Collection of plant material
Dry roots of Piper longum (PL), were purchased from the local market and identified by the Botanist, Department of Botany, S.V.University, Tirupati. Voucher specimens (Herbarium Accession Number 713) were deposited in the herbarium, Department of Botany, S.V. University, Tirupati.
Hexane, ethyl acetate, methanol and aqueous extracts
Hexane, ethyl acetate and methanol extracts were prepared by successive solvent extraction of PL root powder in soxhlet apparatus at 68°C-70°C. The filtrates obtained were distilled and concentrated under reduced pressure at low temperature (40°C to 45°C) in Buchi rotavapor R-200 and finally freeze dried. The yields of the hexane, ethyl acetate and methanol extracts were 38%, 15% and 21% w/w respectively. To prepare aqueous extract the root powder was soaked in distilled water in a glass jar for 48h at room temperature and the solvent was filtered. This was repeated 3–4 times until the filtrate gave no coloration. The filtrate was concentrated to dryness under reduced pressure in Buchi Rotavapor R-200 and finally freeze dried. The yield of the extract was 22% (w/w). All the extracts were stored at 0°C in airtight containers until needed for further studies.
Induction of diabetes
Diabetes was induced in male Wistar albino rats aged 2–3 months (180–200 g body weight) by intraperitoneal administration of STZ (single dose of 50 mg/kg b.w.) dissolved in freshly prepared 0.01M citrate buffer, pH 4.5 [13]. After 72 h rats with marked hyperglycemia (FBG ≥250 mg/dl) were selected and used for the study. All the animals were allowed free access to tap water and pellet diet and maintained at room temperature in plastic cages.
This study was approved by Institute’s Animal Ethics Committee vide Resolution no: 08/2011-20129(i)/a/CPCSEA/IAEC/SVU/CHA-SAN/dt.25.09.2011.
Experimental design
Evaluation of antihyperglycemic effect of different extracts of Piper longum root (Plr) in normal and STZ-induced diabetic rats (Short term study)
The animals were divided into six groups and each group consisted of six rats:
Group 1: Untreated normal rats
Group 2: Untreated diabetic rats
Group 3: Diabetic rats treated with 200 mg Plr.hexane extract /kg b.w.
Group 4: Diabetic rats treated with 200 mg Plr.ethylacetate extract /kg b.w.
Group 5: Diabetic rats treated with 200 mg Plr.methanolic extract/kg b.w.
Group 6: Diabetic rats treated with 200 mg Plr. Aqueous extract /kg b.w.
After an overnight fast the diabetic treated rat groups received the ethyl acetate, methanol, aqueous extracts (dissolved in 1 ml of distilled water) and hexane extract (dissolved in 1 ml of 5% Tween 80) by gastric intubation using a force feeding needle. Untreated normal and diabetic rats were fed distilled water alone. Blood samples were collected from the tail vein at 0, 1, 2, 3, 4, 5 and 6 h after the administration of Plr extracts and blood glucose levels were determined by using glucose oxidase–peroxidise reactive strips.
Evaluation of antihyperglycemic activity of PlrAqe in normal and STZ induced diabetic rats - dose dependent study (Short term study)
The animals were divided into 9 groups and each group consisted of six rats:
Group 1: Untreated normal rats
Group 2: Untreated diabetic rats
Group 3: Normal rats treated with 200 mg PlrAqe/kg b.w.
Group 4: Normal rats treated with 300 mg PlrAqe/kg b.w.
Group 5: Normal rats treated with 400 mg PlrAqe/kg b.w.
Group 6: Diabetic rats treated with 200 mg PlrAqe/kg b.w.
Group 7: Diabetic rats treated with 300 mg PlrAqe/kg b.w.
Group 8: Diabetic rats treated with 400 mg PlrAqe/kg b.w.
Group 9: Diabetic rats treated with 0.02 g glibenclamide/kg b.w.
After an overnight fast diabetic treated groups received PlrAqe suspended in distilled water in respective doses, where as untreated normal and diabetic rat groups were fed with distilled water alone with force feeding needle. Blood samples were collected from the tail vein at 0, 1, 2, 3, 4, 5 and 6 h after the administration of PlrAqe and blood glucose levels were determined by using glucose oxidase–peroxidase reactive strips.
Phytochemical analysis was carried out in the PlrAqe by different methods of phytochemical analysis [14].
Effect of PlrAqe on oral glucose tolerance (OGT) in normal rats
The rats were divided into three groups, with 6 animals (n = 6) in each group.
Group 1: Normal untreated rats
Group 2: Normal rats treated with 0.02 g glibenclamide/kg b.w
Group 3: Normal rats treated with 200 mg PlrAqe/kg b.w.
After an overnight fast group 2 & Group 3 rats were fed with glibenclamide and PlrAqe respectively. Normal untreated rats (group 1) were fed with distilled water alone. Thereafter, following 30 min of post extract and drug administration all the animals were fed with glucose (2 g/kg.b.wt). Blood samples were collected from tail veins prior to dosing and after 30, 60, 90 and 120 min of glucose administration. FBG levels were analyzed using glucose-oxidase-peroxidase reactive strips (Accu-chek, Roche Diabnostics, GmbH, Germany) [15].
Effect of long term treatment with PlrAqe on glycemic control, lipid profile, hepatic and renal function markers in diabetic rats
The rats were divided into 5 groups and each group consisted of 6 rats.
Group 1: Normal untreated rats.
Group 2: Normal rats treated with 200 mg PlrAqe /kg b.w/day.
Group 3: Diabetic untreated rats.
Group 4: Diabetic rats treated with 200 mg PlrAqe /kg b.w/day.
Group 5: Diabetic rats treated with 0.02g of glibenclamide/kg b.w/day.
PlrAqe or glibenclamide was administered to the rats every day morning for 30 days by gastric intubation using oral gavage. Blood samples were collected from tail veins before the start of the treatment and on 10th, 20th and 30th days of the treatment and fasting blood glucose levels were estimated. All the five groups of rats were sacrificed on the 30th day after an overnight fast, by anesthetizing with anesthetic ether and further by cervical dislocation and then blood, liver and kidney were collected and immediately stored at -20°C till further analysis. Body weights of all the animals were recorded prior to the treatment and sacrifice.
Analytical procedures
Estimation of blood glucose was carried out by glucose oxidase–peroxidase method [16]. The estimation of protein was carried out by the Lowry method [17]. HbA1c was estimated by the method of Eross et al. [18]. Estimation of serum cholesterol was carried out by Zlatkis method [19]. Serum triglycerides were estimated by Foster and Dunn method [20] and HDL-cholesterol was estimated by Burstein method [21]. The VLDL cholesterol was calculated using the formula, TG/5 mg/dl. The serum LDL cholesterol was calculated by Friedwald formula [22]. Atherogenic index was calculated by using the formula, TC-HDL-C/HDL-C [23]. Plasma SGOT and SGPT activities were determined by Reitman and Frankel method [24]. Activity of serum alkaline phosphatase (ALP) was determined by p-nitro phenyl phosphate method [25]. Serum creatinine & Serum urea levels were measured by Jaffe’s and diacetyl monoxime methods respectively [26, 27].
Statistical analysis
The results were expressed as mean ± S.D. The statistical analysis of results was carried out using Student t-test and one-way analysis (ANOVA) followed by DMRT.