Bheemesh Vangalapati et al.

/ Journal of Pharmacy Research 2014,8(5),610-613

Research Article
ISSN: 0974-6943

Available online through

www.jpronline.info

Total phenolic content and free radical scavenging activity of Pterocarpus marsupium
heartwood & Tribulus terrestris dry fruits: An in vitro comparative study
Bheemesh Vangalapati, Poornima A Manjrekar*, Anupama Hegde
Department of Biochemistry, Kasturba Medical College, Mangalore, Manipal University, Karnataka, India.
Received on:25-04-2014; Revised on: 30-04-2014; Accepted on:06-05-2014
ABSTRACT
Background: Majority of the disorders like diabetes, atherosclerosis, cancer and neurodegenerative diseases are linked to oxidative stress.
Antioxidants derived from plant sources offer resistance against the oxidative stress by way of their free radicals scavenging & lipid
peroxidation inhibitory actions. Present study was designed to compare the free radical scavenging activity in relation to the total phenolic
content of P. marsupium heartwood & T. terrestris dry fruits. Methods: Total phenolic content of the extracts was determined by using
Folin-Ciocalteu method following a slightly modified method of Singleton et al by comparing with standard Gallic acid. The ability to
scavenge 1, 1-diphenyl-2-picryl-hydrazyl(DPPH) radical is measured by a decrease in the absorbance at 517 nm as described by Hou et al
comparing with standard Ascorbic acid. Results: Phenolic contents ranging from 45 to 160 and 5 to 25mg of Gallic acid equivalents (GAE)
per gram of extract were present in P. marsupium & T. terrestris respectively. At 100g concentration P. marsupium & T. terrestris produced
85.9 & 67.5% of DPPH scavenging activity respectively and the standard ascorbic acid(10g) produced 90% scavenging activity.
Conclusion: The present study concludes that both the plant extracts have shown significant in vitro free radical scavenging activity.
Among them, aqueous extract of P. marsupium heartwood exhibited better activity which can be attributed to the presence of high phenolic
content. Hence these herbal constituents are of adjuvant importance in the management of various free-radical mediated pathological
conditions.
Key words: Oxidative stress, free radical scavenging, DPPH, P. marsupium, T. terrestris
INTRODUCTION:
Reactive oxygen species (ROS) such as hydroxyl radicals, superoxide anions and nitric oxide inactivate the enzymes and damage important cellular component causing injury through covalent binding
and lipid peroxidation. Majority of the disorders like diabetes, atherosclerosis, cancer and neurodegenerative disorders are linked to
oxidative stress1. Antioxidants are agents which offer resistance
against the oxidative stress by scavenging the free radicals, inhibits
lipid peroxidation2. Phenolic compounds are known for their scavenging potential due to the presence of hydroxyl groups. It has
been discovered that several phenolic antioxidants such as
flavanoids, tannins, coumarins, xanthones and procyanidins scavenge radicals dose dependently, therefore they are observed as
promising therapeutic agents for free radical pathologies3 and also
plant materials have been believed to be less toxic with minimum
side effects than synthetic drugs. Medicinal plants are easy to
access and also economic for the poor people.

out India. Traditionally it is used as an aphrodisiac4, diuretic &

anthelmintic as well as to treat edema, ocular infections & abdominal
distention5. Hypolipidemic & hypoglycemic activities have been reported with aqueous extract of T. terrestris dry fruits6.
P. marsupium is a large tree commonly found in hilly regions, especially in parts of the Western Ghats in Karnataka-Kerala region. It is
commonly known as Indian Kino or Vijayasar. It is used in traditional
medicine for the treatment of diabetes mellitus7. P. marsupium has
also been documented to help in regeneration of pancreatic betacells8. Many phenolic compounds such as marsupin, pterospin &
pterostilbene have been shown to possess significant antidiabetic
activity in diabetic rats9. The present study was designed to compare
the free radical scavenging activity in relation to the total phenolic
content of P. marsupium heartwood & T. terrestris dry fruits.

T. terrestris, commonly known as Gokharu and puncture vine

MATERIALSAND METHODS:
belongs to the family Zygophyllaceae, widely distributed through
*Corresponding author.
Dr. Poornima A Manjrekar,
Professor & Head,
Department of Biochemistry,
Kasturba Medical College, Mangalore,
Manipal University, Karnataka, India.

Plant Material:
Sample of T. terrestris dry fruit aqueous extract was procured from
Himalaya Drug Company, New Delhi. Heartwood of P. marsupium
was purchased from Alvas herbal pharmacy, Moodbidri,
Karnataka,India and taxonomically identified by the Botanist.

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Preparation of plant Extract:
One part (30 gm.) of dry coarse powder of P. marsupium heartwood
was boiled in sixteen parts of water for 15 minutes at 50 C. It was
filtered through muslin cloth and filtrate was kept for flash evaporation in rotary vacuum flash evaporator at 5 rpm (75C) for seven
hours. Remaining residue was collected from round bottom flask and
dried in heating mantle for one hour to obtain semi-solid form of
extract. Total yield was 5.9 grams. It was aliquoted and stored at -4C
to prevent contamination.
Chemicals:
1, 1- diphenyl-2- picrylhydrazyl (DPPH) was purchased from Sigma
Chemical Co. Ltd. All other chemicals and reagents used were of
analytical grade.
Total Phenolic content estimation:
The total phenolic content of the both extracts was determined by
using Folin-Ciocalteu reagent following a slightly modified method
of Singleton et al10. Gallic acid was used as a reference standard for
plotting calibration curve.
Principle: Phenolic compounds undergo redox reaction with
phosphomolydbic acid and phosphotungstic acid present in FolinCiocalteau reagent in alkaline medium and produce blue colored complex.0.5 mL of the plant extract (100 g/mL) was mixed with 2 mL of the
Folin-Ciocalteu reagent (diluted 1:10 with de-ionized water) and neutralized with 4 mL of sodium carbonate solution (7.5%, w/v). The
reaction mixture was incubated at room temperature (302C) for 30
min with recurrent shaking for the blue color development. The absorbance was measured at 765 nm using double beam UV-VIS spectrophotometer. Phenolic content was determined from the standard
curve of gallic acid. Phenols expressed as milligrams/gram gallic acid
equivalent (GAE) of dry extract.
1, 1- diphenyl-2- picrylhydrazyl (DPPH) scavenging activity:
The DPPH free radical scavenging activity of the both extracts was
determined by the modified method of Hou et al11.
Principle: Antioxidants react with the stable DPPH radical and convert into 1, 1- diphenyl-2-picryl hydrazine. Ability to scavenge the
DPPH radical is measured by a decrease in the absorbance. Aliquots
containing various concentrations (2100g/ml) of P. marsupium &
T. terrestris in the final volume of 1 ml were mixed with 1 ml of 0.05 mM
DPPH, equal amount of methanol served as control.Ascorbic acid (2100g/ml) was used as standard. Reaction mixtures were incubated at
37C for 20 min& the absorbance was recorded at 517 nm.
The capability of DPPH free radical scavenging activity was

calculated using the following equation: DPPH scavenging effect

(%) = [(A0-A1)/A0) 100],
where A0 is the absorbance of the control and A1 is the absorbance of
standard/test.
STATISTICAL ANALYSIS:
The data is expressed as Mean Standard Deviation (S.D.) and
between the groups analysis was performed by using ANOVA in
SPSS Version 16, p < 0.05 is considered as significant.
RESULTS:
Total phenol Estimation:
From the Gallic acid standard graph the amount of phenols were
calculated and expressed as milligrams of phenol per gram of the
sample. The total phenolic content was calculated as gallic acid equivalent (GAE) by using the following formula: T=C xV/M. T is the total
phenolic content in mg/g of the extracts as GAE, C is the gallic acid
concentration obtained from the calibration curve in mg/ml, V is the
volume of the extract in ml and M is the weight of the extract in grams.
Phenolic contents ranging from 45 to 160 and 5 to 25mg of GAE per
gram of extract were present in P. marsupium heartwood and
T. terrestris dry fruit extract respectively (Figure-1).

Total phenolic content estimation

Absorbance of G.A

Absorbance of P.M

Absorbance of T.T

Linear (Absorbance of G.A)

1.8
1.6
1.4
1.2
1

R =0.9994

0.8
0.6
0.4
0.2
0
20

40

60

80

100

Figure-1: Phenolic content estimation

DPPH free radical scavenging:
Various concentrations (2, 4,6,8,10,20,40,60,80,100g) of aqueous
extracts of P. marsupium and T. terrestris were tested for DPPH free
radical scavenging activity. It was observed that the test compound
scavenged free radicals in a concentration dependent manner. At
100g concentration P. marsupium & T. terrestris produced 85.9 &
67.5% of DPPH scavenging activity respectively. Ascorbic acid
(100g) as standard produced 90.3 % DPPH free radical scavenging
activity.

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Bheemesh Vangalapati et al. / Journal of Pharmacy Research 2014,8(5),610-613

pterospin were also responsible for decreasing plasma glucose levels in diabetic rats .T. terrestris dry fruits contain Saponins,
flavonoids and some phenolic compounds, these substances could
significantly reduce the level of serum glucose and also produce
some antioxidant activity. Both are considered to be good antidiabetic agents but P. marsipium takes the lead due to its potential
antioxidant activity compared to T.terrestris.

DPPH Free radical scavenging actvity

P.Marsupium

T.terrestris

ascorbic acid

% SCAVENGING ACTIVITY

100
80
60
40
20
0
2

10

20

40

60

80

100

Concentration in MICROGRAM/ML

Figure-2: Graphical representation of DPPH free radical scavenging activity

DISCUSSION:
In a normal cell there is an appropriate pro-oxidant and antioxidant
balance. However, this balance can be shifted towards the
pro-oxidant when production of reactive oxygen species is increased
or when levels of antioxidants are diminished, state is known as
oxidative stress.Free radicals are the chemical bodies that can exist
separately with one or more unpaired electrons.The production of
free radicals can bring about many reactions which can lead to extensive tissue damage if the stress is prolonged. Proteins, lipids and
DNA are prone to the free radicals induced damage. Antioxidants
acts by neutralizing the free radicals or protecting the antioxidant
defense mechanisms. Antioxidant effectiveness is measured in terms
of the inhibition of suitable substrate oxidation. Various methods are
available for determining free radical scavenging effects.
In this study, aqueous extracts of P. marsupium heartwood and
T. terrestris fruits were tested to determine the total phenolic contents and their ability to scavenge the DPPH free radicals by in vitro
method. As reported in the earlier studies12our results also suggests
that T.terrestris dry fruit has low phenolic content when compared to
P.marsupium heartwood. Due to high phenolic content in
P.marsupium, it possess potent antioxidant activity as evident from
its increased scavenging activity. There was significant difference (P
<0.003) between three groups in their free radical scavenging
activity.Among two extracts, P. marsupium exhibited the highest
scavenging activity of 85.9% at a concentration of 100 g/ml. Further
increase in concentrations of P. marsupium and Ascorbic acid
attained saturation in their scavenging activity whereas T. terrestris
shown a concentration dependent activity (Figure-2). IC50 values
for P. marsupium and T .terrestris was 30g and 70g respectively.
Demonstration of the strong antioxidant activity of this extract
would certainly increase its potential as an antidiabetic drug. Major
phenolic constituents of P. marsupium are pterostilbene and

CONCLUSION:
The present study concludes that both the plant extracts have shown
significant invitro free radical scavenging activity. Among them, aqueous extract of P. marsupium heartwood exhibited better activity which
can be attributed to the presence of high phenolic content. Hence
these herbal constituents are of adjuvant importance in the management of various free-radical mediated pathological conditions.
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Source of support: Nil, Conflict of interest: None Declared

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