5.
Deoxyribose (10mM):- 0.134 gms of deoxyribose dissolved in
100ml of distilled water.
6.
Phosphate buffer (50mM, pH 7.4):- 6.80gms of Potassium dihydrogen
phosphate and 7.10gms of disodium hydrogen phosphate are dissolved in
sufficient distilled water to produced 1000ml.
7.
10% Trichloroacetic acid: 10gms of Trichloroacetic acid were
dissolved in 100ml of distilled water.
8.
0.5% Thiobarbituric acid (TBA): 0.5 gms of TBA dissolved in 100ml of
0.025M NaoH.
9.
DPPH Solution [90µM]: Dissolve 35.48 mg of DPPH in 10 ml
methanol to get a concentration of 3.548mg/ml or 0.348µg/ml. 1ml of the
solution is diluted to 100 ml with methanol to get a concentration of
35.48µg/ml.
Working Standard and Test solutions:
a.
Test solution for DPPH Method: 50 mg of ethanolic extract was dissolved in 10 ml of
methanol and volume was made up to 50ml to give 1mg/ml stock solution. The
above stock solution was further diluted to get the working test solution in
the concentration range of 10 - 100µg/ml.
b.
Standard solution for DPPH
Method: Curcumin and Ascorbic acid
solutions (10-100µg/ml) were prepared in methanol as similar to test solutions.
c.
Test solution f or Hydroxyl scavenging by
EDTA method: 10 mg of
ethanolic extract was dissolved in sufficient quantity of methanol and volume
was made up to 100ml to give 100µg/mL of the stock solution. The above stock
solution was further diluted to get the working test solution in the
concentration range of 1-10µg/mL.
d.
Standard solution for Hydroxyl scavenging by
EDTA method: Catechin and
Vitamin E standard solutions [1-10µg/mL] were prepared in methanol as similar
as test solutions.
Procedure
for Detemination of Hydroxyl Radical Scavenging Activity by EDTA: Stock solutions of all reagents were
prepared in doubly distilled water. The assay was performed by adding 0.1ml of
EDTA, 0.01 ml of FeCl3, 0.1ml
of Hydrogen peroxide, 0.35 ml of
deoxyribose, 1 ml of the extract/ working standard solution [1-10 mcg/mL], 0.3
ml of phosphate buffer [pH 7.4, 50Mm] and 0.1ml of ascorbic acid in sequential
order of addition. The mixture was then incubated at 370 C for 60
min. 1ml of incubated reaction mixture was mixed with 1 ml of 1% Thiobarbituric
acid [TBA] and 1 ml of 5% Trichloro acetic acid in NaOH(0.025M) to develop the
pink colored chromogen and absorbance was measured at 532nm. Deoxyribose
degradation was measured as TBARS and percentage inhibition was calculated. % RSC
was calculated by the following formula:
Where,
%RSC=Radical scavenging capacity.
Ablank = Absorbance of reagent
blank.
Asample=Absorbance of sample.
From RSC values, the IC50 values
was calculated which represents the concentration of scavenging compound that
was 50% neutralization.IC50 values were obtained by linear
regression method using % activity in y-axis and concentration in x-axis. RSC
of ethanolic extract was determined and the activity was compared with standard
Catechin and Vit.E.
Procedure
for Determination of Antioxidant activity by DPPH Method: A commercially available and stable free
radical DPPH (Sigma chemicals, U.S.A), soluble in methanol was used. DPPH in
its radical from has an absorbance peak at 517nm, which disappears on reduction
by an antioxidant compound. 1ml of different concentrations of the
extract/standard (10-100µg/ml) were added to 2ml of freshly prepared methanolic
solution of 90µM DPPH and the volume was made up to 4 ml with methanol. The
reaction mixtures were kept at room temperature in the dark and after 1 hr the
absorbance was measured at 517nm using spectrophotometer. A blank was performed excluding the
extract. The optical density of the
sample, and the blank was measured by comparing the methanol.
Curcumin and ascorbic acids were used as
standards. The percentage inhibition of
DPPH in reaction mixture was calculated by comparing with the blank. % RSC was
calculated by the following formula:
Where,
%RSC=Radical scavenging capacity.
Ablank= Absorbance of reagent
blank. Asamlpe=Absorbance of sample.
From RSC values, the IC50 values
was calculated which represents the concentration of scavenging compound that
was 50% neutralization.IC50 values were obtained by linear
regression method using % activity in y-axis and concentration in x-axis. RSC
of ethanolic extract, water soluble ethanolic extract, and water insoluble
ethanolic extract were determined and the activity was compared with standard
ascorbic acid and Curcumin.
RESULTS
AND DISCUSSION:
Hydroxyl radical scavenging activity was
measured by studying the competition between deoxyribose and test compound for
hydroxyl radical generated from the Fe3+/ ascorbate/EDTA/H2O2
system. The hydroxyl radical attacks deoxyribose, which results in
thiobarbituric acid reacting substance (TBARS) formation4 based on
the inhibition rate of 2-deoxyibose oxidation by hydroxyl radical. The scavenging capacity for hydroxyl radical
was measured according to the modified method of Halliwell 5
Among the all oxygen radicals, the hydroxyl
radical (OH) is the most reactive and damages diverse biomolecules. Hydroxyl
radicals were generated in a reaction mixture containing ascorbate, H2O2
and Fe3+, EDTA at pH 7.4 and measured b their ability to degrade the
sugar deoxyribose into fragments that on heating with TBA at low pH from a pink
chromogen. Ethanolic extract of A.sclerocarpa
bark showed high hydroxyl radical scavenging activity that intensify with
increasing concentration, reacting 84% at 9µg/ml while at an equal concentration
the standard vitamin E and catechin caused 75% and 67 % inhibition of hydroxyl
radical.
Table 2(a) and 2(b) shows the scavenging
capacity of Alphonsea sclerocarpa bark extracts and standards. The decreasing order of antioxidant effect
(expressed as IC50) is ethanolic extract 0.7 µg/ml, vitamin E
0.8µg/ml and Catechin 4.6µg/ml. It was
observed that the IC50 of Alphonsea sclerocarpa bark extracts
was higher than that of standards.
The free radical scavenging activity of Alphonsea
sclerocarpa bark extracts was measured by 2,2-diphenyl-1-picryl
hydrazyl [DPPH] using the method of Aquino et al5. DPPH is a free
radical compound that has been widely used to test the free radical scavenging
ability of various samples and method requires relatively short time compared
with others. The reduction capacity of DPPH radical was determined by
decreasing it absorbance at 518nm induced by antioxidants. The decreased
absorbance by DPPH is due to progressive reaction between free radicals and
antioxidant molecules which results in scavenging of radical by donation of
hydrogen. It is visually noticeable by decrease in intensity of purple color.
Hence, the DPPH free radical is generally used as a substrate to evaluate
antioxidant activity. Stable free radical DPPH was effectively scavenged by
ethanolic extract Alphonsea sclerocarpa barks and the inhibition was found to
be dose-dependent. Table 1(a) and 1(b) illustrates the scavenging activity of Alphonsea
sclerocarpa bark extracts, Curcumin and ascorbic acid on the DPPH
radicals and the effects increase with increasing concentration range
10-100ug/mL. The scavenging activity of Alphonsea sclerocarpa bark extracts
and standards on the DPPH radical decreased in the order: Ascorbic
Acic>Curcumin>ethanolic extract were of 96%, 94%, and 92% at the
concentration of 50µg/mL respectively. The concentration of Alphonsea
sclerocarpa bark extract, resulting in 50% inhibition of the free
radical-IC50 was 23 µg/mL and those of standards curcumin and ascorbic acid was
10µg/mL and 7 µg/mL.
CONCLUSION:
The present study revealed that the bark
extract of A.sclerocarpa possess the
significant antioxidant activity and the further pharmacological investigations
are required for evaluation of potential benefits as natural antioxidant in
different experimental models.
REFERENCES:
1. R.E. Fries, in Die naturlichen
Pflanzenfamilien,” vol 17 aII. Ed. By A. Engler and K. Prantl, Duncker and
Humblot, Berlin, 1959.
2.
Alklaloids of Alphonsea
sclerocarpa Dragana Tacić, G. Percy Wannigama, Bruce K. Cassels,
André Cavé J. Nat. Prod., 1987, 50 (3), pp 518–519.
3.
Elizabeth S, and Rao MNA, Int.J Pharm 58, 1990, 237.
4.
Kuchnady E and Rao MNA, Int.J Pharmacognosy 58,
1990, 237.
5.
Aquino R, Moraelli S, Lauro M R, Abdo S, Saija A,
and Tomaino A, J. of Natural Products,
64, 2001, 1019
Received on 13.09.2009
Accepted on 15.09.2009
© A&V Publication all right reserved
Research J. Pharmacology and
Pharmacodynamics 2009; 1(2): 66-69