Identification,activity Evaluation And Biosynthetic Pathways Analysis Of The Differential Constituents In Aqueous-Methanol Extracts Of Polygonum Multiflorum Thunb

Based on the quality evaluation of Polygonum multiflorum in 13 regions of China,the fingerprints of aqueous-methanol extracts of Polygonum multiflorum from Deqing and Chongqing were carried out,then the differential constituents were screened using metabonomics techniques.Effects of the extracts and differential constituents of Polygonum multiflorum on cell apoptosis,cell cycle,immune phagocytosis,inflammatory response and chronic toxicity were explored respectively.Effects of differential constituents on CYP450 activities of human and mice liver microsomes were evaluated.Genes of biosynthesis-related enzymes of differential constituents were screened by transcriptome data,and the biosynthesis pathways were speculated based on the correlation analysis between the expression levels of the biosynthesis-related enzymes and the contents of different components.Furthermore,the key steps of the biosynthetic pathway were verified by biocatalysis which could provide a novel insight and experimental basis for germplasm resources evaluation.The results are mainly as follows:(1)50 compounds of Polygonum multiflorum including stilbenes,quinones and so on were determined.The results of metabonomics showed that the contents of stilbenes in Deqing Radix Polygonum multiflori were higher than Chongqing’s,yet the contents of terpenoids decreased significantly.2,3,5,4’-tetrahydroxystilbene-2-O-β-D-glucoside(THSG),polydatin,emodin-8-O-β-D-glucoside and emodin were screened and identified as the differential constituents.Especially,the relative content of THSG in Polygonum multiflorum of Deqing is 40 times than Chongqing’s.(2)The aqueous-methanol extracts of Polygonum multiflorum from Chongqing could inhibit the proliferation of HepG2 and LO2 cell lines,and the proliferation inhibition rates were higher than Deqing’s.THSG and other stilbenes had no cytotoxicity to the both cells.Only emodin had inhibitory effects on the proliferation of HepG2 and LO2 cells in a concentration-dependent manner.It induced apoptosis of LO2 cells by inhibiting the G0/G1 phase of cell cycles in a concentration-dependent manner.The expressions of Caspase-3 and Caspase-9 were increased whereas Bcl-2 expression decreased after treatment with emodin.(3)THSG and polydatin inhibited LPS-induced NO production in Raw246.7 cells in high concentrations.Emodin at 10μM could inhibit the LPS-induced NO production in Raw246.7 cells and weaken the effect on the phagocytosis.It inhibited IL-1β and TNF-α genes and proteins expression in cells in concentration-dependent manner.The expression of phosphorylated P38 in cells was decreased significantly after treatment with emodin.(4)The body weight of the experimental mice decreased significantly after oral administration of emodin for 30 days.There were no significant difference in the expression of inflammatory factors IL-1β,IL-6 and TNF-α.The results showed that only high doses of emodin caused liver and kidney injury in mice.(5)THSG had remarekable inhibitory effects on CYP3A4,and its IC50 value was 23.22 μM.Emodin had sigificant inhibitory effects on CYP1A2,2C19 and 2E1 of human liver microsomes,their corresponding IC50 values were 4.01,33.72 and 33.39μM,respectively.The consequences of enzyme activities showed that there were differences between human and mice liver microsomes.Especially,the inhibitory effects of the above components on CYP2C19 and 2E1 were varied considerably.(6)The results of CCA of differential constituents and 17 differential gene expression levels between Deqing and Chongqing samples showed that 6 enzyme genes associated with THSG and other stilbene components including phenylalanine ammonia-lyase Unigene 452,resveratrol stilbene synthase CL9535,resveratrol hydroxylase Unigene 8034,etc.Emodin and other anthraquinones were negatively correlated with the genes above,and the positive correlated genes were mainly polyketase CL3367,glycosidase Unigene 30227 and Unigene 12334.(7)Biocatalytic experimental results showed that the crude enzyme of Polygonum multiflorum could catalyze the polyketide reaction of 4-coumaryl-coenzyme A and malonyl-coenzyme A to generate resveratrol,then hydroxylate resveratrol to generate THSG aglycone,and produced THSG after glycosylation.Resveratrol was also glycosylated directly in crude enzyme to generate polydatin.CONLUSION:(1)THSG,polydatin,emodin-8-O-beta-D-glucoside and emodin were identified as the main differential constituents of Polygonum multiflorum.THSG and polydatin might have no hepatocyte toxicity,while emodin might have potentially toxic effects.(2)The variations in the contents of these differential constituents were correlated with the expression of 9 key enzymes in Polygonum multiflorum such as resveratrol stilbene synthase and resveratrol hydroxylase.(3)The differential constituents THSG and other stilbenes of Polygonum multiflorum have biosynthetic competition with anthraquinones such as emodin.The biosynthesis of THSG is a branch of phenylalanine metabolism,which is produced by the biosynthesis pathway of resveratrol,then hydroxylation follow-up glucosidization.