Comparison of the Antioxidant Potency of Four Triterpenes of Centella asiatica against Oxidative Stress

Abstract

We comparatively evaluated the antioxidant properties of key triterpenes from Centella asiatica, including asiatic acid (AA), asiaticoside, madecassic acid, and madecassoside, in several cell types, including skin fibroblasts, macrophages, hepatocytes, and endothelial cells, under conditions promoting oxidative stress. AA conferred the highest viability on Hs68 cells exposed to ultraviolet B (UVB) irradiation. Triterpene pretreatment attenuated the UVB-induced generation of reactive oxygen species (ROS) and malondialdehyde (MDA), as well as the UVB-induced depletion of glutathione (GSH) in skin fibroblasts. AA most potently inhibited UVB-induced MMP generation, resulting in increased intracellular collagen levels. Pretreatment with triterpenes, particularly AA, significantly improved cell viability and attenuated TBHP-induced levels of ROS, alanine aminotransferase, and aspartate aminotransferase in HepG2 cells. Triterpenes attenuated ROS levels and reduced MDA and GSH expression in EA.hy926 cells. In RAW264.7 macrophages, production of nitric oxide, tumor necrosis factor-α, and interleukin-6 (indicators of LPS-induced oxidative damage) was significantly reduced by treatment with any of the triterpenes. Statistical analyses of triterpene biological activities using principal component analysis and hierarchical clustering revealed that AA exerted the greatest overall influence and showed remarkable activity in Hs68 and HepG2 cells.

Keywords: Centella asiatica; antioxidant activity; oxidative stress; triterpenes.

Figure 1

Structures of asiaticoside, madecassoside, asiatic acid, and madecassic acid.

Figure 2

Effects of triterpenes (10 μM) on cell viability (A), protective effect (B), UVB-induced reactive oxygen species production (C), GSH depletion (D), and MDA production (E) in Hs68 cells. Values are expressed as means ± SD (n = 3). Different letters indicate significant (p < 0.05) differences based on Duncan’s multiple range test level. CON, control; UVB, ultraviolet B; GSH, glutathione; MDA, malondialdehyde; AA, asiatic acid; AD, asiaticoside; MA, madecassic acid; MD, madecassoside.

Figure 3

Effects of triterpenes (10 μM) on UVB-induced MMP-1 (A), MMP-3 (B), and collagen (C) production in Hs68 cells. Values are means ± SD (n = 3). Different letters indicate significant (p < 0.05) differences based on Duncan’s multiple range test level. CON, control; UVB, ultraviolet B; MMP-1, matrix metalloproteinase-1; MMP-3, matrix metalloproteinase-3; AA, asiatic acid; AD, asiaticoside; MA, madecassic acid; MD, madecassoside.

Figure 4

Effects of triterpenes (5 μM) on cytotoxicity (A), protective effects (B), reactive oxygen species production (C), ALT (D), and AST (E) in HepG2 cells. Values are expressed as means ± SD (n = 3). Different letters indicate significant (p < 0.05) differences based on Duncan’s multiple range test level. CON, control; TBHP, tert-butyl hydroperoxide; ALT, alanine transaminase; AST, aspartate aminotransferase; AA, asiatic acid; AD, asiaticoside; MA, madecassic acid; MD, madecassoside; ns, not significant.

Figure 5

Effects of triterpenes (2.5 μM) on cell viability (A) and their protective effects (B) in H₂O₂-induced EA.hy926 cells. Effects of triterpenes (2.5 μM) on H₂O₂-induced ROS production (C), GSH depletion (D), and MDA production (E) in EA.hy926 cells. Effects of triterpenes (2.5 μM) on NO generation (F) in EA.hy926 cells. Values are expressed as means ± SD (n = 3). Different letters indicate significant (p < 0.05) differences based on Duncan’s multiple range test level. CON, control; H₂O₂, hydrogen peroxide; GSH, glutathione; MDA, malondialdehyde; NO, nitric oxide; AA, asiatic acid; AD, asiaticoside; MA, madecassic acid; MD, madecassoside; ns, not significant.

Figure 6

Effects of triterpenes (10 μM) on cell viability (A), NO production (B), TNF-α (C), and IL-6 (D) in LPS-induced RAW264.7 cells. Values are expressed as means ± SD (n = 3). Different letters indicate significant (p < 0.05) differences based on Duncan’s multiple range test level. CON, control; NO, nitric oxide; LPS, lipopolysaccharide; TNF-α, tumor necrosis factor-α; IL-6, interleukin-6; AA, asiatic acid; AD, asiaticoside; MA, madecassic acid; MD, madecassoside; ns, not significant.

Figure 7

Two-dimensional scatter diagram of principal component analysis based on triterpene biological activities. AA, asiatic acid; AD, asiaticoside; MA, madecassic acid; MD, madecassoside; EA, EA.hy926 cells; HG, HepG2 cells; HS, Hs68 cells; RW, Raw264.7 macrophages; GSH, glutathione; ROS, reactive oxygen species; NO, nitric oxide; TNF-a, tumor necrosis factor-α; C, collagen; AST, aspartate aminotransferase PE, protective effect; IL-6, interleukin-6; MMP3, matrix metalloproteinase-3; ALT, alanine aminotransferase; MDA, malondialdehyde; MMP1, matrix metalloproteinase-1.

Figure 8

Hierarchical clustering analysis of triterpenes based on triterpene biological activities. AA, asiatic acid; AD, asiaticoside; MA, madecassic acid; MD, madecassoside; EA, EA.hy926 cells; HG, HepG2 cells; HS, Hs68 cells; RW, Raw264.7 macrophages; GSH, glutathione; ROS, reactive oxygen species; NO, nitric oxide; TNF-a, tumor necrosis factor-α; C, collagen; AST, aspartate aminotransferase PE, protective effect; IL-6, interleukin-6; MMP3, matrix metalloproteinase-3; ALT, alanine aminotransferase; MDA, malondialdehyde; MMP1, matrix metalloproteinase-1.