Indian Journal of Pharmacology 2003; 35:

373-378

RESEARCH PAPER

P. SANTHAKUMARI et al.

MODULATION OF OXIDATIVE STRESS PARAMETERS BY TREATMENT WITH

PIPER BETLE LEAF IN STREPTOZOTOCIN INDUCED DIABETIC RATS

P. SANTHAKUMARI, A. PRAKASAM, K.V. PUGALENDI

Department of Biochemistry, Faculty of Science, Annamalai University, Annamalainagar-608 002. Tamilnadu
Manuscript Received: 26.10.2002
ABSTRACT

Revised: 27.5.2003

Accepted: 21.6.2003

Objectives: To study the effect of oral administration of Piper betle leaf powder suspension on lipid
peroxidation and antioxidants in streptozotocin (STZ) diabetic rats.
Methods: Male Wistar strain rats were orally administered the leaf suspension of P. betle, (75 and 150
mg/kg body weight) for 30 days. Plasma and erythrocytes were separated out and the liver and kidney
were homogenized in ice-cold buffer and the assays of thiobarbituric acid reactive substances (TBARS),
hydroperoxides, glutathione (GSH), superoxide dismutase (SOD EC 1.11.1.1), catalase (CAT EC 1.11.1.6)
and glutathione peroxidase (GPx EC 1.11.1.9) were performed in the supernatant obtained from liver
and kidney of control and STZ diabetic rats. Plasma TBARS, hydroperoxides, ascorbic acid and alphatocopherol were measured.
Results: Oral administration of P. betle (75 and 150 mg/kg body weight) for 30 days resulted in a
significant reduction in plasma thiobarbituric acid reactive substances (TBARS), hydroperoxides, alpha-tocopherol and significant improvement in glutathione, superoxide dismutase, catalase and glutathione peroxidase in the liver and kidney of STZ diabetic rats when compared with untreated diabetic
rats. The antioxidant effect of P. betle at 75 mg/kg for 30 days was found to be comparable to
glibenclamide in diabetic rats.
Conclusion: The leaf suspension of P. betle 75 mg/kg body weight showed significant antioxidant
effects in STZ diabetic rats.

KEY WORDS

Antioxidants

diabetes mellitus

lipid peroxidation

INTRODUCTION
Since the time of Charaka and Susruta many herbal
medicines in different oral formulations have been
recommended for the treatment of diabetes mellitus
(Madhumeha). Extracts of drugs from plant sources
such as Allium sativum (garlic), Azadirachta indica
(neem), Vinca rosea (nayantara), Gymnema
sylvestra (meshashringe), Trigonella foenum
graecum (fenugreek), Momordica charantia (bitter
gourd), Ficus bengalensis (banyan), Eugenia
jambolana (black berry), Ocimum sanctum Linn
(tulsi) and Eclipta alba (karichalankanni) are some
of the plants reported to posses antihyperglycemic
activity in experimental animals1-3.
Correspondence: K.V. Pugalendi
E-mail: drkvp232@rediffmail.com

Many unknown and lesser-known plants are used

in folk and tribal medicinal practices in India. The
medicinal values of these plants are not known much
to the scientific world. P. betle (Family Piperaceae)
is one such a plant of the several ingredients in a
chew commonly known as pan. The betel plant is a
slender, aromatic creeper, rooting at the nodes. The
branches of the plant are swollen at the nodes. The
plant has alternate, heart shaped, smooth, shining
and long stalked leaves with pointed apex 4. Medicinally P. betle is an aromatic, carminative, stimulant
and astringent used as a preventive for worms and
in snake bite4. Juice of the leaves is dropped into
eyes in painful infection and night blindness. Essential oil from leaves of this plant has been used for

PIPER BETLE AND OXIDATIVE STRESS PARAMETERS

the treatment of respiratory catarrhs and as antiseptic

and the fruit is employed with honey as a remedy for
cough5. Preliminary investigation in our laboratory
showed that oral administration of aqueous suspension of P. betle at a dose of 75 mg/ kg body weight
showed better sugar reduction than 150 mg /kg body
weight (unpublished data). Further, comprehensive
search of literature revealed that very limited work
has been carried out to explore the effect of P. betle
on lipid peroxidation and antioxidants status. Hence,
the present study was undertaken to investigate the
antioxidant potential of the medicinally important herb
and the tissue lipid peroxidation status.
MATERIALS AND METHODS
Plant material: Piper betle.Linn (Syn:Chavica betle
Miq.) popularly known as "vetrelei" in Tamil, "Betel"
in English and "Nagavalli" in Sanskrit was purchased
from the local market, Cuddalore district, Tamil Nadu,
India. The plant was authenticated by a Taxonomist
in the Department of Botany, Annamalai University
and herbarium specimens were submitted
(AU.3898). The authenticated plant leaves were used
for the preparation of extract.
Preparation of plant material: Fresh leaves were
collected and air-dried in shade at room temperature. The dried leaves were powdered mechanically
and sieved using a fine muslin cloth. The fine powdered leaves were kept separately in airtight containers until the time of use.
Drugs and chemicals: Streptozotocin (STZ) was
obtained from Sigma chemical company. All other
chemicals used were of analytical grade.
Experimental animals: Male albino Wistar rats
(150-200 g) bred in the Central Animal House, Rajah Muthiah Medical College, Annamalai University,
were used in this study. The animals were fed on a
pellet diet (Hindustan Lever, India) and water ad libitum. The animals were maintained in their respective groups for 30 days. All studies were conducted
in accordance with the National Institute of Health
Guide for the Care and Use of Laboratory Animals6
and the experiments were carried out as per the Institutional Ethics Committee.
Experimental induction of diabetes: Adult (9
weeks old) male Wistar rats were made diabetic

374

with an intraperitoneal injection of streptozotocin

(STZ, 50 mg/kg body weight) dissolved in citrate
buffer (0.1 M, pH 4.5). Streptozotocin injected animals exhibited massive glycosuria and hyperglycaemia within a few days. Diabetes was confirmed
in STZ rats by measuring the fasting blood glucose
concentration, 96 h after the injection with STZ. Albino rats with blood glucose level above 200 mg/dL
were considered to be diabetic and were used in
this experiment. Six rats were injected with 2% gum
acacia alone that served as control.
Experimental design: After the induction of diabetes the rats were divided into six groups of six animals each.
Group I

- Control rats received vehicle solution

(2% gum acacia)

Group II

- Diabetic control

Group III

- Normal rats received P. betle (75 mg/

kg body weight) leaf powder in 2%
gum acacia

Croup IV

- Diabetic rats given P. betle leaf powder (75 mg/ kg body weight) in 2%
gum acacia

Croup V

- Diabetic rats given P. betle leaf powder (150 mg/ kg body weight) in 2%
gum acacia.

Group VI

- Diabetic rats received glibenclamide

(600 g / kg body weight) as aqueous
solution.

The vehicle and drugs were administered orally by

an intragastric tube daily for 30 days. After 30 days
of treatment, the rats were fasted overnight and sacrificed by cervical decapitation. The plasma was
separated for the estimation of thiobarbituric acid
reactive substances (TBARS)7, hydroperoxides8 ,
ascorbic acid9 and - tocopherol10 and the liver and
kidney were dissected out and washed with ice-cold
saline immediately. A portion of the tissue was
homogenized using a Potter Elvejham homogenizer
and the extract was used for the estimation of
TBARS 11, hydroperoxides 8, glutathione12 superoxide
dismutase13 (SOD, EC 1.11.1.1), catalase14 (CAT, EC
1.11.1.6) glutathione peroxidase15 (GPx EC 1.11.1.9)
and tissue protein16.

375

P. SANTHAKUMARI et al.

Table 1. Effect of Piper betle on TBARS, hydroperoxides, ascorbic acid and -tocopherol in the plasma of normal and experimental animals after 30 days treatment.
Groups

Normal ( 2% gum acacia)

TBARS
(nmoles/ml)

Hydroperoxides
(nmoles/ml)

Ascorbic acid
(mg/dL)

-tocopherol
(mg/dL)

1.70+0.15a

0.44+0.04a

1.8+0.17a

1.8+0.34a

Normal + Piper betle

(75 mg/kg)

1.68+0.22

0.43+0.01

1.7+0.10

1.6+0.30a

Diabetic control

3.76+0.50b

0.78+0.08b

0.7+0.08b

3.1+0.30b

Diabetic + Piper betle

(75 mg/kg)

1.80+0.41

0.48+0.05

1.2+0.10

2.2+0.26c

Diabetic + Piper betle

(150 mg/kg)

2.17+0.61c

0.56+0.04d

1.1+0.10d

2.3+0.47c

Diabetic + Glibenclamide
(600 g/kg)

1.75+0.35a

0.45+0.02a

1.4+0.10c

1.9+0.27a

4.12
<0.05

3.86
<0.05

6.44
<0.05

7.22
<0.05

F
P

Values are mean+SD of 6 rats from each group. df=5, 30. V alues with different superscripts (a, b, c and d) differ significantly with
each other at P<0.05. (Duncans multiple range test).

Statistical analysis: Values were represented as

mean+SD. Data were analysed using one-way
analysis of variance (ANOVA) and group means were
compared using Duncans multiple range test. P
values <0.05 were considered significant.
RESULTS
Table 1 and 2 show the levels of TBARS, hydroperoxides, ascorbic acid and alpha tocopherol in plasma,
liver and kidney of control and experimental animals. A significant elevation in plasma and tissue
TBARS, hydroperoxides were observed in diabetic
rats when compared with control rats. There was a
significant reduction in plasma ascorbic acid and elevated -tocopherol in diabetic rats compared to
control rats. Oral administration of P. betle (75 and
150 mg/kg body weight) for 30 days shows significant reduction in TBARS, hydroperoxides, -tocopherol with a significant elevation in ascorbic acid in
drug treated diabetic rats.
Table 3 shows the concentration of GSH and the
activities of enzymatic antioxidants such as SOD,
CAT and GPx in the liver and kidney of control and
experimental animals. Activities of these enzymes
and GSH level significantly decreased in diabetic animals when compared with control rats. Oral admin-

istration of P. betle (75 and 150 mg / kg body weight)

significantly reversed these enzymes to near normal value. Rats administered with P. betle (75 mg/kg
body weight) alone did not produce any significant
alteration in plasma and tissue antioxidant parameters.
DISCUSSION
Lipid peroxidation is a free radical induced process
leading to oxidative deterioration of polyunsaturated
fatty acids. Under physiologic conditions, low concentrations of lipid peroxides are found in tissues.
Karpen et al 17 observed an elevated level of lipid
peroxides in the plasma of diabetic rats and lipid
peroxidation is one of the characteristic features of
chronic diabetes. Lipid peroxide mediated tissue
damage has been observed in the development of
both type 1 and 2 diabetes. Nakakimura and Mizuno18
have reported that the concentration of lipid peroxides increases in the kidney of diabetic rats and an
increased level of TBARS is an index of lipid
peroxidation. Our results show that in diabetic control animals the levels of TBARS and hydroperoxides were high in plasma, liver and kidney, due to
increased lipid peroxidation. In P. betle and
glibenclamide treated diabetic rats, the TBARS and
hydroperoxides levels were low in plasma, liver and

PIPER BETLE AND OXIDATIVE STRESS PARAMETERS

376

Table 2. Effect of Piper betle on TBARS and hydroperoxides in the liver and kidney of normal and experimental animals after 30
days treatment.
TBARS
(mmoles/100 g tissue)

Groups

Hydroperoxides
(mmoles/100 g tissue)

Liver

Kidney

Liver

Kidney

Normal ( 2% gum acacia)

0.760.09a

1.450.18a

72.33.06a

51.363.38a

Normal + Piper betle

(75 mg/kg)

0.780.05a

1.570.11a

70.66.56a

50.54.01a

Diabetic control

1.870.33b

2.700.40b

97.65.26b

75.014.03b

Diabetic + Piper betle

(75 mg/kg)

0.870.04

1.850.07

79.22.79

57.74.78c

Diabetic + Piper betle

(150 mg/kg)

1.280.12d

2.100.15d

83.14.03d

63.25.60d

Diabetic + Glibenclamide
(600 g / kg)

0.830.04a,

1.610.11a, c

75.433.46a

54.22.41a

6.12
<0.05

12.36
<0.05

10.04
<0.05

F
P

5.06
<0.05

Values are mean+SD of 6 rats from each group. df=5,30. V alues with different superscripts (a, b, c and d) differ significantly with
each other at P<0.05. (Duncans multiple range test).

Table 3. Effect of Piper betle on GSH, SOD, CAT and GPx in liver and kidney of normal and experimental animals after 30 days
treatment.
GSH
(mg/100 g tissue)

SOD
(UnitA/mg protein)

Catalase
(UnitB/mg protein)

GPx
(UnitC/mg protein)

Groups
Liver

Kidney

Liver

Kidney

Liver

Kidney

Liver

Kidney

Normal
52.0+4.38a
(2% gum acacia)

33.7+6.02a

6.80+0.25a 16.9+1.38a

75.2 + 1.46a 33.1+1.64a

8.54+1.03a 6.30+0.53a

Normal+P. betle 50.6+4.0a

(75 mg/kg)

34.6+4.13a

6.91+0.22a 17.1+1.51a

76.7 + 1.13a 34.2+1.96a

8.60+0.39a 6.40+0.50a

Diabetic control 28.0+3.38b

18.7+1.70b

3.41+0.09b 10.1+0.91b

44.9 + 1.38b 21.1+5.05b

4.81+0.45b 4.08+0.41b

26.8+0.94

7.57+0.33c 5.22+0.30c , d

24.0+2.26

5.01+0.34

Diabetic+P. betle 32.6+4.45d

(150 mg/kg)

20.0+2.84d

5.94+0.20d 13.7+1.02c

41.4 + 0.95d 23.1+1.80d

5.29+0.60d 4.53+0.57c , d

46.3+0.98a

30.6+1.96a

6.48+0.24a 15.9+1.37a , c 65.9 + 3.10e 30.1+2.43a

8.01+0.25a 5.90+ 0.21a,c

9.64
<0.05

8.76
<0.05

4.67
<0.05

5.24
<0.05

F
P

6.40
<0.05

53.7 + 2.94

Diabetic+P. betle 38.5+4.35

(75 mg/kg)

Diabetic+
Glibenclamide
(600 g/kg)

14.6+1.32

11.12
<0.05

7.42
<0.05

5.31
<0.05

Values are mean+SD of 6 rats from each group. df=5,30

A One unit of activity was taken as the enzyme reaction which gave 50% inhibition of nitroblue tetrazolium (NBT) reduction in one
minute.
B - moles of hydrogen peroxide consumed/min.
C - g of glutathione consumed/min.
Value with different superscripts (a, b, c, d and e) differ significantly with each other at P< 0.05. (Duncans multiple range test).

377

P. SANTHAKUMARI et al.

kidney, which may be due to the free radical scavenging action of active ingredients in P. betle .
Choudhary and Kale19 have reported that P. betle
extract inhibited the radiation induced lipid
peroxidation process effectively and that could be
attributed to its ability to scavenge free radicals involved in initiation and propagation steps.
The most important antioxidant in the cell membrane
is -tocopherol20. It interrupts the chain reaction of
lipid peroxidation by reacting with lipid peroxyl radicals, thus protecting the cell structures against damage21. Increased level of -tocopherol found in the
STZ diabetic rats as compared with control rats in
our study may be due to the release of membrane
bound -tocopherol from damaged cell membrane
since it is water insoluble. Takeneka et al 22 have reported increased levels of alpha tocopherol in the
liver of diabetic rats in spite of increased susceptibility to oxidation.
SOD protects tissues against oxygen free radicals
by catalyzing the removal of superoxide radical
(O2-), which damages the membrane and biological structures 23. Catalase has been shown to be responsible for the detoxification of significant amounts
of H2O224. SOD and catalase are the two major scavenging enzymes that remove the toxic free radicals
in vivo. Reduced activities of SOD and catalase in
liver and kidney have been observed during diabetes and this may result in a number of deleterious
effects due to the accumulation of superoxide radicals (O2-) and hydrogen peroxide25. P. betle and
glibenclamide treated rats showed decreased lipid
peroxidation that is associated with increased activity of SOD and CAT.
GPx catalyzes the reduction of H2O2 to H2O and O2
at the expense of GSH. GPx activity is also reduced
in diabetic condition. This may be due to inactivation
of the enzyme involved in disposal of oxygen species and also insufficient availability of GSH26. The
present study also observed the depleted levels of
GSH in STZ diabetic rats and elevation of GPx after
treatment with Piper betle leaf suspension and
glibenclamide.
Our study corroborates the study of Choudhary and
Kale19 who have indicated the protective effects of P.
betle and its potential to elevate the antioxidant status in radiation induced lipid peroxidation.

The ability of P. betle on tissue lipid peroxidation

and antioxidant status in diabetic animals has not
been studied before. The result of this study shows
that a daily administration of an aqueous suspension of P. betle for 30 days provides better antioxidant potential and protection against tissue lipid
peroxidation.
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PP SURYA KUMARI PRIZE

INDIAN PHARMACOLOGICAL SOCIETY
PP Surya Kumari prize is awarded by IPS every year for the best research paper published in
any journal on "diabetes mellitus, other endocrinal and metabolic disorders" in the last five
years. The prize is open to Indian scientists working in Indian laboratories. The award is
presented to the winner at the annual conference of IPS.
Those who wish to compete for the prize for the year 2004 may submit five reprints/copies of
the paper (published in 1999-2003) to the Chief Editor, Indian Journal of Pharmacology at the
following address on or before 31st March, 2004.
E-mail : ijp@jipmer.edu
Phone : 0413-2271969
Fax
: 0413-2272067

378

Prof. R. Raveendran
Convener, PP Surya Kumari Prize,
The Chief Editor-IJP,
Department of Pharmacology,
JIPMER, Pondicherry - 605 006. India