Sheshachala forest (Rayalaseema region, Andhra Pradesh, India), which lie geographically in the South Eastern Ghats are known for the rich heritage of flora where the tribes use Piper longum roots to treat the diabetes mellitus . Hence our study was aimed to find out the scientific evidence for the safe use of the roots of Piper longum to treat DM. Earlier, Shanmugam Manoharan et al. demonstrated that the ethanolic extract of dried fruits of Piper longum has potent antihyperglycemic and antilipidperoxidative activity in alloxan induced diabetic rats . In the present study, we have selected the roots of Piper longum for evaluating antihyperglycemic and antihyperlipidemic activity since the phytochemical constituents of fruits and roots are different. In our study STZ was used to induce diabetes mellitus in rats. At low dose, STZ (50 mg/kg b.w) partially destructs the beta cells resulting in insufficient insulin secretion causing type 2 diabetes . It is widely accepted animal model and reported to resemble human hyperglycemic non ketotic diabetes mellitus , is often associated with kidney hypertrophy which may contribute to end stage renal damage, hepatotoxicity, oxidative stress and hypercholesterolemia [32, 33].
During the short term study the aqueous and methanolic extracts produced significant antihyperglycemic activity at a dosage of 200 mg/kg b.w in diabetic treated rats. Aqueous extract is more potent than methonolic extract. The hexane and ethylacetate extracts did not show significant antihyperglycemic activity, may be due to the lack of phytochemical constituents like alkaloids and glycosides which are present in methanolic and aqueous extracts.
During the dose dependent study, the aqueous extract at a dosage of 200 mg/kg b.w has produced maximum antihyperglycemic activity in diabetic rats. The higher doses of PlrAqe (300 & 400 mg/kg b.w) could not produce the predictable higher antihyperglycemic effect due to the presence of some other substances in the aqueous extract, which interfere with the antihyperglycemic effect . So, the long term study was carried out with the dose of 200 mg PlrAqe/kg b.w.
The oral glucose tolerance test also confirmed blood glucose lowering activity of PlrAqe. The onset of antihyperglycemic action was observed from 60 min of the treatment and a steady state increase in the action continued up to 120 min. The PlrAqe may be involved in enhancement of glucose utilization, so blood glucose levels were significantly decreased in glucose loaded rats.
The loss in body weights observed in STZ induced diabetic rat group (after a period of 30 days) may be due to muscle wasting and loss of tissue proteins upon induction of diabetes with STZ [35, 36]. The gain in body weight was observed both in normal treated and diabetic treated groups. PlAqe treatment in diabetic treated group for 30 days resulted in a significant (66%) reduction in their FBG levels and these effects were higher than those of the standard oral hypoglycemic agent glibenclamide. Earlier Santhakumari et.al and Kaleem et.al reported 26% and 52% reduction in the FBG levels of diabetic rats treated with aqueous extracts of Piper betle leaves and Piper nigrum seeds respectively for 30 days.
HbA1c is used as a marker for estimating the degree of protein glycation in diabetes mellitus. HbA1c was found to increase in patients with diabetes mellitus and the amount of increase is directly proportional to the fasting blood glucose level . In diabetic condition, the excess glucose present in the blood reacts with haemoglobin to form HbA1c . Hence HbA1c levels were elevated and total haemoglobin levels were depleted in untreated diabetic rats. HbA1c levels were well regulated near to normal levels in PlrAqe treated diabetic group, this could be due to an improvement in insulin secretion upon PlrAqe treatment. Piper betle which belongs to same genus has been reported to have the ability to reduce HbA1C levels in diabetic rats .
Diabetes mellitus is usually associated with prominent levels of serum lipids and such an increase causes the risk factor for coronary heart diseases . A variety of alterations in metabolic and regulatory mechanisms, due to insulin deficiency or due to insulin resistance are responsible for the observed accumulation of lipids . STZ-induced diabetes also developed hyperlipidemia which is in agreement with our previous observations [34, 42]. In the present study, the PlrAqe significantly reduced the TC, TG, LDL-C and VLDL-C levels with an increase of HDL-C in treated diabetic rats compared to untreated diabetic rats (Figure 2). This may be due to the insulinotropic effect or insulin secretagogue activity of this extract. PlrAqe treated diabetic rats showed decrease in atherogenic index and increase in percentage of protection against atherogenicity. Decrease in atherogenic index is due to increase in HDL-C levels after the treatment. HDL-C is known to play an important role in the transport of cholesterol from peripheral cells to the liver by a pathway termed reverse cholesterol transport, and is considered to be a cardio protective lipid. The existence of negative correlation between HDL-C and atherosclerosis resulted in improvement in the percentage of protection against atherogenicity in STZ induced diabetic treated rats .
Under hyperglycemic condition disturbances in carbohydrate, lipid and protein metabolisms together with oxidative stress are likely to affect hepatic and renal functions. Hence our study was also focused to know the protective activity of PlrAqe against hepatic and renal damage caused by diabetes. In the present study serum enzymes such as SGOT, SGPT and ALP were used in the evaluation of hepatic damage (Figure 3). In diabetic rats an increase in these enzyme activities reflects active liver damage. Increased levels of SGOT and SGPT under insulin deficiency  have been related with increased gluconeogenesis and ketogenesis during diabetes. Moreover, increased levels of these enzymes together with ALP and Acid phosphatase (ACP) are reported to be associated with liver dysfunction and leakage into blood stream in diabetes . Oral administration of PlrAqe in diabetic rats resulted in reduction in the activities of these enzymes in serum compared to the diabetic untreated group.
In our study elevated levels of serum urea and creatinine were observed in diabetic untreated rats, which are considered as significant markers of renal dysfunction . Negative nitrogen balance with enhanced tissue proteolysis and decreased protein synthesis can contribute to increased serum urea and creatinine levels, indicating impaired renal functions in diabetic animals . After the treatment with PlrAqe a significant reduction in the levels of urea and creatinine were observed in the diabetic treated rats. It indicates that PlrAqe is preventing the renal damage in diabetic rats.