| The Jin-Xuan capsule,consisted of the extracts of Lonicera japonica(Jinyinhua)and Scrophularia ningpoensis(Xuanshen),was developed on the basis of our investigation on"Mailuoning injection" for the prevention and treatment of ischemic heart disease.In this paper,the HPLC specific chromatogram of the raw medicine of "Jin-Xuan capsule"was established.Under the guidance of the HPLC specific chromatogram,27 compounds were obtained through various chromatographic techniques,such as D101 macroporous adsorption resin column chromatography(CC),silica gel CC,Sephadex LH-20 CC,ODS CC and PHPLC.On the basis of extensive analyses of spectroscopic evidences,their structures were identified as secoxyloganin(1),secologanin dimethyl acetal(2),epi-vogeloside(3),vogeloside(4),1-oxo-6-(?-D-glucopyranosyloxy)-5-vinyl-4,4a,5,6-tetrahydro-1H,3H-pyrano[3,4-c]pyran-3-sulfonic acid(5),8-oxo-3-(P-D-glucopyranosyloxy)-4-vinyl-4,4a,5,6,7,8-hexahydro-3H-pyrano[3,4-c]pyridine-6-sulfonic acid(6),(E)-aldosecologanin(7),7-ketologanin(8),8-epiloganin(9),loganin(10),morroniside(11),kingside(12),harpagide(13),harpagoside(14),3-caffeoylquinic acid(15),5-caffeoylquinic acid(16),4-caffeoylquinic acid(17),5-caffeoylquinic acid ester methyl(18),3,4-dicaffeoylquinic acid(19),3,5-dicaffeoylquinic acid(20),4,5-dicaffeoylquinic acid(21),3,5-dicaffeoylquinic acid ester methyl(22),4,5-dicaffeoylquinic acid ester methyl(23),trans-caffeic acid(24),cinnamic acid(25),angoroside C(26),rutin(27).The cardioprotective effect of compounds(1-2,7,15-23,26)on both H2O2-induced and hypoxia-induced injury of cardiomyocytes was evaluated by MTT assay.The results showed that 3,4-dicaffeoylquinic acid(19),3,5-dicaffeoylquinic acid(20),4,5-dicaffeoylquinic acid(21),3,5-dicaffeoylquinic acid ester methyl(22)and 4,5-dicaffeoylquinic acid ester methyl(23)with a concentration of 10-3 mol/l showed significantly cardioprotective effect.Furthermore,a rapid identification method based on ultra-high-performance liquid chromatography coupled with high resolution electrospray ionization mass spectrometry(UPLC-HR-ESI-MS)was established.Based on the information of UV spectrum,retention time of HPLC and fragmentation of MS,the structures and source of 24 peaks were elucidated.The further investigation on the chemical constituents of Scrophularia ningpoensis led to the isolation of 53 compounds through various chromatographic techniques.Their structures were elucidated as scrophularianine A(1),scrophularianine B(2),scrophularianine C(3),indole-3-carboxaldehyde(4),scrophularianoid A(5),scrophularianoid B(6),harpagide(7),harpagoside(8),8-O-p-coumaroylharpagide(9),8-0-(2-hydroxycinnamoyl)harpagide(10),8-O-E-p-methoxycinnamoylharpagide(11),8-O-feruloylharpagide(12),6'-O-p-coumaroylharpagide'(13),6'-O-feruloyharpagide(14),unduloside ?(15),lasianthoside 11(16),tricyclohumuladiol(17),7-hydroxyterpineol(18),acteoside isomer(19),1-(3-hydroxy-4-methoxyphenyl)ethyl-3-a-L-rhamnopyranosyl-6-caffeoyl-?-D-Glucopyranoside(20),isomartynoside(21),acteoside(22),jionoside D(23),martynoside(24),campneoside I(25),angoroside C(26),sibirioside A(27),sibirioside B(28),arrillatose B(29),oct-1-en-3-yl arabinopyranosyl-(1?6)-?-glucopyranoside(30),1-octen-3-ol 3-O-?-D-xylopyranosyl(1 ?6)-?-D-glucopyranoside(31),2-phenethyl P-primeverosdie(32),4-ethyl-5-nitrobenzene-1,2-diol(33),4-methyl-5-nitrobenzene-1,2-diol(34),3-methyl-4-nitrobenzene-1,2-diol(35),4'-hydroxyacetophenone(36),3'-hydroxy acetophenone(37),4'-hydroxy-3'-methoxy-acetophenone(38),3',5-dimethoxy-4'-hydroxyacetophenone(39),protocatechuic acid methyl ester(40),vanillin(41),4-hydroxybenzaldehyde(42),4-methylcatechol(43),homovanillyl alcohol(44),2,6-dimethoxy-4-(methoxymethyl)phenol(45),8-hydroxycoumarin(46),6-hydroxy-2,3-dihydroinden-l-one(47),trans-cinnamic acid(48),trans-4,hydroxycinnamic acid methyl ester(49),4-methoxycinnamic acid(50),traps-4-methoxycinnamic acid methyl ester(51),trans-caffeic acid methyl ester(52),ferulic acid methyl ester(53).Among them,compounds 1-3 were new unusual monoterpene pyridine alkaloids with cyclopenta[c]pyridine skeleton reported from the genus Scrophularia for the first time,compounds 5-6 were new iridoid glycosides,and compound 33 was new phenolic compound.Thirty compounds(1-4,8-9,11-13,17-18,21,26,33-34,36-39,41-50,52)against H2O2-induced injury of cardiomyocytes was evaluated by MTT assay and TUNEL assay.The myocardial protective bioassay indicated that 4-ethyl-5-nitrobenzene-1,2-diol(33),4-methyl-5-nitrobenzene-1,2-diol(34),4-methylcatechol(43),and trans-caffeic acid methyl ester(52)with adjacent phenolic hydroxyl groups in the structures,showed potent cardioprotective activity,but other compounds were inactive.This result suggests that the existence and position of phenolic hydroxyl groups maybe play an important role in the cardioprotective effect against H2O2-induced injury of cardiomyocytes.This work preliminaryly revealed the cardioprotective constituents of "Jin-Xuan capsule"and Scrophularia ningpoensis,and provided a theoretical basis for further investigation on quality control. |
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