Study On Separation And Purification Of Oleanolic Acid From Brazil Ginseng By Macroporous Resin

Objective: Blumea aromatic（Wall.）DC.,a member of Composita e,is a perennial herb,and its whole plant can be used to cure eczema,s kin itchy,dermitis and rheumatoid（arthritis）et al.The main chemical co mponents of Blumea aromatic（Wall.）DC.are flavonoids,volatile oils,terp enoids and steroids.There are some studies about its chemical component s,but isolated compounds does not accurately reflect its pharmacological effects.Besides,in our early activity study on Blumea aromatic（Wall.）DC.study,we found that the ethyl acetate fraction had a good anti-hepatocar cinoma activity.Therefore,this study aimed to research the chemical com position of ethyl acetate and preliminarily study on the new screened com pounds’ activity of anti-hepatocarcinoma in vitro.Methods: 50 kg of herb were first soaked with 500 L 80% alco hol for 7 days.The herb was filtered to take the filtrate.The herbs were soaked with 400 L 80% ethanol for 7 days and repeated twice and combined with three filters of the liquid.After recovered solvent,3.12 kg of ethanol extract was obtained.And then Ethanol extract was suspended in water,and extracted successively with ethyl acetate and petroleum ether o ne by one.After recovered solvent,602.78 g of ethyl acetate site extract was obtained.The Blumea aromatic（Wall.）DC.was separated and purifie d by silica gel,Dextran gel chromatography,ODS,sephadex LH-20 gel,MCI gel.The 1H-NMR、¹³C-NMR、MS and references analysis were used for identification of structure.Cell proliferation viability was determined by MTT assay.Hep3 B and Hep G2 cells were treated with various concen trations（1、5、10、30、70、100、150,200 μmol/L）of Compound 4 and Compound 14 for 24,48 and 72 hours,and cisplatin（75 μmol/L）was us ed as positive control.The OD value was measured by the enzyme-labele d instrument,and calculated the half-maximal inhibitory concentration（IC50）value by the Bliss method.Results: We isolated and identified 16 compounds in total,including Kaempferol⑴,5,7-dihydroxy-4’,6-dimethoxyflavone⑵,luteolin⑶,（20R）-2α,3α,20-trihydroxy-16β-acetylpregnane⑷（new）,5-dihydroxy-16β-acetyl-1,（2R）-5α-2α,3α,16β,20-tetrahydroxypregnane⑸,3,5,7,4’-tetrahydroxy-6-methoxyflavo ne⑹,β-sitosterol⑺,β-carotene⑻,3,3’,4’-O-tetramethyl quercetin⑼,3’,4’,7-t rimethoxy quercetin⑽,Ainasin⑾,no gallon Ethyl acetate⑿,gallic acid⒀,7-hydroxyspruceanol⒁（new）,apigenin⒂,coumaric acid⒃.MTT assay showed that hepatocarcinoma cells（Hep3B and HepG2）were inhibited bycompound 4 and compound 4.The IC50 of Compound 4 to Hep3 B and Hep G2 at 48 h respectively were 78.15 μmol/ L-1and 151.24 μmol/ L-1;72 h was 55.27 μmol·L-1 and 126.64 μmol·L-1.The IC50 of Compound 14 to Hep3 B and HepG2 at 48 h respectively were 121.44 μmol·L-1 and 193.82 μmol·L-1;72 h was 135.80 μmol·L-1 and 223.78 μmol·L-1Conclusions: Among the 16 compounds,there are 10 compounds isol ated from Blumea aromatic（Wall.）DC.for the first time,including 6-di methoxyflavone⑵,（20R）-2α,3α,20-trihydroxy-16β-acetylpregnane⑷（new）,5-dihydroxy-16β-acetyl-1,（2R）-5α-2α,3α,16β,20-tetrahydroxypregnane⑸,3,5,7,4’-tetrahydroxy-6-methoxyflavone⑹,3,3’,4’-O-tetramethyl quercetin⑼,3’,4’,7-t rimethoxy quercetin⑽,no gallon Ethyl acetate⑿,7-hydroxyspruceanol⒁（n ew）,apigenin⒂,coumaric acid⒃.compound 4 and compound 14 can sig nificantly inhibit the proliferation of Hep3 B and HepG2 in a does-depende ntmanner.