Antioxidant mechanism of black garlic extract involving nuclear factor erythroid 2-like factor 2 pathway

Abstract

Backgroun/objectives: Although studies have revealed that black garlic is a potent antioxidant, its antioxidant mechanism remains unclear. The objective of this study was to determine black garlic's antioxidant activities and possible antioxidant mechanisms related to nuclear factor erythroid 2-like factor 2 (Nrf2)-Keap1 complex.

Methods/materials: After four weeks of feeding rats with a normal fat diet (NF), a high-fat diet (HF), a high-fat diet with 0.5% black garlic extract (HF+BGE 0.5), a high-fat diet with 1.0% black garlic extract (HF+BGE 1.0), or a high-fat diet with 1.5% black garlic extract (HF+BGE 1.5), plasma concentrations of glucose, insulin,homeostatic model assessment of insulin resistance (HOMA-IR) were determined. As oxidative stress indices, plasma concentrations of thiobarbituric acid reactive substances (TBARS) and 8-isoprostaglandin F2α (8-iso-PGF) were determined. To measure antioxidant capacities, plasma total antioxidant capacity (TAC) and activities of antioxidant enzymes in plasma and liver were determined. The mRNA expression levels of antioxidant related proteins such as Nrf2, NAD(P)H: quinone-oxidoreductase-1 (NQO1), heme oxygenase-1 (HO-1), glutathione reductase (GR), and glutathione S-transferase alpha 2 (GSTA2) were examined.

Results: Plasma glucose level, plasma insulin level, and HOMA-IR in black garlic supplemented groups were significantly (P < 0.05) lower than those in the HF group without dose-dependent effect. Plasma TBARS concentration and TAC in the HF+BGE 1.5 group were significantly decreased compared to those of the HF group. The activities of catalase and glutathione peroxidase were significantly (P < 0.05) increased in the HF+BGE 1.0 and HF+BGE 1.5 groups compared to those of the HF group. The mRNA expression levels of hepatic Nrf2, NQO1, HO-1, and GSTA2 were significantly (P < 0.05) increased in the HF with BGE groups compared to those in the HF group.

Conclusions: The improvements of blood glucose homeostasis and antioxidant systems in rats fed with black garlic extract were related to mRNA expression levels of Nrf2 related genes.

Keywords: Garlic; antioxidant; glucose; insulin; oxidative stress.

Fig. 1. TBARS concentration s after the supplementation of black garlic extracts.

Rats were fed with 5 different diets for 5 weeks; NF, Normal fat (7% fat); HF, High-fat (20% fat + 1% cholesterol); HF+BGE 0.5, High-fat + 0.5% black garlic extract (BGE); HF+BGE 1.0, High-fat + 1.0% BGE; HF+BGE 1.5, High-fat + 1.5% BGE. Each bar represents the mean ± SD. Different letters above each bar indicate significant differences among groups at α = 0.05 as determined by Duncan's multiple range test.

Fig. 2. 8-iso-PGF₂ changes after the supplementation of black garlic extracts.

Rats were fed with 5 different diets for 5 weeks; NF, Normal fat (7% fat); HF, High-fat (20% fat + 1% cholesterol); HF+BGE 0.5, High-fat + 0.5% black garlic extract (BGE); HF+BGE 1.0, High-fat + 1.0% BGE; HF+BGE 1.5, High-fat + 1.5% BGE. Each bar represents the mean ± SD. NS above the bars indicate no significant differences among groups at α = 0.05 as determined by Duncan's multiple range test.

Fig. 3. Total antioxidant capacity changes after the supplementation of black garlic extracts.

Rats were fed with 5 different diets for 5 weeks; NF, Normal fat (7% fat); HF, High-fat (20% fat + 1% cholesterol); HF+BGE 0.5, High-fat + 0.5% black garlic extract (BGE); HF+BGE 1.0, High-fat + 1.0% BGE; HF+BGE 1.5, High-fat + 1.5% BGE. Each bar represents the mean ± SD. Different letters above each bar indicate significant differences among groups at α = 0.05 as determined by Duncan's multiple range test.

Fig. 4. Effect of black garlic extract on mRNA expression of transcription factor and antioxidants in liver of rats.

Rats were fed with 5 different diets for 5 weeks; NF, Normal fat (7% fat); HF, High-fat (20% fat + 1% cholesterol); HF+BGE 0.5, High-fat + 0.5% black garlic extract (BGE); HF+BGE 1.0, High-fat + 1.0% BGE; HF+BGE 1.5, High-fat + 1.5% BGE. Total RNA was isolated using TRI-reagent, and cDNA was synthesized using 3 µg of total RNA with SuperScript II reverse transcriptase. Real-time PCR was performed using SYBR green and standard procedures to assess the mRNA expression of the primer in liver samples obtained from each group. An Applied Biosystems StepOne softwere v2.1 was used. Each bar represents the mean ± SD of three independent experiments. Different letters above each bar indicate significant differences among the groups at α = 0.05 as determined by Duncan's multiple range tests. GR, glutathione reductase; NQO1, NAD(P)H:quinone-oxidoreductase-1; HO-1, heme oxygenase-1; Nrf2, nuclear factor erythroid 2-like factor; GSTA2, glutathione S-transferase alpha 2.