ANTIOXIDANT SCREENING OF Averrhoa bilimbi (Kamias)

Cananga odorata (Ylang-Ylang), and Plumiera alba (Calachuchi)

USING 2,2-diphenyl 1-picrylhydrazyl DPPH ASSAY

Ruth T. Libag1, Maria Jacquilyn F. Ancheta2, Gail P. Igaya2 and Maribel V. Tolentino3

Department of Chemistry Angeles University Foundation, 2 College of Pharmacy

1

Our Lady of Fatima University, 3College of Engineering Angeles University Foundation

Scientific evidence suggests that antioxidants reduce risk for

chronic diseases including cancer and heart disease. Primary sources of
naturally occurring antioxidants are whole grains, fruits and vegetable
while the synthetic antioxidants currently used have been found to exhibit
various health effects. It is for this reason that the researchers focused
on natural antioxidants present in medicinal plants. The samples used in
the study were the ethanolic extracts of Kamias, Ilang-ilang and
Calachuchi using 2,2-diphenyl 1-picrylhydrazyl (DPPH) assay. All have
antioxidant activity against the free radical DPPH at varying amounts. The
leaves of Kamias have the highest antioxidant activity, followed by the
flowers of ilang ilang and calachuchi. All of the samples were initially
measured at 0.125 mg/ml crude ethanolic extract and read at 520 nm
using spectrophotometry. The free radical scavenging activities of the
plant samples using the parameter EC50 also coincide with the amount of
antioxidants present expressed in percentage. Thus, Kamias has the
highest EC50, followed by ilang ilang and calachuchi, respectively. The
results showed that the given plant samples are potential sources of
antioxidants.

Antioxidant compounds in food play an important role as a health-

protecting factor. Scientific evidence suggests that antioxidants reduce risk for
chronic diseases including cancer and heart disease. Primary sources of
naturally occurring antioxidants are whole grains, fruits and vegetables. The
synthetic antioxidants currently used have been found to exhibit various health
effects. That is why researchers focused on natural antioxidants present in
medicinal plants (Castro 2006).

Previous epidemiological and experimental evidence suggest that

antioxidants positively influence the defence of the body against cancer. Since
cancer can be induced by damage to DNA, deoxyribonucleic acid, it is postulated
that free radicals produced from normal chemical reactions may be responsible
for the development of some human cancers. Because antioxidants can
neutralize free radicals or unpaired electrons, they are now of considerable
importance as chemopreventive agents (Beecher 2000).

The main characteristic of an antioxidant is its ability to trap free radicals.

Highly reactive free radicals and oxygen species are present in biological
systems from a wide variety of sources. These free radicals may oxidize nucleic
acids, proteins, lipids or DNA and can initiate degenerative disease. Antioxidant
compounds like phenolic acids, polyphenols and flavonoids scavenge free
radicals such as peroxide, hydroperoxide or lipid peroxyl and thus inhibit the
oxidative mechanisms that lead to degenerative diseases (Prakash 2001).

Free radicals and oxidants can trigger lipid peroxidation, as well as the
oxidation of proteins and DNA, causing extensive damage to body cells.
Oxidative stress resulted from an imbalance of oxidising species and natural
antioxidants in the body has been thought to have contributed to aging, cell
apoptosis, and severe diseases such as cancer, Parkinson’s disease,
Alzheimer’s disease, and even cardiovascular disorders. Epidemiological studies
and intervention trials on prevention of cancer and cardiovascular disease in
people taking antioxidant supplements are suggestive that dietary intake of
antioxidants can help free radicals and oxidants and protect the body against
diseases. It is also clear that most of the dietary antioxidants have low or
minimal toxicity, and that intake can be increased without adverse effects (Frei
1994).

There are different kinds of antioxidant test like ABTS (2,2’-azonobis-3[-

ethylbenzthiazoline-6-sulphonic acid]), DPPH (1-1-diphenyl-2-picrylhydrazyl),
ORAC(oxygen radical absorbance capacity), β-carotene–linoleate model system,
this test is to determine the free-radical scavenging activity of sample or plant
sample. One such method that is currently popular is based upon the use of the
stable free radical diphenylpicrylhydrazyl (Castro et. al. 2006).

DPPH free radical (1,1-diphenyl-2-picryl-hydrazyl) DPPH and reduced

form the molecule of 1,1-diphenyl-2-picryl-hydrazyl(α,α-diphenyl-β-
picrylhydrazyl) DPPH is characterised as a stable free radical by
virtue of the delocalisation of the spare electron over the molecule as
a whole, so that the molecules do not dimerise, as would be the case
with most other free radicals. The delocalisation also give rise to deep violet
color, characterised by an absorption band in ethanol solution centered at
520nm.
When a solution of DPPH is mixed with that of a substance that can
donate a hydrogen atom, then this gives rise to the reduced form with the violet
color from the picryl group still present). Representing the DPPH radical by Z
and the donor molecule by AH, the primary reaction is Z + AH = ZH+ A [1]
where ZH is the reduced form and A is free radical produced in this first step.
This latter radical will then undergo further reactions which control the overall
stoichiometry,that is, the number of molecule of DPPH reduce by one molecule
of the reactant. The reaction [1] is therefore intended to provide the link with
the reactions taking place in an oxidaizing system,such as the autoxidation of a
lipid or other unsaturated substance; the DPPH molecule Z is thus intended to
represent the free radicals formed in the system whose activity is to be
suppressed by the substance AH. (Molyneux 2004)
 As oxidative stress might be an important part of many human diseases,
the use of antioxidants in pharmacology is intensively studied, particularly as
treatments for stroke and neurodegenerative diseases. Antioxidants are also
widely used as ingredients in dietary supplements in the hope of maintaining
health and preventing diseases such as cancer and coronary heart disease.
Although some studies have suggested antioxidant supplements have health
benefits, other large clinical trials did not detect any benefit for the formulations
tested, and excess supplementation may be harmful.
In addition, there has been growing interest in natural antioxidant because
they have greater application in food industry for increasing the stability and
shelf-life of food products (Suja 2004).
It is in this basis that the researchers under take to determine the
antioxidant properties on plants and flowers readily available, they were able to
identify three plant samples. Among these are the following, Plumeria alba
(Calachuchi), Averrhoa bilimbi (Kamias), and Cananga odorata (Ilang Ilang).

METHODOLOGY

Collection of Plant Samples

The leaves and flowers were brought to the National Museum and were
properly identified and were given authentication by professional botanists.

After the collection of plant samples, fresh water was used to clean and
wipe off dirt and contaminants on the samples. Plant specimens were air dried
through room temperature to make sure that moisture was properly evaporated
off and confine the important constituents of the plant samples.

Extraction

To extract the organic constituents from the plant samples, the dried
materials were cut into smaller pieces and were grounded. By the use of
solvent extraction method, diluted 80% ethyl alcohol was used to completely
submerge the material. After forty eight hours of submerging materials to
solvent, the extracts were collected through filtration. To ensure that all
important constituents of the materials were collected, washing is also done by
the use of fresh portions of alcohol. Plant residues were discarded.

Rotary Evaporation

To concentrate the filtrate, rotary evaporation was used. After which, the
exact volume of the concentrated extracts were measured.

Preparation of Vitamin C Calibration Curve for DPPH Assay

 Standard Vitamin C solutions were prepares starting at an initial
concentration of 0.25mg/mL. This standards were used to establish a calibration
curve that was used as the basis of conclusion whether the plant sample has an
antioxidant activity or none.

The DPPH Assay was patterned on the procedure used by Molyneux.

The DPPH reagent was prepared by dissolving 0.0100g of DPPH in 80% ethanol
to make 250mL and a concentration 100µM.

Preparation of Calibration Curve for Vitamin C.

Volume Tub Tub Tub Tub Tub Tub Tub

in mL e1 e2 e3 e4 e5 e6 e7
DPPH
2 2 2 2 2 2 2
Solution
Vitamin
C 0.10 0.20 0.40 0.60 0.80 1.00 1.10
Standard
Distilled
1.90 1.80 1.60 1.40 1.20 1.00 0.90
Water
Total
4.00 4.00 4.00 4.00 4.00 4.00 4.00
Volume

The absorbances of the Vitamin C standards were taken at 520 nm using

a spectrophotometer. DPPH alone was used as blank.
Diagram of Methodology
RESULTS AND DISCUSSION

The following graph shown below shows the regression equation of the
calibration curve for Vitamin C.

Graph 1. Vitamin C Standard Calibration Curve

0 .3 5
y = -3 .0 2 7 4 x +0 .2 8 3
0.3
R2 = 0 .9 5 0 3
0 .2 5
Absorbance (520 nm)

0.2

0 .1 5
c
0.1

0 .0 5

0
0 0 .0 2 0 .0 4 0 .0 6 0 .0 8 0.1 0.1 2
-0 .05
Concentration of Vitamin C in mg/mL

Graph 1 above shows a good correlation between absorbance and

Vitamin C concentration with an R2 = 0.9503 while the slope of the line gives a
regression equation of y = -3.0374x + 0.283 (y=mx+b) and expressed as AEAC.
Thus, the slope for the calibration curve is reliable for computing the antioxidant
capacity of the crude ethanolic extracts of Calachuchi, Kamias, and Ilang-ilang.

Table 2 shows the mean absorbances of the crude ethanolic extracts at 0.125
mg/mL concentration against 100 μM DPPH. It is noted from table 1 that the
absorbance of the blank reads 0.3095 at 520nm.

Table 2. Plant Sample Mean Absorbances & Antioxidant Activity in mg/mL

Plant Sample Mean mg/mL
 Absorbance Antioxidant
Calachuchi 0.214 0.0228
Kamias 0.105 0.0588
Ilang-ilang 0.170 0.0373

Using the regression equation from graph 1 (y = - 3.0274 x + 0.283)

with R2 = 0.9503. The Ascorbic Acid Equivalent Antioxidant Capacity (AEAC)
were taken. The table above showed that Kamias has the highest antioxidant
capacity against DPPH with an AEAC of 0.0588 mg/mL, followed by Ilang-ilang
and Calachuchi at 0.0373 and 0.0228 mg/mL, respectively.

The concentration of Antioxidant in the crude extracts were computed

using the regression equation from the vitamin C calibration curve using the
formula of the slope of the line, y = mx + b

Where: y = the absorbance of samples

m = slope of the line
b = blank absorbance
x = the antioxidant activity in mg/mL

Graph 2. The Antioxidant Activities of Plant Extracts Against DPPH

0.0 7 0 0

0 . 0 60 0
Amount of Antioxidants in mg/mL

0 . 0 50 0

0 . 0 40 0

0.0 3 0 0

0.0 2 0 0

0.0 1 0 0

0.0 0 0 0
Calachuchi Kam ias Ilang-ilang
Pla nt Sa m ple s

A 100 μL sample was taken from the 2.5 μL/mL initial concentration of the
crude ethanolic extracts and was added to 1.9 mL distilled water to make the
resulting concentration of extract to 0.125 mg/mL. From this concentration of
extract, the percentage of antioxidant present was computed and presented in
Graph 3.

Graph 3. Percentage of Antioxidant Present in 0.125 mg/mL Crude

Samples

50 . 0 0 %
45. 0 0 %
40 . 0 0 %
Percentage of antioxidant

3 5. 0 0 %
3 0. 0 0%
2 5. 0 0 %
2 0. 0 0%
1 5. 0 0 %
1 0. 0 0%
5. 0 0 %
0.0 0%
Calachuchi K am ias Ilang-ilang
Pla nt Sa m ple s

Graph 3 above showed corresponding percentage of antioxidant present

in 0.125mg/mL crude samples. The results showed that Kamias has the highest
antioxidant content at 47.04%. This means that almost half of the crude extract
of Kamias has antioxidant capacity. Ilang-ilang follows at 29.84% and
Calachuchi at 18.24% at the given concentration of extracts.

Percentage antioxidant present in each sample were taken using the

following formula
%Ao = (Ao Concentration ÷ 0.125 mg/mL) x 100

Another parameter which tests the antioxidant capacity of the samples is

by computing its effective concentration, EC50 , also known as IC 50 , is defined as
the concentration of substrate that causes 50% loss of the DPPH activity.
 Graph 4. EC50 of the Crude Extracts

7 0 .0 0

60 . 0 0

50 . 0 0
EC 50 Value

40 . 0 0

3 0 .0 0

2 0 .0 0

1 0 .0 0

0 .0 0
Calachuchi Kamias Ilang-ilang
Plant Sample

Percent inhibition expressed as EC50 values at 0.125 mg/mL of crude

extract showed that Kamias has the highest inhibitory concentration and was
able to inhibit DPPH resulting to 66.07% loss. This is followed by Ilang-ilang at
45.07% and Calachuchi at 30.86% at the concentration of 0.125mg/mL crude
ethanolic extracts of the samples.
EC50 (Q) for each of the sample was computed using the following formula.

Q = 100 ( Ao – Ac )
Ao

Where: Ao = the absorbance of positive control containing DPPH only

Ac = absorbance of solution containing extracts and DPPH.

CONCLUSION

All the plant samples namely Kamias, Ilang-ilang and Calachuchi have
antioxidant activity against the free radical DPPH at varying amounts. With the
leaves of Kamias having the highest antioxidant activity, followed by the flowers
of ilang ilang and calachuchi. Consequently, the given samples Kamias, Ilang-
ilang and Calachuchi could be used as a natural source of antioxidant since all of
them gave antioxidant capacities in varying degrees starting at 0.125mg/ml
concentration of the crude ethanolic extracts. The free radical scavenging
activities of the plant samples using the parameter EC50 also coincide with the
amount of antioxidants present expressed in percentage. Thus, Kamias has the
highest EC50, followed by ilang ilang and calachuchi, respectively. The results
showed that the given plant samples are potential sources of antioxidants.

REFERENCES

Beecher, G. (2000). Measurement of “new” health related food components.

Food Composition Laboratory, USDA/ARS/FCL Beltsvelle MD 20705.

Castro, I A. et.al. (2006). 2,2 –Diphenyl-1-picrylhydrazil free radical scavenging

activity of antioxidant mixtures evaluated by response surface
methodology. International Journal of Food Science and Technology
2006, 41 (Supplemental 1), 59-67.

Castro, I.A. et.al. (2006). Free Radical Scavenger and antioxidant capacity
corrrelation of alpha-tocopherol and Trolox measured by three in vitro
methodologies. Intenational Journal of Food Science and Nutrition, 57,
75-82.

Molyneux, P. (2004). The use of the stable free radical diphenylpicrylhydrazyl

(DPPH) for estimating antioxidant activity. Songklanakarin J. Sci.
Technol., 2004, 26(2) : 211-219

Prakash, A. (2001) Antioxidant Activity. Medallion Laboratories: Analytical

Progress 2001, Vol 19 No. 2.

Frei, B. (1994). San Diego: Academic Press. Natural antioxidants in human

health and disease.

Suja, K.P., Jayalekshmy, A. & Arumughan, C. (2004). Free radical scavenging

behavior of antioxidant compounds of Sesame (Sesamum indicum L.) in
DPPH System. Journal of Agricultural and Food Chemistry,52, 912-915.