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Sujith & Darwin, Int. J. Res. Phytochem. Pharmacol.

, 1(2), 2011, 39-44

ISSN: 2231-010X
Research Article
www.ijrpp.pharmascope.org

Evaluation of Free Radical Scavenging Potential of methanolic extract of Butea


Frondosa Leaves
K. Sujith*, C. Ronald Darwin
Department of Pharmacology, K.K.College of Pharmacy, Chennai, India

ABSTRACT

The methanolic extract of Buteafrondosa leaves was screened for invitro antioxidant activities by using different in
vitro scavenging models .Safer antioxidants suitable for long term use are needed to prevent or stop the progres-
sion of free radical mediated disorders such as arthritis, hemorrhagic shock, atherosclerosis, diabetes, hepatic
injury, aging, neurodegenerative diseases and carcinogenesis. Antioxidant activity of methanolic extract of Butea-
frondosa leaves were assessed using DPPH, hydroxyl, hydrogen peroxide ,nitric oxide in vitro scavenging mod-
els.The extract was also studied for metal chelating activity , reducing power , total phenolic compounds and assay
2+
on lipid peroxidation induced by Fe -ascorbate system using rat liver homogenate. The methanolic extract of Bu-
teafrondosa leaves exhibited significant scavenging activity in DPPH, hydroxyl, hydrogen peroxide ,nitric oxide in
vitro scavenging models and also showed significant metal chelating activity.The reducing power of the extract
increased in dose dependant manner. The extract showed marked protection in lipid peroxidation induced by
Fe2+-ascorbate system using rat liver homogenate. The methanolic extract of Buteafrondosa leaves shows signifi-
cant antioxidant activity in different in vitro models. Thus augmenting the wide use of plant in the indigenous sys-
tem of medicine for free radical mediated disease.
Keywords: Antioxidant; free radicals; Butea frondosa
INTRODUCTION Plant material
Buteafrondosa (Papilionaceae) is an erect tree found The plant material of Butea frondosa leaves used for
commonly throughout the hills of South India, grow investigation was collected from Pathanamthitta dis-
gregariously in open grassland, and scattered in mixed trict of Kerala and the leaves of Buteafrondosa was
forests. The leaves of Buteafrondosa are traditionally identified and authenticated by Dr. Sasikala Ethirajulu,
used in inflammatory condition, liver disease, skin dis- research officer (pharmacognosy) Central Research
ease, worm infestation and haemorrhoids (kritikar., Institute (Siddha), Chennai and voucher specimen of
2000, kapoor., 1990). Buteafronodosa leaves are the plant has been be deposited in the herbarium of
shown to possess antinflammatory activity (Mengi et- the department.
al., 1999). However, there are no scientific reports on
Preparation of extracts
the antioxidant activity of Butea frondosa leaves. Free
radicals are the fundamental cause of different kinds of The leaves of Butea frondosa were shade-dried and
disease. They cause biochemical damage in cells and powdered to a coarse powder. The powder was passed
tissues which results in several disease such as arteri- through a 40-mesh sieve and were subjected to conti-
osclerosis , liver disease, diabetes mellitus, inflamma- nuous hot extraction in Soxhlet apparatus with petro-
tion , renal failure, aging and cancer (Devasagayam etal leum ether (60% v/v) and the marc left after petroleum
.,2003). The present study was undertaken to investi- ether extraction were dried and extracted with metha-
gate antioxidant and free radical scavenging activity of nol (90% v/v) in Soxhlet apparatus. The extract was
methanolic extract of Butea frondosa leaves by using evaporated under reduced pressure using rotary eva-
in-vitro assay methods. porator until all the solvent had been removed to give
an extract sample with a yield of 9.7% w/w. Methanolic
Materials and Methods
extract of Butea frondosa (MEBF) were used for identi-
fication of phytochemical constituents and for in vitro
* Corresponding Author antioxidant studies.
Email: sruthiadoor@gmail.com
Preliminary pytochemical analysis
Received on: 16-01-2011
Revised on: 03-03-2011 Preliminary phytochemical investigation was con-
Accepted on: 18-05-2011 ducted as per procedure described by (Kokate, 1994).

©JK Welfare & Pharmascope Foundation | International Journal of Research in Phytochemistry & Pharmacology 39
Sujith & Darwin, Int. J. Res. Phytochem. Pharmacol., 1(2), 2011, 39-44

Table 1: Free radical scavenging activity of MEBF Table 3: Hydrogen Peroxide Scavenging activity of
by DPPH reduction MEBF

Sl. Concentration IC50 Value Sl. Treatment % protection of lysis


Inhibition (%)
No. (g/ml) (g/ml) No. (g/ml) 90 min 120 min
1. 800 76.27  0.37 8.62 
1 Control 14.62  0.53
2. 600 67.47  0.30 0.213
3. 400 55.58  0.197 383.91  0.94 64.23  35.14 
2. MEBF (100) * *
0.321 0.532
4. 200 42.50  0.24
69.04  53.42 
5. 100 24.91 + 0.44 3. MEBF (200)
0.682* 0.131*
Values are mean  SEM of 5 replicates Standard Vi- 39.04 
51.94  0.30
*
4. *
Evaluation of Antioxidant activity of the Butea fron- tamin E (100) 0.73
dosa Values are Mean  SEM of 5 replicates. *p<0.001
DPPH Radical Scavenging Activity vs control.
o
incubated at 37 C in a constant shaker bath at 150
DPPH scavenging activity was measured by spectro-
cycles/minute. Aliquots (1 ml) in duplicate were with-
photometric method (Mary et al., 2002). To a metha-
drawn at different time interval. To one aliquot 4 ml of
nolic solution of DPPH (100 µM, 2.95 ml), 0.05 ml of
cold saline was added in order to stop the reaction, 4
test compounds dissolved in methanol was added at
ml of distill water was added to the other aliquot to
different concentration (100-1000 µg/ml). Equal
produce 100% lysis of RBC. The two aliquots were cen-
amount of methanol was added to the control. Absor-
trifuged separately and optical density of supernatant
bance was recorded at 517 nm.
liquid measured spectrophotometrically at 540 nm. To
Hydroxyl Radical Scavenging activity evaluate antioxidant potential of MEBF (100, 200
µg/ml) and vitamin E (100 µg/ml) are added to the sus-
The hydroxyl radical scavenging activity was measured
pension 30 min prior to addition of enzyme and sub-
by studying the competition between deoxyribose and
strates (Gulcin etal., 2002).
the extract for hydroxyl radicals generated from the
Fe3+/ ascorbate / EDTA / H2O2 system (Guyton.,1997). Nitric Oxide Scavenging
The reaction mixture contained deoxyribose (2.8mM),
Nitric oxide scavenging activity was measured by using
Fecl3 (0.1mM), EDTA (0.1mM, H2O2 (1mM), ascorbate
a spectrophotometer. Sodium nitroprusside (5mM) in
(0.1mM), KH2PO4-KOH buffer (20mM, pH 7.4) and vari-
phosphate buffered saline was mixed with different
ous concentrations (100-300 µg/ml) and mannitol (100
concentrations of the extract (100-1000 µg/ml) and
µg/ml) of the drug in a final volume of 1 ml. The reac-
vitamin E (100 - 1000 µg/ml) dissolved in methanol and
tion mixture was incubated for 1 hr at 37oC. Deoxyri-
incubated at 25°C for 30 min. A control without test
bose degradation was measured at 532 nm
compound but with equivalent amount of methanol
Hydrogen peroxide scavenging activity was taken. After 30 min 1.5 ml of the incubation solu-
tion were removed and diluted with 1.5 ml of Griess
Blood specimen was collected from the orbital plexus
reagent (1% sulphanilamide, 2% phosphoric acid, and
of rats, centrifuged for 5 min at 1000 rpm and subse-
0.1% napthyl ethylene diamine dihyrochloride). The
quently washed with physiological saline for four
absorbance of the chromophore formed during diazo-
times. The cell suspension was then transferred quanti-
tization of the nitrite with sulphanilamide and subse-
tatively to different flasks containing 0.1 U/ml glucose
quent coupling with napthylethylene diamine was
oxidase 0.5 mg/ml glucose in a manner so that the final
measured at 546 nm (Balakrishnan etal., 2002).
erythrocyte concentrated was 1% and the flask were
Determination of reducing power
Table 2: Hydroxyl radical scavenging activity of
MEBF 10mg of methanolic extract of Butea frondosa in 1ml of
distilled water was mixed with phosphate buffer (2.5
Sl. ml, 0.2 M, pH 6.6) and potassium ferricyanide [K3
Concentration (g/ml) Inhibition (%) Fe(CN)6] (2.5 ml, 1%). The mixture was incubated at
No.
50°C for 20 min. A portion (2.5 ml) of trichloroacetic
1 100 27.04  0.21
acid (15%) was added to the mixture, which was then
2 200 30.90  0.15 centrifuged at 3000 rpm for 10 min. The upper layer of
3. 300 45.22  0.45 the solution (2.5 ml) was mixed with distilled water
(2.5 ml) and ferric chloride, (0.5 ml, 0.1%), and the ab-
4. Mannitol 100 63.17  0.92
sorbance was measured at 700 nm. Increased absor-
Values are Mean  SEM of 5 replicates. bance of the reaction mixture indicates increased re-

40 ©JK Welfare & Pharmascope Foundation | International Journal of Research in Phytochemistry & Pharmacology
Sujith & Darwin, Int. J. Res. Phytochem. Pharmacol., 1(2), 2011, 39-44

Table 4: Nitric Oxide scavenging activity of MEBF and Vitamin E

Sl. No. Concentration (g/ml) Inhibition (%) IC50 Value (g/ml)


1. MEBF 800 67.40  0.37
2. 600 58.29  0.31
3. 400 35.52  0.43 561.31  1.65
4. 200 20.99  0.39
5. 100 11.17 + 0.50
6. Vitamin E 800 85.36  0.735
7. 600 79.47  0.155
8. 400 59.48  0.035 331.98  4.72
9. 200 40.44  0.358
10. 100 24.80  1.23
Values are mean  SEM of 5 replicates
ducing power. The reducing power of the standard pyrocatechol as a standard (Tripathi etal.,2001). 0.1 ml
drug (Butylated hydroxyl toluene) was also determined of extract solution in a volumetric flask is diluted with
by the above described procedure. (Nagulendran etal., distilled water (46 ml). About 1 ml of Folin-Ciotalteu
2007). reagent was added and a content of the flask was
mixed thoroughly. After 3 min, 3ml of sodium bicarbo-
Table 5: Determination of reducing power of nate (2%) was added, and then the mixture was al-
MEBF lowed to stand for 2hrs with intermittent shaking. The
Sl. Concentration absorbance was measured at 760nm. The concentra-
Absorbance tion of total phenolic compounds in the Butea frondosa
No. (g/ml)
is determined as µg of pyrocatechol equivalent by us-
1 Control 0.0734  0.001
ing an equation that was obtained from standard pyro-
1.065  0.002
*
2. MEBF 500
catechol graph. The equation is given below.
0.9102  0.002
*
3. 375
4. 250 0.6488  0.002* Absorbance = 0.001 X pyrocatechol (µg) + 0.0033
5. 125 0.424  0.002 *
Metal chelating activity
6. BHT 500 0.821  0.001*
Metal chelation property of the extract for Ferric ion
7. 375 0.813  0.001*
(Fe3+) was estimated by using thiocyanate method.
8. 250 0.491  0.002*
Here different concentrations (1:2.5 to 1:10 ratio) and
9. 125 0.410  0.001* EDTA (1:10 ratio) of the extract were mixed with a
Values are Mean  SEM of 5 parallel measure- fixed concentration of ferric chloride (10 µg). The mix-
ments. *p<0.001 vs control. Spectrophotometric ture was incubated for 30 min. At the end of the incu-
deduction of the Fe+3 – Fe+2 transformation; Buty- bation, 1 ml of potassium thiocyanate (25%) was added
lated hydroxytoluene (BHT) and absorbance of ferric-thiocyanate complex (reddish
brown complex) was measured at 450 nm. The results
Determination of total phenolic compounds
were compared with standard EDTA ratio. Metal chela-
Total soluble phenolic compounds in methanolic ex- tion property of the extract for Ferrous ion (Fe2+) was
tract of Butea frondosa were determined by Folin- estimated by using 2,2'-bipyridyl method. Here differ-
Ciotalteu reagent according to following method using ent concentrations of the extract were mixed with a
Table 6: Effect of MEBF on Fe2+ / Fe3+ metal chelation

Absorbance at % Chelation of Absorbance at


Iron: Drug % Chelation of Fe3+
525 nm Fe2+ 460 nm
(control )1:00 0.308 0 1.02 0
1:0.25 0.276 10.19  0.241* 0.890 12.74  0.097*
1:0.5 0.254 17.52  0.563* 0.812 20.33  0.133*
1:1 0.224 27.13  0.614 *
0.754 26.03  0.105*
1:2.5 0.216 29.60  0.415* 0.720 29.34  0.118*
1:5 0.204 33.50  0.416 *
0.685 32.8  0.183*
49.54  0.528 51.87  0.099
* *
1:10 0.159 0.490
79.86  0.562 85.38  0.069
* *
EDTA (1:10) 0.062 0.149
Values are Mean  SEM of 5 parallel measurements. *p<0.001 vs control

©JK Welfare & Pharmascope Foundation | International Journal of Research in Phytochemistry & Pharmacology 41
Sujith & Darwin, Int. J. Res. Phytochem. Pharmacol., 1(2), 2011, 39-44

Table 7: Effect of MEBF on inhibition of lipid pe- Antioxidant studies


roxidation – Induction by Fe3+/ascorbate DPPH Scavenging Reduction
Reduction of DDPH radicals due to the scavenging abili-
Sl. Concentration (g
% Inhibition ty of MEBF can be observed by decrease at 517 nm.
No. /ml)
IC50 value of MEBF was found to be 383.91  0.94
1 25 16.18  0.131 g/ml. Results are shown in Table 1.
2 50 22.276  0.515
Hydroxyl Radical Scavenging Activity
3 100 36.75  0.853
The scavenging ability of MEBF on hydroxyl radical is
4 200 42.04  0.424 shown in Table -2. MEBF was capable of scavenging
5 300 51.954  0.424 hydroxyl radical in a dose – dependent manner. It ex-
6 Vitamin E – 100 65.04 + 0.210 hibited a scavenging activity (27.04, 30.90, 45.22%) on
hydroxyl radical at dose of 100 g, 200 g, 300 g re-
Values are Mean  SEM of 5 replicates
spectively at same time mannitol (100 g) exhibited
fixed concentration of ferrous sulphate (10µg). The 63.17% hydroxy radical scavenging activity.
mixture was incubated for 30 min. At the end of the
incubation, 2 ml of 2,2'-bipyridyl (1mM) was added and Hydrogen peroxide scavenging activity
absorbance of ferrous-bipyridyl complex (pink- Haemolysis of RBC, induced by H2O2 sysem was signifi-
coloured complex) was measured at 525 nm. The re- cantly (p<0.001) inhibited by MEBF at all treated doses,
sults were compared with EDTA (Comporti etal.,1965). when compared with that of control. The percentage
Assay of lipid peroxidation inhibition of lysis by MEBF increased in a dose depen-
dent manner. Results were comparable with the stan-
Inhibition of Lipid Peroxide Formation Induced by dard (p<0.001). Results are shown in Table -3.
Fe2+-Ascorbate System
Nitric oxide scavenging activity of MEBF
Reaction mixture (0.5ml) containing 25% rat liver ho-
mogenate (0.1 ml) w/v in Tris-HCl buffer (40 mM, pH The scavenging of nitric oxide by the MEBF was con-
7.0), potassium chloride (30 mM), ferrous ion (0.16 centration dependent and the IC50 value was found to
mM) and ascorbic acid (0.06 mM) was incubated for be 561.31  1.65 g/ml. MEBF was capable of scaveng-
1hr at 37ºC in the presence and absence different con- ing nitric oxide radical in a dose – dependent manner.
centrations (25 – 300 µg/ml) of the extract and vitamin It exhibited a scavenging activity (20.99, 35.52, 58.29,
E (100 µg/ml). For this 0.4 ml of the reaction mixture 67.40%) on nitric oxide radical at dose of (200, 400,
was treated with sodium dodecyl sulphate (SDS-0.2 ml, 600, 800 g) and IC50 value of vitamin E was found to
8.1%), thiobarbituric acid (TBA-1.5 ml, 0.8%) and acetic be 331.98  4.72 g/ml. Results are shown in Table -4.
acid (1.5 ml, 2.5% of pH 3.5). The mixture (4 ml) was Reducing power
taken kept in a water bath at 95ºC for 1 hr. After cool-
ing 1 ml of distilled water and 5 ml of a mixture of n- Reducing power of MEBF increased with increased
butanol and pyridine (15:1 v/v) were added and shaken concentration of test compound. All the doses of MEBF
vigorously. After centrifugation, the chromophore was have showed higher activities than control and the
measured at 532 nm. The percentage inhibition of lipid difference was statistically very significant (p<0.001).
peroxidation was determined by comparing the result Results were comparable with standard (BHT)
of control and test compounds (Sen etal.,1992). (p<0.001). Results are shown in Table -5.
Statistical Analysis Determination of total phenolic compounds
The data on in-vitro studies were reported as mean ± In MEBF (1 mg), 146.9  1.15g pyrocatechol equiva-
Standard deviation (n=5). For determining the statistic- lent of phenol was detected.
al significance, standard error mean and analysis of Effect of MEBF on Fe2+/Fe3+ metal chelation
variance (ANOVA) was employed (Bolton.,1997).
MEBF significantly (P<0.001) chelated Fe2+ (49.54%)
RESULTS 3+
and Fe (51.87%) at 1:10 ratio of iron: MEBF chelating
Preliminary phytochemical screening ability for metal transition ions (Fe2+, Fe3+) increased in
a dose dependent manner. In similar conditions, EDTA
The methanolic extract showed the presence of alkalo- exhibited 79.86% chelation for Fe2+ and 85.38% for Fe3+
ids, carbohydrates, protein, tannins, phenols, flavono- respectively, which is significant (P<0.001) when com-
ids and terpenes. pared with the control. Results are shown in Table -6.
2+
Lipid peroxidation induced by Fe / ascorbate system
The extract was found to inhibit Fe2+ / ascorbate sys-
tem induced lipid peroxidation in rat liver homogenate
42 ©JK Welfare & Pharmascope Foundation | International Journal of Research in Phytochemistry & Pharmacology
Sujith & Darwin, Int. J. Res. Phytochem. Pharmacol., 1(2), 2011, 39-44

in a dose dependent manner. It exhibited inhibition ing of transition metal ion catalysts, decompositon of
16.18, 22.27, 36.75, 42.04, 51.95% inhibition at dose of peroxides, prevention of cations, and radical scaveng-
25, 50, 100, 200, 300 g respectively at the same time ing.
vitamin E (100 g) inhibited 65.04% lipid peroxide for- Phenols are very important plant constituents because
mation. Results are shown in Table- 7. of their scavenging ability due to their hydroxyl groups,
DISCUSSION this made us to investigate the amount of total phenol-
ic compounds in extract. In the MEBF (1mg), 146.9 ug
The free radical scavenging activity of the plant was pyrocatechol equivalent of phenols was detected the
evaluated based on the ability to quench the synthetic
phenolic compounds may contribute directly to anti-
DPPH. This assay provided useful information on the
oxidative action.
reactivity of the compounds with stable free radicals,
because of the odd number of electrons. DPPH shows a Lipid peroxidation(LPO) has been implicated in the pa-
strong absorption band at 517nm in visible spectrum thogenesis of number of diseases including diabetes. It
(deep violet colour) as the electron becomes paired off is well established that bioenzymes are very much sus-
in the presence of free radical scavenger the absorp- pectible to LPO which is considered to be the starting
tion vanishes and the resulting discoloration (absor- point of many toxic as well as as degenerative process..
bance) stoichiometric with respect to the number of The extract showed a significant protection against
electron taken up. The bleaching of DPPH absorption 2+
Fe /ascorbic acid induced LPO that could be caused by
representative of the capacity of the test compound to absence of ferryl-perferyl complex. It is generally as-
scavenge free radical independently (Hu etal.,2007).
sumed that ability of the plant phenolic compounds
Hydroxyl radical is a principle contributor for tissue such as flavanoids, to chelate ions in the LPO system is
damage. The formation of hydroxyl radical from fenton very important for their antioxidant property. There-
reaction was quantified using 2,deoxy -D-ribose degra- fore, an attempt was made to determine the role of
dation. Studies with MEBF have revealed significant ion chelation, since the inhibtion of Fe2+/ascorbic acid
hydroxyl scavenging activity in invitro. induced LPO could also be due to chelating the iron.
Our extract showed chelating effect against ferric (Fe3+)
H2O2 can generate hydroxyl radical via fenton reaction.
and ferrous (Fe2+). So it could be concluded from the
In addition H2O2 can easily cross the cell membrane
present study, the extract offers protection against
and exerts an injurious effect of tissue through a num-
Fe2+/ascorbic acid induced lipid peroxidation. The pre-
ber of different mechanisms, such as perturbing intra-
liminary phytochemical analysis of our present investi-
cellular Ca2+ monostat, increased intracellular ATP in- gation revealed the presence of flavonoids. Active con-
ducing DNA damage and inducing aptotosis. Glucose
stituent of plant extract such as flavonoids are re-
oxidase in presence of glucose produces H2O2. H2O2
ported to posses antioxidant potential. The results of in
thus liberated by glucose oxidase may cause direct
vitro experiment suggest that Butea frondosa use in
oxidative attack on cell membrane and increases lipid
Indian system of medicine may be due to their antioxi-
rupture to lipid bilayer, osmotic fragility, aggregation of dant and free radical scavenging activity.
membrane proteins and decrease activity of mem-
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44 ©JK Welfare & Pharmascope Foundation | International Journal of Research in Phytochemistry & Pharmacology