Study On The Antioxidant Effect In Vitro And Molecular Mechanism Of Apoptosis Induced-by Herbacetin

Herbacetin （HER） is a natural flavonoid compound that can be extracted from Ramose ScouringRush Herb, Rhodiola rosea and cotton. By far, its biological and pharmacological activities lackcorresponding attention.Antioxidant is widely used in food and pharmaceutical industries and natural antioxidant is paidmore and more attention. In order to provide a theory of the application of herbacetin in naturalantioxidant field, the antioxidant activity of herbacetin in vitro was evaluated by determining theabilities to scavenge DPPH, hydroxyl free radical and inhibit Cu²⁺-H₂O₂/AAPH-induced proteindamage and carbonylation. The results showed that10-100μmol/L herbacetin could significantlyscavenge DPPH, hydroxyl free radical and the median inhibitory concentrations （IC50values） ofherbacetin were49.28and219.2μmol/L, respectively. Meanwhile, herbacetin could significantlyinhibit Cu²⁺-H₂O₂/AAPH-induced protein damage and carbonylation in a dose-dependent manner.Additionally, in this study, the apoptotic effect of HER against the human hepatoma cell line（HepG2） was investigated in order to provide preliminary experimental evidences for supporting thepossibility of HER to be considered as one of the novel pharmacological treatment strategies in livercancer. MTT assay was used to measure the effect of herbacetin on HepG2cell viability. Fluorescentdyes （AO-EB/DAPI） and DNA ladder extraction kit were applied to investigate the apoptotic effect ofherbacetin on HepG2cells. The mitochondria related apoptotic proteins （Bcl-2, Bax, PARP, caspase-3,cytochrome-c） were determined to explore the apoptotic molecular mechanism of herbacetin onHepG2cells. The results indicated that, after treatment for48h, lower dose of HER （25μmol/L） hasno effect on the viability of HepG2cells. However, at higher concentrations （50-200μmol/L）, HERtreatment resulted in significant decrease in cell viability. Meanwhile, herbacetin induced nuclearshrinkage, DNA fragmentation, and poly （ADP-ribose） polymerase （PARP） cleavage in HepG2cellswith the same concentrations. Compared to the control group,25-200μmol/L herbacetin increased theexpression of Bax, caspase-3, PGC-1α, cytochrome-c, induced oxidative stress and suppressed thelevel of phosphorylated Akt in HepG2cells. Pretreatment HepG2cells with5mmol/L NAC, anexogenous free radical scavenger, significantly decreased the herbacetin-induced apoptotisis.