Study On Isolation And Identification Of Active Ingredient From Penthorum Chinense Pursh And Its Bioactivities And Extraction Technology

Penthorum chinense Pursh (P. chinense P.) is a member of Penthorum Gronov Ex L(Penthoroideae (Van Tiegh) Engl. Subfamily of Saxifragaceae). The Penthoroideae (VanTiegh) Engl. Subfamily has only one genus and about three plant, only one of which exists inChina. P. chinense P is a Miao nationality herbal drug used in Chinese folk medicine forremedy forjaundice, oedema and traumatic injury, cholecystitis, adiposis hepatica andinfectious hepatitis. P. chinense P is widely distributed in China and eastern Asian, and havebeen domesticated and large-scale cultivation in Sichuan province. Gansu a prepared Chinesemedicine from the extract of P. chinense has been used in clinic for treating hepatitis andproven to be effective for several years So it is an urgent need for a comprehensiveunderstanding of its active components and pharmacological effects to control product qualityand further reasearch.Different models, Antioxidant (Reductive potential, DPPH, Superoxide anion, Hydroxylradicals and lipid peroxidation), Anti-liver cancer cell (BEL-7402&SMMC-7721cells),Protecting Liver by CCl4injuried L02liver cell, were used to evaluate the activity of differentsolvents extract from P. chinense P. The activity of Ethanol (EtOH) extract was stronger thanthe aqueous extract, which showed the active ingredient of P. chinense P was more easilyextracted by alcohol. Considering others, the70%EtOH was elected to be extraction solvent.70%ethanol extract of P. chinense P. was hierarchical extraction by petroleum ether (PE),ethyl acetate (EA), butanol (n-BuOH) and water (W2) according to priority for four parts,which was respectively evaluated by antioxidant, anti-liver cancer and liver protection modelas above. The results show that70%EtOH’s active ingredients have got better separation afterfractional extraction, and its main active ingredient is concentrated in the EA and n-BuOHfraction, and PE part almost has no activity. Known to all, its main active ingredients wereflavonoids, saponins etc. moderately polar substances. Overall, EA part was more reactive,thus ethyl acetate fraction was elected for further separation..The active site of EA part was separated and purified by various column chromatography(including silica gel column, Sephdeax LH-20gel column and ODS column) and the preparedHPLC, and was gained four componds.Their structure was identified by massspectrometry(MS), nuclear magnetic resonance (1H-NMR spectra,13C-NMR, andtwo-dimensional spectrum HMBC, etc.) and the literatures comparison. They are respectively,A3:-sitosterol(115.3mg), D1:Quercetin(22.4mg), G2:Pinocembrin-7-O-[3-O-galloyl-4",6" -hexahydroxydiphenoyl]--glucose(598.5mg), and G3: Thonningianins A(345.5mg), amongwhich A3is a sterol, and the others are flavonoid, besides G2and G3are rare ellagic tanninflavonoids. And G2and G3are the main active ingredient isolated from P. chinense P, thecontent of which are much higher than other methods. Thonningianins A.is firstly isolatedfrom P. chinense P., also firstly from Saxifragaceae. Three flavonoids were showed goodactivities of antioxidant and anti-liver cancer. The DPPH antioxidant of G3, G2is superior tothe positive control:VC; the activity of the flavonoids, especially G3&G2, are far strongerthan the positive control5-FU in the anti-liver cancer (SMMC-7721) experiments, and theirIC50order is that IC50(μmol/l) G3(52.17)<G2(112.82)<D1(554.09)<5-FU (1902.01). It canbe inferred that the main active ingredient of P. chinense P. is flavonoids, especially ellagictannin flavonoids.The isolation and activities results indicated that the flavonoids of P. chinense P. is the mainactive ingredient, so the extraction process of total flavonoids from P. chinense P. wasoptimized. Firstly investigated the feasibility of the method, based on the single factorexperiments to determine the affecting factors of ultrasonic extraction of total flavonoids.According to the Box-Benhnken central composite experimental design principle, Responsesurface analysis methodology was used to optimize the sepration conditions. The optimizedresults is extraction temperature of50°C, extraction time of70min, the ethanol concentrationof60%, liquid to solid ratio of20:1, and the yield was7.21%. The results was verifid andconsistent with the theory, and have some application value.