the antioxidant
potential (using 2,2-diphenyl1-picrylhydrazyl
(DPPH)
radical scavenging assay) of
the crude extract obtained
from decoction using distilled
H2O; and
2. carry
out
phytochemical
screening on the crude
extract.

1.3. Significance of the Study

The study aims to present
proofs the A. bilimbis ability to act
as antioxidant and other native
beliefs
upon
its
usage
as
treatments for some diseases. The
study
also
investigates
the
possibility for producing cheaper
antioxidant
and
antibacterial
medicine with the plant A. bilimbi.
1.4. Scope and Limitation
The study was conducted to
determine the antioxidant property
and the presence of secondary
metabolites of Averrhoa bilimbi.
Experiments were conducted in the
research facilities of the University
Of San Carlos (USC)-Technological
Center, Department of Chemistry.

The experiments were done within

a span of approximately two weeks
only. Plant samples were collected
from
the
premises
of
USC,
Talamban, Cebu City, Philippines.
The extraction process was
based on decoction in distilled H2O
as the solvent.
Phytochemical
screening was done to classify the
secondary metabolites present in
the crude extract of Averrhoa
bilimbi. The antioxidant potential
was evaluated using 2,3-diphenyl-picrylhydrazyl
(DPPH)
radical
scavenging activity.
The extraction method based on
sequential soaking method in the
following
solvents:
n-hexane,
dichloromethane
(DCM)
and
ethanol were not done. The
isolation of the extract and the
characterization
via
Fourier
Transform Infrared Spectroscopy
(FT-IR) were not done.

CHAPTER 2: REVIEW OF
RELATED LITERATURE
2.1. Averrhoa bilimbi Linn
Classification:
Kingdom
Plantae
(Plants)

Subkingdo Tracheobionta
(Vascular Plants)
m
Superdivisi Spermatophyta
(Seed Plants)
on
Division
Magnoliophyta
(Flowering Plants)

Class
Subclass
Order
Family
Genus
Species

Dicotyledonae
Rosidae
Oxalidales
Oxalidaceae
Averrhoa
Bilimbi L.

Description:
Averrhoa bilimbi is a tree that
grows about 13 meters tall with
compound leaves of 14-38 yellowgreen, hairy leaflets. The flowers of
the plant usually grow directly out
of the trunk of the tree, and are
cream colored with red dots about
1-2 cm. long. The fruits are oblong
with yellow-green, glossy outer
skin. The fruits have a slightly
acidic taste.
Geographic Distribution:
This
plant
most
likely
originated in Moluccas, Indonesia.
The species is now found in
Philippines, Sri Lanka, Bangladesh,
Maldives, Malaysia, and to some
other Southeast Asian countries.
Alternative/Folk Medicinal Uses:
The leaves are applied as a
paste on itches, mumps, and
rheumatism, and on skin eruptions.
They are also applied on bites of
poisonous creatures. A leaf infusion
is a remedy for coughs and is taken
after childbirth as tonic. A paste of
pickled bilimbis is smeared all over
the body to hasten recovery after a
fever.
2.2. Free
radicals
Antioxidants

and

Free radicals are atoms that

contain an unpaired electron and
can be formed when oxygen
interacts with certain molecules.
Once formed, these highly reactive
radicals can start a chain reaction.
The worst scenario that could
happen is when they would react
with important cellular components
such
as
DNA,
or
the
cell
membrane. Cells may function
poorly or die if this occurs. To
prevent this kind of damage, the
body has a defense system of
antioxidants.
Antioxidants are molecules
that can safely interact with free
radicals to stop the chain reaction
before further damage to the cells
may happen.

2.3. Antioxidant
Potential
Assays
The DPPH assay method is a
stable free radical, which is based
on reduction of 2,2-diphenyl-1picryl-hydrazyl(DPPH).
Nitrogencentered radicals such as DPPH
react with phenols by means of two
different mechanisms: (i) a direct
abstraction of H-atom of the phenol
and (ii) an electron transfer process
from the hydroxyl group of phenols
(OH) or its phenoxide anion (O2-)
to DPPH.
(i)
OH + DPPH -> O + DPPH
(ii)
O + DPPH -> products
The contribution of either of the
pathways depends on the nature of

the solvent and/or the redox

potentials of the species involved.
The reduction of phenols and
abstraction of phenol-H atom
mechanism
is
predominant
generally in non-polar solvents, but
on the other hand, in polar solvents
such as methanol or ethanol the
electron
transfer
mechanism
becomes crucial as it forms strong
hydrogen bonds with the phenol
molecules. The DPPH has been
used extensively as a substrate to
evaluate antioxidant activity of
biological as well as chemical
substance as it is a useful reagent
for investigating the free radical
scavenging activities of many
compounds.
Another one is Reducing
Power. The reducing power of any
compound is usually used as an
indicator of its electron-donating
ability, which is an important
mechanism
for
testing
its
antioxidative
activities
as
a
function of reducing power, which
increases
with
increasing
concentration in all the samples
(Duh, 1997).

CHAPTER
METHODOLOGY

3:

Collection and Preparation of

Samples
Leaves, stems, and fruits of
A. bilimbi were randomly collected
from the premises of University of

San Carlos, Talamban, Cebu City,

Philippines on May 14, 2015. The
leaves and stems were air dried for
four days. The fruits were chopped
and were put in the freezer for
three days and were then freeze
dried for 24 hours.
EXTRACTION
Samples of ten grams of each
part were decoctated.
Decoction. Weighed sample
was mixed with 200 mL water and
boiled for 10 minutes.
Then, the crude extract was
then cooled and filtered the
unwanted particles and substances
of the crude extract. The residual
from filtering process was only the
liquid crude extract.
PHYTOCHEMICAL SCREENING FOR
KAMIAS
The crude extracts of A.
bilimbi were screened for the
presence of Polyphenols
Tannins, Saponins, Terpenoids,
Steroids, Flavonoids, Alkaloids,
Anthocyanin, Carotenoids, and
Cardiac Glycosides. See specific
procedures presented in Appendix
A.
TEST FOR ANTIOXIDANT POTENTIAL
The antioxidant potential of
the two fractions per stem, root,
and fruit crude extracts were
measured
via
DPPH
assay
according to the method of Lim et
al(2007).
3 mL of DMSO was
added to all three trials on each

fraction. The solution with 0.0267g

of DPPH in ethanol and 9 mL was
added to each 1 mL trial to get 50
ppm concentration. After one hour
of incubation, absorbance of each
trial was evaluated and recorded.
Percent
radical
scavenging
actuvuty
of
each
trial
was
calculated using the formula:
% radical scavenging activity =
[(Acontrol - Asample)/Acontrol] x 100
Such that Asample is the
absorbance added to the ethanolic
DPPH solution, while Acontrol is the
absorbance of DMSO to the
ethanolic DPPH solution.
CHAPTER 4 : RESULTS
The
phytochemical
screening
showed the presence of saponins
and cardiac glycosides on the fruit
crude extracts. The stem crude
extracts was screened to be
positive with Tannin, sapponin, and
terpenoids. And the root crude
extracts was screened and showed
to be positive in tannins and
saponins. Refer to Appendix B for
more detailed readings.
The parts were tested on their
antioxidant potential. 18.58 %
average absorbance showed from
New batch of fruit crude sample
test with an outlier. The new batch
of stem crude sample showed
12.17%
average
absorbance
without any outlier. And the New
batch of root crude extract
revealed
12.42%
average
absorbance without any outlier.

CHAPTER 5 : DISCUSSION
The presence of tannin in stem and
root of A. bilimbi could prove the
antioxidant, and anticarcinogenic
potential of the plant. And as to the
plant, toxicity of tannin could serve
as protection from living organisms
in all ratio.
Saponin which is present in three
tested parts of A. bilimbi could lead
the antibacterial, and antimicrobial
potential which is to shield against
intruding bacteria and fungi into
the plant.
Presence of Terpenoid in stem of
the plant could support the ability
of the plant to prevent bacteria
from entering the plant.
Cardiac glycosides present in the
fruit of plant could support the
availability for A. bilimbi as cure
and prevention for few heart
ailments.
All plant parts have significant
antioxidant potential. The fruit of
the plant revealed the most
potential on antioxidant activity.
CHAPTER 6 : CONCLUSION
The presence of tannin, saponin,
terpenoid, and cardiac glycosides
support the effectiveness of A.
bilimbi, commonly called as iba.
The folk usage of the plant as
medicine was effective. In fact, the
study proved that the folkly unused
stem of the plants can now be
effectively
used
also
for

medication. This plant can be

produced as less expensive and
commerial medicine after further
research. The plant can be used
effectively as antibacterial and
antimicrobial medication as well as
prevention and cure for heart
ailments. Hence, much further
study needs to be performed to
confirm
the
safety
and
effectiveness of production of the
Iba plant as heart ailments
medicine.
Appendix A: Phytochemical
Screening
Polyphenols compounds
A vial containing 2mL of the crude
extract was added with 2-3 drops
of 1% ferric chloride. Formation of
a green, purple, blue or black
solutions would imply the presence
of phenolic compounds.
Tannin
Dropwise 15% FeCl3 solution was
added to 2 mL of the extract in a
vial.
Formation
of
blue-black
precipitate would indicate the
presence of hydrolysable tannins
while brownish-green precipitate
would indicate condensed tannins.
Saponin
2.5 mL of extract with 5 mL of
distilled water was diluted in a test
tube. Heat to boiling and allow
cooling. Shaken vigorously to
observe frothing which would

suggest the presence of saponins.

Observe.
The results were verified by adding
2 drops of corn oil to the solution
followed by vigorous shaking. The
presence of emulsion at the frothwater interface was observed.
Terpenoids
Warning: Be careful in handling
concentrated
sulfuric
acid
(corrosive).
5 mL of the extract was evaporated
in an evaporating dish to dryness
using
a
water
bath.
When
completely dry, 2 mL of chloroform
was added, the test tube was
inclined
then
added
slowly
concentrated sulfuric acid and let it
stand. The formation of a reddish
brown coloration at the interface
was observed.
Steroids
5 mL of the crude extract was
evaporated to dryness in an
evaporating dish using water bath.
When
completely
dry,
the
remaining solid was dissolved in 2
mL of acetic anhydride. Added with
dropwise of concentrated sulfuric
acid. Formation of an emerald
green solution would indicate the
presence of steroids.
Flavonoids
Dropwise of 2 mL of 1M sodium
hydroxide was added to 2 mL
sample extract in a test tube.
Observed. Added with a few drops

of 0.6M hydrochloric acid until

colorless. A yellow to orange
solution with sodium hydroxide that
would turn colorless upon addition
of the acid would denote the
presence of flavonoids.
5 mL of the extract was
evaporated to dryness in an
evaporating dish using a water
bath.
When
completely
dry,
concentrated sulfuric acid was
added dropwise. Flavones and
flavonols
dissolved
into
concentrated sulfuric acid would
produce a deep yellow colored
solution. Chalones and aurones
would produce red or red-bluish
solution
and flavavones give
orange to red colors.
Alkaloids
3 drops of Wagners reagent ( 2 g
of iodine and 6 g of potassium
iodide in 100 mL water) was added
to 2 mL sample extract in a vial.
Formation of blue-black precipitate
would confirm the presence of
alkaloids.
Anthocyanin
2 mL of 2N HCl and ammonia was
added to 2mL of the extract in a
test tube. Pink solution to blue
violet would indicate the presence
of anthocyanin.
Carotenoid
5 mL of chloroform was added to 2
mL of the extract in a test tube and
shake vigorously. The mixture was

filtered and added dropwise with

85% sulfuric acid. Blue color in the
interface
would
indicate
the
presence of carotenoids.
Cardiac glycosides
1 drop of 15% FeCl3 was added to 2
mL of the crude extract in the vial.
This then, was followed by layer of
the solution with 1 mL of
concentrated
sulfuric
acid.
Formation of a brown ring at the
interface
would
indicate
the
presence of cardiac glycosides.
Appendix B: Phytochemical
Screening Results
Averhhoa bilimbi Fruits
Secondar Trial 1 Trial 2
Final
y
Metabolit
e
Polyphen
Negati
ols
ve
Tannins
Negati
ve
Saponins
+
+
Positiv
e
Terpenoid
Negati
s
ve
Steroids
Negati
ve
Flavonoid
Negati
s
ve
Alkaloids
Negati
ve
Anthocya
Negati
nin
ve
Carotenoi
Negati
ds
ve
Cardiac
+
+
Positiv

Glycoside
s

Averhhoa bilimbi Stems

Secondar Trial 1 Trial 2
Final
y
Metabolit
e
Polyphen
Negati
ols
ve
Tannins
+
+
Positiv
e
Saponins
+
+
Positiv
e
Terpenoid
+
+
Positiv
s
e
Steroids
Negati
ve
Flavonoid
Negati
s
ve
Alkaloids
Negati
ve
Anthocya
Negati
nin
ve
Carotenoi
Negati
ds
ve
Cardiac
Negati
Glycoside
ve
s

Averhhoa bilimbi Fruits

Secondar Trial 1 Trial 2
Final
y
Metabolit
e
Polyphen
Negati
ols
ve
Tannins
+
+
Positiv
e
Saponins
+
+
Positiv

Terpenoid
s
Steroids

Flavonoid
s
Alkaloids

Anthocya
nin
Carotenoi
ds
Cardiac
Glycoside
s

e
Negati
ve
Negati
ve
Negati
ve
Negati
ve
Negati
ve
Negati
ve
Negati
ve

Appendix C: DPPH Scavenging

Activity
Results
Averrhoa bilimbi Fruits
Set
Trial
Abs.
%
1
1.150 -9.73 %
0.935
10.78
52
%
Old
ppm
1.174
-12.02
Batch
%
(DPPH
2
1.088 -3.82 %
0.0267
1.019 2.77 %
52
g)
1.221 -16.51
ppm
%
Contr
1.048
ol
1
0.843
35.10
New
52
%
1.230
5.31
%
Batch
ppm
0.905
30.3%
(DPPH
%
0.0267g)
2
1.230 5.31 %
1.084
16.55
52
%
ppm
1.054
18.86

New
Batch

Contr 1.299
ol
Aver 18.58
age
%

Averrhoa bilimbi Stems

Set
Trial
Abs.
%
1
1.053 6.98 %
1.031 8.92 %
48
1.010
10.78
Old
ppm
%
Batch
2
1.043 7.86 %
(DPPH
52
0.0269
1.007
11.04
ppm
g)
%
Contr
1.132
ol
1
0.952
15.15
52
%
0.981
12.57
New
ppm
%
Batch
0.952
15.15
(DPPH
%
0.0273g)
2
1.019 9.18 %
1.012 9.80 %
48
0.997
11.14
ppm
%
Contr
1.122
ol
New
avera
12.17%
Batch
ge
Averrhoa bilimbi Leaves
Set
Trial
Abs.
%
1
1.037 7.98 %
1.055 6.39 %
64
1.019 9.58 %
Old
ppm
2
1.064 5.59 %
Batch
1.169 -3.73 %
56
(DPPH
1.015 9.34 %
ppm
0.0267
Contr 1.127
g)

New
Batch
(DPPH
0.0267g)

ol
1
52pp
m
2
52
ppm

Contr
ol
New
batch

Avera
ge

1.055

13.17
%
1.160 4.53 %
1.050
13.58
%
0.957
21.23
%
0.965
20.58
%
0.940
22.63
%
1.215
12.42%

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