| Background and ObjectiveThere were about 19.3 million new cases of cancer in 2020,and 9.9 million people died of cancer according to the "Global Cancer Statistics" report.The mortality rates of liver cancer and gastric cancer ranked third and fourth in cancer-related mortality rates.Glioma is the most common malignant brain tumor leading to significant morbidity and mortality in all age groups,which is the number one cause of cancer-related mortality in children aged 0-14 years.For the treatment of malignant tumors,surgical resection is mainly used in clinical treatment,and chemotherapy and radiotherapy are supplemented.However,surgical treatment with the high risk is easy to relapse,radiotherapy and chemotherapy in the treatment of tumors are accompanied by a high incidence of adverse reactions,and chemotherapy drugs generally have the disadvantages of poor prognosis,easy development of drug resistance and low clinical efficacy.Therefore,there is an urgent need to find safe and effective anti-tumor drugs.Paris polyphylla Smith var.yunnanensis(Franch.)Hand.-Mazz.and Paris polyphylla Smith var.chinensis(Franch.)Hara,which are stipulated as Chinese medicine in the 2020 edition of Chinese Pharmacopoeia,have been greatly reduced in recent years due to natural and human factors.Paris fargesii var.petiolata(Baker ex C.H.Wright)Wang et Tang is a variety of Paris with strong rhizome and high medicinal value.Studies have shown that the main pharmacodynamic substance of Paris is steroidal saponins,also known as Chonglou saponins,which has many biological activities such as anti-tumor,anti-inflammatory,antibacterial,hemostasis and others.Among them,the anti-tumor effect is a research hotspot.In this study,chemical extraction and separation methods were used to purify the chemical constituents of P.fargesii var.petiolata,and their structures were identified by spectroscopic techniques.All compounds were evaluated to screen out those with anti-tumor effects.Through the analysis of the chemical constituents of the plant,the preliminary theoretical basis was provided for its replacement of the officinal plant,and the scientific basis was provided for the future quality standard formulation of the medicinal material.Methods1.Isolation and identification of chemical constituentsTotal saponins were extracted with water-saturated n-butanol from 70%ethanol extract of rhizomes of P.fargesii var.petiolata which were purified by column chromatographies of silica gel,ODS,Sephadex LH-20 and semi-preparative HPLC,and their structures were identified by chemical methods and spectroscopic analysis(NMR,MS).2.Anti-tumor activity of the obtained compoundsThe effects of obtained compounds on the proliferation of tumor cells were evaluated to screen out ones with anti-tumor activity by CCK-8 method,which was used to calculate the IC50 values of the compounds against human glioma cells(U87,U251),human liver cancer cells(HepG2)and human gastric cancer cells(SGC-7901).Effects of the new compound with anti-tumor effect on the apoptosis and cell cycle arrest of four tumor cells were detected by flow cytometry.Results1.20 steroidal saponins were obtained,of which 7 were new compounds.Twenty steroidal saponins were acquired by chromatography and spectrum analysis for the first time,which identified as pennogenin-3-O-α-L-rhamnopyranosyl-(1→4)-[α-Lrhamnopyranosyl-(1→2)]-β-D-glucopyranoside(1),polyphyllin VI(2),pennogenin-3-O-βD-xylopyranosyl-(1→5)-α-L-arabinofuranosyl-(1→4))-[α-L-rhamnopyranosyl-1→2]-β-Dglucopyranoside(3),pennogenin-3-O-β-D-apiofuranosyl-(1→4)-[α-L-rhamnopyranosyl(1→2)]-β-D-glucopyranoside(4),polyphyllin H(5),polyphyllin I(6),polyphyllin V(7),26-O-β-D-glucopyranosyl-3β,22α,26-trihydroxy-(25R)-furost-5-en-3-O-β-D-glucopyranosyl-(1→3)-[α-L-rhamnopyranosyl-(1→2)]-β-D-glucopyranoside(8),26-O-β-D-glucopyranosyl-3β,17α,22α,26-tetrahydroxy-(25R)-furost-5-en-3-O-β-D-xylopyranosyl-(1→5)-α-Larabinofuranosyl-(1 4)-[α-L-rhamnopyranosyl-(1→2)]-β-D-glucopyranoside(9),dichotomin(10),parisyunnanoside A(11),saponin Th(12),26-O-β-D-glucopyranosyl-3β,17α,22a,26-tetrahydroxy-(25R)-furost-5-en-3-O-β-D-glucopyranoside-(1→3)-[α-L-rhamnopyranosyl-(1→2)]-β-D-glucopyranoside(13),26-O-β-D-glucopyranosyl-3β,26-dihydroxy(25R)-cholest-5,17(20)-dien-16,22-dione-3-O-β-D-glucopyranosyl-(1→5)-α-L-arabinofuranosyl-(1→4)-[α-L-rhamnopyranosyl-(1→2)]-β-D-glucopyranoside(14),26-O-β-D-glucopyranosyl-3β,26-dihydroxy-(25R)-cholest-5,17(20)-dien-16,22-dione-3-O-β-D-xylopyranosyl-(1→5)-α-L-arabinofuranosyl-(1→4))-[α-L-rhamnopyranosyl-1→2]-β-D-glucopyranoside(15),26-O-β-D-glucopyranosyl-3β,26-dihydroxy-(25R)-cholest-5,17(20)-dien16,22-dione-3-O-α-L-arabinofuranosyl-(1→4)-[α-L-rhamnopyranosyl-(1→2)]-β-D-glucopyranoside(16),26-O-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl-3β,26-dihydroxy(25R)-cholest-5,17(20)-dien-16,22-dione-3-O-α-L-arabinopyranosyl-(1→4)-[α-L-rhamnopyranosyl-(1→2)]-β-D-glucopyranoside(17),26-O-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl-3β,22α,26-trihydroxy-(23R,25R)-16,23-cyclocholest-5,17(20)-dien-22-one-3O-α-L-arabinofuranosyl-(1→4)-[α-L-rhamnopyranosyl-(1→2)]-β-D-glucopyranoside(18),parispseudoside(19),ypsiyunnoside A(20).Among them,the seven new compounds are saponins 4,9,13-15,17 and 18,respectively.2.Screening out 7 compounds with anti-tumor activity.The results of CCK-8 experiment showed that compounds 1-7 had different degrees of inhibitory effect on U87,U251,HepG2 and SGC-7901 cells,and new compound 4 had a good cytotoxicity against four human cancer cells with IC50 values of 8.02±0.45,6.31±0.37,8.24±0.57 and 6.20 ± 0.79 μmol/L.The results of flow cytometry showed that new compound 4 induced apoptosis of U87,U251,HepG2 and SGC-7901 cells,and arrested the cell cycle of U87,HepG2 and SGC-7901 in S phase.Conclusions1.Study the chemical compositions of P.fargesii var.petiolata deeplyTwenty steroidal compounds were identified from the plant for the first time,including seven isospirosterols,six furosterols and seven cholestanols.2.Seven new compounds with novel structuresAmong the 20 steroidal saponins,7 new compounds were identified.The sugar moiety of the compound 4 possessed a D-apiofuranosyl,which was rare in Chonglou saponins.The aglycone of compound 18 possessed a rare skeleton of 6/6/6/5/5 pentacyclic condensed cholestanol which was hardly seen in nature and firstly found from the genus.3.New compound 4 with a good anti-tumor effectThe anti-tumor effect of 20 compounds were evaluated by in vitro anti-tumor activity experiment.New compound 4 with good anti-tumor effect was obtained,preliminary activity of which was studied and the theoretical basis was provided for the further study. |
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