Content uploaded by Dr Pandurangan
Author content
All content in this area was uploaded by Dr Pandurangan on Jul 06, 2014
Content may be subject to copyright.
IJPR | January-March | 59
International Journal of Pharmaceutical Research
2012, Volume 4, Issue 1. 59-62.
ISSN 0975-2366
Research Article
Anti-hyperglycemic potential of Rourea minor roots in streptozotocin (STZ) induced diabetic rats.
Anu
Chaudhary*
1
, Anil Bhandari
2
, A.Pandurangan
1
1
Department of Pharmacy Bharat Institute of Technology, Partapur, By-Pass road, Meerut-250103, India
2
Jodhpur
pharmacy college, Jodhpur, Rajasthan, India, 342003
*Corresponding author: E-mail ID: anuman.82@rediffmail.com
Tel.: +91-121-2440480
Received: 14/07/2011, Revised: 16/081/2011,02/09/2011
ABSTRACT
Rourea minor (Gaertn.) Aubl. (Connaraceae), a woody vine (liana), is used extensively in the indigenous system of medicine
as an anti-diabetic agent. The current investigation focuses on the serum insulin augmentation and anti-hyperglycemic
property of methanolic extract of Rourea minor roots on streptozotocin induced diabetic rats. Type 2. diabetes was induced
by administering intraperitoneal injection of a Freshly prepared STZ solution (60 mg/Kg og body weight) in 0.1M cold
citrate buffer to the overnight fasted rats. The diabetes induced animals were fed with plant extract at the increasing dosage
of 100mg, 200mg and 400mg of body wt. The combined plant extracts administrated animals revealed a significant (P<0.05)
increment of serum insulin levels and higher reduction in hyperglycemia when compared to the diabetic control rats
(P<0.05).
KEYWORDS: Rourea minor, streptozotocin (STZ), hyperglycemia, glucose tolerance test.
INTRODUCTION:
The present day life pattern predisposes human to the
risks of diabetes mellitus, obesity, stress and such life style
diseases. Diabetes mellitus is a major public health
problem. According to WHO reports, more than 176
million patients suffer worldwide and it is estimated that in
2025, there will be about 300 million patients living with
this condition. The increase is expected to be 42% in
developed countries and 70% in developing countries [1].
Diabetes mellitus is a group of metabolic alterations
characterized by hyperglycemia resulting from defects in
insulin secretion, action or both. It has already been
established that chronic hyperglycemia of diabetes is
associated with long term damage, dysfunction and
eventually the failure of organs, especially the eyes,
kidneys, nerves, heart and blood vessels [2]. It is the fourth
leading cause of death in the most developed countries and
there is substantial evidence that it is epidemic in many
developing and newly industrialized nations. Diabetes
mellitus is a syndrome resulting from a variable interaction
and environmental factors and is characterized by depleted
insulin secretion, hyperglycemia and altered metabolism of
lipid, carbohydrates and proteins, in addition to damaged β-
cells of pancreas and increased risk of complications of
vascular diseases [3].
Streptozotocin induction of diabetes is an experimental
model widely used to study glycemic changes in plasma.
There are more then 1200 plants species worldwide that are
used in the treatment of diabetes mellitus and a substantial
number of plants have shown effective hypoglycemic
activity after laboratory testing [4]. Rourea minor (Gaertn.)
Aubl. (Connaraceae), a woody vine (liana), is known under
a number of synonyms: Aegiceras minus Gaertn., Connarus
microphyllus (Hook. et Arn.) Planch., Pterotum
procumbens Lour., R. santaloides (Vahl) W. et A., R.
caudatum Planch., R. microphylla (Hook. Et Arn.) Planch.,
and Santaloides microphyllum (Hook. Et Arn.) Schell [5,
6].
The leaves of this plant have been used in Chinese folk
medicine as a styptic to treat minor abrasions and lesions
[7]. Phytochemically, it has been reported to contain
triterpenes steroids, quinones, flavanes, flavones,
anthracenediones, and fatty acids [8, 9].Keeping all these
facts in view, the present study was carried out to test the
antihyperglycaemiuc potential of methanolic extract of
Rourea minor roots.
MATERIALS AND METHODS:
Collection of Plant material
The roots of Rourea minor (Connaraceae), were
purchased from local vendor and identified by Dr. K.
Madhava Chetty, Department of Botany, Sri Venkateswara
University, Tirupati, Andhra Pradesh, India. A voucher
specimen (BIT/Anu/2009/MI-520) was deposited in the
herbarium of School of Pharmacy, Bharat Institute of
Technology, Meerut, UP,
Plants extract preparation
Dried powdered roots of Rourea minor were defatted
with petroleum ether (60-80
0
C) and extracted with
methanol by soxhelation. After exhaustive extraction, the
solvent was collected and filtered. The solvent was
concentrated under reduce pressure at 50-55°C and
concentrated methanol extract were stored in vacuum
desiccator.
Animals
Adult male Wistar rats weighing around 180-200g
were kept in polypropylene cages (three in each cage) at an
ambient temperature of 25±2
0
C and 55-65% relative
humidity 12±1 hr light and dark schedule was maintained in
the animal house till the animals were acclimatized to the
laboratory conditions, and were fed with commercially
available rat chow (Hindustan Lever Ltd., Bangalore. India)
and had free access to water ad libitum. The experiments
were designed and conducted in accordance with the
institutional guidelines and approved by Institutional
Chaudhary et al / International Journal of Pharmaceutical Research 2012 4(2) 59-62
60 | IJPR | January-March
Animal Ethics Committee (Regn No:
1147/ab/07/CPCSEA).
Acute toxicity
Acute toxicity study was performed as per OECD-423
guidelines [10]. Albino Wistar rats of either sex were used.
Overnight fasted rats received test drugs at a dose of 2000
mg/Kg of body weight orally. Then the animals were
observed continuously once in half an hour for the next 4
hours and then after 24 hours for general behavioral,
neurologic and autonomic profiles and to find out the
mortality. The extract found to be safe up to the dose of
2000 mg/kg body weight.
Oral glucose tolerance test:
The oral glucose tolerance test was performed in
overnight fasted normal animals [11]. Rats divided into five
groups (n=6) were administered 2% Tween 80, methanolic
extract of rourea minor at dose level of 100 mg/kg, 200
mg/kg, 400 mg/kg and glibenclamide (500 μg/kg). Glucose
(2 g/kg) was fed 30 min after the administration of
methanolic extracts. Blood was withdrawn from the retro-
orbital sinus at 0, 30, 60, 90 and 120 min of methanolic
extract administration. Fasting serum glucose levels were
estimated by the Radio Immuno Assay kit (BRAC,
Mumbai).
Normoglycemic study
For normoglycemic study, Rats divided into seven
groups (n=6) were administered 2% Tween 80, methanolic
extract of madhuca indica at dose level of 150 mg/kg, 300
mg/kg and methanolic extract of rourea minor at dose level
of 100 mg/kg, 200 mg/kg, 400 mg/kg and glibenclamide
(500 μg/kg). Glucose (2 g/kg) was fed 30 min after the
administration of methanolic extracts [12]. Blood glucose
levels were estimated on days 0, 4, 8 and 12.
Experimental induction of diabetes
Diabetes was induced by administering intraperitoneal
injection of a freshly prepared STZ solution (60 mg/kg of
body weight) in 0.1M cold citrate buffer pH 4.5 to the
overnight fasted rats [13]. The animals were allowed to
drink 5% glucose solution to overcome the drug induced
hyperglycemia [14]. Because of the STZ instability in
aqueous media, the solution is made using cold citrate
buffer immediately before administration. The control rats
were injected with citrate buffer alone as placebo. Animals
with blood glucose values above 250 mg/dL on day 3 of
STZ injection were considered as diabetic rats. The
treatment was started after day 5 of diabetes induction and
was considered as day 1 of treatment.
Statistical analysis:
Statistical analysis was carried out by using one way
ANOVA as in standard statistical software package of
social science (SPSS). The values were considered
significant at p< 0.05. The studies conducted into six
groups of six animals, as follows:
Experimental design
Blood sampling: At the end of day 12, blood samples were
collected from the inner canthus of the eye under light ether
anesthesia using capillary tubes (Micro Hemocrit
Capillaries, Mucaps). Blood was collected into fresh vials
containing anticoagulant antiserum and separated in a
centrifuge at 2000 rpm for 2 min. Serum insulin levels were
estimated by the Radio Immuno Assay kit supplied by the
Board of Radiation and Isotope Research, Bhaba Atomic
Research Centre (BRAC), Mumbai, India.
Groups Treatment(once daily)
Route of
administration
Normal
control
Tween 80 Oral
Diabetic
control
STZ 60mg/kg i.p
Diabetic +
Treatment
Dose I
Extract( 100 mg/kg) Oral
Diabetic +
Treatment
Dose II
Extract( 200 mg/kg) Oral
Diabetic +
Treatment
Dose III
Extract( 400 mg/kg) Oral
Diabetic+
Standard
Glibenclamide(500μg/kg
body weight)
Oral
RESULTS AND DISCUSSION
Acute toxicity studies revealed the non toxic nature of
the methanolic extracts at the three dose levels. There were
no morphological changes like distress, hair loss,
restlessness, convulsions, laxative effects, coma, weight
loss etc. At the end of treatment period, there was no
lethality or toxic reaction at any of the doses selected.
Streptozotocin is a broad spectrum antibiotic obtained from
Streptomyces achromogenes. STZ causes massive
reduction in insulin release via destruction of β cells of the
islets of Langerhans and thereby induces hyperglycemia
[15]. In the present study, the antihyperglycemic activity of
methanolic root extract of Rourea minor was assessed in
normal and STZ induced diabetic rats. Oral administration
of a single dose of methanolic root extract of Rourea minor
caused a significant decrease in serum glucose level in
normal rats.
Table 1. Effect of methanolic extracts of Rourea minor roots on serum glucose level (mg/dL) on glucose tolerance test
in glucose loaded rats
Group Treatment 0 min 30 min 60 min 90 min 120 min
1. Control (vehicle) 89.2±2.9 108.3±1.1 100.2*±1.4 103.7± 2.1 96.3*±1.6
2. MRM (100 mg/kg) 85.7±1.26 132.7±3.19 121.7±2.45 95.4±4.26 82.6±1.75
3. MRM (200 mg/kg) 86.6±2.11 117.6±2.15 107.7±3.25* 85.3±4.72** 74.6±2.10
4. MRM (400 mg/kg) 87.3±1.44** 121.4±3.15 119.1±2.14 99.4±3.45 97.3±1.76**
5. Glibenclamide (500 µg/kg) 83.4±1.5 80.1±2.7 79.5*±1.1 75.1*±2.1 73.6*±2.9
Values are expressed as mean ± SEM; n=6 in duplicate for each treatment;
*statistically significant difference from the corresponding zero time value; P<0.05
Chaudhary et al / International Journal of Pharmaceutical Research 2012 4(2) 59-62
IJPR | January-March | 61
Table 2. Effect of methanolic extracts of Rourea minor roots on serum glucose level (mg/dL) in normal fasted animals
Group Treatment Day 0 Day 4 Day 8 Day 12
1 Control (vehicle 79.4±2.2 78.4±3.3 76.1±36 76.3±2.4
2 MRM (100 mg/kg) 75.6±3.45 66.4±3.21 62.2±5.31 61.1±1.44
3 MRM (200 mg/kg) 73.5±1.78 * 64.1±2.46** 63.4±2.36 58.9±1.35
4 MRM (400 mg/kg) 73.2±2.71 59.7±5.39 51.7±3.15* 51.6±3.05
5 Glibenclamide (500 µg/kg) 80.1±2.3 74.4±2.3 66.7*±3.2 59.6*±1.7
Values are expressed as mean ± SEM; n=6 in duplicate for each treatment;
*statistically significant difference from the corresponding zero time value; P<0.05
A dose of 400 mg/kg and 200 mg/Kg of methanolic
extract produced maximum glucose lowering effect,
whereas 100 mg/kg of methanolic extract showed a
significant hypoglycemic effect throughout the study
period. For glucose tolerance test, In all groups except for
glibenclamide, at 30 min of initiating glucose tolerance test,
blood glucose concentration was higher than at zero time
but decreased significantly (Table 1) from 30 min to 120
min. Methanolic extracts of Rourea minor were enhancing
glucose utilisation, thus the blood glucose level was
significantly decreased in glucose loaded rats and these
effects were dose dependent.In normoglycaemic rats, the
doses of 100, 200 and 400 mg/Kg of methanolic extracts of
Rourea minor roots reduced hyperglycaemia on days 4, 8
and 12 of treatment (Table 2). A significant hypoglycaemic
activity was found on day 12 with 100, 200 and 400 mg/Kg
of Rourea minor. The main mechanism by which the
extracts bring the hypoglycemic effects most probably
involves stimulation of peripheral glucose consumption.
After oral administration of 100, 200 and 400 mg/Kg of
methanolic extracts of Rourea minor, a significant
reduction was observed in the blood glucose level of STZ
induced diabetic rats. A dose dependent effect was seen
with doses of 100, 200 and 400 mg/Kg body weight
throughout the study period (Table 3).Furthermore, the
glycemia profile observed in the glibenclamide group
indicates that the extract of Rourea minor acts on the liver
or on peripheral glucose consumption [16].
The glibenclamide effects on glucose can be attributed
to the enhanced activity of the β cells of the pancreas,
resulting in secretion of a large amount of insulin. These
results have indicated that some drugs may also be effective
in NIDDM. The significant hypoglycemic effects of Rourea
minor roots in diabetic rats indicate that this effect can be
mediated by stimulation of glucose utilization by peripheral
tissues.
In diabetes the increased blood sugar levels might be
due to either insulin resistance of the body cells or
decreased secretion of insulin from beta cells manifest in
the decreased serum insulin levels [17].
The reduction in the serum insulin levels in the STZ
treated rats might be attributed to the reduced secretion of
the hormone which might be due to the damage of the beta
cells of endocrine pancreas. Similar studies were recorded
earlier in the STZ treated rats, the levels of serum insulin
significantly reduced [18]. Nitric oxide has been
demonstrated to participate in the beta cell damage during
STZ induced diabetes [19].
CONCLUSION:
The present study suggests that the methanolic extract of
Rourea minor had hypoglycemic effect revealed by
increased serum insulin levels and therefore attribute to
therapeutic value of the methanolic extract of Rourea minor
to combat the diabetic condition in rats. Further studies are
in progress to isolate the active principle(s) and elucidate
the exact mechanism of action of Rourea minor root.
REFERENCE
1. King H, Aubert RF, and Hermann WH. Global burden
of diabetes, 1995-2025: prevalence, numerical
estimates, and projections. Diabetes Care 1998; 21:
1414–1431.
2. Huang THW, Peng G, Kota BP, Li GQ, Yamahara J,
Roufogalis BD and Li Y. Anti-diabetic action of
punica granatum flower extract: activation of PPAR-γ
and identification of an active component. Toxicol.
Appl. Pharmacol 2005; 207:160-169.
3. Davis SN, Granner. Insulin, Oral Hypoglycemic
Agents, and the pharmacology of the Endocrine
pancreas, In: Hardman JG, Limbird LE, Molinoff PB,
Ruddon RW and AG Gilman’s: The Pharmacological
basis of therapeutics. 9
th
ed., Chap.60, New York, The
McGraw-Hill Companies Inc. 1487-1518 (1996).
4. Eddouks M, Maghrani M, Michel JB. Hypoglycemic
effect of Triticum repens P. Beauv in normal and
diabetic rats. J. of Ethno pharmacol 2005; 102,228-
232.
5. Wu ZY, Yin WQ, Bao SY, Tao DD, Yuan SH, Deng
XF,Yuan SX, You HZ, Lin Q. In Index Florae
Yunnanensis, Kunming, Yuannan, PR China, first ed,
Tomus I, 1984; pp. 869.
6. Wu ZY, Raven PH, In Flora of China. Sciences Press,
Beijing and Missouri Botanical Garden Press, St.
Louis, 2003;437.
Table:3 Effect of Methanolic bark extract of madhuca indica on serum glucose level(mg/dl) in streptozotocin induced
diabetic rats
Group Treatment Week 0 Week 1 Week 2 Week 3
1. Normal Control 128.86±1.71 141.32±2.03 130.13*±3.52 117.37±1.16
2. Diabetic control 190.81±2.68 309.55±4.48 380.05±5.13 411.91±5.02
5. MRM (100 mg/kg) 339.3±1.22 397.1±11.15
**
289.7±13.29
**
276.7±14.23
*
6. MRM (200 mg/kg) 319.7±5.31 273.5±12.24 253.5±12.21
**
161.5±11.51*
7. MRM (400 mg/kg) 316.5±5.41 243.7±10.45
**
216.6±10.73
*
120.6±11.2
*
8.. Glibenclamide (500 µg/kg) 385.5±2.02 302.8*±3.32 190.3*±3.68 132.6*±4.87
Values are expressed as mean ± SEM; n=6 in duplicate for each treatment;
*statistically significant difference from the corresponding zero time value; P<0.05
Chaudhary et al / International Journal of Pharmaceutical Research 2012 4(2) 59-62
62 | IJPR | January-March
7. Jiangsu Medical College. In: Zhongyaodacidian (A
Dictionary of Traditional Chinese Medicines), first ed.
Shanghai Science and Technology Publishers,
Shanghai, PR China, 1986; 1616.
8. Ramiah, N, Prasad NBR, Abraham, K,. Rapanone and
leucopelargonidin from the roots of Rourea
santaloides. Journal of Institute of Chemists (India)
1976; 48 (Pt. 4), 196–197.
9. Jiang JQ, Fang SD, Xu CF, Luo JT. Chemical
constituents of Rourea microphylla (Hook. et Arn)
Planch. Zhiwu Xuebao 1990; 32, 376–379.
10. Organisation for Economic Cooperation and
Development. OECD Guidelines for the testing of
chemicals. OECD guideline 425: Acute oral toxicity:
up and down procedure. June 1998.
11. Boner-Weir S, Deery D, Leahy JL, Weir GC.
Compensatory growth of pancreatic cells in adult rats
after short term glucose infusion. Diabetes 1989;
38:49-53.
12. Rakesh B, Sanjay J, Deep Q, Amit J, Giriraj S, Ravi
G. Antidiabetic activity of aqueous root extract of
Ichnocarpus frutescens in streptozotocin-nicotinamide
induced type-II diabetes in rats. Indian J Pharmacol
2008(40); 1:19-22.
13. Sun Q, Sekar N, Goldwaser I, Genshonov E,
Fridkin M, Shechter Y. Vanadate restores glucose
6 phosphate in diabetic rats: a mechanism to enhance
glucose metabolism. Am.J physiol Endocrinol
metab.2000 Aug; 279(2):E403-410.
14. Ramachandran B, Ravi K, Narayanan V, Kandaswamy
M, Subramanian S. Protective effect of macrocyclic
binuclear oxovanadium complex on oxidative stress in
pancreas of sreptozotocin induced diabetic rats. Chem-
Biol Interact 2004; 149:9-21.
15. Rakieten N, Rakieten ML, Nandkarni MV. Studies on
the diabetogenic action of streptozotocin. Cancer
Chemother Rep. 1963; 29:91-98.
16. Kavalali G, Tansali H, Goksel S, Hatemi H.
Hypoglycaemic activity of Urtica pilurifera in
sreptozotocin induced diabetic rats. J Ethnopharmacol
2002; 84:241-245.
17. Mohammad Ali E, Razeih Y. Hypoglycemic effect
of Teucrium polium: studies with rat pancreatic islets.
J. of Ethnopharmacol 2004; 95(1):27-30.
18. Yoon JW, Ray UR. Perspectives on the role of viruses
in insulin dependent diabetes. Diabetes care 1985;
Sep-Oct; 8Suppl. 1:39-44.
19. Duran Reges G, Pascoe Lira D, Vilar Rojas C,
Medina Navarro R. Diabetogenic effect of STZ
diminishes with loss of Nitric oxide role of ultra violet
and carboxy PT10. Pharmacology 2004; 71(1); 17-24.