Indian Journal of Experimental Biology

Vol. 42, October 2004, pp. 989-992

Antioxidant effect of Boerhavia diffusa L. in tissues of alloxan induced

diabetic rats
M Amarnath Satheesh & L Pari *
Department of Biochemistry, Faculty of Science, Annamalai University, Annamalainagar 608 002, India
Received 6 February 2004; revised 27 May 2004

Administration of B. diffusa leaf extract (BLEt; 200 mg/kg) for 4 weeks resulted in a significant reduction in thiobarbutric
acid reactive substances and hydroperoxides, with a significant increase in reduced glutathione, superoxide dismutase, catalase,
glutathione peroxidase and glutathione -5- transferase in liver and kidney of alloxan induced diabetic rats. The results suggest
that BLEt has remarkable antidiabetic activity and can improve antioxidant status in alloxan induced diabetic rats.
Keywords: Boerhavia diffusa, Alloxan diabetes, Lipid peroxidation, Enzymic antioxjdants
IPC Code: Int Cl 7 A61P

The harmful influence of diabetes mellitus on
metabolism of tissues and organ is well known.
Insulin is a major anabolic hormone in the body, and
therefore, derangement of insulin function affects not
only glucose metabolism but also fat and protein
metabolism in the majority of tissues I. Glucose
control plays an important role in the pro-oxidant
lantioxidant balance. Macromolecules such as
molecules of extra cellular matrix, lipoproteins and
deoxy ribonucleic acid are also damaged by free
radicals in diabetes mellitus 2 .
The roots of Boerhavia diffusa L. possess diuretic
action 3 , anti-inflammatorl, antifibrinolytic 5 , anti-
convulsant6 and hepatoprotective activities 7,8. Its leaf
extract has hypoglycemic effects 9• In the present
communication, the effects of B. diffusa leaf extract
(BLEt) on antioxidant status in liver and kidney of
alloxan diabetic rats are reported.
Materials and Methods
Plant material--Boerhavia diffusa leaves were
collected freshly from Chidambaram, Cuddalore
district. The plant was identified at the herbarium of
Botany Department of the Annamalai University. A
voucher specimen (No. 2865) was deposited.
Preparation of plant extract-B. diffusa leaves
(500g) were chopped into small pieces, extracted with
1500 ml water by the method of continuous hot
*Correspondent author
Phone: +91-4144-238343
Fax: +91-4144-238145
E-mail : paribalaji@rediffmail.com
extraction at 60°C for 6 hr and evaporated. A dark
semi-solid (greenish-black) material was obtained
(22.5 g). It was stored at 4°C until used. When
needed, the residual extract was suspended in distilled
water and used in the studylO.
Animals-Albino rats weighing 160-200g body
weight were obtained from the Central Animal House,
Department of Experimental Medicine, Rajah
Muthiah Medical College, Annamalai University. All
animal experiments were approved by the ethical
committee (Vide. No: 64, 2002), Annamalai
University and were in accordance with the guidelines
of the National Institute of Nutrition, Indian Council
of Medical Research, Hyderabad, India. Before and
during the experiment, rats were fed with normal
laboratory pellet diet (Lipton India Ltd, India) and
water ad libitum. After randomization into various
groups, the rats were acclimatized for a period of 2-3
days in the new environment before initiation of
experiment.
Chemicals-Alloxan monohydrate was purchased
from BDH Chemicals Limited, Poole, England.
Boehringer Mannheim GmbH Kit (ELISA-Principle)
was used for insulin assay. All the biochemicals and
chemicals used in the experiment were of analytical
grade and purchased locally.
Induction of experimental diabetes-The rats were
injected with alloxan monohydrate dissolved in sterile
normal saline at a dose of 140 mglkg body weight,
ipll. After 2 weeks, rats with moderate diabetes
having glycosuria (indicated by Benedict's qualitative
990 INDIAN J EXP BIOL, OCTOBER 2004

test) and moderate hyperglycemia (200 - 280 mg/dl) Statistical analysis-Statistical analysis was done
were used for the experiment. by analysis of variance (ANOY A) followed by
Experimental design----1n the experiment, a total of Duncans Multiple Range Test (DMRT).
30 rats (18 diabetic surviving rats, 12 normal rats)
were used. The rats were divided into following 5 Results and Discussion
groups of 6 each after the induction of alloxan The results are shown in Tables 1-3. The BLEt
diabetes: leaves extract produced a marked decrease in blood
Group 1: Normal untreated rats. glucose at 200mg/kg body weight in normal as well as
Group 2: Normal rats given BLEt 200 mg/kg body in alloxan diabetic rats after 4 weeks treatment. These
weight in aqueous solution daily using an findings are in agreement with those reported by
intragastric tube for 4 weeks. 9
Chude et a1 • The antidiabetic effect of BLEt may be
Group 3: Diabetic control. due to increased release of insulin from the existing ~
Group 4: Diabetic rats given BLEt 200 mg/kg body cells of pancreas similar to that observed after
weight 9 in aqueous solution daily using an glibenclamide administration.
intragastric tube for 4 weeks. Lipid peroxidation is one of the characteristic
Group 5: Diabetic rats given glibenclamide 600 Ilg/kg features of chronic diabetes. It has been observed that
body weight '2 in aqueous solution daily insulin secretion is closely associated with
using an intragastric tube for 4 weeks. lipoxygenase derived peroxides 21 ,22. The reduction of
two electrons from alloxan gives dialuric acid, which
Sample collection--At the end of 4 weeks, the
undergoes oxidation and leads to generation of O 2',
animals were deprived of food overnight and
sacrificed by decapitation. Fasting blood samples Table I-Changes in levels of blood glucose and plasma insulin 0
were collected in fresh vials containing sodium normal and experimental animals
fluoride and potassium oxalate (anticoagulant agent) (Values are given as mean ± SD for 6 rats in each group]
for the estimation of glucose. Plasma was separated
Groups Fasting blood Plasma insulin
for the estimation of insulin. Liver and kidney were
glucose (mg/dl) (JlUlml)
dissected out, washed in ice-cold saline, patted dry
Normal 91. 99 ± 6.28" 17.18±0.84"
and weighed.
Normal + BLEt 81.0 I ± 5.83 b 19.76 ± 1.18 b
Biochemical measurements-Fasting blood Diabetic control 257_18 ± 12.54c 4.92 ±0.30e
glucoselJ, thiobarbituric acid reactive substances Diabetic + BLEt 129.92 ± 8.03 d 10040 ±0.63 u
(TBARS)14, hydroperoxides ls , reduced glutathione Diabetic + glibenclamide 135.70±9.9I d 9.74 ±0.57 d
(GSH)16, superoxide dismutase (SOD)I7, catalase 's , Values not sharing a common superscript letter differ significantl
glutathione peroxidase (GPX)19 and glutathione-S- at P<0.05 (DMRT).
transferase (GSTio were determined. Duncan procedure, Range for the level 2.91, 3.06, 3.16, 3.22.

Table 2-Changes in levels of TBARS, hydroperoxides and reduced glutathione in liver and kidney of normal and experimental animals
[Values are given as mean ± SD for 6 rats in each group]

Groups TBARS Hydroperoxide Reduced glutathione

(mM/IOOg tissue) (mg / 100 mg tissue)
Normal Liver 0.87± 0.03 a 76.17 ± 2.79 a 46.91±2.08'
Kidney 1.59± 0.07 ab 56.35 ± 2.23 a 31.08 ± 2.15'
Normal +BLEt Liver 0.82±0.02 a n.07±3.25 a 49.95± 2.91'
Kidney 1.51±0.06a 52.09±2.17 b 34.83 ± 2.48"
Diabetic control Liver 2.04 ± O.ll b 101.70 ±6.05 b 23.35±0.80b
Kidney 2.25 ± 0.19 c 79.14±4A9c 20.53 ± 0.90b
Diabetic + BLEt Liver 1.36 ± 0.05 c 84.18 ± 4.83 c 41.29± 1.86c
Kidney 1.73±0.llbd 62.75 ± 3.07 d 26.83 ± 1.46c
Diabetic + Glibenclamide Liver 1.59 ± O.06d 90.03 ± 4.32d 39.66 ± 1.43c
Kidney 1.86 ±0.12d 66.94 ± 3.24d 25.08 ± 1.27c
Values not sharing a common superscript letter differ significantly at P<0.05 (DMRT).
Duncan procedure, Range for the level 2.91, 3.06, 3.16, 3.22.
 SATHEESH & PARI: ANTIOXIDANT EFFECT OF BOERHA VIA DIFFUSA 991

Table 3------(:hanges in activities of catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione-S-
transferase (GST) in liver of normal and experimental animals
[Values are given as mean ± S D for 6 rats in each group]

Groups Catalase A Superoxide Glutathione Glutathione-S-

dismutaseB peroxidasec transferase D
Normal Liver 72.03 ± 4.39· 6.33 ± 0.30' 6.57 ± 0.32" 6.19 ± 0.44"
Kidney 33.78 ± 2.00" 14.53 ± o.n" 4.66 ± 0.27" 5.50 ± 0.23"
Normal + BLEt Liver 73.63±4.87" 6.52±0.39" 6.82±O.41 " 6.65±0.48 b
Kidney 35.11±2.35 " 15.78±O.82 b 5.26±0.32 b 5.89± 3.35 b
Diabetic control Liver 44.97 ± 2.oob 4.16 ± O.13 b 4.45 ± 0.17 b 3.24 ± 0.18 e
Kidney 23.19 ± 0.75 b 9.86 ± O.44e 2.56 ± O.lle 2.63 ± 0.12e
Diabetic + BLEt Liver 66.37 ± 3.02c 5.85 ± 0.20e 5.68 ± 0.23c 5.64 ± 0.30d
Kidney 28.40± 1.61 c 13.80 ± 0.64" 4 .oo± 0.20d 4.83 ± 0.20d
Diabetic + glibenclamide Liver 62.71 ± 2.80c 5.63 ± 0.18c 5. 12± 0.20d 4.96 ± 0.25 d
Kidney 25 .57 ± l.35 d 11 .57 ± 0.52d 3.55 ± 0. 1ge 3.77 ± O.13 e
Values not sharing a common superscript letter differ significantly at P<0.05 (DMRT).
Duncan procedure, Range for the level 2.91 ,3.06,3.16, 3.22.
A = ~ mole of H 20 2 consumed / min/mg protein
B =One unit of activity was taken as the enzyme reaction which gave 50% inhibition of NBT reduction in one min
C = ~g of GSH consumed / min/mg protein
D = ~ moles of CDNB - GSH conjugate formed / min/mg protein

H20 2 and OH·23 . Dialuric acid has been observed to
stimulate lipid peroxidation in vitro. In this context, a
marked increase in the concentration of TBARS and
hydroperoxides were observed in liver and kidney of
diabetic rats. Increased lipid peroxide concentration in
the liver and kidney of diabetic animals has already
been reported 24 • Administration of BLEt and
glibenclamide significantly decreased the levels of
TBARS and hydroperoxides in diabetic rats.
Glutathione (GSH), a tripeptide present in all the
cells is an important antioxidant25 . Decreased
glutathione levels in diabetes has been considered to
be an indicator of increased oxidative stress26 • GSH
also functions as free radical scavenger and in the
repair of radical caused biological damage27 .28 • A
decrease was observed in GSH in liver and kidney
during diabetes. The decrease in GSH level represents
increased utilization due to oxidative stress29 •
Administration of BLEt and glibenclamide increased
the content of GSH in liver and kidney of diabetic
rats.
SOD is an important defense enzyme which
catalyses the dismutation of superoxide radicals 3o•
Catalase is a hemeprotein which catalyses the
reduction of hydrogen peroxides and protects the
tissues from highly reactive hydroxyl radicals 3J •
Therefore, reduction in the activity of these enzymes
(SOD, CAT) result in a number of deleterious effects
due to the accumulation of superoxide anion radicals
and hydrogen peroxide. Administration of BLEt and
glibenclamide increased the activities of SOD and
catalase in diabetic rats.
The activities of GPx and GST were observed to
decrease significantly in diabetic rats. Both GPx, an
enzyme with selenium, and GST catalyse the
reduction of hydrogen peroxide and hydroperoxides
to non-toxic products 32 • The decreased activities of
these enzymes result in the involvement of deleterious
oxidative changes due to the accumulation of toxic
products. Administration of BLEt and glibenclamide
increased the activities of GPx and GST in diabetic
liver and kidney.
The B. diffusa leaves are rich in alkaloids and
sterols including ursolic acid, hypoxanthine-9-L-
arabinofuranoside, punarnavine 1 and 2, myricyl
alcohol, myristic acid and quinolizidine alkaloids 33 •
These compounds may be responsible for the
antioxidant and antidiabetic activity of B. diffusa
leaves, which may be attributed to its protective
action on lipid peroxidation and to the enhancing
effect on cellular antioxidant defense contributing to
the protection against oxidative damage III
alloxanized diabetes.
References
1 Newsholme E A & Lech A R, Biochemistry for the medical
sciences, (Wiley, Chichester) 1983,832.
2 Bonnefont Rousselot D, Bastarad J P, Jaudon M C &
Delattre J, Consequences of diabetic status on the
oxidant/antioxidant balance, Diabetes Metab, 26 (2000) 163.
3 Singh R P, Shokala K P, Pandey B L, Singh R G, Usha S, &
992 INDIAN J EXP BlOL, OCTOBER 2004
Singh R F, Recent approach in clinical and experimental
evaluation of diuretic action of punarnava (Boerhaavia
diffusa) with special effect to nephrotic syndrome, J Res Ind
Med,II(I)(1992)29 .
4 Bhalla T N, Gupta M B & Bhargava K P, Anti-inflammatory
activity of Boerhaavia diffusa L, J Res Ind Med, 6(1) (1971)
11.
5 Jain G K, Khanna N M, Punarnavoside: A new
anti fibrinolytic agent from Boerhaavia diffusa L, Indian J
Chem A, 28(3) (1989) 163.
6 Mudgal V, Studies on medical properties of Convolvulus
phuricaulis and Boerhaavia diffusa. Planta Med, 28(1)
(1975) 62.
7 Chandan B K, Sharma A K & Anand K K, Boerhaavia
diffusa: A study of its hepatoprotective activity, J
Ethnopharmacol, 31 (1991) 299.
8 Rawat A K S, Mehrotra S, Tripathi S C & Shome V,
Hepatoprotective activity of Boerhaavia diffusa L. roots, a
popular Indian ethnomedicine, J Elhnopharmacol, 56 (1997)
61.
9 Chude M A, Orisakwe 0 E, Afonne 0 J, Gamaniel K S,
Vongtaa 0 H & Obi E, Hypoglycemic effect of the aqueous
extract of Boerhaavia diffusa leaves, Indian J Phamzacol, 33
(2001) 215.
10 Jain S R, Hypoglycemic principle in the Musa sapientum and
its isolation, Planta Med, 1 (1968) 43.
II Saraswathi P R & Swaminathan G, Effect of sodium
molybdate on carbohydrate metabolism enzymes in alloxan
induced diabetic rats, J Nutr Biochem, 13 (2002) 21.
12 Pari L & Vma M J, Hypoglycemic effect of Musa sapientum
L. in alloxan-induced diabetic rats, J Ethnopharmacol, 68
(1999) 321.
13 Sasaki T, Matsy S & Sonae A, Effect of acetic acid
concentration on the colour reaction in the O-toluidine boric
acid method for blood glucose, Rinsho Kagaku, 1 (1972)
346.
14 Fraga C G, Leibovitz B E & Toppel A L, Lipid peroxidation
measured as TBARS in tissue slices: Characterization and
comparison with homogenates and micro somes, Free Radic
Bioi Med, 4 (1988) 155.
15 Jiang Z Y, Hunt J V & Wolff S D, Ferrous ion oxidatiC" 'l in
the presence of xylenol orange for detection of lipid
hydroperoxide in low density lipoprotein, Anal Biochem, 202
(1992) 384.
16 Ellman G L, Tissue sulfllydryl groups, Arch Biochem
Biophys, 82 (1959) 70.
17 Kakkar P, Das B & Viswanathan P N, A modified
spectrophotometric assay of superoxide dismutase, Indian J
Biochem Biophys, 21 (1984) 130.
18 Sinha K A, Colorimetric assay of catalase, Annal Biochem,
47 (1972) 389.
19 Rotruck J T, Pope A L, Ganther H E & Swanson A B,
Selenuim: Biochemical role as a component of glutathione
peroxidase, Science, 179 (1984) 588.
20 Habig W R, Pbst M J & Jakpoly W B, Glutathione
transferase. A first enzymatic step in mercapturic acid
formation, J Bioi Chem, 249 (1974) 7130.
21 Walsh M F & Pek S B, Possible role of endogenous
arachidonic acid metabolites in stimulated release of insulin
and glucagon from the isolated, perfused rat pancreas,
Diabetes, 33 (1984) 929.
22 Metz S A, Oxygenation products of arachidonic acid: Third
messengers for insulin release, Prostaglandins, 27 (1984)
147.
23 Winterbourn C C & Munday R, Glutathione-mediated redox
cycling of alloxan, Biochem Pharmacol, 38 (1989) 271 .
24 Nakakimura H & Mizuno K, Studies on lipid peroxidation in
biological systems II. Hyperlipoperoxidemia in mice induced
by alloxan, Chem Pharm Bull, 28 (1980) 2207.
25 Lu S C, Regulation of hepatic glutathione synthesis: Current
concepts and controversies, FASEB J, (1999) /169.
26 Mc Lennan S V, Heffernan S, Wright L, Rae C, Fisher E,
Yue D K & Turtle J R. Changes in hepatic glutathione
metabolism in diabetes, Diabetes, 40 (1991) 344.
27 Meister A, New aspects of glutathione biochemistry and
transport selective alterations of glutathione metabolism,
NUlr Rev 42 (1984) 397.
28 Nicotera P & Orrenius S, Role of thiols in protection against
biological reactive intermediates, Adv Exp Med Bioi, 197
(1986) 41.
29 Anuradha C V & Selvam R, Effect of oral methionine on
tissue lipid peroxidation and antioxidants in alloxan induced
diabetic rats, J Nutr Biochem, 4 (1993) 212.
30 Mc Cord J M, Keele B B & Fridovich I, An enzyme based
theory of obligate anaerobiosis; The physiological functions
of superoxide dismutase, PNAS, VSA , 68 (1976) 1024.
31 Chopra R N, Chopra I C, Handa K L & Kapur L D,
Medicinal plants in diabetes in Indigenous drugs of India ,
edited by P Gupta, 2nd edn, (V N Dhar & Sons Ltd, Calcutta,
India) 1958, 314.
32 Bruce A, Freeman D & James C, Biology of Diseases - Free
radicals and tissue injury, Lab Invest, 47 (1982) 412.
33 Sharma P C, Yelne M B & Dennis T J, Database 011
medicinal plants used in Ayurveda, Vol I, (Central Council
for Research in Ayurveda & Sidha, New Delhi), 2000, xiii,
528.
,
'{'