| Paris polyphylla Smith var.chinensis（Franch.）Hara.,a plant of genus Paris,together with P.polyphylla Smith var.yunnanensis（Franch.）Hand.-Mazz.is used as the origin plant of Paridis rhizoma described in the Chinese Pharmacopoeia（2020 edition）.The current situation of resource shortage in Paridis Rhizoma promotes the study of non-medicinal parts.In the early stage,the flavonoids were isolated from the above ground part of PPC plants via many methods.Further research has found that the aerial parts of PPC were also abundant steroidal saponins.The structure of furostanol saponin is similar;the sugar unit is many,the polarity is large,the chromatographic behavior exists the phenomenon of co-outflow,especially for the C25 isomer with C22-OH,there is a special separation difficulty.The literature showed that the C22-OH of furostanol saponin was active and easy to solvation and difficult to separate.It is of great significance to study its transformation mechanism.Based on this,the composition of the aboveground part of PPC was studied in this experiment using silica gel column chromatography,ODS column,Flash rapid preparation,preparation of high performance liquid phase,13 compounds were isolated from this experiment.Their structures were identified as neoprotodioscin（1）;methyl-protodioscin（2a）;（23α,25R）-,23,27-dihydroxyspirost-5-en-3-O-α-L-rhamnopyranosyl-（1→2）-[(β-D-glucopyranosyl-（1→3）]-β-D-glucopyranoside,parispolyside J（3）;（23α,25S）-,23,27-dihydroxyspirost-5-en-3-O-α-L-rhamnopyranosyl-（1→2）-[(β-D-glucopyranosyl-（1→3）]-β-D-glucopyranoside,parispolyside K（4）;Aculeatiside（A）（5）;（25R）-26-O-β-D-glucopyranosyl-furost-5-en-3β,22α,26-triol-3-O-α-L-rhamnopyranosyl-（1→2）-[β-D-glucopyranosyl-（1→4）-α-L-rhamnopyranosyl-（1→4）]-β-D-glucopyranoside,parispolyside I（6）;neoprotogracillin（7）;proto-gracillin（8）;（25R）-27-O-β-D-glucopyranosyl-5-en-3β,27-dihydroxyspirost-3-O-α-L-rhamnopyranosyl-（1→2）-[（β-D-glucopyranosyl-（1→4））-α-L-rhamnopyranosyl-（1→4）]-β-D-glucopyranoside,parispolyside L（9）;（25R）-27-O-β-D-glucopyranosyl-5-en-3β,27-dihydroxyspirost-3-O-α-L-rhamnopyranosyl-（1→2）-[(β-D-glucopyranosyl-（1→3）]-β-D-glucopyrano-side,parispolyside M（10）;neosolanigroside Y6（11）;solanigroside Y6（12）.Of which compound 3,4,6,9,10,11 are new compounds.The furostanol saponin 1,7 and 12 were first isolated from this genus and compounds 2,5,and 8 was first isolated from this plant.Compounds 1,7,11 was 25S configuration and first isolated from this genus.During the separation and purification process,this study yielded 6derived methylation products,compounds neoprotodioscin（1a）;methylprotodioscin（2a）;methylneoprotogracillin（7a）;methylproto-gracillin（8a）;methylneosolanigroside Y6（11a）;methylsolanigroside Y6（12a）.Among them,11a and 12a were first isolated from this genus.Compounds 1a and 7a were first isolated from this plant.The separation strategy of furostanol saponin C25 isomers was established:furostanol saponin with C22-OH dissolved in methanol were heated to convert into their methylated products,methanol-water system was used as mobile phase to prepare and separate them,the50%acetonitrile was used to convert them into hydroxyl form.The separation of the two isomers was greatly improved after methylat-ion,which was beneficial to the separation and purification of furostanol saponin andprovided a new idea for the separation and purification of furostanol saponin.See the literature,previously divided from plants to some C22 methylated furostanol saponins,in order to verify the presence form of furostanol saponins in plants,the furostanol saponin was heated in methanol,anhydrous ethanol and 50%acetonitrile.And the solvation products were analyzed by HPLC and UPLC-QTOF/MS.As a result,the C22 of furostanol saponin in the form of hydroxylation can be converted to C22 position as ethyl and methyl.Mass data analyzed the products of methylation andethylization,with the molecular weight increasing 14 and 28 on the basis of prototype furostanol saponin,[M+14/28+HCOO]-,[M-14/28-H]-,[M-H]-,fully verifies the solvation effectallowing for the existence of furostanol saponinin prototype and solvation products in different solvents.It is shown that the furostanol saponin were existing in the form of prototype and methoxy and ethoxy in the process of extracting methanol or ethanolas solvent.It is fully demonstrated that furostanol saponinis derived by prototype,methylation and ethylation in plants.The methylation and ethylation products are artificial derivatives.The literature indicates that higher active spirostanol glycosides are known to be formed from furostanol glycosides during postharvest treatment and storage in fresh plants,furostanol saponin 26-O-β-glucosidase（F26G）involved in this conversion.The preliminary research found that the content of spirostanol glycosides in the hot air dry ing was higher than that of the shaded drying,whether this does with the inclusion of F26G-mediated furosteroidal saponins to spirosteroidal saponins is worth further study.The extraction of F26G from rhizome of PPC and its reaction conditions with substrate were investigated and optimized.The crude enzyme was finally extracted from sodium hydrogen phosphate-citric acid as p H 6.After 4 times dilution,this crude enzyme reacts with 10 mmol·L-1substrates in 15min and the temperaturewas are50℃.The F26G reacts with furostanol saponin isolated from laboratory,detecting the products and verifying the conversion mechanism of furostanol saponin with HPLC.The transformation mechanism of furostanol saponin was preliminarily investigated,and the determination of different drying methods,different drying temperatures and different drying times showed that drying promoted the conversion to some extent,and the drying method was most obvious,which may be related to crude enzyme,that is,the drying treatment affects the transformation of furostanol saponin to spirostanol saponin by affecting the vitality of F26G enzyme. |
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