Antioxidant Capacity-Related Preventive Effects of Shoumei (Slightly Fermented Camellia sinensis) Polyphenols against Hepatic Injury

Abstract

Shoumei is a kind of white tea (slightly fermented Camellia sinensis) that is rich in polyphenols. In this study, polyphenols were extracted from Shoumei. High-performance liquid chromatography (HPLC) showed that the polyphenols included mainly gallic acid, catechin, hyperoside, and sulfuretin. In an in vitro experiment, H₂O₂ was used to induce oxidative damage in human normal hepatic L-02 cells. In an animal experiment, CCl₄ was used to induce liver injury. The in vitro results showed that Shoumei polyphenols inhibited oxidative damage in normal hepatic L-02 cells, and the in vivo results showed that the polyphenols effectively reduced liver index values in mice with liver injury. The polyphenols also decreased aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), triglyceride (TG), total cholesterol (TC), blood urea nitrogen (BUN), nitric oxide (NO), malondialdehyde (MDA), interleukin 6 (IL-6), interleukin 12 (IL-12), tumour necrosis factor alpha (TNF-α), and interferon gamma (IFN-γ) levels and increased albumin (ALB), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) levels in the serum of mice with liver injury. Furthermore, pathological observation showed that the Shoumei polyphenols reduced CCl₄-induced hepatocyte damage. qRT-PCR and Western blotting showed that the polyphenols upregulated the mRNA and protein expression of neuronal nitric oxide synthase (nNOS), endothelial nitric oxide synthase (eNOS), manganese- (Mn-) SOD, copper/zinc- (Cu/Zn-) SOD, CAT, and inhibitor of nuclear factor kappa B (NF-κB) alpha (IκB-α) and downregulated the expression of inducible nitric oxide synthase (iNOS) and NF-κB p65. The Shoumei polyphenols had a preventive effect against CCl₄-induced mouse liver injury equivalent to that of silymarin. The four polyphenols identified as the key substances responsible for this effect mediated the effect through their antioxidant capacity. These results suggest that Shoumei polyphenols are high-quality natural products with liver-protective effects.

Figure 1

Polyphenol extract constituents of Shoumei. (a) Standard chromatograms. (b) Chromatograms of lotus leaf polyphenols. 1: Gallic acid; 2: catechin; 3: hyperoside; 4: sulfuretin.

Figure 2

Effects of Shoumei polyphenols on survival rate of human normal hepatic L-02 cells. Values presented are the mean ± standard deviation (N = 10/group). Sample data in each group come from normal distribution, and the difference in variance between the two groups was significant (P > 0.05). ^a-eMean values with different letters over the bar are significantly different (P < 0.05) according to Tukey's honestly significant difference. Normal: untreated L-02 cells; oxidative damage: hydrogen peroxide-treated L-02 cells.

Figure 3

Effects of Shoumei polyphenols on survival rate of hydrogen peroxide-induced oxidative damage in human normal hepatic L-02 cells. Values presented are the mean ± standard deviation (N = 10/group). Sample data in each group come from normal distribution, and the difference in variance between the two groups was significant (P > 0.05). ^a-eMean values with different letters over the bar are significantly different (P < 0.05) according to Tukey's honestly significant difference. Normal: untreated L-02 cells; oxidative damage: hydrogen peroxide-treated L-02 cells.

Figure 4

Effects of Shoumei polyphenols on SOD, GSH-Px, and CAT activities in hydrogen peroxide-induced oxidative damage in human normal hepatic L-02 cells. Values presented are the mean ± standard deviation (N = 10/group). Sample data in each group come from normal distribution, and the difference in variance between the two groups was significant (P > 0.05). ^a-eMean values with different letters over the bar are significantly different (P < 0.05) according to Tukey's honestly significant difference. Normal: untreated L-02 cells; oxidative damage: hydrogen peroxide-treated L-02 cells.

Figure 5

IκB-α and NF-κB mRNA expression in hydrogen peroxide-induced oxidative damage in human normal hepatic L-02 cells. Values presented are the mean ± standard deviation (N = 10/group). Sample data in each group come from normal distribution, and the difference in variance between the two groups was significant (P > 0.05). ^a-eMean values with different letters over the bar are significantly different (P < 0.05) according to Tukey's honestly significant difference. Normal: untreated L-02 cells; oxidative damage: hydrogen peroxide-treated L-02 cells.

Figure 6

Histopathological observation of the liver of mice with CCl₄-induced hepatic damage. Normal: untreated mice; model: CCl₄-treated mice; SPL: 100 mg/kg Shoumei polyphenols and CCl₄-treated mice; SPH: 200 mg/kg Shoumei polyphenols and CCl₄-treated mice; silymarin: 200 mg/kg silymarin and CCl₄-treated mice.

Figure 7

nNOS, eNOS, and iNOS mRNA (a) and protein (b) expression in liver tissue of mice with CCl₄-induced hepatic damage. Values presented are the mean ± standard deviation (N = 10/group). Sample data in each group come from normal distribution, and the difference in variance between the two groups was significant (P > 0.05). ^a-eMean values with different letters over the bar are significantly different (P < 0.05) according to Tukey's honestly significant difference. Normal: untreated mice; model: CCl₄-treated mice; SPL: 100 mg/kg Shoumei polyphenols and CCl₄-treated mice; SPH: 200 mg/kg Shoumei polyphenols and CCl₄-treated mice; silymarin: 200 mg/kg silymarin and CCl₄-treated mice.

Figure 8

CAT, Gu/Zn-SOD, and Mn-SOD mRNA (a) and protein (b) expression in liver tissue of mice with CCl₄-induced hepatic damage. Values presented are the mean ± standard deviation (N = 10/group). Sample data in each group come from normal distribution, and the difference in variance between the two groups was significant (P > 0.05). ^a-eMean values with different letters over the bar are significantly different (P < 0.05) according to Tukey's honestly significant difference. Normal: untreated mice; model: CCl₄-treated mice; SPL: 100 mg/kg Shoumei polyphenols and CCl₄-treated mice; SPH: 200 mg/kg Shoumei polyphenols and CCl₄-treated mice; silymarin: 200 mg/kg silymarin and CCl₄-treated mice.

Figure 9

IκB-α and NF-κB mRNA (a) and protein (b) expression in liver tissue of mice with CCl₄-induced hepatic damage. Values presented are the mean ± standard deviation (N = 10/group). Sample data in each group come from normal distribution, and the difference in variance between the two groups was significant (P > 0.05). ^a-eMean values with different letters over the bar are significantly different (P < 0.05) according to Tukey's honestly significant difference. Normal: untreated mice; model: CCl₄-treated mice; SPL: 100 mg/kg Shoumei polyphenols and CCl₄-treated mice; SPH: 200 mg/kg Shoumei polyphenols and CCl₄-treated mice; silymarin: 200 mg/kg silymarin and CCl₄-treated mice.