Screening Of Active Ingredients Targeting CYP1A1 Enzyme To Alleviate Benzo(a)pyrene Toxicity And Product Development

Polyphenol compounds are secondary metabolites of higher plants,which have antioxidant,anti-inflammatory,anti-cancer and bacteriostatic activities,and participate in the regulation of a variety of metabolic pathways.Benzo(a)pyrene(BaP),a polycyclic aromatic hydrocarbon consisting of five benzene rings,which has slightly toxic.But its metabolic end product benzo(a)pyrene-7,8-diol-9,10-epoxide(BPDE)has strong genetic toxicity and carcinogenicity.BPDE can induce DNA damage,which is the main reason for the toxicity of BaP.Cytochrome P450 1A1(CYP1A1)is the most important metabolic enzyme in the activation of BaP,so the metabolism of BaP can be blocked and therefore the toxicity of BaP can be reduced by inhibiting the activity of CYP1A1.The exploration of the effective inhibitors of CYP1A1 in diet and the study of the mechanism of action between them is of great guiding significance for the alleviating toxicity of BaP by dietary functional components.In this study,300 common plant polyphenols and their derivatives were collected based on the interaction between polyphenols and CYP1A1 enzyme,and the binding energy database of plant polyphenols and CYP1A1 enzyme was constructed through molecular docking.Two polyphenols with the best effect of reducing the toxicity of BaP were screened out virtually: neohesperidin(NH)and neohesperidin dihydrochalcone(NHDC).In order to verify the reliability of the screening results,the binding properties with CYP1A1 and its detoxification ability of BaP were analyzed by cytotoxicity assays,comet assays,prokaryotic expression and in vitro binding experiments.Based on the screening results,the selected polyphenols were protected by embedment in cyclodextrin to improve their digestion stabilities and corresponding functional foods were developed which can alleviate the toxicity of BaP.The main results are as follows:1.Virtual screening and establishment of docking energy database of CYP1A1 and polyphenols: the structural data of 300 common polyphenols were collected.After pretreatment,the binding energy database of CYP1A1 and polyphenols was established by molecular docking.CYP1A1 was used as the target,and the potential effectors NH and NHDC were screened by molecular docking.Through docking analysis,it was found that NH and NHDC could statically bind to the active center of CYP1A1 through hydrogen bonding,hydrophobicity and van der Waals force,and inhibit their enzyme activity competitively.2.In vitro interaction between polyphenols and proteins.The prokaryotic expression vector was constructed by CYP1A1 gene sequence,and the CYP1A1 protein was expressed by EScherichia coli BL-21(DE3)and obtained by renaturation of inclusion body protein.The enzyme activity detection experiments in vitro showed that the combination of NH and NHDC with CYP1A1 in vitro significantly inhibited the activity of the enzyme.With the increase of concentration,the inhibition rate of NH on CYP1A1 enzyme activity was 0.24%-10.46% higher than that of NHDC.3.Verification of protective effect of polyphenols on HepG2 cells.HepG2 cells were treated with BaP to construct an injury model,and the cell survival rates were detected by thiazole blue(MTT)assays to verify the protective effect of polyphenols on BaP-induced cell injury.The protective effect of screened polyphenols on DNA damage caused by BaP was verified by comet assays.The results showed that NH and NHDC had a dose-dependent protective effect on BaP-induced injury.4.Active ingredient embedment and product development: NH and NHDC are unstable under physiological conditions and have poor water solubility at room temperature,so their bioavailability is limited.In this study,cyclodextrin was used to encapsulate the active ingredients and a new functional food was developed to alleviate the toxicity of BaP.Overall,this study constructed a polyphenol structure information database,targeted at the binding characteristics of polyphenols and CYP1A1,screened and verified the potential effectors of CYP1A1 enzyme through molecular docking,prokaryotic expression and cell experiments.Embedded the screened polyphenols on this basis,and developed relevant functional foods.This study proposed a new method to search for effective inhibitors of CYP1A1,which provided a new thought and theoretical basis for the development of new functional foods.