Antidiabetic activity of Rhinacanthins-rich extract and Rhinacanthin-C

Abstract

Rhinacanthins-rich extract (RRE) is a semi purified leaf extract that contains 60% w/w of rhinacanthin-C (RC) obtained from Rhinacanthus nasutus leaf, a popular medicinal plant used in Thai traditional medicine and an herbal drink in Taiwan and China. RRE was prepared by microwave assisted green extraction method along with a simple step of fractionation using Amberlite® column. RC, rhinacanthin- D (RD) and rhinacanthin-N (RN) were isolated from the RRE using silica gel column chromatography. In this study, RRE and its marker compounds were investigated for their antidiabetic potential using in vitro a-glucosidase inhibition, superoxide scavenging, antiglycation, glucose uptake stimulation and adipogenic inhibition assays. An in vivo experiment using nicotinamide-streptozotocin induced diabetic rats was also performed to validate and compare the hypoglycemic and hypolipidemic activity of RRE with its major constituent RC. Moreover, in silico studies were conducted to predict structure activity relationship, pharmacokinetic and toxicity profile of rhinacanthins. RRE (IC50 value of 25.0 μg/mL) exhibited a-glucosidase inhibitory activity nearly equal to that of RC (IC50 value of 22.6 μg/mL) but stronger than that of RD (IC50 value of 71.5 μg/mL) and the standard drug, acarbose (IC50 value of 395.4 μg/mL), while RN was inactive. Kinetic studies revealed that both RRE and RC exhibited noncompetitive a-glucosidase inhibitory activity, while combinations of either RRE or rhinacanthin-C with acarbose (competitive inhibitor) at low concentrations (IC50, IC50 and IC50) showed a synergistic inhibitory effect. In silico studies identified the binding mode of RC highlighting the formation of both polar and apolar contacts of ligand with a-glucosidase. Superoxide scavenging activity was performed by cyclic voltammetry and fructose mediated human serum albumin glycation model was used for antiglycation activity. Molecular interaction studies were conducted to identify the structure activity relationships of rhinacanthins. On the basis of kinetic measurements, RRE exhibited the most potent antioxidant activity via E,C; mechanism, with an ICso value of 8.0 μg/mL, antioxidant capacity of 39439 M and binding constant of 45709 M''. Antiglycation assay showed that RRE exhibited almost same glycation inhibitory effect to that of RC, with ICso values of 39.7, and 37.3 μg/mL, respectively, but higher than that of RD (IC50 of 50.4 μg/mL), RN (IC50 of 89.5 μg/mL) as well as the positive control, rutin (IC50 of 41.5 μg/mL). The potent superoxide scavenging and albumin glycation inhibitory effect of RRE rationalized its therapeutic application in various chronic diseases especially in the complications of diabetes. Obesity is one of the imperative dynamic in the incidence and intensification of type 2 diabetes mellitus (T2DM). R. nasutus leaf extracts are previously reported for their antidiabetic and antiobesity potential. In the present study, RRE and its marker compounds have been evaluated for glucose uptake stimulatory and antiadipogenic activities in 3T3-L1 and L6 cells. Glucose uptake stimulation in both 3T3-L1 and L6 cells was performed by quantification of residual glucose in the media using glucose oxidase kit. Antiadipogenic activity in 3T3-L1 adipocytes was performed by intracellular lipids quantification using oil red O dye. At the highest effective dose, RRE (20 μg/mL) exhibited satisfactory glucose uptake stimulatory effect in 3T3-L1 adipocytes that is nearly equal to RN (20 μg/mL) and the positive control insulin (0.58 μg/mL), but higher than RC (20 μg/mL) and RD (20 μg/mL). In addition, treatments of L6 myotubes showed that RRE (2.5 μg/mL) exhibited potent and same glucose uptake stimulation (>80%) to RC (2.5 μg/mL) and the standard drugs, insulin (2.90 μg/mL) and metformin (219.5 μg/mL), but higher than RD (2.5 μg/mL) and RN (2.5 μg/mL). Furthermore, RRE (20 μg/mL) exhibited potent antiadipogenic effect in 3T3-L1 adipocytes, which was equivalent to RC (20 μg/mL) but higher than RD (20 μg/mL) and RN (20 μg/mL). In animal experiments, RRE (24.11 mg/kg equivalent to 15 mg/kg RC content), RC (15 mg/kg) and the standard drug glibenclamide (600 μg/kg) were comparatively assessed for their hypoglycemic and hypolipidemic activity in nicotinamide-streptozotocin induced diabetic rats for 28 days. RRE and RC significantly reduced the fasting blood glucose, HbA1c and food/water intake, while increased the insulin level and body weight in diabetic rats without affecting the normal rats. The serum lipid, liver and kidney biomarkers were markedly normalized by both RRE and RC in diabetic rats without affecting the normal rats. Moreover, the histopathology of pancreas revealed that RRE and RC evidently restored the islets of Langerhans in diabetic rats. The overall results indicated that RRE has significantly comparable antidiabetic potential to that of RC. Furthermore, the in silico pharmacokinetic and toxicity analysis predicts that RC is orally non-toxic, non- carcinogenic and non-mutagenic with a decent bioavailability. The undertaken study suggests that RRE could be used as an effective natural remedy in the treatment of diabetes.