Study On The Chemical Constituents And Fingerprint Of The Peels From Paris Polyphylla.var.yunnanensis

Objective:By studying the chemical constituents and establishing the HPLC fingerprint of the peels of Paris polyphylla.var.yunnanensis,and clarifying the main compounds,and establishing the HPLC fingerprint,to provide theoretical basis for the effective development and rational utilization of the peels of Paris polyphylla.var.yunnanensis.Methods:samples Preparation:The dried peels 21 kg of Paris polyphylla.var.yunnanensis was taken and crushed.The coarse powder（all of which passed through No.1 screen and no more than 20%of the"fine powder"passed through No.3 screen）was obtained and placed in a 50 L glass column and soaked with 2 times 70%ethanol overnight.Then,percolated with 10 times the amount of 70%ethanol,the extraction was concentrated in a single-effect external circulation evaporator until it had no alcohol flavor,Adjusted the alcohol content of 50L of solvent to 30%,left for overnight,and then centrifuged through a tubular centrifuge.The precipitation and supernatant parts were obtained.The two portions were concentrated and dried,obtained precipitate and supernatant dry extract.Study chemical compositions:The extract was mixed with silica gel,Then silica gel column chromatography,Sephadex LH-20,RP-C18 column chromatography and semi-preparative HPLC column chromatography was used to separate and purify the chemical constituents,and the structures of the compounds were identified by13C-NMR,1H-NMR,DEPT,MS spectroscopic data with references.Fingerprint research:According to the"Guidelines for Validation of Analytical Methods for Drug Quality Standards"in the fourth part of Chinese Pharmacopoeia（2020 Edition）,HPLC was used to study the fingerprint of the peels of Paris polyphylla.var.Yunnanensis,the chromatographic fingerprint similarity evaluation system（version 2.0）was used to process the chromatographic data.Results:1.Samples preparation:21 kg of dried pericarp of the peels Tower（batch:20201109）was taken.Through percolation extraction,centrifugal separation and drying,90 g of centrifuge precipitated dry extract of peels Tower pericarp ext ract and 3.34 kg of supernatant dry extract were obtained.2.Chemical composition research:By means of macroporous adsorption resi n column chromatography,silica gel column chromatography,Sephadex LH-20,RP-C18 column chromatography,semi-preparative HPLC column chromatograp hy and other modern separation methods,the chemical components of the two extracts were separated and purified,and 24 compounds were isolated from the peel extract of Paris polyphylla.var.Yunnanensis.The structures of 23 compo unds were identified by 13C-NMR,1H-NMR,DEPT,MS,named diosgenin-3-O-α-L-rhamnopyranosyl（1→4）-α-L-rhamnopyranosyl（1→4）-[α-L-rhamnopyranosyl（1→2）]-β-D-glucopyranoside（1）,pennogenin（2）,4-butyl-5-ethyl-3,4-dihydropyran-2-on e（3）,kaempferol（4）,Quercetin（5）,Isorhamnetin（6）,20-hydroxyecdysone（7）,isorha mnetin-3-O-β-D-glucopyranoside（8）,dioscin（9）,kaempferol-3-O-β-D-galactopyranos ide（10）,kaempferol-3-O-β-D-glucopyranoside（11）,isoquercitrin（12）,Hyperin（13）,kaempferol-3-O-β-D-galactopyranosyl-（l→6）-β-D-glucopyranoside（14）,isorhamneti n-3-O-β-D-galactopyranosyl-（l→6）-β-D-glucopyranoside（15）,isorhamnetin-3-O-β-D-gentiobioside（16）,polyphyllosideⅣ（17）,（25R）-17α,27-dihydroxy-spirost-5-en-3β-yl-O-α-L-rhamnopyranosyl-（1→2）-O-[O-α-L-rhamnopyranosyl-（1→4）-α-L-rhamnop yranosyl-（1→4）]-β-D-glucopyranoside.（ypsilandroside L）（18）,polyphyllosideⅢ（19）,pregna-5,16-dien-3β-ol-20-one-3β-O-α-L-rhamnopyranosyl-（1→2）-[α-L-rhamn opyranosyl-（1→4）]-β-D-glucopyranoside（20）,pregna-5,16-dien-3β-ol-20-one-3β-O-α-L-rhamnopyranosyl-（1→2）-[α-L-arabinopyranosyl-（1→4）]-β-D-glucopyranoside（21）,pregna-5,16-dien-3β-ol-20-one-3β-O-α-L-rhamnopyranosyl-（1→2）-[α-L-rhamnop yranosyl-（1→4）-α-L-rhamnopyranosyl-（1→4）]-β-D-glucopyranoside（22）,Aethioside（23）.3.study on fingerprint:According to the relevant guiding technical princip les of pharmacopoeia,HPLC fingerprint of Fructus dipterygoides L.was establi shed by fingerprint method research and methodology verification.The fingerpri nt had high isolation and good reproducibility.Nine batches of Fructus diptery goides L.were determined,and the similarity of each batch was between 0.903 and 0.980.Sixteen common peaks were identified,and nine chromatographic peaks were identified as kaempferol-3-O-β-D-galactopyranosyl-（l→6）-β-D-glucop yranoside,isorhamnetin-3-O-β-D-galactopyranosyl-（l→6）-β-D-glucopyranoside,iso rhamnetin-3-O-β-D-gentiobioside,isorhamnetin-3-O-β-D-glucopyranoside,polyphyl linⅦ,polyphyllin PA,polyphyllin H,polyphyllinⅡ,polyphyllinⅢ,kaempfer ol-3-O-β-D-galactopyranosyl-（l→6）-β-D-glucopyranoside,isorhamnetin-3-O-β-D-ga lactopyranosyl-（l→6）-β-D-glucopyranoside were isolated from genus Paris for th e first time.Conclusions:1.By studying the chemical constituents of the peel extract of Dipterocarpus dipterocarpus,24 compounds were isolated and 23 compounds were identified,including 11 flavonoids,6 steroid saponins,1 sapogenin,3 C₂₁steroid compounds,1insect hormone and 1 other compound,among which 6 compounds were isolated from Dipterocarpus dipterocarpus for the first time,which basically clarified the main chemical constituents of the peel of Dipterocarpus dipterocarpus.2.In this study,HPLC method was used for the first time to establish the fingerprint of chemical constituents of fructus dipterygium L.peel.The method has high sensitivity,specificity and reproducibility,which can provide a basis for quality control of the peels of Paris polyphylla.var.yunnanensis.3.The above research provides a reference for further development,utilization and quality control of the peels of Paris polyphylla.var.yunnanensis.