B-175 Phytochemicals in K. pinnata and the Antioxidant Activity of Combined K. pinnata and Metformin Preparations on Normal and Diabetic Skeletal Muscle Cells

Abstract

Abstract Background Diabetes Mellitus is associated with oxidative stress, which may play a central role in developing diabetic complications. With the rise in healthcare costs, more people globally are choosing to complement their pharmacological regimen with dietary supplements from natural products. In this study, the compound composition of K. pinnata and the effects of combined preparations of K. pinnata and metformin on antioxidant activity in human skeletal muscle myoblasts (HSMM) and human diabetic skeletal muscle myoblasts (DHSMM) were investigated. Methods K. pinnata leaves were crushed and extracted with hot water, then gravity filtered and freeze-dried. A 1 mL aliquot of the Kalanchoe pinnata extract solution was utilized to determine the chemical composition of the herbal extract. Liquid chromatography-mass spectrometry (LC-MS) was run in negative ion mode, with the K. pinnata extract analyzed in triplicate. A computer program further analyzed the LC-MS data to determine the chemical components in K. pinnata aqueous extract. A database algorithm within Compound Discoverer was used to identify the compounds in K. pinnata aqueous extract. For subsequent cell culture experiments, the K. pinnata leaf extract was weighed and combined with aqueous metformin solutions to prepare various combinatorial treatments. Human skeletal muscle myoblast cells and diabetic human skeletal muscle myoblast cells (Lonza Inc, Walkersville, MD) were cultured and incubated at 37 °C with 5% CO2. A CCK-8 cytotoxicity assay was performed to determine cellular viability, while trypan blue was used to assess the cellular proliferation of in-vitro cells. The effects of combinatorial preparations of K. pinnata and metformin on antioxidant indices were determined in HSMM, DHSMM, and HSMM H2O2 stress-induced cells. Results Liquid chromatography-mass spectrometry (LC-MS) showed that K. pinnata contains biologically active flavanols. The main compounds identified in locally grown K. pinnata were quercetin, kaempferol, apigenin, epigallocatechin gallate (EGCG), and avicularin. Antioxidant results indicated that a combinatorial preparation of K. pinnata with metformin might modulate antioxidant responses by increasing the enzymatic activity of superoxide dismutase and increasing levels of reduced glutathione. A combination of 50 μM and 150 μg/mL of metformin and K. pinnata resulted in a significant increase in reduced glutathione levels in non-diabetic and diabetic human skeletal muscle myoblasts and H2O2 stress-induced human skeletal muscle myoblasts. Additionally, a K. pinnata treatment (400 µg/mL) alone significantly increased catalase (CAT) activity for non-diabetic and diabetic human skeletal muscle myoblasts and an H2O2 stress-induced human skeletal muscle myoblast cell line while significantly lowering malondialdehyde (MDA) levels. The antioxidant activity of the treatment options was more effective at promoting cell viability after 24 h of treatment vs 72 h. Conclusion These treatment options show promise, especially after 24 h for treating oxidative stress-mediated pathophysiological complications associated with type II diabetes.