Antioxidant activity of a new multiflorane-type triterpene from Cucurbita argyrosperma seeds and their protective role in hydrogen peroxide induced oxidative stress

Background: Cucurbita argyrosperma seeds have acquired a reputation as an herbal remedy to treat various diseases because this plant is a predominant source of natural compounds with powerful anti-inflammatory and antioxidant properties, and supplementation with seeds improves oxidative stress. Previous studies indicated that an imbalance between H2O2 production and elimination capacity is responsible for β-cell vulnerability thereby making β-cells a susceptible target for pathogens. The aim of this investigation was to evaluate the protective effects of one new multiflorane-type triterpene 3β-trans-caffeoyloxymultiflor-8-ene-7α,12β, 18 β-triol (1) from MeOH extract from C. argyrosperma, on rat pancreatic β cells (INS-1 cells) exposed to hydrogen peroxide (H2O2) induced oxidative stress conditions. Methods: The chemical structure of the novel triterpene, which was identified as 3β-trans-caffeoyloxymultiflor-8-ene-7α,12β, 18 β-triol (1), was established based on the interpretation of spectroscopic analyses. The antioxidant activities of 1 were led by the detected radical scavenging potential of 2,2-dyphenyl-1-picrylhydrazyl (DPPH) and 3.1 2,2′-Azino-bis(3-Ethylbenzothiazoline-6-Sulfonic Acid) ABTS. The assays were conducted on INS-1 cell lines exposed to increasing concentrations of 1 at 5, 10 and 20 µg/mL and H2O2 at 250 µM. Then the experiments, cell viability, cell integrity (LDH; lactate dehydrogenase release), mitochondrial function (ATP analysis), ROS formation, lipid peroxidation (MDA), and caspase-3, 9 activities were measured in the cells. We also determined the effect of 1 on antioxidant enzyme levels and cytotoxicity in pancreatic β cells under oxidant conditions. Results: The results showed that triterpene displayed high free-radical-scavenging activity, which is similar to that of standard antioxidants used. Concentrations of 5, 10, and 20 μg/mL protected INS-1 cells against H2O2 induced cytotoxicity with a decrease in cell death and a marked increase in cell viability as well as sustained cellular functionality (ATP). Antioxidant enzymes such as glutathione peroxidase (GPx), glutathione reduced (GSH), catalase (CAT), superoxide dismutase (SOD), and the non-antioxidant enzyme (GSH) increased in INS-1 cells with 1 pre-treatment. MDA in pancreatic cells was ameliorated by 1 pre-treatment reducing intracellular reactive oxygen species level. Findings also demonstrated that H2O2 induced apoptosis in INS-1 cells and produced modulation of the caspase-3, 9 expressions in INS-1 cells exposed to 1. Exposure to 1 significantly inhibited ROS and apoptosis, reducing β cell dysfunction under oxidant conditions. Conclusions: Triterpene consequently could be a promising natural antioxidant for use in maintaining the integrity of pancreatic β-cells exposed to oxidative stress conditions and being able to participate in the control type 2 diabetes.