Dihydromyricetin dhm

Measurements of the activity of the recombinant DeDFR1 and DeDFR2 were performed according to the methods of Petit et al. and Katsu et al. with minor modifications [25 (link), 35 (link)]. A 500-μL reaction mixture consisted of 1.6 mM NADPH, 50 mM Tris-HCl (pH 7.0), 0.8 mM substrate, and 5 mg of total protein. Three dihydroflavonols were selected as experimental substrates: dihydrokaempferol (DHK), dihydroquercetin (DHQ) and dihydromyricetin (DHM) (Sigma-Aldrich). Each reaction was terminated after 30 min at 30°C by extraction with ethyl acetate. Because the obtained leucoanthocyanidins were unstable in solution, the residues were dissolved after evaporation in 200 μL of n-BuOH-HCl (95:5) and incubated at 95°C for 30 min to form anthocyanidins. The HPLC analysis of the DFR products was similar to the anthocyanidin detection described above. The process was repeated at least three times for each sample.

Hep3B cells derived from American Type Culture Collection (ATCC, Rockville, MD, USA) were grown in Dulbecco’s modified Eagle medium (DMEM, Life Technologies, Carlsbad, CA, USA) with 10% fetal bovine serum (FBS, Gibco, Australia). The cells were incubated in an incubator containing 95% air and 5% CO₂ at 37 °C. Dihydromyricetin (DHM) obtained from Sigma-Aldrich (St Louis, MO, USA) was dissolved in dimethylsulfoxide (DMSO, Sigma-Aldrich) and then different concentrations of DHM were used to treat Hep3B cells. Nedaplatin (NDP, Nichi-Iko Pharmaceutical Co., Ltd.) was solubilized in sterilized H2O. DMSO served as a control group.