Vitex agnus-castus L. (Verbenaceae) Improves the Liver Lipid Metabolism and Redox State of Ovariectomized Rats

Abstract

Vitex agnus-castus (VAC) is a plant that has recently been used to treat the symptoms of menopause, by its actions on the central nervous system. However, little is known about its actions on disturbances in lipid metabolism and nonalcoholic fat liver disease (NAFLD), frequently associated with menopause. Ovariectomized (OVX) rats exhibit increased adiposity and NAFLD 13 weeks after ovary removal and were used as animal models of estrogen deficiency. The rats were treated with crude extract (CE) and a butanolic fraction of VAC (ButF) and displayed the beneficial effects of a reduction in the adiposity index and a complete reversion of NAFLD. NAFLD reversion was accompanied by a general improvement in the liver redox status. The activities of some antioxidant enzymes were restored and the mitochondrial hydrogen peroxide production was significantly reduced in animals treated with CE and the ButF. It can be concluded that the CE and ButF from Vitex agnus-castus were effective in preventing NAFLD and oxidative stress, which are frequent causes of abnormal liver functions in the postmenopausal period.

Figure 1

High performance liquid chromatography (HPLC) of standard AGN (Panel (a)) CE (panel (b)) and ButF (panel (c)). HPLC traces of 10 μg standard AGN, CE, or ButF. The retention times of AGN and other majors peaks measured at 254 nm are indicated.

Figure 2

Liver histochemical analysis. Rat liver slices were frozen at −80°C, sectioned with a cryostat and stained for lipids using Sudan III. The images were captured at 40x magnification. In contrast to the control (panel (a)), OVX + CE (panel (c)), and OVX + ButF (panel (d)), the liver of OVX (panel (b)) contained considerable amounts of lipid inclusions (orange) as indicated by the arrows. Calibration bar: 50 μm.

Figure 3

Gravimetric determination of liver total lipid content in liver. The liver fragments (approximately 1.0 g) from control, OVX, OVX + CE, and OVX + ButF rats were homogenized in a 2 : 1 chloroform-methanol mixture for the gravimetric determination of hepatic total lipids and the results are expressed as g/100 g wet liver weight (n = 6-7). The vertical bars represent the standard errors. The letter indicates significant differences between the values as calculated via ANOVA (^a P < 0.001 OVX versus control, OVX + CE, and OVX + ButF).

Figure 4

Mitochondrial and peroxisomal capacity to oxidize fatty acids. The liver mitochondrial β-oxidation capacity (panel (a)) was determined polarographically in the presence of 100 μM 2,4-DNP. Mitochondria (0.6–1.0 mg/mL) were incubated in a total volume of 2.0 mL. Reactions were initiated by the addition of the following: 20 μM octanoyl-CoA + 2.0 mM L-carnitine (Oct-CoA), 20 μM palmitoyl-CoA + 2.0 mM L-carnitine (Palm-CoA), or 20 μM palmitoyl-L-carnitine (Palm-L-Carn). The peroxisomal palmitoyl-CoA oxidase activity (panel (b)) from control, OVX, OVX + CE, and OVX + ButF rats were measured via fluorimetry (excitation, 503 nm; emission, 529 nm) based on the oxidation of DCFH-DA by H₂O₂ into DCF in a reaction catalyzed by exogenous peroxidase. The reactions were initiated by the addition of 30 μM palmitoyl-CoA (palm-CoA). The values are expressed as the means of 6–10 individual experiments with different mitochondrial and peroxisomal preparations. The vertical bars represent the standard errors and statistical significance was evaluated using ANOVA (P < 0.05).

Figure 5

Mitochondrial ROS generation and ROS scavenger systems. The liver mitochondrial oxidative status was evaluated by assessing mitochondrial ROS generation and the ROS scavenging system. Mitochondrial H₂O₂ generation (panel (a), pmol DCF/min × mg protein; n = 5–8); mitochondrial GSH levels (panel (b), μg GSH/mg protein; n = 6–10); MnSOD activity (panel (c), U SOD/mg protein; n = 5-6); mitochondrial GR activity (panel (d), nmol NADPH oxidized/min × mg protein; n = 7–9); GPx1 activity (panel (e), nmol NADPH oxidized/min × mg protein; n = 6–8), and NNT activity (panel (f), mmol APADH produced/min × mg protein; n = 4-5) were evaluated. The vertical bars represent the standard error. The letters indicate significant differences between the values as determined by ANOVA (P < 0.05): letter a indicates the mean values different from the control; letter b indicates the mean values different from the OVX and OVX + CE; letter c indicates the mean values different from the control, OVX + CE, and OVX + ButF.

Figure 6

Evaluation of the liver redox status. The liver oxidative status was evaluated by assessing the liver GSH levels. GSH (panel (a), μg GSH/mg protein; n = 6–10); liver lipid peroxidation using the TBARS method (panel (b), nmol MDA/mg protein; n = 7–9); total Cu,ZnSOD activity (panel (c)) and KCN-sensitive Cu,ZnSOD (panel (d)) were measured and expressed as U SOD/mg protein (n = 5-6); CAT activity (panel (e), μmol H₂O₂ consumed/min × mg protein; n = 6–10); GR activity (panel (f), NADPH oxidized/min × mg protein; n = 7–9); G6PD activity (panel (g), NADPH produced/min × mg protein; n = 6–9) and GPx3 activity (panel (h), NADPH oxidized/min × mg protein; n = 7–9) were evaluated. The vertical bars represent the standard error. The letters indicate significant differences between the values as determined using ANOVA (^a P < 0.05 OVX versus control, OVX + CE, and OVX + ButF).