Study On Chemical Components Of Mulberry Cell Culture And Glycosyltransferase Of Astragalus Mongolic

This dissertation consists of two parts:the chemical constituents of cell suspension cultures of Morus alba L in part Ⅰ,and studies on glycosyltransferases from Astragalus membranaceus(Fisch.)Bge.var.mongholicus(Bge.)Hsiao in part Ⅱ.Part I Studies on chemical constituents of cell suspension cultures of Morus alba LMorus alba L has important economic value and a long history of drug use.Its root bark,branches,leaves and fruits can be used as medicine,which has good pharmacological activity.In the 1990s,Japanese scientists isolated and identified a variety of structural types of compounds from mulberry root bark and its suspension cultured cells with many significant biological activities,which attracted extensive attention.In our previous work,several adducts with anti-inflammatory activity have been isolated and identified from mulberry suspension cultured cells.In order to obtain more compounds with novel structure and bioactivity,the chemical constituents of ethyl acetate fraction of 78%ethanol extract from mulberry suspension cultured cells were studied.The ethyl acetate fraction of 78%ethanol extract of mulberry suspension cultured cells was separated and purified by various chromatographic techniques(silica gel column chromatography,C18 column chromatography,sephadex LH-20 gel column chromatography,normal/reverse phase semi-preparative HPLC,etc.).Eleven Diels-Alder adducts were isolated and identified by various spectroscopic techniques(IR,UV,MS,NMR,CD).Wherein compounds 1 and 2 are novel compounds,and compounds 3-5,7 were obtained from mulberry cell cultures for the first time.Their stuctures were identified as mongolicin H(1),morbilisin J(2),mongolicin F(3),mulberrofuran G(4),artonin D(5),kuwanon R(6),morbilisin C(7),mulberrofuran E(8),chalmoracin(9),mulberrofuran F(10)and kuwanon J(11).The cytotoxicity and antidiabetic activity of Diels-Alder adducts were evaluated in vitro.The results showed that compounds 1-11 exhibited potent cytotoxicity with IC50 values of 2.09-63.2 μM,and showed significant inhibitory activity against protein tyrosine phosphatase 1B(PTP1B)enzyme with an inhibitory rate of 78.2-96.3%at 10 μM.In addition,compound 5 displayed significant inhibitory effect on xanthine oxidase(XOD)by 92.9%at 10 μM.Part Ⅱ Studies on glycosyltransferases from Astragalus membranaceus(Fisch.)Bge.var.mongholicus(Bge.)HsiaoThe dry roots of A.membranaceus(Fisch.)Bge.is a traditional Chinese medicine with various pharmacological activities.The Chinese pharmacopoeia collects dry roots of A.membranaceus Bge.var.mongholicus(Bge.)Hsiao or A.membranaceus(Fisch.)Bge.The cultivation and management of A.membranaceus is more difficult,and value is not as good as A.membranaceus Bge.var.mongholicus,thus A.membranaceus Bge.var.mongholicus accounts for more than 80%of A.membranaceus cultivation.Glycosyltransferases are ubiquitous in animals,plants,and microorganisms,and can catalyze the transfer of sugars from donors to receptors with high efficiency,regio-and stereo-selectivity.Glycosylation can not only enrich the structural diversity of natural products,but also improve their water solubility,pharmacological activities and so on.Glycosylation can be synthesized by enzymatic method,which has attracted much attention because of its mild conditions and specificity.A.membranaceus contains various triterpenoid saponins,the main structural type is cycloastragenol-type triterpenoid saponins.AstragalosideⅣ is the main active ingredient of A.membranaceus,but the glycosyltransferase responsible for glycosylation in astragaloside Ⅳ biosynthesis pathway has not been reported.In order to elucidate the glycosylation mechanism of astragaloside Ⅳ,gene cloning,heterogenous expression and functional identification were investigated baed on the transcriptome sequencing.In this work,19 glycosyltransferase genes(AmUGT1-AmUGT19)were cloned from fresh roots of A.mongholicus.The enzyme assay results showed that AmUGT7 and AmUGT14 can catalyze 3-O-xylosylation cycloastragenol,and AmUGT7 and AmUGT15 can catalyze 6-O-glucosylation of 3-O-cycloastragenol xyloside.We speculate that the glycosylation of astragaloside might occur after cytoskeleton and P450s oxidation,and AmUGT7 might be responsible for 3-O-xylosylation and AmUGT15 might be responsible for 6-O-glucosylation of 3-O-cycloastragenol xyloside.