INDIRECT ANTIOXIDANT ACTIVITY ASSOCIATED WITH A NOVEL PHENOLIC ANTIOXIDANT DERIVED FROM THE SOFT TISSUES OF THE PACIFIC OYSTER, CRASSOSTREA GIGAS

Kenji YOSHIIKE¹, Hirotoshi FUDA², Hitoshi CHIBA², Mitsugu WATANABE¹

¹Watanabe Oyster Laboratory Co., Ltd.and 490-3, Shimoongata-Cho, Hachioji, Tokyo, Japan

²Faculty of Health Science, Hokkaido University, Sapporo 060-0812, Japan

e-mail: gakujutsu@oyster.co.jp

Key words: indirect antioxidant, Keap1, Nrf2, phase II enzymes, 3,5-dihydroxy-4-methoxybenzyl alcohol,

Background and objective:

We identified a novel phenolic antioxidant, 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA), in the soft tissues of the Pacific oyster. DHMBA is an amphiphilic antioxidant and it was confirmed to exhibit antioxidant activity in the brain and various organs. Our previous studies showed DHMBA has antioxidant, the stress-relieving and other effects of the DHMBA-containing fraction in mice with abnormal hyperactivity of the hypothalamic–pituitary–adrenal axis induced by oxidative stress in the brain. From the results of these studies, the concentration at which DHMBA exerts its efficacy was clearly lower than that of other antioxidants. Indirect antioxidant induced expression of the enzymatic antioxidant gene. In addition, the findings suggested that DHMBA has both direct and indirect antioxidant functions. We therefore examined the effects of DHMBA on the Keap1-Nrf2 pathway, which is an important biological defense mechanism against oxidative stress.

Methods:

Human liver cancer cells (C3A) and a reporter gene assay were used to evaluate activation of the Keap1-Nrf2 pathway by DHMBA. A Dual-Glo^® Luciferase Assay with FuGENE^® HD Transfection Reagent, and two luciferin reporter vectors were used to measure luciferin activity. cDNA was synthesized using the DoScript^TM Reverse Transcription System.Real-time polymerase chain reaction was then used to measure the expression of Nrf2 target genes in the samples.

Results:

The reporter gene assay revealed that DHMBA significantly increased luciferin activity in a dose-dependent manner (P < 0.001), while chlorogenic acid had no effect on luciferin activity. DHMBA also significantly induced expression of the Nrf2 target genes SOD1 (P < 0.001), GPx (P < 0.001) and CAT (P < 0.01) in a dose-dependent manner.

Conclusion:

DHMBA was found to have indirect antioxidant properties in the activation of the Keap1-Nrf2 pathway and the induction of phase II enzymes.