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S I N G A P O R E M E D I C A L J O U R N A L
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Singapore Med J 2000; Vol 41(1):
Effects of an Ethanolic
Extract of Gynura procumbens on Serum Glucose, Cholesterol and
Triglyceride Levels in Normal and Streptozotocin-Induced Diabetic Rats
Aim of Study: The aim was to demonstrate the effects of the leaves of Gynura procumbens (Lour.) Merr. on blood sugar and lipid levels in experimental animals.
Methodology: We obtained an ethanolic extract of the leaves of G. procumbens and monitored the effects of an oral administration of (i) different single doses of the extract on oral glucose tolerance in streptozotocin-induced diabetic and normal rats and (ii) fourteen doses over 7 days on serum cholesterol and triglyceride levels in streptozotocin-induced diabetic rats. Metformin and glibenclamide were used as positive control drugs.
Results: The extract, at single doses of 50, 150 and 300 mg/kg orally, significantly suppressed the elevated serum glucose levels in diabetic rats; 150 mg/kg was found to be the optimum hypoglycaemic dose. The extract however did not significantly suppress the elevated serum glucose levels in normal rats, unlike glibenclamide. Metformin, but not glibenclamide, improved glucose tolerance in the diabetic rats. When the optimum dose was given to diabetic rats for 7 days, the extract significantly reduced serum cholesterol and triglyceride levels in these rats.
Conclusion: These results indicate that the leaves of G. procumbens may have biguanide-like activity.
Keywords: Gynura procumbens, diabetic rat, cholesterol, triglyceride, oral glucose tolerance test
Gynura procumbens is found in various parts of Southeast Asia. It has been used for the treatment of eruptive fevers, rash and kidney disease(1). Recently, the leaves of this plant have been used as folk medicine to control diabetes mellitus and hyperlipidemia. However by reviewing the current literature, we know of no previous research on the pharmacological properties of this plant. Therefore experiments were carried out to test the antidiabetic and antihyperlipidemic activities of this plant.
The oral glucose tolerance test (OGTT) is a well-accepted and frequently used assay to screen hypoglycaemic activity(2). Streptozotocin (STZ) is a valuable agent for the experimental production of diabetes, and it is less toxic than other chemical agents inducing diabetes. The diabetogenic effect of STZ is the direct result of irreversible damage to pancreatic b cells, allowing degranulation and loss of insulin secretion. So the STZ-induced diabetic animal is one of the animal models of human insulin-dependent diabetes mellitus (IDDM)(3,4). Diabetes mellitus is a disease with profound effects on lipid metabolism. Insulin affects mammalian lipid metabolism in several ways, eg. it inhibits the activity of lipoprotein lipase, therefore it decreases the mobilisation of free fatty acids from the peripheral fat depots. On the other hand, it stimulates the synthesis of fatty acids in the liver, adipose tissue and intestine. The STZ-induced diabetic animal is thus considered as an animal model of hyperlipidemia(5).
The present study was undertaken to evaluate the possible hypoglycaemic activity of the ethanolic extract of G. procumbens leaves in the normal and the STZ-induced diabetic rats. The effects of the extract on serum total cholesterol (TC) and triglyceride (TG) levels were also examined in the diabetic rats. To check the safety of taking this plant, acute toxicity and behavioural changes were observed.
MATERIALS AND METHODS
The fresh leaves of G. procumbens (1 kg) were blended and extracted with 95% alcohol (1.5L) until exhaustion. After filtration with cotton wool, the mixture was centrifuged at 10,000 g for 20 min. The supernatant was concentrated to 0.5L at 38°C by a rotavapor (Buchi Labortechnik AG, Switzerland). This solution was then freeze-dried, yielding 16.38 g of light green powder. The extract was suspended in distilled water before use.
2) Repeated administration of the extract in diabetic rats: 6 diabetic rats were treated orally with 150 mg/kg extract twice daily (at 9.00 am and 6.00 pm) for 7 days. Another 6 diabetic rats were given orally an equal volume of distilled water and served as controls. These diabetic rats were used for the determination of serum total cholesterol and triglyceride as well as liver P450 content. Food and water were given ad libitum; the amount consumed as well as the body weight were recorded daily.
3) Acute toxicity: The BALB/c mice were used to investigate the possible toxic effect of the extract. The extract was given orally to two different groups of BALB/c mice (n = 4) at a dose of 1 g/kg and 5 g/kg of body weight, respectively. All mice were observed for 8 hours after administration of the extract to check for toxic symptoms. They were kept under observation for 7 days. The extract, antidiabetic drugs and their respective vehicles were administered orally by gavage to rats.
Collection of blood
Serum TC and TG
of liver microsomal P450 content
2) Measurement of protein content in microsomes. Protein content in each microsomal suspension was determined by Lowry’s method(9).
3) Spectrophotometric measurement of liver microsomal P450 content. An aliquot of microsomal preparation of 1 mg protein/mL was obtained by adding 0.5 mL of 1 M potassium phosphate buffer and the required volume of 1.15% KC1. The modified technique of Omura and Sato(10) was adopted in this assay to eliminate the absorption peak at 420 nm due to contamination by haemoglobin in the sample. The microsomal preparation was placed in two cuvettes and initially saturated with carbon monoxide. A small amount of sodium dithionite (not more than 2 mg) was added to the sample cuvette only. The microsomal P450 content was then determined from the difference in absorbance values between the dithionite-reduced and control microsomal preparations using a Shimadzu UV-2100 dual-beam spectrophotometer. The molar extinction coefficient of microsomal P450 at the cmax of 450 nm was 91 mM-1 cm-1.
Body weight, food
and water intake
OGTT in diabetic
At 60 mins after glucose load, serum glucose levels in all the animals reached a peak. At 120 mins, the three doses of the extract produced significantly lower serum glucose levels compared to the vehicle. A maximum decrease of 15.8% was observed with 150 mg/kg of extract; this dose also produced a significant decrease in serum glucose at 60 mins. The reference drug, metformin (500 mg/kg) caused a significant decrease in serum glucose levels at 30, 60 and 120 mins compared with the vehicle. However the serum glucose levels in the glibenclamide (5 mg/kg)-treated group throughout the study were similar to those of the vehicle-treated group.
OGTT in normal
Serum TC And TG
P450 content and acute toxicity study
BALB/c mice given 1 g/kg and 5 g/kg body weight of the extract of G.procumbens did not show any visible signs of toxicity, eg. excitement, restlessness, respiratory distress, convulsion, or coma. Moreover, they remained alive and well for up to 7 days. They also maintained their body weights during this period. The mean body weights of BALB/c mice given 1 g/kg and 5 g/kg extract were 22.1 g and 20.2 g on day 0, and 22.8 g and 20.2 g on day 7, respectively.
In the present study, the hypoglycaemic activity of the ethanol extract of G.procumbens was evaluated in the normal and STZ-induced diabetic rats, a model of human insulin-dependent diabetes, using the OGTT. The extract improved the glucose tolerance in STZ-induced diabetic rats, but not in normal rats. Under the same conditions, metformin produced a significant glucose clearance in STZ-induced diabetic rats, but glibenclamide did not. Furthermore, after 7 days administration of the extract, the serum TC and TG levels of the extract-treated diabetic rats were significantly lower than those of the control rats.
It is well known that sulfonylureas and biguanides are the major oral hypoglycaemic agents used worldwide. Glibenclamide, a sulfonylurea derivative, causes hypoglycaemia by stimulating pancreatic b cells to release more insulin, and inhibiting glucagon secretion. As these effects require a functional pancreas, it can lower blood sugar levels in non-diabetic subjects(12). In contrast to sulfonylureas, metformin, a biguanide derivative, has no such stimulating activity. Its blood glucose-lowering action dose not depend on functional pancreatic b cells, being due to its extrapancreatic actions, such as 1) increase of glucose utilisation and 2) reduced hepatic gluconeogenesis(12). Metformin can also ameliorate abnormalities in lipid levels, causing a decrease in circulating triglyceride and total cholesterol concentrations(13).
In view of the similarity between the effects of G. procumbens and metformin rather than glibenclamide, it seems that the hypoglycaemic effect of G. procumbens may be due to a biguanide-like activity.
P450 is an enzyme that is responsible for drug metabolism and detoxification in the liver(14). As P450 was not altered by extract treatment in the present experiment, G. procumbens is unlikely to produce a pharmacokinetic interaction with other drugs taken together. The extract was also non-toxic to the laboratory mice.
In conclusion, our studies show that the ethanolic extract of G. procumbens leaves has antihyperglycaemic and antihyperlipidaemic activities in diabetic rats. Further pharmacological and phytochemical investigations are being done to identify the active compound(s), and also to elucidate the mechanism(s) of action.
The authors wish to thank the National University of Singapore for the research grant (RP 60329) and a research scholarship awarded to Dr X F Zhang.