| Research background and purposeThe rhizoma of genus Paris of（family Melanthiaceae）,possesses highly medicinal value since ancient times,such as dispelling snake poison,clearing heat,relieving pain,etc.according to the market survey,almost all the rhizome of Paris,are in the folk circulation and used as Paridis Rhizoma.Recently studies have shown that steroidal saponins are the main active ingredient in Paris,which has attracted the attention of many researchers for its significant antitumor activity.Paris polyphylla var.latifolia is a variety of Paris which has been studied by our research group previously and few compounds have been reported.In order to elaborate the chemical composition of Paris polyphylla var.latifolia,the n-butanol extraction part of the rhizome of this plant was studied.Firstly,the compounds were isolated and purified by chromatography methods,and their structures were identified by spectroscopic and chemical techniques.And then the cytotoxic activity of the isolated compounds was evaluated by CCK-8 method.Finally,the anti-glioma effect and the underlying mechanism of the compound which exhibited significant cytotoxicity was preliminarily explored.The purpose of this research was to supplement the chemical composition of Paris polyphylla var.latifolia,to provide a theoretical basis for the supplement resource of Paris,and to provide a scientific research basis for its quality control and management in the future.By mining the structure of biodiversity,it provides lead compounds for anti-tumor drugs.Method1.Study on chemical constituents of n-Butanol extract of alcohol extract from Paris polyphylla var.latifoliaThe rhizome of Paris polyphylla var.latifolia was crushed and extracted by ethanol under reflux,then partitioned by petroleum ether and n-butanol,successively.The n-butanol extract was isolated and purified by silica gel column chromatography,gel column chromatography,medium pressure preparative liquid chromatography and semi-preparative high performance liquid chromatography.The structures of the compounds were identified by chemical means and spectroscopic techniques.2.Cytotoxic activity screening,target prediction and anti-glioma mechanism exploration of the obtained compoundsThe effects of these compounds on the proliferation of tumor cells（He La,Capan-2,U251,LN229,Hep G2）were evaluated by CCK-8 method,and the compounds with cytotoxic activity were screened.Meanwhile,SMILES of saponins against glioma were collected and predicted by Swiss Target Prediction.PPI protein interaction and Cytoscape interaction network were constructed.KEGG and GO enrichment were used to predict potential targets of saponins against glioma.The effect of compound 9 on apoptosis of five kinds of tumor cells was detected by flow cytometry,and the signaling pathway of compound 9 on apoptosis of glioma cells LN229 was verified by Western blot.Results1.37 Compounds were isolated and identified,including 4 new compounds and 1 new natural productA total of 37 compounds,chonglouside H（1）,polyphyllinⅥ（2）,polyphyllin H（3）,diosgenin-3-O-α-L-arabinofuranosyl-（1→4）-β-D-glucopyranoside（4）,pennogenin-3-O-β-D-glucuropyranosyl-（1→3）-[α-L-rhamnopyranosyl-（1→2）]-β-D-glucopyranoside（5）,polyphyllinⅤ（6）,pennogenin-3-O-α-L-rhamnopyranosyl-（1→2）-β-D-xylopyranosyl-（1→4）-β-D-glucopyranoside（7）,polyphyllinⅡ（8）,diosgenin-3-O-β-D-fructofuranosyl-（1→6）-[α-L-rhamnopyranosyl-（1→2）]-β-D-glucopyranoside（9）,diosgenin-3-O-β-D-fructofuranosyl-（1→6）-[α-L-arabinofuranosyl-（1→4）]-[α-L-rhamnopyranosyl-（1→2）]-β-D-glucopyranoside（10）,polyphyllinⅠ（11）,pennogenin-3-O-β-D-xylopyranosyl-（1→5）-α-L-arabinofuranosyl-（1→4）-[α-L-rhamnopyranosyl-1→2]-β-D-glucopyranoside（12）,paris saponinⅦ（13）,26-O-β-D-glucopyranosyl-（25R）-3β,20α,26-trihydroxyfurostan-5,22-diene-3-O-α-L-rhamnopyranosyl-（1→2）-β-D-glucopyranoside（14）,26-O-β-D-glucopyranosyl-（25R）-22-methoxy-furost-5-en-3β,26-diol-3-O-α-L-rhamnopyranosyl-（1→2）-β-D-glucopyranoside（15）,parisaponin I（16）,protogracillin（17）,hypoglaucin H（18）,spongipregnolosides A（19）,26-O-β-D-glucopyranosyl-nuatigenin-3-O-α-L-rhamnopyranosyl-（1→4）-[α-L-rhamnopyranosyl-（1→2）]-β-D-glucopyranoside（20）,pregna-5,16-diene-3β-ol-20-one 3-O-α-L-arabinofuranosyl-（1→4）-[α-L-rhamnopyranosyl-（1→2）]-β-D-glucopyranoside（21）,chonglouoside SL-8（22）,pallidifloside D（23）,3β,16β-dihydroxy-pregn-5,20-dien-22-carboxylicacid-γlactone-3-O-α-L-arabinopyranosyl-（1→4）-O-[α-L-rhamnopyranosyl-（1→2）]-β-D-glucopyranoside（24）,aethioside A（25）,parispolyside E（26）,parispseudoside A（27）,parispseudoside B（28）,parisyunnanosides I（29）,24-O-β-D-galactopyranosyl-1β,3β,21,23,24-pentol-（23S,24S）-spirost-5,25（27）-diene-1-O-α-L-rhamnopyranosyl-（1→2）-[β-D-xylopyranosyl-（1→3）]-β-D-glucopyranoside（30）,（8R,9R,10S,6Z）-trihydroxyoctadec-6-enoicacid（31）,（10E,9S,12S,13S）-trihydroxyoctadec-10-enoic acid（32）,（9S,10R,11R,12Z,15Z）-9,10,11-trihydroxyoctadeca-12,15-dienoic acid（33）,9,10,11-trihydroxyoctadec-12-enoic acid（34）,（2S）-1-O-palmitoyl-3-O-β-D-galactopyranosylglycerol（35）,gingerglycolipid B（36）,gingerglycolipid A（37）were extracted and identified by chromatographic separation and spectral analysis.Four of the new compounds are 9,10,24,30,and one new natural product is 21.2.13 Compounds with cytotoxic activity were screened,and the anti-glioma effect of new compound 9 was preliminarily exploredA total of 13 steroid saponins（1–13）with cytotoxic activity were screened based on the half inhibitory concentration of compounds on tumor cells.Among them,the new compound 9 exhibited significant cytotoxic activities on five tumor cells（He La,U251,LN229,Hep G2,Capan-2）,and IC50values were 3.30±0.38,3.85±0.44,4.18±0.31,4.32±0.51,3.26±0.34μM,respectively.A total of 151 common targets were obtained by summarizing the Paris saponin and glioma targets,and 33 main targets were obtained by PPI construction and MCODE screening.GO analysis showed that the signaling pathway of transmembrane receptor protein tyrosine kinase was mainly in the biological process,the cell composition was mainly the receptor,and the molecular biological function was mainly the plasma membrane signal receptor complex and the kinase binding.KEGG analysis indicated that the EGFR-related signaling pathway may be the potential target of Paris saponins against glioma.Flow cytometry showed that apoptosis of five tumor cells could be induced by the new compound 9.The results of Western blot indicated that compound 9 could significantly up-regulate the expression of Bax,Cleaved caspase-3,Cytochrome C,and significantly down-regulate the expression of Bcl-2,p-EGFR,p-Akt,p-m TOR,and p-PI3K.There was no significant difference in the protein expression levels of PI3K,Akt,m TOR and EGFR.ConclusionResearch conclusion1.In-depth study of the chemical composition of Paris polyphylla var.latifoliaA total of 37 compounds were isolated and identified,including 19 steroid saponins,6 C-21 steroids,4 cholestanol saponins,1 deformable sprirostanol saponins and 7 non-saponins.Except for 4 compounds（1,2,8,13）,they were isolated from this plant for the first time.2.All the new compounds had certain novelty,and new compound 9 showed anti-glioma activityThe new compounds 9 and 10 are the first fructosyl-containing steroid saponins found in family Melanthiaceae,and the second and third cases of fructosyl-containing steroid saponins reported in nature,and both compounds 9 and 10 have certain antitumor activity in vitro.The new compound 24 has the relatively rare parent nuclear structure of C-21 steroid derivatives and compound 30 has the rare spirosterane parent nuclear structure of polysaccharide chains.The potential targets of isosprirosterol saponins against glioma were predicted by network pharmacology.Compound 9 might induce the apoptosis of glioma cells LN229by regulating EGFR/PI3K/Akt/m TOR pathway in vitro. |
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