Anti-Stress and Glial Differentiation Effects of a Novel Combination of Cucurbitacin B and Withanone (CucWi-N): Experimental Evidence

Abstract

Background: Natural extracts and compounds used in traditional home medicine are known for their safety and a variety of health promoting and therapeutic potentials. In contrast to the single molecule mediated targets, the combinational therapies are preferred for their multi-functional and limited toxic regimens and may be useful for disease therapeutics as well as to increase the quality of life during a variety of environmental stresses.

Purpose: We aimed to combine the active ingredients of Chinese (Helicteres angustifolia) and Indian (Withania somnifera) ginsengs to develop a natural, efficient, and welfare combinatorial mixture with high anti-stress and glial differentiation potentials.

Methods: Using cultured cells as a model system, we developed a combination of active ingredients of Chinese (Cucurbitacin B [Cuc]) and Indian (Withanone [Wi-N]) ginsengs. Eleven chemical models of environmental stresses were used. Cytotoxicity studies were performed using human skin fibroblast for anti-stress and rat glioma cells for glial differentiation effects.

Results: We demonstrate that the novel combination of Cuc and Wi-N, CucWi-N, was non-toxic to normal cells. It caused stress protection in assays using normal human fibroblasts subjected to a variety of stresses. Of note, cells showed remarkable protection against oxidative and UV stresses and marked by decrease in DNA damage and reactive oxygen species. We examined and found the glial differentiation potential of CucWi-N in rat glioblastoma cells. CucWi-N clearly induced differentiation phenotype, well-marked with upregulation of GAP43, MAP2, and GFAP, which have been shown to play a key role in glial differentiation.

Conclusion: These data demonstrate anti-stress and glial differentiation potential of CucWi-N (a novel combination of Cuc and Wi-N) that could be recruited in nutraceutical and pharmaceutical avenues and hence warrant further evaluation and mechanistic studies.

Keywords: Cucurbitacin B; Glial differentiation; Stress; Therapeutics; Withanone.

Fig. 1.

Anti-stress potential of CucWi-N in normal skin fibroblasts (TIG-3). a Dose response of cells to Cuc and CucWi-N during 48 h treatment; (b), dose titration and determination of IC50 of 48 h H2O2 treatment; (c), dose titration and determination of IC50 of 48 h UV treatment; (d), tabulated chemical/physical models of environmental stresses; (e), viability of control and CucWi-N-treated cells exposed to stresses as indicated by numbered with reference to d; (f), graphical representation of the percent fold change in cell viability of cells exposed to stresses and recovery in CucWi-N supplemented medium with respect to the ones recovered in normal medium. CucWi-N, Cucurbitacin B and Withanone. * p < 0.05, ** p < 0.01, *** p < 0.001.

Fig. 2.

Effect of CucWi-N on stress-induced increase in DNA damage and ROS levels in normal skin fibroblasts (TIG-3). a Comet assay images of control and CucWi-N-treated TIG-3 cells with/without UV stress (5 mJ/cm2). Length of the “tail” corresponds to the extent of DNA damage. Whereas UV-treated cells showed increase in tail DNA, CucWi-N treatment caused decrease in tail length; (b), ROS assay images of control and H2O2 (600 µM) stressed cells recovered either in control or CucWi-N-supplemented medium. CucWi-N-treated cells showed protection against H2O2-induced increase in ROS. Quantitation of (a) and (b) is shown on the right panels, respectively. * p < 0.05, ** p < 0.01, *** p < 0.001.

Fig. 3.

Effect of CucWi-N on viability and differentiation phenotypes of rat glial fibroblasts (C6 cells). a Cytotoxicity and dose response of C6 cells to CucWi-N: IC01 (low), IC10 (medium) and IC50 (high) were determined for 24 and 48 h treatment regimens; (b), Morphology of cells treated with indicated doses of CucWi-N. Phase contrast, GFP, and crystal violet-stained images are shown. * p < 0.05, ** p < 0.01, *** p < 0.001.

Fig. 4.

Effect of CucWi-N on markers for glial differentiation. a Immunostained images of CucWi-N treated C6 cells demonstrating a dose-dependent increase in glial cell differentiating protein markers GAP43, MAP2, and GFAP; (b), immunoblotting images of CucWi-N treated C6 cell lysates demonstrating dose-dependent increase in glial cell differentiating protein markers (top) with quantitation (bottom). * p < 0.05, ** p < 0.01, *** p < 0.001.