| Objective(s):Research the chemical constituents and biological activities of three medicinal plants of Orchidaceae,Pleione praecox(J.E.Smith)D.Don,Pleione maculata(Lindl.)Lindl.,Cremastra appendiculata(D.Don)Makino.Method:Separation of compounds is carried out by solvent extraction,silica gel column chromatography,RP-18 reversed column chromatography,MCI column chromatography,macroporous resin D101,semi-preparative HPLC,preparative TLC,polyamide column chromatography,Sephadex LH-20 column chromatography and other separation techniques.Identify the structure of the obtained compound by nuclear magnetic resonance,infrared spectroscopy,ultraviolet spectroscopy,circular dichroism and mass spectroscopy methods.The bioactive research included the protective activity of SH-SY5Y cells,cytotoxic activity and protective the damaged PC12 cells.Results:From Pleione praecox,33 compounds were isolated and identified,including 9 new compounds:2,6-dihydroxy-5,8-dimethoxyphenanthrene(1),3,7-dihy-droxy-2,4,6-trimethoxy-9,10-dihydrophenanthrene(2),1,2’,7,7’-tetrahydroxy-4,4’-dimethoxy-9’,10’-dihydro-2,1’-biphenanthrene(3),1,1’,7,7’-tetrahydroxy-4,4’,8,8’-tetramethoxy-2,2’-biphenanthrene(4),2,2’,7,7’-tetrahydroxy-5,5’-dimethoxy-1,1’-biphenanthrene(5),Pleioneprarine A(6),Pleioneprarine B(7),7-hydroxy-2,3-dimeth-oxy-1-(p-hydroxybenzyl)dihydrophenanthrene(8),3’-hydroxy-5-methoxy-3-O(3"-methoxybenzyl)bibenzyl(9).The main chemical components are phenanthrene compounds,dihydrophenanthrene compounds,phenanthrene dimers,bibenzyl compounds and phenylpropanoids.Pharmacological studies have found that among the compounds 1-25,new compounds 4,6,8,9 and known compound 22 have cytotoxicity at 1 μM and 50 μM;new compound 7 shows cytotoxicity at 50 μM;known compounds 13,15,and 19 and 23 showed cytotoxicity at a concentration of 1μM.On this basis of this,the antioxidant activity of compounds 1～25 on SH-SY5Y cells induced by hydrogen peroxide was also studied.The new compounds 3,4,5,8 and the known compounds 10,12,15,22,and 24 show good anti-oxidative damage effects at 50 μM,and the cell survival rate is equivalent to the positive control EGCG;the known compound 15 and 23 showed strong anti-oxidative damage at 1μM,and the cell survival rate was above 80%,the difference was statistically significant;the dimer compounds of phenanthrene and bibenzyl 18～21 showed strong anti-oxidative damage at 1μM and 50 μM.Compounds 18～21 had good anti-oxidative damage effect,and their activity were close to the positive control substance,and expressed potential development and application value.From Pleione maculata,38 compounds were isolated and identified,including 7 new compounds:2,3,7-trihydroxyl-4,8-dimethoxy-phenanthrene(1),3’,3,4"-tri-hydroxy-5-methoxy-2’-(p-hydroxybenzyl)-bibenzyl(2),3,6’,4"-trihydroxy-5-meth-oxy-2’-(4"-hydroxybenzyl)bibenzyl(3),3,3’-dihydroxy-5-methoxy-6-(3",4"-dihy-droxybenzyl)bibenzyl(4),12-(4-Hydroxy--3-methoxyphenyl)-11-hydroxymethyl-3-methoxy-9,10,11,12-tetra-hydrophenanthro[2,1-b]furan-7-ol(5),12-(4-methoxy-3-hydroxy-phenyl)-11-acetoxyroxymethyl-4-methoxy-9,10,11,12-tetrahydrophe-nanthro[2,1-b]furan-7-ol(6),2’,4,7,7’-tetrahydroxy-2,4’-dimethoxy-9,9’,10,10’-tetrahydro-1,1’-biphenanthrene(7).The main chemical constituents are phenanthr-ene compounds,dihydrophenanthrene compounds,bibenzyl compounds,aromatic compounds and steroid compounds.Some compounds were tested for the inhibitory activity of human lung cancer cell lines(A549),human breast cancer drug-sensitive cell lines(MCF-7/S),and human ovarian cancer cell lines(SKOV-3).The new compound 2 was tested on three cell lines.It has good inhibitory activity,with IC50 of 1.57,12.73,0.12 μM,and its effect is stronger than the positive control cisplatin;compound 30 also has good inhibitory activity against 3 tumor cell lines,and with IC50 of 2.45,6.83,4.23 μM,respectively.From Cremastra appendiculata,45 compounds were isolated and identified,among which 3 are new compounds:4’,4"-dihydroxy-2’,3’-dimethoxydihydro-chalcone(2),2,6-dihydroxy-8-methoxy-1,4-phenanthraquinone(2),Cremastrappenoid A(3),the main chemical components are bibenzyl,phenanthrenequinone,phenanthrene compounds,terpenes compounds and steroids.The remaining 42 are known compounds.Among them,compounds 4-6,9,11～19,22,23,25,31～34,36,37,41,43,45 were isolated from this plant for the first time.Pharmacological studies found that among compounds 1-21,all compounds except compounds 15,18 inhibited cell activity at 1 μM and 50 μM.Compounds 7,10,and 20 showed cytotoxicity at 1 μM,and compounds 6,9,12,13,and 16 showed cytotoxicity at 50 μM.Compounds 1,2,3,4,5,8,11,14,17,19 and 21 showed cytotoxicity at 1 μM and 50 μM.On the basis of this,the antioxidant activity of compounds 1-21 on SH-SY5Y cells induced by hydrogen peroxide was studied.It was found that,in addition to compounds 10,15,and 18,new compound 1 and known compound 7 showed cytotoxicity at 1 μM;known compounds 12,13,and 16 showed cytotoxicity at 50 μM;new compounds 2,3 and known compound 4,5,8,11,14,17,19,20,21 inhibited cell activity at 1 μM and 50 μM.It was also found that the new compound 2 showed higher antioxidant damage at 1 μM and 50 μM.The cell survival rate is more than 60%;the new compound 3 exhibits a high anti-oxidative damage effect at 50 μM,and the cell survival rate is more than 90%;compounds 5 and 7 showed better effects at 1 μM with high anti-oxidant damage,among which the cell survival rate is above 60%,the difference is statistically significant.Further research also found that compound 1 could reduce the expression of MDA at a dose of 0.1 μM,and increase the expression of SOD,reduce the expression of MDA,NO,and nNOS at a dose of 1 μM and 10 μM(p<0.05,p<0.01),The difference is statistically significant.Compound 2 could increase the expression of SOD and decrease the expression of MDA at a dose of 0.1 μM,and increase the expression of SOD,decrease the expression of MDA,NO,and nNOS at a dose of 1 μM and 10 μM.The difference is statistically significant,and academic significance(p<0.05,p<0.01).The new compounds and some known compounds have been tested for the inhibitory activity of human lung cancer cell lines(A549),human breast cancer drug-sensitive cell lines(MCF-7/S),and human ovarian cancer cell lines(SKOV-3).The research results show that the new compound 2 has good inhibitory activity on MCF-7/S and SKOV-3 cell lines,with IC50 of 5.73 and 3.95 μM,respectively;compound 37 is known to have strong inhibitory activity against 3 cell lines,with IC50 of 3.07,2.19,2.70 μM;Compounds 42 and 43 showed good inhibitory activity against MCF-7/S cell lines,with IC50 of 2.16 and 5.09 μM,respectively;the above effects are stronger than the positive control cisplatin.Conclusion(s):From three orchidaceous medicinal plants,116 compounds were isolated and identified,of which 19 are new compounds.Their structure types include the basic framework types of orchids:phenanthrene,bibenzyl,dimer phenanthrene,dimer dihydrophenanthrene,and benzyl substituted phenanthrene and bibenzyl compounds with benzyl groups.In addition,some other compounds such as terpenoids,lignans and esters were also obtained in the research.The phenanthrene and bibenzyl compounds isolated from the Pleione praecox showed high anti-oxidative damage to the antioxidant damage of SH-SY5Y cells induced by hydrogen peroxide.Except for the two esters compounds,other compounds have varying degrees of protection against SH-SY5Y cell damage.Structure-activity analysis showed that dimer phenanthrene,which is stronger than benzyl-substituted bibenzyl,followed by benzyl-substituted phenanthrene,and esters have no activity.The dimer phenanthrene compound isolated from the Pleione maculata has strong cytotoxic activity.The tumor cell inhibitory activity test was performed on 7 new compounds,and it was found that the new compound 2 has strong inhibitory activity against three tumor cell lines;some known compounds were tested for the inhibitory activity of three tumor cell lines,and the results showed that compound 30 has good inhibitory activity on tumor cell lines A549,MCF-7/S and SKOV-3,and its effect is stronger than the positive control cisplatin.The new compound 3 isolated from Cremastra appendiculata showed antioxidant activity against SH-SY5Y cells induced by hydrogen peroxide at 50 μM;the new compound 1 and 2 played anti-oxidant effect by increasing SOD,reducing the expression of MDA,NO,nNOS,so as to protect damaged nerve cells.Dimer phenanthrene compounds 42,43 have a strong inhibitory effect on tumor cell lines(MCF-7/S).In this study,for the first time,the chemical components of the medicinal plants of the genus Pleione praecox and the Pleione maculata were isolated and identified,and the biological activities of the compounds were reported.Systematic chemistry and biological activity research were performed on the toxic medicinal plant of Cremastra appendiculata.This research has enriched the phytochemistry and biological activity of Orchid family. |
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