| Paris polyphylla Smith var.yunnanensis(Franch.)Hand.-Mazz.(PPY)and P.polyphylla Smith var.chinensis(Franch.)Hara(PPC)are the only medicinal resources of Paridis Rhizoma(PR)recorded in the Chinese Pharmacopoeia 2020 edition.In the records of Materia Medica,PPC was used as the medicinal material source of PR for a long time.After the Ming Dynasty,PPC and PPY were all recorded in Materia Medica.The research on chemical composition mainly focuses on PPY,less attention is paid to PPY,and the main steroidal saponins content determination results of multiple batches samples showed that PPC mainly contained(proto)diosgenyl saponins and PPY contained(proto)pennogenyl saponins,the activities of different steroidal saponin sub-types have not been studied.PR needs a long period of growth to accumulate steroidal saponins to reach the medicinal standard.The analysis of key genes in biosynthetic pathways of steroidal saponins in plant is the basis for regulating the accumulation of steroidal saponins and clarifying the quality formation of PR.Based on the questions mentioned above,the main research contents of this thesis were listed as follows:1.Composition characterization of PPC and PPY based on off-line 2D-LCIn view of problems of co-elution of components and difficulty in finding trace components with conventional mass spectrometry analysis,an off-line two-dimensional liquid chromatography separation system was established.The samples were first separated by a HILIC column and collected,and then different parts were analyzed by a C18 column.Comparison with traditional chromatographic separations,off-line 2D-LC system greatly improved the peak capacity and reduced the co-elution of components.Finally,444 compounds were identified from the rhizomes of PPC and PPY,including 394 steroidal saponins,5 phytosterols,14 nitrogen-containing compounds and 6 phenylglycosides.Among the identified components,there were 306 potential new compounds,mainly furostanol saponins and polyhydroxylated steroidal saponins.PPC and PPY also had different type of furostanol saponins based on the comparison of parts samples.2.Studies on the constituents and bioactivities of PPCLC-MS guided isolated of 50%ethanol elution part of macroporous resin of PPC was used to obtain new compounds.Twenty-six compounds were isolated by silica gel VLC,ODS,Flash rapid preparation chromatography and preparative HPLC.Their structures were elucidated by combination of phycicochemical properties and detailed spectroscopic,including 17 steroidal saponins and 9 cholestanes.Among them,compounds 1-3 and 6-15 are new compounds,and compounds 4,5 are new compounds predicted by LC/MS in the previous stage and this is the first time to accurately identify them by NMR data.These compounds include one new furostanol saponins and seven polyhydroxylated steroidal saponins,which verified the results of mass spectrometry identification.LPS induced RAW264.7 cells model,human liver cancer HepG2 cells and in vitro platelet aggregation experiment was used to evaluated anti-inflammatory,antitumor and hemostatic activities of part of compounds.The results showed that compounds 20 and 24 showed certain NO inhibitory activities,with IC50 of 61.35 μM and 37.23 μM,respectively;compounds 5,18,19 and 24 showed moderate cytotoxicity to HepG2 cells,with IC50 of 22.35,9.43,15.36 and 24.54 μM,respectively;polyphyllin Ⅶ、polyphyllin H、17hydrogracillin and polyphyllin Ⅱ had direct platelet aggregation-inducing activity at dose of 50 μM.3.Preparation and bioactivities evaluation of steroidal saponin sub-types in PPCThe composition of furostanol-type was clarified by component separation,in order to analyze the pharmacological activities of different types of steroidal saponin,steroidal saponin sub-types in PPC were prepared by macroporous resin and Flash rapid preparative chromatography.The hemostatic activity was evaluated by mice cutting tail and induced platelet aggregation test;RAW264.7 cells induced by LPS and COX-2 inhibition experiment were used to evaluated anti-inflammatory activity.The results showed that furostanol-type,pennogenyl-type and diosgenyl-type all could significantly shorten the bleeding time,and pennogenyl-type was the best,which could shorten the time of TT,but had no effect on PT and FIB.In addition,pennogenyl-type and can directly induce platelet aggregation at concentration of 25 μg/mL,while diosgenyl-type was effective at a dose of 50 μg/mL.The anti-inflammatory experiment showed that all sub-types had inhibitory effect on COX-2,and the furostanol-type was the best,but these three sub-types had no effect on LPS induced RAW264.7 cells to release NO and TNF-α.According to the activity evaluation of different steroidal saponin sub-types,the hemostatic activity of the pennogenyl-type was better than that of the diosgenyl-type,and the furostanol-type do not induce platelet aggregation in vitro,but had in vivo hemostatic activities.Therefore,the reasons for the differences in vitro and in vivo hemostatic activities of furostanol-type need to be further studied.4.Biosynthetic pathway and key genes screening of steroidal saponins in PPCThe rhizomes,stems and leaves of PPC and PPY were analyzed by plant metabolomics technology,and the chemical profiles difference between the two species were further clarified.OPLS-DA analysis of rhizomes and leaves of PPC showed that they also had different steroidal profiles,rhizomes mainly contained pennogenyl saponins,but leaves mainly contained diosgenyl saponins,which provided the possibility to screen the key CYP450 genes involved in pennogenin biosynthesis form the transcriptome data of different parts of PPC 125.16 GB data were obtained from transcriptome sequencing of fibrous roots,stems,leaves and seeds of PPC.After de novo assembly,167 genes involved in the biosynthesis pathways of steroidal saponin in PPC were identified,which were divided into 22 metabolic enzymes.Based on the annotation of Pfam database,90 full-length CYP450 genes were identified.By analyzing the expression level of CYP450 gene in different parts,6 highly expressed genes in fibrous roots and seeds compared with leave were screened,which provided a basis for further gene function verification.In sum,the composition of PPC was further enriched,and the results of bioactivities of different steroidal saponin sub-types can provide a basis for the quality control of PR.The genes involved in the biosynthesis pathways of steroidal saponins were screened from the transcriptome data of different parts of PPC,which provides a basis for gene function verification and elucidating the mechanism of quality formation of PR. |
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