Studies On The Saponins From Hylomecon Japonica (Thunb.) Prantl & Kündig (?)

BackgroundHylomecon japonica(Thunb.)Prantl&Kundig is a perennial herbaceous plant which belongs to the genera of Hylomecon;the race of Chelidoninae Reichenb,the family of Papaveraceae,widely distributed in the northeast,east and middle area of China.The roots or rhizomes are often used to treat rheumatism and bruises as a traditional Chinese medicine for a long history.Various kinds of chemical constituents were previously isolated from Hylomecon japonica,including alkaloids,flavonoids,megastigmoids and saponins,and an extract of the plants has been found to exhibit a wide rang of bioactivities including antibacterial,anti-inflammatory and anti-tumor activitiesObjective1.To establish an experimental method for the determination of total saponins(THS)and Hylomeconoside A in Hylomecon japonica;To optimize the extraction technology of THS2.To isolate saponins from Hylomecon japonica and identify their structures.3.To explore the biological activities of saponins from Hylomecon japonica and screen the main active components of the saponinsMethod:1.The extraction technology of THS was optimized by Box-Benhnken experiment design with four groups of factors:ethanol concentration,extraction time,extraction times and liquid-solid ratio that to preliminarily explore the extraction technology of THS.2.Isolation and identification of monomer saponins from Hylomecon japonica The dried whole plants were soaked with 50%EtOH(liquid-solid ratio 12:1)for three days and repeated two times.The extraction was chromatographed on D101 column and the 50%EtOH eluate were collected,yielding crude saponins.The crude saponins was chromatographed on HP-20 column and eluted with 20-45%ethanol,yielding several Constituents.Constituents were separated by through thin layer chromatography,silica gel column chromatography and HPLC.The structures of monomer saponins from Hylomecon japonica were identified by analysis of NMR,HRESI-MS spectrometry date and GC spectroscopic data.3.The MTT method was used in this study to investigate the cell viability effect of saponins of Hylomecon japonica on A549,AGS,Hela,Huh7,HT-29 and K562.The Annexin V/FITC-PI kit and flow cytometry was used to analysis the apoptotic effect of saponins on those cells.Results:1.The optimal extraction technology of THS was obtained,which were 50%of ethanol concentration,3 days of extraction time,2 times of extraction times,12:1 of liquid-solid ratio.2.Eighteen triterpenoid saponins were isolated from the EtOH extract of Hylomecon japonica and the structures of seventeen of them were identified on the basis of spectroscopic and chemical evidences.Among them,compounds 1-1 5 were undescribed compounds and named as Hylomeconoside C-Q,and compounds 16-17 were isolated from Hylomecon japonica for the first time,and the structures of that seventeen compounds were respectively:Hylomeconoside C:3-O-?-D-galactopyranosyl-(1?2)-?-D-glucuronopyranosyl gypsogenin 28-O-?-L-rhamnopyranosyl-(1?2)-?-L-arabinopyranosideHylomeconoside D:3-O-?-D-galactopyranosyl-(1?2)-?-D-glucuronopyranosyl gypsogenin 28-O-?-D-xylopyranosyl-(1?4)-?-L-rhamnopyranosyl-(1?2)-?-L-arabinopyranosideHylomeconoside E:3-O-?-D-galactopyranosyl-(1?2)-[?-L-arabinopyranosyl-(1?3)]-?-D-glucuronopyra nosyl gypsogenin 28-O-?-D-glucopyranosyl-(1?3)-[?-D-xylopyranosyl-(1?4)]-?-L-rhamnopyranosyl-(1?2)-?-L-arabinopyranosideHylomeconoside F:3-O-?-D-galactopyranosyl-(1?2)-?-D-glucuronopyranosyl gypsogemn 28-O-?-D-galactopyranosyl-(1?3)-[?-D-xylopyranosyl-(1?4)]-?-L-rhamnopyranosy 1-(1?2)-?-D-fucopyranosideHylomeconoside G:3-O-?-L-rhamnopyranosyl-(1?3)-[?-D-galactopyranosyl-(1?4)]-?-D-glucuronopyra nosyl quillaic acid 28-O-?-D-galactopyranosyl-(1?3)-[?-D-xylopyranosyl-(1?4)]-?-L-rhamnopyranosy 1-(1?2)-?-D-fucopyranosideHylomeconoside H:3-O-?-L-rhamnopyranosyl-(1?3)-[?-D-galactopyranosyl-(1?4)]-?-D-glucuronopyra nosyl quillaic acid 28-O-?-D-xylopyranosyl-(1?3)-?-D-xylopyranosyl-(1?4)-?-L-rhamnopyranosyl-(1?2)-?-D-quinovopyranosideHylomeconoside I:3-O-?-D-galactopyranosyl-(1?2)-?-D-glucuronopyranosyl gypsogenin 28-O-?-L-arabinopyranosyl-(1?3)-[?-D-xylopyranosyl-(1?4)]-?-L-rhamnopyranosy 1-(1?2)-?-L-arabinopyranosideHylomeconoside J:3-O-?-D-galactopyranosyl-(1?2)-?-D-glucuronopyranosyl gypsogenin 28-O-?-D-galactopyranosyl-(1?3)-[?-D-xylopyranosyl-(1?4)]-?-L-rhamnopyranosy 1-(1?2)-?-D-galactopyranosideHylomeconoside K:3-O-?-D-galactopyranosyl-(1?2)-?-D-glucuronopyranosyl gypsogemn 28-O-?-D-galactopyranosyl-(1?3)-?-L-rhamnopyranosyl-(1?2)-?-L-arabinopyranos ideHylomeconoside L:3-O-?-D-galactopyranosyl-(1?2)-?-D-glucuronopyranosyl gypsogemn 28-O-?-D-xylopyranosyl-(1?4)-?-L-rhamnopyranosyl-(1?2)-?-D-quinovopyranosid eHylomeconoside M:3-O-?-D-glucuronopyranosyl gypsogenin 28-O-?-D-xylopyranosyl-(1?3)-?-D-xylopyranosyl-(1?4)-?-L-rhamnopyranosyl-(1?2)-?-D-quinovopyranosideHylomeconoside N:3-O-D-xylopyranosyl-(1?3)-?-D-glucuronopyranosyl gypsogenin 28-O-?-D-xylopyranosyl-(1?4)-?-L-rhamnopyranosyl-(1?2)-?-D-quinovopyranosid eHylomeconoside O:3-O-?-D-galactopyranosyl-(1?2)-[a-L-rhamnopyranosyl-(1?3)]-?-D-glucuronopyra nosyl quillaic acid 28-O-?-D-xylopyranosyl-(1?3)-?-D-xylopyranosyl-(1?4)-?-L-rhamnopyranosyl-(1?2)-?-D-fucopyranosideHylomeconoside P:3-O-?-D-galactopyranosyl-(1?2)-[a-L-rhamnopyranosyl-(1?3)]-?-D-glucuronopyra nosyl gypsogenin 28-O-?-D-xylopyranosyl-(1?3)-?-D-xylopyranosyl-(1?4)-?-L-rhamnopyranosyl-(1?2)-?-D-galactopyranosideHylomeconoside Q:3-O-?-D-glucopyranosyl-(1?2)-?-D-glucuronopyranosyl gypsogenin 28-O-?-D-galactopyranosyl-(1?3)-[?-D-xylopyranosyl-(1?4)]-?-L-rhamnopyranosy 1-(1?2)-?-L-arabinopyranosideSaponins 16:3-O-?-D-galactopyranosyl-(1?2)-[a-L-arabinopyranosyl-(1?3)]-?-D-glucuronopyra nosyl gypsogenin 28-O-?-D-glucopyranosyl-(1?3)-[?-D-xylopyranosyl-(1?4)]-?-L-rhamnopyranosyl-(1?2)-?-D-fucopyranosideSaponins 17:3-O-?-D-galactopyranosyl-(1?2)-?-D-glucuronopyranosyl gypsogemn 28-O-?-D-glucopyranosyl-(1?3)[[?-D-xylopyranosyl-(1?4)]-?-L-rhamnopyranosyl-(1?2)-?-D-fucopyranside 3.THS extract exhibited moderate cytotoxicity against A549,AGS,Hela,Huh7,HT-29 and K562 cell lines.The cytotoxicity of monomer saponins against those cell lines were selective,some of the saponins showed obvious inhibitory activity,among them,Hylomeconoside J and Saponins 16 had the most obvious inhibitory effect on AGS cell line activity,with IC50 value of 6.01 ?M and 3.66 ?M,respectively,while some of the saponins showed no inhibitory activity in the rang of 10-100?M concentration.Flow cytometry was used to detect the apoptosis of tumor cells and the result showed that the saponins improved the apoptosis rate and it was dose-dependent.Conclusion:1.It was reasonable to use the Box-Benhnken experimental design to optimize the extraction technology of THS and the optimal extraction conditions obtained can be used to guide the practice.2.Fifteen undescribed saponins,as well as two known saponins were isolated,which provided theoretical basis for the further study of saponins in the whole plant.3.Saponins from Hylomecon japonica showed good cytotoxic activity in vitro experiments,which provided experimental basis for the further study of pharmacological activities of Hylomecon japonica.