Studies On Bioactive Constituents Of Three Natural Medicines-Pericarps Of Sapindus Rarak, Flowers Of Chrysanthemum Morifolium Ramat. ’Huaiju’ Cv.Nov. And Punica Granatum

By means of bioassay-guided separation,the chemical constituents and pharmacological activity of three natural medicines,the pericarps of Sapindus rarak DC.,flowers of Chrysanthemum morifolium Ramat.’huaiju’ cv.nov.and Punica granatum Linn.,were studied for the purpose of providing detailed chemical and pharmacological information for the development of the traditional medicinal materials and their clinical application.The following results were obtained.1.Anti-hyperlipidemic constituents from the pericarps of Sapindus rarak DC.Using the pancreatic lipase screening model in vitro,the methanolic extract and the methanol-eluted fraction of the pericarps of S.rarak were found to show pancreatic lipase inhibitory activity(IC₅₀ = ca.614,420μg/mL,respectively),and were also found to suppress plasma triglyceride（TG） elevation in olive oil-treated mice at 500 mg/kg p.o.By bioassay-guided separation,seven new oleanane-type triterpene oligoglycosides（1-7） were isolated together with sixteen known compounds including twelve saponins（8-19） and four sesquiterpene glycosides （20-23） from the active fraction（MeOH-eluted fraction）.On the basis of chemical and spectroscopic evidence,they were elucidated as raraoside A（1）,rarasaponinⅠ（2）,Ⅱ（3）,Ⅲ（4）,Ⅳ（5）,Ⅴ（6）,andⅥ（7）,sapindoside B（8）,hederagenin 3-O-（2-O-acetyl-β-D-xylopyranosyl）-（1→3）-α-L-rhamnopyranosyl-（1→2）-α-L-arabinopyranoside（9）,hishoushi-saponin Ee（10）,mukurozi-saponin E₁（11）,hederagenin 3-O-（3,4-di-O-acetyl-β-D-xylopyranosyl）-（1→3）-α-L-rhamnopyra-nosyl -（1→2）-α-L-arabinopyranoside（12）,hederagenin 3-O-α-L-arabinopyranosyl-（1→3）-α-L-rhamnopyranosyl -（1→2）-α-L-arabinopyranoside（13）,hederagenin 3-O-（2,4-di-O-α-L-arabinopyra-nosyl） -（1→3）-α-L-rhamnopyranosyl-（1→2）-α-L-arabinopyranoside（14）,mukurozi-saponin G（15）, hederagenin 3-O-α-L-arabinofuranosyl-（1→3）-α-L-rhamnopyranosyl-（1→2）-α-L-arabinopyranoside （16）,hederagenin 3-O-（3,5-di-O-α-L-arabinofuranosyl）-（1→3）-α-L-rhamnopyranosyl-（1→2）-α-L-arabinopyranoside （17）,mukurozi-saponins Y₁（18）,mukurozi-saponins Y₂（19）,mukuroziosideâ… a （20）,mukuroziosideâ…¡a（21）,mukuroziosideâ… b（22） and mukuroziosideâ…¡b（23）.Except for new compounds 1-7,compounds 9,10,13,14 and 16-22 were obtained for the first time from this plant.The pancreatic lipase inhibitory activity of the isolated compounds,thirteen triterpene saponins and three acyclic sesquiterpene glycosides,were determined.The inhibitory potency(IC₅₀) order of saponin constituents was 11（100μM）＞10（117μM）＞17（121μM）＞8（125μM） =16 （125μM）＞12（129μM）＞13（130μM）＞2（131μM）＞1（151μM）＞15（166μM ）＞3（172μM）＞9（300μM）＞4（576μM）;Acyclic sesquiterpene glycosides（20,21 and 23） showed little inhibitory activity.Based on the above-mentioned results,the structure-activity relationships of triterpene saponins of the pericarps of S.rarak for inhibition of pancreatic lipase activity were elucidated as follows.The terminal-β-D-xylopyranosyl enhanced the inhibitory activity in comparison with terminal-α-L-arabinopyranosyl.For the saponins with arabinose as terminal suggar moiety,α-L-arabinofuranose showed more potent inhibitory activity thanα-L-arabinopyranose.The number and position of acetyls at the terminal-glycosyl also differentially affected the inhibitory activity.In conclusion for this part of the study,the pericarps of S.rarak mainly contain triterpene saponins and acyclic sesquiterpene glycosides.Among them,triterpene saponins potently inhibit pancreatic lipase activity in vitro and the plasma TG elevation in vivo,which are the major active constituents responsible for the anti-hyperlipidemic activity of the pericarps of S.rarak.The investigation in this part provided helpful information of potential candidates for the development of novel anti-hyperlipidemic drugs.2.Anti-allergic constituents from the flowers of Chrysanthemum morifolium Ramat.’huaiju’ cv.nov.Using RBL-2H3 cells model in vitro and compound 48/80 induced scratching reaction in mice, the 75%ethanol extract of Chrysanthemum morifolium Ramat.’huaiju’ cv.nov.（Huai-ju） were found to show potent anti-allergic activities both in vivo and in vitro.The 60%ethanol-eluted fraction and 95%ethanol-eluted one were confirmed as the active fractions due to both highest inhibitory activity of degranulation in antigen-induced RBL-2H3 cells(IC₅₀= ca.6.8,16μg/mL, respectively).By means of bioassay-guided separation,a new flavonoid derivative（1） were isolated together with nineteen known compounds including fourteen flavonoids（2-15） from the active fraction （60%ethanol-eluted fraction）,and four caffeoyl-quinic acid derivatives（16-19） were isolated from 30%ethanol-eluted fraction.On the basis of chemical and spectroscopic evidence,they were elucidated as diosmetin 7-（6"-O-p-hydroxyphenylacetyl）-O-β-D-glucopyranoside（1）,luteolin（2）, apigenin（3）,acacetin（4）,diosmetin（5）,diosmetin 7-O-β-o-glucoside（6）,luteolin 7-O-β-D-glucoside （7）,apigenin 7-O-β-D-glucoside（8）,acacetin 7-O-β-D-glucoside（9）,acacetin 7-O-（6"-O-acetyl） -β-D-glucoside（10）,scolimoside（11）,diosmin（12）,linarin（13）,eriodictyol（14）,hesperetin 7-O-β-D-glucoside（15）,3,5-dicaffeoyl-quinic acid（16）,1,3-dicaffeoyl-epi-quinic acid（17）, macranthoin F（18） and chlorogenic acid（19）.Except for the new compound 1,compounds 11,12 and 18 were obtained for the first time from the Chrysanthemum sp.HPLC-DAD fingerprinting profile analysis indicates that there are great differences between Huai-ju and Hang-bai-ju in composition and contents of four flavone aglycones,diosmetin,luteolin, apigenin and acacetin,and that diosmetin and acacetin were charateristic constituents only existed in Huai-ju,but not in Hang-bai-ju.Thus,the anti-allergic activity of the four flavones aglycones was investigated.They all potently inhibited the release ofβ-hexomsanidase and TNF-αstimulated by either antigen or calcium ionophore A23187 from RBL-2H3 cells.Luteolin showed the most potent inhibitory activity for the release ofβ-hexomsanidase stimulated by antigen or calcium ionophore A23187(IC₅₀ =2.8,3.8μM),while that of diosmetin for the release of TNF-αstimulated by antigen was the most potent(IC₅₀=3.4μM).Some anti-allergic mechanism of diosmetin was studied for the first time.It was implicated with the inhibition of the increase in intracellular free Ca²⁺ levels([Ca²⁺]_i),determined with Fluo 3/AM probe.Diosmetin inhibited antigen-induced increase in[Ca²⁺]_i and calcium ionophore A23187 induced degranulation,with the IC₅₀ values of 3.6μM and 5.1μM,respectively.On the other hand,A23187 induced increase in[Ca²⁺]_i could not be attenuated by diosmetin.These findings suggested that the inhibitory target of diosmetin for the early-phase of hypersensitivity involved in the degranulation processes both before and after increase in[Ca²⁺]_i in RBL-2H3 cells. Furthermore,diosmetin also suppressed the expression of mRNA of cytokines TNF-αand IL-4 in antigen-stimulated RBL-2H3 cells,determined by RT-PCR,which indicated the inhibitory target of diosmetin at late-phase in RBL-2H3 cells.In conclusion for this part of the study,the flowers of Chrysanthemum morifolium Ramat. ’huaiju’ cv.nov.（Huai-ju） mainly contain flavonoids and caffeoyl-quinic acid derivatives.Among them,flavone aglycone constituents are the major active constituents responsible for anti-allergic activity of the flower herb.And the potent anti-allergic activity of diosmetin suggested that it may be a potential candidate for developing novel anti-allergic drugs.3.Constituents with tumor necrosis factor-αinhibitory activity from the flowers of Punica granatum Linn.Using L929 cells screening model in vitro,the methanolic extract and the ethyl acetatesoluble fraction of the flowers of P.granatum were found to show inhibitory effect on tumor necrosis factor-α（TNF-α,1 ng/mL）-induced cytotoxicity in L929 cells.By means of bioassayguided separation,a new taraxastane-type triterpene（1）,was isolated from the active fraction（ethyl acetate-soluble fraction） together with nine known compounds,including four triterpenes（2-5）, two galloyl glucoses（6,7） and two flavones（8,9）.On the basis of chemical and spectro- scopic evidence,they were elucidated as punicanolic acid（1）,oleanolic acid（2）,ursolic acid（3）,maslinic acid（4）,asiatic acid（5）,1,2,6-tri-O-galloyl-β-D-glucopyranoside（6）,1,2-di-O-galloyl-4,6-O-（S）-hexahydroxydiphenoyl-β-D-glucopyranoside（7）,luteolin（8） and tricetin（9）.Except for the new compound 1,compounds 6,7 and 9 were obtained for the first time from the Punica sp. The TNF-αinhibitory activity of the isolated compounds were investigated.Among them, punicanolic acid（1）,oleanolic acid（2）,maslinic acid（4）,1,2,6-tri-O-galloyl-β-D-glucopyranoside （6）,1,2-di-O-galloyl-4,6-O-（S）-hexahydroxydiphenoyl-β-D-glucopyranoside（7） and luteolin（8） were found to show significant inhibitory activities on TNF-α-induced cytotoxicity in L929 cells at 30μM or 100μM.Among them,punicanolic acid showed higher potency than the positive compound piperine.In conclusion for this part of the study,the flowers of P.granatum mainly contain triterpene acids,galloyl glucoses and flavones.And the potent inhibitory effect of punicanolic acid on TNF-α-induced cytotoxicity in L929 cells suggested that it may be a potential candidate for developing new type of hepatoprotective drugs with the property of lowering the sensitivity to TNF-α.