Hepatoprotective Effect of Steroidal Glycosides From Dioscorea villosa on Hydrogen Peroxide-Induced Hepatotoxicity in HepG2 Cells

Abstract

Dioscorea villosa, commonly known as "Wild Yam" and native to North America, is well documented for its pharmacological properties due to the presence of steroidal glycosides. However, the hepatoprotective potential of these compounds has not been studied so far. The present investigation was aimed to study the hepatoprotective effect of the steroidal glycosides from D. villosa against H₂O₂, a known hepatotoxin, in human liver cell line (HepG2). Cytotoxicity assessment was carried out in cells exposed to various concentrations (10-50 μM) of compounds for 24 h using MTT assay and morphological changes. All tested compounds were known and among them, spirostans (zingiberensis saponin I, dioscin, deltonin and progenin III) were found to be cytotoxic whereas, furostans (huangjiangsu A, pseudoprotodioscin, methyl protobioside, protodioscin, and protodeltonin) were non-cytotoxic. Further, HepG2 cells were pretreated with biologically safe concentrations (10, 30, and 50 μM) of non-cytotoxic compounds and then cytotoxic (0.25 mM) concentration of H₂O₂. After 24 h, cell viability was assessed by MTT and NRU assays, while morphological changes were observed under the microscope. The results showed that treatment of HepG2 cells with compounds prior to H₂O₂ exposure effectively increased cell viability in a concentration-dependent manner. Furthermore, huangjiangsu A, pseudoprotodioscin, methyl protobioside, protodioscin, and protodeltonin at 50 μM increased GSH level and decreased intracellular ROS generation against H₂O₂-induced damages. The results from this study revealed that compounds isolated from D. villosa have hepatoprotective potential against H₂O₂-induced cytotoxicity and ROS generation and could be promising as potential therapeutic agents for liver diseases.

Keywords: Dioscorea villosa; Dioscoreaceae; H2O2; ROS generation; cytotoxicity; steroidal glycosides.

FIGURE 1

Structures of isolated compounds from Dioscorea villosa.

FIGURE 2

Cytotoxicity assessment by MTT assay in HepG2 cells following the exposure of different concentrations of compounds isolated from Dioscorea villosa. All values are given as mean ± SD. (A) Non-cytotoxic compounds and (B) Cytotoxic compounds. ^∗p < 0.05, ^∗∗p < 0.01 versus control.

FIGURE 3

Representative images of the morphological changes in HepG2 cells after the exposure of the compounds isolated from Dioscorea villosa. All the images were acquired using a phase contrast inverted microscope at 20× magnification. Each scale bar = 1 mm.

FIGURE 4

Cytotoxicity assessments by MTT assay in HepG2 cells following the exposure of various concentrations of H₂O₂ for 24 h. All values are given as mean ± SD. ^∗p < 0.05, ^∗∗p < 0.01 versus control.

FIGURE 5

Assessment of protective potential of compounds on cell viability of HepG2 cells by (A) MTT assay and (B) NRU assay. Cells were exposed to 10, 30, and 50 μM of non-cytotoxic compounds for 24 h. Then the cells were exposed to H₂O₂ at 0.25 mM for 24 h. All values are given as mean ± SD. ^∗p < 0.05, ^∗∗p < 0.01 versus H₂O₂.

FIGURE 6

Morphological changes observed in HepG2 cells. Cells were pre-exposed with non-cytotoxic compounds for 24 h and then H₂O₂ (0.25 mM) for 24 h. Images were acquired using a phase contrast inverted microscope at 20× magnification. Each scale bar = 1 mm.

FIGURE 7

Protective potential of compounds isolated from Dioscorea villosa on depletion in glutathione level. HepG2 cells were exposed to 50 μM of non-cytotoxic compounds for 24 h prior to the addition of H₂O₂ for 24 h. All values are represented as mean ± SD. (i) Control; (ii) H₂O₂ (0.25 mM); (iii) Huangjiangsu A (50 μM) + H₂O₂ (0.25 mM); (iv) Pseudoprotodioscin (50 μM) + H₂O₂ (0.25 mM); (v) Methyl protobioside (50 μM) + H₂O₂ (0.25 mM); (vi) Protodioscin (50 μM) + H₂O₂ (0.25 mM); and (vii) Protodeltonin (50 μM) + H₂O₂ (0.25 mM). #p < 0.01 versus control, ^∗p < 0.05 and ^∗∗p < 0.01 versus H₂O₂ exposure.

FIGURE 8

(A) H₂O₂-induced ROS generation and ameliorative effect of pre-treatment of compounds in HepG2 cells and (B) percent change in ROS generation. ROS generation was evaluated using dichlorodihydrofluorescein diacetate (DCFH-DA) dye. (i) Untreated control; (ii) H₂O₂ (0.25 mM); (iii) Huangjiangsu A (50 μM) + H₂O₂ (0.25 mM); (iv) Pseudoprotodioscin (50 μM) + H₂O₂ (0.25 mM); (v) Methyl protobioside (50 μM) + H₂O₂ (0.25 mM); (vi) Protodioscin (50 μM) + H₂O₂ (0.25 mM); and (vii) Protodeltonin (50 μM) + H₂O₂ (0.25 mM). ^#p < 0.01 versus control, ^∗p < 0.05 versus H₂O₂ exposure. Each scale bar = 1 mm.