ANTIDIABETIC, ANTIOXIDANT, AND ANTI-INFLAMMATORY EFFECTS OF A NOVEL POLYHERBAL FORMULATION IN DIABETES MELLITUS

Abstract

Diabetes mellitus is characterized by hyperglycemia, oxidative stress and chronic inflammation. Medicinal plants with complementary phytochemical profiles provide a promising multi-targeted strategy, but reproducible analytical standardization and integrated in vitro evaluations are often lacking. To prepare and standardize a polyherbal formulation (PHF) containing Withania somnifera, Tinospora cordifolia and Gymnema sylvestre and to evaluate its in vitro antioxidant, antidiabetic enzyme inhibition and anti-inflammatory properties using validated analytical and biochemical methods. Extracts of authenticated plant materials were prepared in 70% ethanol, concentrated and blended in a 2:2:1 (WS:TC:GS) ratio. Standardization included organoleptic assessment, pharmacopeial physicochemical parameters, total phenolic and flavonoid content, HPTLC fingerprinting and validated HPLC quantification of marker compounds. Antioxidant activity was measured by DPPH, ABTS, FRAP and nitric oxide scavenging assays. In vitro antidiabetic potential was assessed by α-amylase, α-glucosidase and DPP-4 inhibition assays. Anti-inflammatory activity was evaluated via protein denaturation inhibition, membrane stabilization (HRBC), and exploratory nitric oxide inhibition in LPS-stimulated RAW 264.7 macrophages. Data were analyzed by ANOVA with Tukey post hoc test. The PHF displayed reproducible physicochemical parameters (moisture 6.2 ± 0.3%, total ash 5.8 ± 0.2%, ethanolic extractive 18.4 ± 0.9%), TPC 124.6 ± 4.5 mg GAE/g and TFC 38.2 ± 1.7 mg QE/g. HPLC quantification yielded withanolide A 1.52 ± 0.06 mg/g, tinospora-equivalent 2.34 ± 0.08 mg/g and gymnemic acid 3.12 ± 0.11 mg/g. Antioxidant IC50s were DPPH 32.4 ± 1.9 µg/mL and ABTS 28.7 ± 1.6 µg/mL; FRAP 910 ± 35 µmol Fe2+/g. Enzyme inhibition IC50s were α-glucosidase 48.6 ± 2.7 µg/mL, α-amylase 85.3 ± 4.4 µg/mL and DPP-4 112.1 ± 6.0 µg/mL. Anti-inflammatory effects included protein denaturation inhibition 68.1 ± 3.2% at 200 µg/mL and reduction of LPS-induced nitric oxide by 42.7 ± 3.5% at 100 µg/mL (P < 0.05). The standardized PHF shows reproducible chemical markers and multi-modal in vitro activities relevant to diabetes management, warranting further preclinical and clinical investigation.