IN VITRO AND IN VIVO EVALUATION OF ISOLATED COMPOUNDS FROM POLYHERBAL EXTRACTS FOR ENZYME INHIBITION IN DIABETES MELLITUS

Narendar Bhojak, Rohit Srivastava, Swetlana Gautam, Vrushali Sanjay Bais, Monika Yadav, Mahesh Kumar Gupra, Renu Solanki*

ABSTRACT

The clinical value of most of the traditional polyherbal preparations in diabetes mellitus is in a disadvantaged
position since they have not been characterized in terms of their active principles and their exact mechanism of
action. The objective of the present exploration was the isolation, characterization, and assessment of the
antidiabetic activity of selected compounds in a polyherbal mixture (Phaseolus vulgaris, Trigonella foenumgraecum,
Panax ginseng, Spinacia oleracea) by means of bio-guided fractionation process aimed at carbohydratedigesting
enzymes. A-glucosidase and a-amylase in vitro inhibition by the methanol extract of the polyherbal
combination was significant. Through bioassay-directed purification, CPH-01, a flavonoid glycoside, and CPH-02,
a derivative of phenolic acid, were purified. Enzymatic tests in vitro showed that CPH-01 was an extremely active
and selective a-glucosidase inhibitor (IC50 = 12.3 uM) with an activity about 5-fold higher than that of the standard
medication acarbose. CPH-01 (100 mg/kg/day 4 weeks) as an oral solution was found to induce remarkable
antihyperglycemic effects in high fat diet/streptozotocin rat model of type 2 diabetic rats, with a significant
reduction in fasting blood glucose (by 58.4%), glucose tolerance and HgA1c. This treatment also alleviated
diabetic dyslipidemia and served as an improvement of hepatic and renal functional markers and enhanced
antioxidant defenses in pancreatic tissue by increasing SOD, CAT and GSH levels and decreasing lipid
peroxidation. A toxicity study in acute oral case had defined a good safety profile of CPH-01 at a limit dose of 2000
mg/kg. Molecular docking experiments showed that CPH-01 is a potent inhibitor due to the presence of strong
hydrogen-bonds with active site residues (Asp215, Glu277, Arg442) in the a-glucosidase active site. Finally, this
study has been able to designate CPH-01 as the novel, potent, and selective a-glucosidase inhibitor, having high in
vivo efficacy and antioxidant capacity, making it a successful polyherbal formulation, and a characterized lead drug
in developing a multi-target phytotherapeutic agent to manage diabetes.

Keywords: Diabetes Mellitus; Polyherbal Extract; Bioactive Compounds; Enzyme Inhibition; ?-Glucosidase; ?-Amylase; In Vivo Antidiabetic Activity; Insulin Sensitivity; Glycemic Control.