In-Vitro Antioxidant
Activity of Kedrostis foetidissima (Jacq) Cogn
G. Gopi1, A. Elumalai2
and P. Jayasri3
1Department
of Pharmaceutics, Mahathi College of Pharmacy, CTM X Road, Chittoor
(Dt), Madanapalle, Andhra
Pradesh, 517 319.
2Department
of Pharmacognosy, Anurag Pharmacy College, Ananthagiri
(V), Kodad (M), Nalgonda (Dt), Andhra Pradesh, 508 206.
3Department
of Pharmacognosy, Santhiram College of Pharmacy, Srinivas
Nagar, Kurnool (Dt), Nandyal,
Andhra Pradesh, 518 501.
ABSTRACT:
The present study is to
evaluate a systemic record of the relative antioxidant activity of Kedrostis foetidissima. The ethanolic extract of Kedrostis foetidissima was
screened for their free radical, hydroxyl radical, superoxide and nitric oxide
scavenging activity. Total antioxidant activities of ethanolic extract were
compared with standard antioxidants ascorbic acid, copper sulphate
2, 6- di-ter-butyl-p-hydroxytoluene (BHT). Results indicate the ethanolic
extract exhibited antioxidant potential of in-vitro screening methods.
The results indicate that ethanolic extract showed moderate activity against
standard drugs
KEYWORDS: Kedrostis foetidissima, in-vitro antioxidant activity, DPHH,
free radical scavenging activity.
INTRODUCTION:
Natural antioxidants present
in the plants scavenge harmful free radicals from our body. Free radical is any
species capable of independent existence that contains one or more unpaired
electrons which reacts with other molecule by taking or giving electrons and
involved in many pathological conditions. It is possible to reduce the risk of
chronic diseases and prevent disease progression by either enhancing the body’s
natural antioxidant defences or by supplementing with
proven dietary antioxidants1. Synthetic antioxidants like butylated hydroxytoluene (BHT)
and butylated hydroxyanisole
(BHA) commonly used in foods have side effect and are carcinogenic2.
Plant polyphenolic compounds, such as flavonoids are described as scavengers of reactive oxygen
species3. Recently, the ability of phenolic
substances including flavonoids and phenolic acids to act as antioxidants has been extensively
investigated4. Most sources of natural antioxidants originate from
plant materials, but the content of polyphenolic
compounds in the roots and pericarp of tropical and
sub-tropical flora have sparsely reported5.
Kedrostis foetidissima (Jacq.) Cogn. (Cucurbitaceae), slender habitus, climbing and producing a rounded caudex.
Fruits are small, about 7 mm in length, red and finely pubescent. It is very effective in the treatment of asthma, chest pain and urinary
tract infection6, diarrhoea, HIV7, small pox and skin diseases8.
With this background, the present study was carried out to evaluate the in
vitro anti-oxidant activity.
MATERIALS AND METHODS
Collection of plant:
The
dried plant of Kedrostis foetidissima was collected from Tirupathi
hills, Andhra Pradesh, India in the month of Dec 2011 and authenticated by Dr.
P. Jayaraman, Plant Anatomical Research Centre
(PARC), Tambaram, Chennai
and the voucher specimen was kept in the Department of
Pharmaceutics, Mahathi College of Pharmacy,
CTM X Road, Chittoor (Dt), Madanapalle, Andhra Pradesh India. The whole plant was
cleaned, air dried and grounded into powdered separately. The dried powdered
plant material was passed through sieve 60 and stored in air tight containers.
Preparation of extract:
The dried powder of Kedrostis foetidissima plant (200gms) was
successively extracted with Ethanol (80%) at room temperature by Soxhlet extraction process. Each time before
extracting with the solvent, dry the powdered material in oven below 50⁰C. Concentrate
the extract at reduced pressure by Rotary Flash Vacuum Evaporator. Weigh the
extract obtained with the solvent and calculate its percentage in terms of the
air-dried weight of the plant material. Further the concentrated extract was
dried in desicator and stored in vacuum sealed air
tight containers.
Free radical scavenging
activity by DPPH:
The stable 1,
1-diphenyl-2-picryl hydrazyl radical (DPPH) was used
for determination of free radical-scavenging activity of the extract. Different
concentrations of each extract were added, at an equal volume, to ethanolic
solution of DPPH (100 μM). After 15 min at room
temperature, the absorbance was recorded at 517 nm. The experiment was repeated
for three times. Vitamin C was used as standard controls. IC50 values denote
the concentration of sample, which is required to scavenge 50% of DPPH free
radicals. The ethanolic solution of ascorbic acid was taken as standard. The
percent reduction in absorbance was calculated from the initial and final
absorbance at each level9.
Calculation of percentage
inhibition
The percentage inhibition of
radical production by the test sample was calculated using the formula:
Inhibitory ratio = (A0-A1) x100/A0
Where,
A0 is the absorbance of control, A1 is the absorbance
with addition of test sample.
Nitric oxide scavenging
activity:
Aqueous solution of sodium nitroprusside spontaneously generates nitric oxide (NO) at
physiological pH, which interacts with oxygen to produce nitric oxide which was
measured calorimetrically. Ethanolic extract of whole plant were served as
sample (0.5ml from 20mg/ml). Three ml of reaction mixture containing sodium nitroprusside (100mM in PBS) and 0.5ml extracts (20mg/ml)
were incubated at 25O C for 150 minutes. Controls were kept without test sample
in an identical manner. After incubation, 0.5 ml of reaction mixture was
removed and 0.5ml of Griess reagent (1%
sulfanilamide, 2% H3PO4, 0.1% naphthalene diamine dihydrochloride was
added. The absorbance of the chromophore formed was
read at 546 nm.The percentage inhibition of nitric
oxide generation was measured by comparing the absorbance values of control and
those of test samples10, 11.
Superoxide radical
scavenging activity:
The extent of superoxide
generation was studied on the basis of inhibition in the production of nitroblue tetrazolium formazon of the superoxide ion by the test area, where they
received uniform illumination for 30 minutes. Again the optical density was
measured at 560nm. The difference in optical density before and after
illumination is the quantum of superoxide production and the percentage of
inhibition by the test sample was calculated by comparing with the optical
density of the control12.
Hydroxyl radical
scavenging activity:
Deoxy-ribose assay was used to detect hydroxyl radical
scavenging activity and iron binding ability of them test sample. Hydroxyl
radicals are generated in a reaction mixture containing ascorbate,
H2O2 and iron–III-EDTA at pH 7.4 and measured by their
ability to degrade the sugar deoxyribose. The extent
of inhibition is dependent on the concentration of the scavenger and its rate
constant for reaction with hydroxyl radicals. A mixture of 0.5ml of 3mM deoxyribose solution, 0.1mM ferric chloride solution, 0.1mM
solution of EDTA, 0.1mM solution of ascorbic acid and 2 mM
solution of phosphate buffer was taken in a series of test tube and final
volume was made up to 3ml. To this 0.5ml of Trichloro
Acetic Acid (TCA) and Thio Barbituric
Acid (TBA), 10-50mg/ml of various concentrations of the test samples were added
to the above mixture and absorbance was measured at 532nm. Concentration of
extracts required for 50 per cent reduction in absorbance was calculated from
the calibration curve of concentration of the extracts Vs per cent reduction in
absorbance. 0.1mM solution of CuSO4 was prepared and used as
reference standard13.
Table 1: Free radicals and Hydroxyl radicals scavenging
activity of Kedrostis foetidissima
|
S.No |
Free radicals scavenging activity |
Standard (Ascorbic Acid) |
Hydroxyl radicals scavenging activity |
Standard (CuSO4) |
||
|
Conc μg/ml |
Mean ± SD |
Mean ± SD |
Conc μg/ml |
Mean ± SD |
||
|
1 |
10 |
41.23±0.4132 |
20.52±0.52 |
10 |
14.54±0.03 |
32.24±0.003 |
|
2 |
20 |
46.87±0.4951 |
34.67±0.56 |
20 |
17.91±0.03 |
46.84±0.003 |
|
3 |
30 |
50.45±0.7412 |
38.15±0.20 |
30 |
18.54±0.04 |
58.96±0.004 |
|
4 |
40 |
54.45±1.2456 |
41.57±0.61 |
40 |
22.73±0.04 |
65.89±0.004 |
|
5 |
50 |
58.21±0.9854 |
63.87±0.57 |
50 |
25.46±0.04 |
72.64±0.04 |
Values are represented in
Mean ±SD of six replicates performed using graph pad prism software using Kolmogorov-smirnov test
Table 2: Nitric oxide and superoxide radicals
scavenging activity of Kedrostis foetidissima
|
S.No |
Super oxide radical % inhibition |
Standard (Butylated Hydroxy toluene) |
Nitric oxide % inhibition |
|||
|
Conc μg/ml |
Mean ± SD |
Mean ± SD |
Conc μg/ml |
Mean ± SD |
||
|
1 |
10 |
36.54±0.004 |
8.52±0.461 |
10 |
17.82±0.004 |
|
|
2 |
20 |
43.61±0.021 |
12.57±0.460 |
20 |
19.87 ±0.005 |
|
|
3 |
50 |
46.67 ±0.462 |
15.49±1.182 |
50 |
22.73±0.004 |
|
|
4 |
100 |
63.62 ±0.005 |
20.71±0.402 |
100 |
27.59±0.519 |
|
|
5 |
150 |
67.34±0.042 |
25.69±0.840 |
150 |
38.14±0.004 |
|
Values are represented in
Mean ±SD of six replicates performed using graph pad prism software using Kolmogorov-smirnov test.
RESULTS AND DISCUSSION:
The crude ethanolic extract
of whole plant of Kedrostis foetidissima exhibited significant inhibition of nitric oxide and
superoxide scavenging activity. The ethanolic extract has shown moderate
activity against standard drugs. The presence of flavonoids
and tannins in the ethanolic extract is likely to be responsible for the
in-vitro antioxidant against different in-vitro screening methods. Flavanoids and tannins are phenolic
compounds and plant phenolic compounds are major
group of compounds that act as primary antioxidants. Scavenging activity for
free radicals of 1.1- diphenyl-2-picrylhydrazyl (DPPH) has been widely used to
evaluate the antioxidant activity of natural products from plant and microbial
sources. Superoxide anion is one of the most representative free radicals. In
cellular oxidation reactions, superoxide radicals have their initial effects
magnified because they produce other kinds of cell-damaging free cells and
oxidizing agents. In biochemical systems, superoxide radical can be converted
into hydrogen peroxide by the action of superoxide dismutase and the H2O2
can subsequently generate extremely reactive hydroxyl radicals in the presence
of certain transition metal ions. Hydroxyl radicals can attack DNA molecules to
cause strand scission .The IC50 values are represented in Table 1 and 2 against
standard drugs. The study reveals in-vitro antioxidant activity of Kedrostis foetidissima
CONCLUSION
Reactive oxygen species
contribute to various pathophysiological conditions
and endogenous defense mechanisms have evolved to offer protection in these
conditions. Determination of the natural antioxidant compounds of plant
extracts will help to develop new drug candidates for antioxidant therapy. The
overall antioxidant activity of Kedrostis foetidissima is due to flavonoids, tannins
and other phytochemical constituents. The active
chemical constituents responsible for in-vitro antioxidant should be isolated
and the results should be compared with in vivo results.
REFERENCES
1.
Stanner S.A., Hughes J., Kelly C.N., Buttriss
J.A. Review of the epidemiological evidence for the antioxidant
hypothesis. Public Health Nutrition. 7; 2000:
401-422.
2.
Branen AL. Toxicological and biochemistry of butylated
hydroxyl anisole and butylated hydroxytoluene.
J of American oil Chemists Society. 52; 1975: 59-63.
3.
Chen S, Hwang J, Deng PSK.
Inhibition of NADPH: Quinone acceptor oxidoreductase by flavones: a structural activity study.
Archives of biochemistry and biophysics. 303; 1993: 72-77.
4.
Rice-Evans C, Miller NJ.
Total antioxidant status in plasma and body fluids. Methods in enzymology. 234; 1994: 279-293.
5.
Elizabeth M, Williamson.
Textbook of Major herbs of Ayurveda. (I); 2002: 7-11.
6.
Giday, M. An Ethnobotanical study of medicinal
plants used by the Zay people in Ethiopia. Skriftserie. 3; 2001: 81-99.
7.
Otieno, J.N., Hosea, K.M.M., Lyaruu, H.V. and Mahunnah, R.L.A. Multi plant or single plant extracts,
which is the most effective for local healing in Tanzania?. Afr. J. Trad. Com. Alt. Med. 5; 2008: 165-72.
8.
Tabuti, J.R.S., Lye, K.A. and Dhillion, S.S.Traditional herbal drugs of Bulamogi,
Uganda: Plants, use and administration. J. Ethnopharmacol.
88; 2003: 19-44.
9.
C.C. Mensor, F.S. Merezes, G.G. Leitan, A.S. Reis, C.S. Carbe and
S.G. Leitao. Screening of Brazilian
plant extracts for antioxidant activity by the use of DPPH free radical method.
Phytother Res. 15, 2007: 127-130.
10. D.C. Garrat. The Quantitative
Analysis of Drugs, Japan, Chapman and Hall. 1964: 456.
11.
M.J. Green and
H.A.O. Hill. Chemistry of dioxygen.
In: Methods in Enzymology, Academic Press, New York. 105; 1984: 21.
12.
J.M. Cord and I. Fridovich. The biology of oxygen radicals. J. Biol. Chem. 1988: 243, 5753-5790.
13.
N. Sreejayan and M.N.A. Rao. Free radical scavenging activity of curcuminoids.
Drug Res. 46; 1996: 169.
Received on 08.03.2012
Modified on 28.04.2012
Accepted
on 08.05.2012
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A&V Publication all right reserved
Research Journal of Pharmacognosy
and Phytochemistry. 4(4): July- August 2012, 209-211