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167]

Research Letter

Antioxidant potential of methanolic extract of Dolichos

biflorus Linn in high fat diet fed rabbits
Dolichos biflorus Linn (Fabaceae), is commonly known as
Kollu in Tamil and horse gram in English. It has been reported
to lower lipids in rats.[1] A high fat diet induces oxidative stress
in the cells by producing reactive oxygen species. Therefore,
in this study, the influence of the Dolichos biflorus extract on
high fat diet (HFD) induced oxidative stress in rabbits, has
been investigated.
Whole plants of D. biflorus were collected from Sankaran
Koil, Tirunelveli district of Tamilnadu, India. Taxonomic
identification was made by the Botanical Survey of Medicinal
Plant Unit, Siddha, Government of India, Palayamkottai,
Tamilnadu. Four month-old whole plants were dried in the
shade, segregated, and pulverized by a mechanical grinder
and passed through a 40 mesh sieve. The powder was extracted
by methanol in Soxhlet apparatus by continuous hot percolation
method. After filtration through Whatmann filter paper No 40,
the filtrate was vacuum dried at 35 to 40oC. The extracts were
stored in screw cap vials at 4oC until further use. The extractive
value of the methanolic extract was 8.13% w/w. The methanolic
extract of D.biflor us was subjected to preliminar y
phytochemical screening to find out the presence of active
principles. The extracts were suspended in 2% tween 80.
New Zealand white rabbits, weighing 900-1050 g were
procured from the central animal house, Rajah Muthiah Medical
College, Annamalai University. The animals were kept in cages,
2 per cage, with 12:12 h light/dark cycle at 25+2 oC. The
animals were maintained on their respective diets and water
ad libitum. Animal ethical committees clearance was obtained
for the study.
Rabbits were divided into the following five groups, with
six rabbits in each group:
Group I : (Control): Standard chow diet.
Group II : High fat diet (HFD)
Group III : High fat diet plus methanolic extract of
D.biflorus (dose I -200 mg/kg body weight)
Group IV : High fat diet plus methanolic extract of
D.biflorus (dose II -400 mg/kg body weight)
Group V : High fat diet plus standard drug atorvastatin
(1.2 mg/kg body weight).
The compositions of the two diets were as follows:
Control diet
Wheat flour 22.5%, roasted Bengal gram powder 60%,
skimmed milk powder 5%, casein 4%, refined oil 4%, salt
mixture with starch 4%, and vitamin and choline mixture 0.5%.
High fat diet
Wheat flour 20.5%, roasted Bengal gram 52.6%, skimmed
milk powder 5%, casein 4%, refined oil 4%, coconut oil 9%,
salt mixture with starch 4%, vitamin and choline mixture 0.5%,
and cholesterol 0.4%.

Rabbits in groups III and IV were orally fed the methanolic

extracts of D. biflorus dose I and dose II, respectively, and
rabbits in group V were fed standard drug atorvastatin. The
D. biflorus extracts and atorvastatin were suspended in 2%
tween 80 separately and fed to the respective groups of rabbits
by oral intubation. At the end of 11 weeks, all the animals
were sacrificed by cervical decapitation after overnight fasting.
Portions of the tissues from liver, heart, and aorta were
blotted, weighed, and homogenized with methanol (3 volumes).
The lipid extract obtained by the method of Folch et al.[2] was
used for the estimation of thiobarbituric acid reactive
substances[3] (TBARS). Another portion of the tissues was
homogenized with phosphate buffer saline and used for the
estimation of reduced glutathione[4] (GSH), catalase[5] (CAT),
and superoxide dismutase[6](SOD).
Results were expressed as mean+SE of 6 rabbits in each
group. One-way analysis of variance (ANOVA) with Scheffes
multiple comparisons test, were used to determine the
statistical significance. P<0.05 was considered significant.
The average body weight was found to have increased in
high fat diet fed rabbits compared with those in control group.
After administration of two doses of D.biflorus extract it was
found to have decreased. [Table 1] TBARS had significantly
increased and GSH had significantly decreased in liver, heart,
and aorta of rabbits fed HFD (Table 1; Group II) compared
with those in control group I. Administration of D.biflorus
extract significantly lowered the level of TBARS and enhanced
the level of GSH. Higher dose of the plant extract was found to
be more effective and showed comparable results with
standard drug atorvastatin on these two parameters. Activities
of antioxidant enzymes, that is, SOD and CAT in different
groups are given in Table 2. These two enzymes showed a
marked reduction in activity in liver, heart, and aorta of rabbits
in group II which had been fed HFD. Supplementation of
D.biflorus extract with HFD significantly improved the activities
of SOD and CAT in the above tissues of rabbits in groups III
and IV as compared with group II. Preliminary phytochemical
study shows that methanolic extract of the experimental plant
contains phytoconstituents, such as, alkaloids, steroids,
flavonoids, and isoflavone.
Elevated levels of TBARS in liver, heart, and aorta in group
II rabbits are a clear manifestation of excessive formation of
free radical and activation of lipid peroxidation. The significant
decline in the level of TBARS, in rabbits administered with
D.biflorus (Groups III and IV), unveils the antioxidant potential
of the D.biflorus extract. Both doses of the D.biflorus extract
(groups III and IV) help to restore the GSH levels near normal.
The higher dose has more effect, which was comparable to
atorvastatin. Increase in GSH concentration in animals treated
with D.biflorus extract may be due to increased activity of the
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| April 2006 | Vol 38 | Issue 2 | 131-2

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Research Letter

Table 1
Effect of methanolic extract of Dolichos biflorus on average body weight changes, tissue TBARS and glutathione in rabbits fed HFD
Body weight (g)
Treatment

Glutathione (mg/ g tissue)

Liver

Heart

Aorta

Liver

Heart

Aorta

(Dose)

Initial weight

Control

1016.66 +
29.85

1149.16 +
32.15b**

42.19 +
0.88b*

40.4 +
0.79b*

41.64 +
1.07 b**

18.65 +
0.78 b*

20.52 +
1.26 b*

14.61 +
0.52 b*

High fat diet (HFD)

1070.18 +
47.25

1281.66 +
49.15 a**

62.70 +
1.01 a*

63.09 +
0.83 a*

61.30 +
0.86a*

7.21 +
0.66 a*

10.65 +
0.91 a*

7.56 +
0.31 a*

HFD+methanolic extract of
D. biflorus (200 mg/kg, b.w./day)

1100.16 +
34.54

1247.50 +
26.38

47.63 +
1.05 a*,b*

41.78 +
0.94b*

41.43 +
1.05b*

13.22 +
1.19 a*,b*

16.29 +
0.95 a**,b*

11.66 +
0.32 a*,b*

HFD+methanolic extract of
D. biflorus (400 mg/kg, b.w./day)

970.33 +
28.16

1106.66 +
20.27 b**

37.48 +
0.98 a*,b*

34.22 +
1.15 a*,b*

34.56 +
1.60 a** ,b*

16.42 +
0.99 b*

21.54 +
0.92b*

12.7 +
0.38 a**,b*

HFD + atorvastatin
(1.2 mg/kg b.w./day)

1041.66 +
30.48

1103.33 +
27.64 b**

41.80 +
1.07 b*

40.37 +
1.18b*

35.78 +
1.18 a** ,b*

13.6 +
0.54 a*,b*

17.61 +
0.94b*

13.21 +
0.43 b*

6.53
<0.001

318.92
<0.001

203.38
<0.001

81.18
<0.001

31.92
<0.001

38.39
<0.001

58.97

<0.001

One-way
ANOVA

Final weight

TBARS (n mol of MDA formed

/ g tissue)

F
P

Values are mean+SEM of 6 rabbits in each group. *P<0.001, **P<0.05; df=4,18. aGroup I compared with groups II, III, IV and V. bGroup II compared with groups I, III, IV and V.

Table 2
Effect of methanol extract of Dolichos biflorus on tissue SOD and CAT in rabbits fed HFD
SOD (unit min/mg/protein)
Treatment (Dose)
Control
High fat diet (HFD)

CAT ( moles of H2O2, consumed min/mg/protein)

Liver

Heart

Aorta

Liver

12.55 + 0.53 b**

15.58 + 0.54 b*

22.09 + 0.60 b*

24.07 + 0.65 b*

14.56 + 0.51b*

13.76 + 0.60 b*

6.94 + 0.38 a*

11.69 + 0.58a*

11.30 + 0.55 a*

7.91 + 0.37a*

5.72 + 0.35a*

9.75 + 0.57 * *

19.50 + 0.64 * *

17.64 + 0.24 * *

7.91 + 0.50 a**

HFD+methanolic extract of
10.26 + 0.43 ** **
D. biflorus (200 mg/kg, b.w./day)

,b

a ,b

a ,b

Heart

a ,b

Aorta

10.61 + 0.16 * *
a ,b

9.50 + 0.15 a*,b *

HFD+methanolic extract of
11.74 + 0.35 b*
D. biflorus (400 mg/kg, b.w./day)

11.65 + 0.91 b*

22.48 + 0.29 b*

22.84 + 0.37 a**,b * 12.75 + 0.4a** ,b* 12.58 + 0.32 a*,b *

HFD + atorvastatin
(1.2 mg/kg b.w./day)

12.55 + 0.17 b*

14.43 + 0.28 b*

23.67 + 0.16 a** ,b*

22.47 + 0.17 b*

13.46 + 0.17b*

12.44 + 0.12 a*,b *

27.36
<0.001

59.35
<0.001

144.60
<0.001

63.88
<0.001

74.37

<0.001

One-way
ANOVA

F
P

98.70
<0.001

Values are mean +SEM of 6 rabbits in each group. *P<0.001, **P<0.05; df=4,18. aGroup I compared with groups II, III, IV and V. bGroup II compared with groups I, III, IV and V.

enzyme, glutathione reductase, which catalyses the conversion

of oxidized glutathione to reduced glutathione in liver. It may
also be due to enhanced synthesis/transport of GSH.
Restoration of the activities of SOD and CAT to near normal,
observed in tissues of rabbits supplemented with D.biflorous
may be due to the removal of toxic intermediates by the plant
extract in HFD fed rabbits. The components of the plant extract
may also directly activate the antioxidant enzymes.[7] It is
concluded that administration of D.biflorus manifests a
protective action against HFD induced oxidative stress in
different tissues in rabbits. However, further studies are needed
to isolate the active principles, elucidate their structures, and
determine their pharmacological activities.
A. Kottai Muthu, S. Sethupathy*,
R. Manavalan, P.K. Karar
Department of Pharmacy, Annamalai University,
Annamalai Nagar. *Division of Biochemistry,
132

Indian J Pharmacol

| April 2006 | Vol 38 | Issue 2 | 131-2

Rajah Muthiah Medical College, Annamalai University,

Annamalai Nagar-608002. India
E-mail: arthik03@yahoo.com

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of total lipids from animals tissues. J Biol Chem 1957;226:497-9.
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pid arachidonate during microsomal lipid peroxidation. Eur J Biochem
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4. Ellmann GL. Tissue sulfahydryl groups. Arch Biochem Biophys 1959;82:70-7.
5. Sinha AK. Colorimetric assay of catalase. Annal Biochem 1972;47:389-94.
6. Kakkar P, Das B, Visvanathan PN. A modified spectrophotometric assay of
SOD. Indian J Biochem Biophys 1984;21:130-2.
7. Jeyakumar SM, Nalini N, Venugopal P Menon. Antioxidant activity of ginger
(zingiber officinale Rose) in rats fed a high fat diet. Med Sci Res 1999;27:341-4.