Hypericum Anti-active Ingredients Of Hypoxia

Four medicinal plants were confirmed to exhibit significant anti-anoxic effect from about 1000 extracts of plants(Yunnan,Guangxi and Sichuan) through the anti-anoxic PC12 cells assay in vitro and the acute normobaric hypoxia assay in vivo.In this thesis,one of the four medicinal plants: Hyperieum japonicum Thunb.ex Murray was selected for the bioactivity-guided isolation with the in vitro anti-anoxic PC12 cells assay.Hypericum japonicum Thunb.ex Murray is widely distributed in Guangdong,Guangxi,Sichuan,Hunan,Fujian,Jiangxi,et al.It has been used as herbal medicine for the treatment on hepatits,diarrhea,intestinal abscess and other diseases.The anti-anoxic effects of the three solvent(CHCl₃,EtOAc and n-BuOH) extract parts and the rest water parts from the 60%ethanol extract of Hypericum japonicum Thunb.ex Murray were tested through the in vitro anti-anoxic PC12 cells assay and the in vivo acute normobaric hypoxia assay.The EtOAc soluble part showed strong anti-anoxic effects,and was carried out on the previous bioactivity-guided isolation.In our bioactivity-guided isolation of the chemical constituents of Hypericum japonicum Thunb.ex Murray,34 compounds were obtained from the EtOAc soluble part of the 60%ethanol extract of the whole herbs of this plant through various chromatographic techniques.Among them, their structures of 31 compounds were elucidated on the basis of color reactions and spectroscopic analysis.The 31 compounds from Hypericum japonicum Thunb.ex Murray are stigmasterol(1),daucosterin(2),bijaponicaxanthone(3),quercetin-7-O-α-L-rahmnoside(4),quercetin-3-O-α-L-rahmnoside(5),quercetin-3-O-β-D-glucoside(6),4,6-dimethyl, 1-O-[α-L-rhamnopyranosyl-(1→6)-β-D- glucopyranosyl]multifidol(7)*,protocatechuic acid(8),gallic acid(9),quercetin(10),kaempferol(11),3-O-methyl quercetin(12),luteolin-7-O-rutinoside (13),apigenin(14),5,7,4'-trihydroxy-3'-methoxy flavone(15),3',4'-dimethoxy-5,7-dihydroxy flavone(16),p-hydroxybenzoic acid(17),(2E)-dihydrokaempferol(18),(2E)-dihydroquercetin (19),5,7,3',4'-tetrahydroxy flavanone(20),p-hydroxycinnamic acid(21),5,4'-dihydroxy-7-methoxy flavanone(22),(2E)-3,5,3',4'-tetrahydroxy-7- methoxy flavanone (23),(2Z)-dihydroquercetin- 3-O-α-L-rahmnoside(24),chlorogenic acid(25),4-O-β-D-glucosyl coumaric acid(26),1,5-dihydroxy-8-methoxy xanthone(27),1,7-dihydroxy-4-methoxy xanthone (28),3,7-dihydroxy-1,2-dihymethoxy xanthone(29),3,6-dihydroxy-1,2,7-trihymethoxy xanthone (30),1,4,7- trihydroxy-3-hymethoxy xanthone(31).Among them,6 compounds are flavanones including compound 18,19,20,22,23,24;10 compounds are flavones including compound 4,5,6, 10,11,12,13,14,15,16;6 compounds are xanthones including compound 3,27,28,29,30,31,7 compounds are phenolic acids including7,8,9,17,21,25,26;and 2 compounds are others including compound 1 and 2;compound 7 is a new compound;compounds 8,12,18,19,24,25,29, 30 are firstly isolated from Hypericum japonicum Thunb.ex Murray.Parts of compounds isolated from Hypericum japonicum Thunb.ex Murray were tested by anti-anoxic PC12 cells assay in vitro.The result showed that compound 4,5,8,9,10,12,15 had better anti-anoxic activities,wherein compound 4,5,10,12 and 15 are flavones;compound 8 and 9 are phenolic acids.Parts of compounds isolated from Hypericum japonicum Thunb.ex Murray were tested by DPPH method in vitro.The result showed that compound 8,10,18,19,25displayed the same potent oxygen radical scavenging activity as the positive control,Vc,wherein compound 8 and 25 are phenolic acids;compound 10 is flavone;compound 18 and 19 are flavanones.The fingerprint chromatomap of the 60%ethanol extract of Hypericum japonicum Thunb.ex Murray has been established using DAD-HPLC.About 90%of the peak areas in the chromatography have been identified by comparing with the standards,8 compounds had been fingered,and the contents of 5 compounds had been determinate.This research will be providing a preliminary direction for the research and development of Hypericum japonicum Thunb.ex Murra in the future.