Analysis Of Lupin Polyphenol Compound And Effect On The Protection Against Oxidative Damage In HepG2 Cells

In this study,lupine polyphenol compounds were extracted from lupin flour by methanol after processes of degreasing,drying and sieving.The chemical characteristics and antioxidant capacity of lupine polyphenol compounds were analyzed.Also,the effect of lupine polyphenol compounds on oxidative damaged HepG2 cells was accessed,to providing theories for the application of lupine polyphenol compounds in cancer therapy.The lupine powder was degreased using n-hexane(1:20,w:v),followed by treatment with 50%methanol(1:10,w:v)for polyphenol extraction,without detected residue of fat and protein.The content of lupine polyphenols and flavones were determined by foline-phenol method and colorimetric method,respectively.Further,HPLC and LCMS-IT-TOF were employed for the detection of lupin polyphenolic species and content.The antioxidant activity of lupine polyphenols was evaluated by DPPH scavenging ability,ABTS scavenging ability and total antioxidant capacity,with water soluble vitamin E as control.Furthermore,the influence of lupine polyphenols on the oxidative damage and apoptosis of HepG2 cells was studied.The optimum concentrations and incubating time of hydrogen peroxide and lupine polyphenols on cells were selected according to cell viability using MTT method.The cells were divided into three groups,including control group(without treatments),model group(treatment with hydrogen peroxide)and drug group(pretreatment with lupine polyphenols and then hydrogen peroxide).The activities of superoxide dismutase(SOD),glutathion peroxidase(GSH-Px)and catalase(CAT),and the release of reactive oxygen(ROS)and malondialdehyde(MDA)were detected for oxidative stress evaluation.Meanwhile,level of mitochondrial membrane potential and stability of lysosomal membrane were determined,as well as DNA damage and cell apoptosis.The mechanism of lupine polyphenols in regulating oxidative stress and apoptosis was revealed by the expressions of related genes.Results are as follows:(1)The concentration of total phenols is 0.58 mmol GAE/100g,and flavone is 1.21 mmol Rutin/100g DM.Specific polyphenol of apigenin glucopy was quantified as 57.88 mg vitexin/100 g DM.(2)The total antioxidant activity of lupine polyphenols is 0.86 mmol TE/100 g DM,with ABTS scavenging ability of 5.95 mmol TE/100 g DM and DPPH scavenging ability of 4.23 mmol TE/100 g DM.(3)HepG2 cells treated with 300 ?mol/L hydrogen peroxide for 4 h exhibited 54.60%viability,which was set as model concentration for oxidative damage.The concentration of lupine polyphenols for cell treatment is 0.02 mg/mL and the incubation time is 48 h,leading to 33.17%increase in cell viability.With pre-incubation of lupine polyphenols,increases in the activity of SOD and the level of mitochondria membrane potential,and decreases in ROS membrane potential,and decreases in ROS and MDA release were observed compared to model group.The up-regulated expressions of oxidative stress associated genes and the reduced DNA damage and apoptosis revealed the protective effect of lupine polyphenols on cells.The current study provided an extraction process for lupine polyphenols with increased purity,and demonstrated the protective role of lupine polyphenol compounds against oxidative damaged HepG2 cells.