2.9 Estimation of in-vitro Inhibitory α-amylase Assay:
In-vitroanti diabetic activity was estimated using α-amylase inhibition assay. The plant extract and standard acarbose solutions were prepared in different concentrations of 20mg/mL, 40mg/mL, 60mg/mL, 80mg/mL, 100mg/mL and 120mg/mL. Absorbance was measured at 540 nm (Razzaque et al. 2020). The inhibitory concentration was calculated from prepared inhibition curve. The %age inhibition of α-amylase activity was measured by,
Where,
Ac = Control’s absorbance
Aₛ = Standard’s absorbance
Aₑ = Extract’s absorbance
2.10 Experimental Animals:
Albino-Wistar rats of either gender (200g-250g) were kept in cages under standard conditions (20–25˚C), fed with standard pellet food and water ad libitum. An ethical approval of the experimental protocol was obtained from the Institutional Research Ethics Committee (IREC-2018-63) of the University of Lahore and the experiments were conducted according to these institutional guidelines.
2.11 Acute Toxicity Study:
Acute oral toxicity was performed according to the Organization of Economic Cooperation and Development (OECD) 423 guidelines. Wistar rats were divided into 4 groups. Rats were administered with single dose of 200mg/kg, 2000mg/kg and 5000mg/kg aqueous-ethanol extract of Cyperus iria each along with normal control group. The dose was administered in 12-h fasted rats through oral gavage. After administration, the rats were carefully observed for behavioral patterns at 0.5,4,16, 24 and 48 hours. After 48 hours, the animals were dissected for the histopathological examination of liver and pancreas.
2.12 Oral Glucose Tolerance Test (OGTT):
24 hours fasted wistar rats of either sex were selected. The blood glucose level was measured and hyperglycemia was induced with glucose solution, followed by the administration of metformin (500 mg/kg) and extract at dose levels of 125, 250 and 500 mg/kg BW. Glucose level was monitored at 0min 30min, 60min, 90min, 120min, 180min and 210min by withdrawing blood sample from the tip of tail vein using a glucometer. Anti-hyperglycemic inhibition was calculated following the protocol of (Amuri et al. 2017)
2.13 Experimental design:
NCG: Negative control group (n = 5) received normal saline (1 ml/ kg) orally
DCG: Diabetic control group (n = 5) diabetes induced by streptozotocin (STZ) (55 mg/kg), nicotinamide (110 mg/kg) intraperitoneal
PCG: Positive control group (n = 5) administered metformin orally in dose of 500mg/kg after inducing diabetes.
E125: Treatment group (n = 5) administered 125mg/kg aqueous-ethanol extract of Cyperus iria after inducing diabetes.
E250: Treatment group (n = 5) that were administered 250 mg/kg aqueous-ethanol extract of Cyperus iria after inducing diabetes.
E500: Treatment group (n = 5) that were administered 500mg/kg aqueous-ethanol extract of Cyperus iria after inducing diabetes.
2.14 Induction of diabetes
Wistar rats were starved over-night. Freshly prepared nicotinamide (110 mg/kg BW) dissolved in normal saline was administered intraperitoneal (IP) and after a 15 min interval STZ (55 mg/kg BW) dissolved in 0.1 molar (M) citrate buffer (pH 4.5) was injected in a single dose intraperitoneally. The rats were given 5% dextrose solution overnight after the STZ administration to avoid hypoglycemic shock. The blood glucose levels were assessed by drawing blood from the tail vein by an Accu-Check glucometer (Roche). After 72 hours of STZ administration, the rats having glucose level exceeding 250mg/dL were selected for the study and considered diabetic (Zafar et al. 2020)
2.15 Anti-Hyperglycemic Activity:
After the induction of diabetes, the rats were monitored for 15 days. The rats were given their accurate, freshly prepared doses by an oral gavage tube, once a day. The fasting blood glucose levels were checked at 0, 3, 6. 9, 12 and 15 days using an Accu-Check glucometer (Roche, Switzerland).
2.16 Estimation of Inflammatory Cytokines:
Pro-inflammatory cytokines were measured in the serum samples of all treated groups. Rat Eliza kits K1052, K4145 and E4801 were used to evaluate Tumor Necrosis Factor alpha (TNF-α), Interleukin-6 (IL-6) and Cyclooxygenase-2 (COX-2) respectively according to manufacturer instructions Bio vision, Inc, California. The Serum samples were allowed to clot for 30 min in serum separator tubes followed by centrifugation at approximately 3000×g for 10 min (Malik et al. 2020).
2.17 Estimation of Antioxidant Enzymes:
The activity of defense antioxidants superoxide dismutase (SOD) and Glutathione S-transferase (GSH-ST) were measured in plasma samples of animals using commercial kits obtained from Nanjing Jiancheng Bioengineering Institute China as per manufacturer’s instructions. Blood sample (3 mL) was collected in heparinized tubes from each rat. It was centrifuged for 10min at 1000 rpm at 4°C. Plasma as supernatant (yellow layer) was cautiously drawn and positioned on icepack (Sharif et al. 2016b).
2.18 Biochemical Parameters:
Overnight fasted rats were sacrificed by euthanasia. Blood was collected by direct heart puncture. The separated serum was stored at -20°C for further analysis of its biochemical parameters including liver function tests, renal function tests and lipid profile (Khan et al. 2020).
2.19 Histopathology
The liver, kidney, and pancreas of treated and control groups were dissected out, and rinsed with ice-cold saline solution. These organs were preserved in 10% neutral formalin solution. Paraffin blocks were prepared after being washed, dehydrated with alcohol and cleared by xylene. A rotary microtome was used for cutting sections with a thickness of 4–5 mm and stained with hematoxylin and eosin. The slides were observed for histopathological changes under light microscope (optika vision lite 2.1) (Sharif et al. 2016a).
2.20 Statistical analysis:
The results are expressed as mean ± SD. The IC50 was calculated by non-linear regression model using Graph Pad Prism 6.0. Significant difference was assessed between diabetic control and experimental groups. One-way analysis of variance (ANOVA) was performed using Graph Pad Prism 5.0 software followed by Tukey’s test and probability level p < 0.05 was considered statistically significant. The oral glucose tolerance test was evaluated by measuring area under the curve (AUC).