Aqeuous extracts of stems of Tinospora cordifolia induces pancreatic regeneration, promotes glucose uptake and reverses the disturbance in

lipid profile in streptozotocin diabetic rats

Supriya Pai, Reena Lobo and Asha Abraham*Department of Biotechnology, St. Aloysius College, Mangalore, 575 003, Karnataka, INDIA.

Running Head: Effect of Tinospora cordifolia stem extracts on STZ diabetic rats

*Address of corresponding author

Dr. Asha Abraham, Department of Biotechnology,

St. Aloysius College, Mangalore, 575 003, Karnataka, INDIA.

E-mail: abraham.asha@gmail.com, Mob. No. +91 9449555802

Fax : 91 0824-2426631.

ABSTRACT

In the present study aqueous extract of stem, leaf and roots of Tinospora cordifolia were

screened for their hypoglycemic effect on normal rats following glucose load. The stem

extract showed significant hypoglycemic effect. The anti diabetic effect of the stem

extracts were further evaluated in streptozotocin diabetic rats. A 2 week oral treatment of

the stem extract at 200mg/kg body weight was found to have hypoglycemic effect in both

moderately and severely diabetic rats. Also their body weight showed improvement. The

stem extract could reverse the disturbances in the lipid profile – total cholesterol and

triglyceride in serum of the experimental animals. Serum insulin levels showed

improvement in the stem extract treated diabetic rats. Our results also show that the stem

extract promotes glucose uptake in peripheral tissues. Partial phytochemical screening of

the leaf extract showed the presence of major classes of phytochemicals like Flavonoids,

Berberine, terpenoids and glycosides. This supports the antihyperglycemic and

antihypercholesteric effect observed. Thus Tinospora cordifolia stem extract could be

used for the development of drugs against diabetes mellitus.

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INTRODUCTION

Tinospora cordifolia commonly known as Guduchi belonging to the family

Menispermaceae, is a perennial climber of weak and fleshy stem found throughout India and

China. According to Ayurveda, this plant is used for treatment of jaundice, rheumatoid arthritis,

diabetes, gout, viral hepatitis and general weakness. The root and stem of T. cordifolia are

prescribed in combination with other drugs as an anti-dote to snake bite and scorpion sting (Zhao

et al., 1991). Dry barks of T. cordifolia have anti-spasmodic, antipyretic (Ikram et al., 1987),

anti-allergic (Nayampalli et al., 1986), anti-inflammatory (Rai et al., 1997), anti-leprotic

(Asthana et al., 2001) and immuno modulatory properties (Kapil et al., 1997).

Aqueous and ethanol extracts of T. cordifolia roots to diabetic rats showed

reduction in blood glucose levels, similar to glibenclamide and insulin (Dhaliwal et

al.,1999). The hypolipidaemic action of Tinospora cordifolia roots in alloxan diabetic

rats was reported by Stanely et al., (1999). Another study carried out by Wadood et

al.,1992 has shown that the leaf extract of Tinospora cordifolia had significant

hypoglycemic effect in normal and alloxan induced diabetic rabbits, however the extract

had showed no significant effect on the total lipid levels in normal or treated rabbits.

Roots of Tinospora cordifolia were shown to have antioxidant activity on experimental

diabetes (Prince et al., 1999 and Stanley et al., 2001). Literature also, suggests that roots

and leaves of T. cordifolia have anti tumor effect (Stanely et al., 2001 and Singh et al.,

2007).

Most of the previous studies were carried out using the leaves and roots of T. cordifolia

on alloxan induced diabetic models. There are no reports on the anti diabetic properties of stem

extracts of this plant. Streptozotocin (STZ) specifically destroys pancreatic beta cells and

therefore is a better than alloxan in inducing diabetes. We have therefore made an attempt to

study the ant diabetic effect of stems of Tinospora cordifolia in streptozotocin induced diabetic

rats.

MATERIALS AND METHODS

Biochemicals and their sources

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All the chemicals used in the study were of analytical grade and purchased locally.

Animals

Adult Male Wistar rats weighing approximately 250g were purchased from local dealer. These

rates were housed in groups of 3 to 4 in separate cages, maintained in good ventilation in the

animal house with 12 hours dark and light cycle. They were fed with lab chew and water ad

libitum. All experiments were conducted in accordance to the ethical committee of the institution.

Plants

The Tinospora cordifolia were collected from the Ayurvedic herb dealers and maintained in the

green house of St. Aloysius College Mangalore.

Oral Glucose Tolerance Test

Aqueous extract of stem, leaf and roots of Tinospora cordifolia fed orally to 24 hours starved

animals of different groups, following glucose load of 3 gm/Kg body weight. Ten animals were

maintained in each group. The blood samples were collected using tail snipping method. The

blood glucose levels were monitored every 30 minutes for a period of 3 hrs using glucose

estimation kit (AGAPPE, India).

Induction of diabetes mellitus in rats

Diabetes was induced by single intra femoral vein injection of streptozotocin

(STZ) (50 mg/ Kg body weight), dissolved in citrate buffered vehicle (pH 4.5) under light

ether anesthesia. A group injected with vehicle alone served as control. The rats were

categorized into five groups. They are as follows- Control, Control + Stem, Diabetic, Diabetic +

Stem, Diabetic + Insulin. Ten animals were maintained in each group.

Preparation of stem extract

Stem was weighed and ground in ethanol and then soaked in a known volume of ethanol

overnight. The ethanol was separated by filtration and was evaporated to dryness using flash

evaporator (with temperature below 350C). Known amount of this residue was dissolved in 5%

Tween- 80. mention the amount correctly

Stem extract therapy

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Control rats injected with only citrate buffer was divided into 2 groups. One group

received oral ethanolic extract of Tinospora cordifolia resuspended in 5% Tween-80 at 200mg/kg

body weight, twice a day. The other group did not receive any treatment. One group of diabetic

rats, received extract of Tinospora cordifolia at 200mg/kg body weight, twice a day. The final

treatment was given twelve hours prior to sacrifice.

Insulin administration

One group of diabetic rats received subcutaneous injection of insulin (mention the brand)

daily during the entire period of experiment. The dosage varied from 1-6 units depending on the

blood glucose level. The final insulin injection was given twelve hours prior to sacrifice. One

diabetic group did not receive any treatment.

Blood glucose and body weight monitoring

Body weight and blood glucose was monitored at alternative days during the period of 15 days of

stem extract treatment.

Sacrifice of rats

Rats were sacrificed by cervical decapitation and tissues were immediately stored at -20°C till

used. The pancreas was immediately fixed in buffered formalin.

In Vitro -Peripheral Glucose Uptake Studies

Peripheral glucose consumption was studied in the rat diaphragm in animals fasted for 24

hours prior to the experiment. The diaphragm from control and diabetic rats are aseptically

isolated into a balance salt solution and cut into small pieces and incubated in the nutrient

solution (mention the ref) with the constant oxygenation and shaking at 37oC for 90 minutes.

Sodium bicarbonate was aerated with CO2 for 3 minutes and then added to the buffer solution.

Glucose was added to the final concentration of 300mg/100ml of buffer solution. Peripheral

glucose uptake in the presence of various concentrations (mention the conc) of stem extract at

was studied and compared with that in presence of insulin.

Estimation of different biochemical parameters in serum

The blood glucose estimation was done by glucose estimation kit (AGAPPE, India). Serum

triglycerides were estimated using triglycerides kit (ERBA, India). Total cholesterol was

estimated using Cholesterol kit (ERBA, India). HDL cholesterol was estimated using HDL-

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Cholesterol kit (ERBA, INDIA). LDL cholesterol was estimated quantitatively by turbidimetric

immunoassay using LDL- cholesterol kit (ERBA, INDIA). The insulin content in serum was

estimated using Bio LINE insulin ELIZA kit (Belgium).

Statistical analysis of data

Statistical valuations were done by ANOVA test using graph pad Instat Version 2 soft ware.

Histopathological study of pancreas

The formalin fixed pancreas was processed for histology. The tissue sections (5 µm

thickness) were stained with haematoxylin-eosin and observed under light microscope at

40x magnification and the image captured using Magnus Image projecting system

(Olympus).

Partial Phytochemical Screening of Ethanolic Extract of Stems of Tinospora cordifolia

The stem extract was evaporated to dryness and dissolved in absolute ethanol and

scanned from 190- 1100 nm using UV- VIS spectrophotometer (Thermospectronic). The

absorption maxima of the peaks obtained were used for the identification of phytochemicals

according to Harborne (1984).

Analysis for alkaloids

The ethanolic fraction of the stem sample was analyzed for the presence of alkaloids

using TLC on silica gel plates. The solvents used were methanol: Concentrated ammonia (200:3).

Detection was done using Dragandroff reagent. This was further confirmed by Mayers test.

TLC for Berberine:

Detection of berberine was done by TLC on silica gel plates using a solvent system of butanol:

acetic acid: water (mention ratio) and detected by UV illumination. The Rf value was measured.

Analysis for terpenoids and phytosterols

Terpenoids and Phytosterols were detected by Salkowski Test and Liebermann- Buchard (LB)

reaction. The presence of terpenoids was further confirmed by two dimensional TLC on silica gel

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using the solvent system benzene: chloroform (1:1) and benzene: ethyl acetate (19: 1) and dected

by using concentrated sulphuric acid.

The ethanolic fraction of the stem sample was also analyzed for the presence of triterpenes using

a solvent system hexane: ethyl acetate (1:1) and n- butanol: 2M NH4OH (10:1) and detected L.B

or Carr Price reagent.

Argentative TLC was used to analyze the presence of phytosterols by using chloroform

as solvent. Detection was done using L.B reagent.

Analysis for saponins

Saponins were also tested for in crude extracts by their ability to haemolyse blood as well as

byTLC on silica gel using the solvents chloroform: methanol: water (13:7:2). Carr price reagent

was used for the detection.

Analysis for cardiac glycosides

Ethyl acetate: methanol: water was the solvent used to analyze cardiac glycosides. Detection was

done by spraying Carr Price reagent (Bernard et al., 1996).

Analysis for phenolics

Presence of Phenolics were ascertained by Ferric chloride test and confirmed by paper

chromatography using Butanol: acetic acid: water (4:1:5) solvent system and detected under UV

illumination. Also, a two dimensional TLC was performed using the solvent system acetic acid:

chloroform (1:9) and ethyl acetate: benzene (9:11). Folin Ciocalteu Phenol reagent was used for

the detection.

Presence of flavanoids was confirmed by Paper chromatography using Concentrated HCl:

acetic Acid: water solvent system (mention ratio) and detected under UV illumination as well as

TLC using butanol: acetic acid: water (11:1:5). Detection was done under UV illumination.

The ethanolic fraction of the stem extract was used for the detection of tannins by using a

solvent system butanol: acetic acid: wate r(mention ratio). Observed under UV illumination.

Detection of inulin

Indole acetic acid reacts with fructose oligosaccharides along with hydrochloric acid when

incubated at 600 C and cooled at room temperature to give blue colour. Stem extract was taken in

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test tube and about 1ml of indole acetic acid was added along with HCl and incubated at 60 0 C

water bath for 20 minutes and kept room temperature.

RESULTS AND DISCUSSION

In the present investigation aqueous extract from the stem of Tinospora cordifolia was

evaluated for its anti diabetic effect on Streptozotocin induced diabetic rats. We investigated the

hypoglycemic effects of the crude extracts of stem, leavers and roots of Tinospora cordifolia in

the normal rats following glucose load. Results showed that in control rats there is significant

increase in hyperglycemia following glucose load which eventually clears off by 150 minutes

(Table No. 1 and Fig. No.1). The leaf extract significantly increases the blood glucose levels

compared to zero time and maintains hyperglycemic condition up to 180 minutes. Our findings

contradict those of Wadood et al., 1992, who reported that T. cordifolia leaf extract had

hypoglycemic effect on normal rats. The root extract treatment could not control hyperglycemia,

however it helped clear the glucose load by 120 minutes i.e. faster than control rats. The stem

extracts prevented hyperglycemia following glucose load. This condition persists up to 6 hours

following glucose load (data not shown). This indicates that the stem extract may have promoted

glucose uptake or may have stimulated insulin release which helped glucose clearance from the

blood. There was a significant increase in the blood glucose levels in STZ treated rats.

STZ selectively destroys pancreatic β cells, resulting in deficiency of insulin. The insulin

deficiency causes hyperglycemia by decreased entry of glucose into the cells, decreased

utilization of glucose by various tissues and increased production of glucose by gluconeogenesis

by the liver (Granner, 2000). Our results showed that there was significant decrease in the blood

glucose level following 12th day of stem extract treatment at 200mg/kg body weight of diabetic

rats. In the case of control rats treated with stem extract, no significant change was noted

compared to controls. There was significant reduction in hyperglycemic state of diabetic rats

treated with insulin (Table No. 2 and Fig. No.2) though it was not as effective as the stem extract

treatment.

Hypoglycemic effect of T. cordifolia roots and leaves has been reported.

The leaf extract of T. cordifolia at 200mg/kg exerted significant hypoglycemic effect in

normal as well as alloxan treated diabetes (Wadood et al., 1992). In Malaysia an aqueous

extract of T. crispa stem is taken orally to treat diabetes mellitus. After 2 weeks of

treatment of extract at 4g/lt of drinking water rats showed improvement in glucose

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tolerance. We detected the presence of Berberine (Table No. 9 and Fig. No.11),

Phytosterols (Table No. 9 and Fig. No. 14) and Terpenoids in the stem extracts (Fig. No.

12). TLC showed the presence of several terpenoids. UV –Vis scan (Table No. 9) showed

that ε carotene, geraniol, α- pinene, α- terpineol, pulegone may be present.

Alkaloid Berberine is reported to exert hypoglycemic effect by an increase in

glucose uptake, increase insulin secretion, increase in levels of glycolytic enzyme and

decrease in levels of gluconeogenic enzymes (Isaac et al., 2006) . Yin, (2003) reported

that berberine could also act in insulin independent manner. Terpenoids (Fabio et al.,

2006) and Phytosterols (Tanaka et al., 2006) are also involved in reduction of blood

glucose.

There was a significant reduction of body weight of STZ induced diabetic rats, compared to the

controls. Stem extract treatment was found to prevent the decrease in body weight in diabetic rats

and in the control rats the weight was maintained constant. Insulin treatment in diabetic rats

prevented the reduction in body weight (Table No. 3 and Fig. No.3). Weight loss is one of the

major symptoms of insulin deficiency. When the maximum level of renal tubular reabsorption of

glucose is exceeded, sugar excreted in the urine. Glucosuria causes substantial loss of calories.

This loss when coupled with the loss of muscle (due to increased protein catabolism and

decreased in protein synthesis) and adipose tissue (due to enhanced lipolysis), results in severe

weight loss, in spite of increased appetite and increased caloric intake (Granner, 2000). The

extract of roots of T. cordifolia is also reported to have prevented decrease in body weight

(Stanely et al., 2000). Perhaps the phytosterols present may have a role to play in the prevention

of decrease in body weight. Reports suggest that phytosterols derived from Aloe Vera showed no

weight reduction due to glucose loss in urine (Tanaka et al., 2006).

Our study showed a significant increase in serum triglycerides/ triacylglycerol (TAG) in

diabetic rats. Elevated serum cholesterol and triglycerides level i.e. hypercholesterimia is one of

the condition observed in diabetics. Impaired carbohydrate utilization in diabetics leads to

accelerated lipolysis, which results in elevated serum levels of TAGs. However, in our results,

total serum cholesterol remained unchanged (Table No. 4 and Fig. No.4) Insulin treatment did not

reverse this. Stem extract treatment to both diabetic and control rats lowered the TAGs as well as

cholesterol levels (Fig. No.5). So further analysis was done to check serum HDL and LDL levels

and our results showed that the HDL levels were decreased in the diabetic rats compared to

control. Stem extract treatment increased HDL level in both normal and diabetic rats compared to

the untreated. Where as LDL level was elevated in diabetic rats compared to control and this was

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seen to be reversed following stem extract treatment (Table No. 5 and Fig. No.6). The health

status of an individual is generally determined by two ratios TC/HDL and LDL/HDL. Lower

value of these ratios indicates better health. Our results clearly indicates that the values of these

ratios lowered both in stem extract treated control and diabetic compared to the untreated rats

(Table No. 6). Thus we can conclude that stem extract has cardioprotective properties.

Phytochemical screening showed that the stem extract of T. cordifolia is rich in

phytosterols. Phytosterols reduce hypercholesteremia systematically. Thus providing a high

degree of deactivation of the harmful effects caused by cholesterol (zoosterols) upon the human

health (Bruno et al., 2006). Phytosterols at intestinal level, link with cholesterol (zoosterols)

and produce non absorbable micelles. Cholesterol reduction in hepatic contents concurs with the

stimulus to capture LDL and produce less VLDL and apo B. We have also detected saponins in

the ethanolic extracts. Saponins are known to possess blood cholesterol lowering activity

(Racheal et al., 2003, Lee et al., 1999). The mechanism proposed is that, it binds to cholesterol

in the intestinal lumen and so cholesterol is less readily absorbed. In addition, it may also bind

bile acids, causing a reduction in its enterohepatic circulation, increasing its fecal excretion. The

bile acid excretion is offset by synthesis from cholesterol in liver and consequent lowering of

plasma cholesterol (Oakenful and Sidhu, 1990). We have also detected inulin in the stem extracts

(Fig. No.17). Studies shows that inulin has lipid lowering effect (Christine et al., 2004). This may

be one of the reasons for causing reduction in the plasma levels of cholesterol and triacylglycerols

(TG). The hypotriglyceridaemic effect is due to a reduction in hepatic re-esterification of fatty

acids, but mainly in the expression and activity of liver lipogenesis, resulting in lower hepatic

secretion rate of TG (Beylot et al., 2005).

Presence of cardiac glycoside was confirmed by TLC (Fig. No.16). Hypertension

is a related complication associated with diabetes mellitus. Singh et al., 2003 reported the

presence of Cordifoliside in Tinospora cordifolia which is known to have hypolipidemic

effect. All cardiac glycosides are highly selective inhibitors of the active transport of Na+

and K+ across cell membranes, by binding to specific site of Na-K-ATPase. This

inhibition causes activation of Na+-Ca++-exchanger and increase of intracellular Ca++

levels, which interact with contractile proteins of myocardial cells and increasing the

contractility of cardiac muscle (www.ibpassociation.org, 2005). It reduces the magnitude of

ventricular remodeling and diastolic wall stress (Capasso et al., 2004). The main action of

the Cardiac Glycosides is to increase the force of cardiac contract- a rise in the

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concentration of intracellular calcium and sodium ion .Thus helps in easy pumping of

blood even in presence of blockage in blood vessels. Thus our results once again confirm

the cardio protective effect of T. cordifolia.

Our results showed that diabetic rats has lower level of insulin compared to the control rats

which is clear indication of β cell destruction while in serum of stem extract treated diabetic rats

the insulin level was significantly higher when compared to untreated diabetics (Table No. 7 and

Fig. No.7). This clearly indicates that certain component in our stem extract might have mediated

regeneration of pancreas and thus insulin secretion. Flavanoids helps in insulin release from the

islets (Hii and Howell, 1985). Berberine is also reported to have similar effect (Li Qin et al.,

2006, Leng et al., 2004). Also, pancreatic regeneration was studied by histology (Fig. No.9). STZ

induced diabetic rats resulted a significant decease in the number of pancreatic islets per unit

area .Also the islets showed signs of necrosis. Insulin treatment as well as stem extract treatment

showed the signs of regeneration. TLC and paper chromatography confirmed the presence of

phenolics and flavonoids (Table No.9 and Fig. No.15). UV- Vis Spectral analysis revealed that

flavones and flavanols like kaempferol, quercetin might be present. Several flavanoids were

implicated in causing the partial regeneration of pancreatic islets in Teucrium polium treated STZ

induced diabetic rats (Mahmood et al., 2003). Our work showed the presence of flavanols and

flavanones in stem extract. Berberine was known to restore the damage of pancreatic tissues in

rats with diabetes mellitus (Li Qin et al., 2006).

Peripheral glucose uptake studies revealed that the ethanolic stem extract significantly

promoted higher glucose uptake in diabetic rat diaphragm compared to control (Table No. 8).

This might be due to the fact that diabetic tissues, starved of glucose may express more glucose

transporters on its cell membranes. Stem extract mediated better uptake than insulin in both the

groups. However, at higher concentration of stem extracts, uptake was slightly lower in control

rat diaphragms. It has been reported that Berberine induces glucose transport via increased GLUT

1 activity but has no effect on its expression (Zhou et al., 2003). This component of the stem may

be reason for better uptake when compared to the untreated. So also triterpenoids are associated

with alteration of AMP activated protein kinase pathway, which is involved in glucose uptake

(Tan et al., 2008). Our results of phytochemical screening show the presence of triterpenoids in

the stem extract.

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CONCLUSION

Our studies showed that the stem extract treatment of Tinospora cordifolia to diabetic rats

brought down the blood glucose levels close to control values, serum insulin level increased and

pancreatic regeneration was observed by the 12 th day of treatment. Stem also promoted glucose

uptake in peripheral tissues. Two mechanisms are involved in decrease in blood glucose level (1)

increase the uptake of glucose by the tissues (2) regeneration of pancreas and thus insulin release.

Phytochemical screening revealed the presence of major class of phytochemicals like of

phenolics, alkaloids, terpenoids and glycosides. Flavanoids particularly quercitin, terpinoids,

alkaloids like berberine are reported to have antihyperglcemic effect. Flavanoids majorly acts as

an antioxidant and may have a role in ameliorating atherosclerotic risk of patients with diabetes

mellitus. Cardiac glycosides are effective in reducing hypertension linked with diabetes.

Phytosterols, alkaloid- berberine are known to have anti hypercholestremic effect. Thus we

conclude that the effective components in Tinospora cordifolia could be purified and effectively

used develop drugs to treat diabetes and its related complications. Yet further studies need to be

done to check the molecular mechanisms involved.

ACKNOWLEDGEMENTS

The authors thank the Management and Principal of St. Aloysius College, Mangalore for

providing the laboratory facilities and other support for this work. Dr. Mohan Kishore,

Karnataka Ayurveda Medical College, Mangalore is gratefully acknowledged for his

suggestions during this work.

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