Preliminary Phytochemical
Screening and Antioxidant Activity of Petroleum Ether Extract of Trichosanthes dioica Roxb (Fruits)
Yogesh Shivhare, Priya
Singh, K. K. Patel and Prashant Soni
Department
of Pharmacognosy, VNS Institute of Pharmacy, Bhopal (M.P.)
ABSTRACT:
In-vitro antioxidant activity
of petroleum ether extract of Trichosanthes dioica was determined by DPPH free radical scavenging
assay, Reducing power assay, Nitric oxide scavenging
activity, Hydrogen peroxide radical scavenging activity using ascorbic acid as
a standard. The IC50 values were calculated for all the
models in order to evaluate the antioxidant efficiency of Trichosanthes dioica extract.
In all the testing a significant correlation existed between concentration of
the extract and % inhibition of free radicals. Obtained results clearly
indicated that petroleum ether extract of Trichosanthes dioica possesses significant antioxidant potential as compared
with standard.
KEYWORDS: Trichosanthes dioica, Nitric oxide, %
Inhibition
INTRODUCTION:
Oxygen
is a crucial module for the survival of humans.1 Under certain situations it
has deleterious effects on the human body.2
Most of the potentially harmful effects of oxygen are due to the formation and
activity of a number of chemical compounds, known as reactive oxygen species
(ROS). These are highly reactive substances and are responsible for various
diseases like CNS disorders, diabetes mellitus and liver diseases.3 Thus, there is the need of
antioxidants to overcome these problems. Recently, interest has increased
considerably in finding naturally occurring antioxidants for use in foods or
medicinal materials to replace synthetic antioxidants, which are being
restricted due to their side effects such as carcinogenicity.4 Herbal plants
have been used for medicinal applications from earliest time, when man began
caring for his body and health. The emphasis of development of new biologically
active molecule has been gradually replaced by use of total herbs as medicine
and food supplements. Now herbal based products have very good present and
future prospects in International market.5
The fruits of the plant Trichosanthes dioica (family- Cucurbitaceae)
commonly known as pointed gourd in English and Parwal
in Hindi, native to the plains of N. India extending to Assam and E. Bengal. For centuries,
Pointed gourd has been used in folk medicine to heal arrange of ailments and
has been credited with many medicinal properties especially laxative, cooling,
and in bilious fever, beneficial in piles, tonic etc.6, 7 By
keeping this view, the present exploration of the petroleum ether extract of
the fruits of Trichosanthes dioica was
undertaken to evaluate its antioxidant potential.
MATERIALS AND METHODS:
Plant material:
The
fresh fruits of Trichosanthes dioica were collected from the local market
of Bhopal and authenticated by Dr. A. S. Yadav,
professor, Government MVM College Bhopal. The fruits were shade dried and
pulverized into moderately coarse powder and then stored in a tightly closed
container for further use.
Preparation of extract:
The
dried powdered plant material (125g) was extracted in soxhlet
apparatus with petroleum ether (60-80şc). The extract was concentrated to a
brown residue (1.9g w/w). This crude extract was used for further investigation
for potential antioxidant properties.
Preliminary phytochemical screening:
Dried
petroleum ether extract was subjected to preliminary phytochemical
investigations for the identification of secondary metabolites by utilizing
standard methods of qualitative test analysis.8, 9
EVALUATION OF
ANTIOXIDANT ACTIVITY:
DPPH scavenging activity:
The
procedure of Brand- Williams10
has been adapted for evaluation of the free radical scavenging capacity of the methanolic extract. Different concentrations
(05-45μg/ml) of petroleum ether extract of Trichosanthes dioica were prepared and 3ml of each
solution was mixed with 1 ml of a 0.1mM DPPH solution. The decrease in
absorbance was measured at 515 nm after 30 minutes of incubation period at room
temperature using a UV Visible spectrophotometer1700. The scavenging activity
of sample extract was expressed as the inhibition of DPPH radical and
calculated according to the following formula with as the control:
Scavenging Activity
(%) = [(A control - A sample) / A control]*100, where A control
(containing adequately diluted DPPH solution) and A sample is the Absorbance with different dilutions
of drug extract. Ascorbic acid was used as reference standard.
Reducing power assay:
Reducing power of
extract of Trichosanthes dioica was
estimated using the protocol reported by Oyaizu11. Different concentrations of petroleum ether extract
of Trichosanthes dioica
(5-25μg/ml) were prepared and 1ml of each solution was mixed with
phosphate buffer (2.5ml, 0.2M, pH 6.8) and potassium ferricyanide
(2.5ml, 1%). The mixture was incubated at 50˚C for 20 min.
To this mixture, 2.5 ml of 10% trichloroacetic acid (
Nitric oxide scavenging activity:
Nitric oxide
scavenging activity was evaluated by the method of Gupta12. 1ml of Sodium nitroprusside (10mM) in phosphate-buffered saline (PBS) was
mixed with 3.0 ml of different concentrations (20 – 120μg/ml) of the petroleum ether
extract of Trichosanthes dioica dissolved in water and incubated at 250 C for 180
min. The samples from the above were reacted with equal volume of Greiss reagent (1% sulphanilamide,
0.1% napthylethylenediamine hydrochloride and 3% of
phosphoric acid). The absorbance of the chromophore
formed during the diazotization of nitrite with sulphanilamide
and subsequent coupling with napthylethylenediamine
was read at 546 nm. The
percentage scavenging was calculated by using the formula: (A control
- A sample)/ A control ×100, where A control is the absorbance of
solution without extract and A sample
is the absorbance with different dilutions
of drug extract. Ascorbic acid was used as reference standard.
Hydrogen peroxide radical scavenging activity:
The ability of the Trichosanthes dioica
extract to scavenge hydrogen peroxide was determined according to the method of
Ruch13. A solution of
hydrogen peroxide (40 mM) was prepared in phosphate
buffer (pH 7.4). The concentration of hydrogen peroxide was determined by
absorption at 230 nm using a UV Visible spectrophotometer 1700. Then hydrogen
peroxide solution (0.6 ml, 40 mM) was mixed to different concentrations (30 – 180μg/ml) of
the petroleum
ether extract dissolved in water The absorbance of
hydrogen peroxide at 230 nm was determined after ten minutes against a blank
solution containing phosphate buffer without hydrogen peroxide. The percentage
scavenging was calculated by using the formula:
(A control
- A sample)/ A control ×100, where
A control is the absorbance of solution
without extract and A sample is the Absorbance with different
dilutions of drug extract. Ascorbic acid was used as reference standard.
STATISTICAL DATA:
Data are presented as the mean ± SEM of each
triplicate test. The analysis was performed by using Dunnett
vs. Control test and by ANOVA. P<0.05 were considered to be statistically
significant.
RESULTS:
Preliminary phytochemical screening:
Preliminary
Phytochemical screening of the petroleum ether
extract of Trichosanthes dioica showed
the presence of triterpenoids and steroids.
DPPH scavenging
activity:
The
results of DPPH radical scavenging activity of the petroleum ether extract of Trichosanthes dioica with
IC50 (% Inhibition) are shown in fig 1.1. The IC50 value
of petroleum ether extract of Trichosanthes dioica and standard (ascorbic acid) was found to be
33µg/ml, and 11µg/ml respectively. The results showed a significant (p
<0.01) decrease in the concentration of DPPH radical due to the scavenging
ability of petroleum ether extract as compared to standard (ascorbic acid).
Reducing power assay:
The
Reducing ability of petroleum ether extract of Trichosanthes dioica and ascorbic acid were shown in
the fig 1.2. The IC50 value of petroleum ether extract of Trichosanthes dioica and
standard (ascorbic acid) was found to be 34µg/ml and 12µg/ml respectively. The
test material (petroleum ether extract of
Trichosanthes dioica)
has shown significant reducing property when compared with standard (ascorbic
acid).
Nitric oxide
scavenging activity:
The
nitric oxide scavenging activity of petroleum ether extract of Trichosanthes dioica and
ascorbic acid were shown in the fig 1.3 which illustrates the % inhibition of nitric oxide generation by petroleum ether extract of Trichosanthes dioica. Ascorbic acid was used as a reference.
The IC50 value of petroleum ether extract and standard (ascorbic acid) was found to be
113µg/ml and 85µg/ml respectively. The results indicate significant (p <0.01)
decrease in the concentration of nitric oxide radical due to the scavenging
ability of petroleum ether extract as compared to standard (ascorbic acid).
Hydrogen peroxide
radical scavenging activity:
Hydrogen peroxide scavenging activity of petroleum ether extract of Trichosanthes dioica and
ascorbic acid were shown in the fig 1.4. It showed significant scavenging
activity of hydroxyl radical generated from H2O2 system.
The IC50 value for petroleum
ether extract was 162µg/ml, whereas 167µg/ml was the value of
ascorbic acid. The results indicated that petroleum ether extract possessed significant antioxidant activity (p<0.01).
DISCUSSION:
DPPH is a constant free radical at room
temperature and accepts an electron or hydrogen radical to develop into a
stable diamagnetic molecule.14
The decrease in absorbance of DPPH radical caused by antioxidants, because of
the reaction between antioxidant molecules and radical, progresses, which
consequences in the scavenging of the radical by hydrogen donation.15 Thus, as expected, petroleum ether extract of Trichosanthes dioica showed DPPH radical scavenging activity in a concentration
dependent manner. In the reducing power assay, the reduction and
consequent formation of the ferrous product is monitored coincident formation
of Perl’s Prussian blue at 700 nm.16
An increasing absorbance is indicative of potent antioxidant activity. In the present work, it was observed that
absorbance of the petroleum ether extract was increased with increase in concentration of test. So, petroleum
ether extract of T. dioica showed concentration dependant reducing capacity. Excess
concentration of NO is associated with several diseases.17, 18 Oxygen reacts with the excess nitric oxide to
generate nitrite and peroxynitrite anions, which act
as free radicals.19,20 In the
present study, the extract competes with oxygen to react with nitric oxide and
thus inhibits generation of the anions. Even though hydrogen
peroxide itself is not very imprudent, it can sometimes cause cytotoxicity by giving augment to hydroxyl radicals in the
cell. Thus, removing H2O2 is very important throughout
food systems. Scavenging of H2O2 by antioxidants may be
due to contribution of electrons to H2O2, thus
neutralizing it to water.21 As the petroleum ether
extract scavenged hydrogen peroxide radical similar to the standard (ascorbic
acid). So it reflects that the plant extract could probably reduce the
formation of hydroxyl radical in-vivo and
can prevent the cellular and oxidative damage.
CONCLUSION:
Hence, the results obtained
from data concludes that petroleum ether extract of Trichosanthes dioica has potent antioxidant activity
which may probably be due to the phytoconstituents
present in the plant or could be a function of either the individual or the
additive effects of the phytoconstituents.
ACKNOWLEDGEMENT:
Mr. Yogesh Shivhare, corresponding author, sincerely thanks to Principal,
VNS Institute of Pharmacy, Bhopal (M.P.) India, for
providing necessary facilities during the course of this work.
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Received on 01.01.2010
Accepted on 24.03.2010
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Research Journal of Pharmacognosy and Phytochemistry. 2(2): March -April 2010, 155-158