Hypericum Anti-tumor Active Ingredient And Method For The Determination

Hypericum japonicum Thunb.,a one-year growth herb,which widely distributes in south drainage area of Changjiang River,in China.At present,the ethanol extract from Hypericum japonicum has been made up to injection and sirup for the treatment of infectious hepatitis and hepatoma.It was reported that the injection of Hypericum japonicum had cytotoxic activity against several tumor cell lines but no cytotoxic activity against normal cell lines.However, bioactivity components responsible for its anticancer activity have not been well investigated up to now.Thus,we have undertaken an investigation on the anti-tumor constituents,as well as development of quality assessment of the plant.The experiments showed that the chloroform extract had remarkable anti-tumor activity in vitro,and its IC₅₀against Bel-7402 and Hela cell lines were 151.4μg.mL^-1 and 174.9μg.mL^-1,respectively.The whole plants of Hypericum japonicum Thunb.(5 kg)were extracted with 80%aqueous alcohol to give an alcoholic extract,which was extracted with chloroform to afford an active chloroform extract.Seven compounds were isolated from the chloroform fraction by silica gel,Sephadex LH-20 and Polyacylamine chromatography and they were identified as follow by chemical reactions and spectral analysis:quercetin-7-O-α-L-rhamnoside,isoquercetrin,tricin,isojacareubin,quercetin, hyperoside and quercetrin.Among them,tricin was isolated from the plant for the first time.The cytotoxic activities of the seven isolated compounds against six tumor cell lines were evaluated by MTT assay.The results showed that tricin and isojacareubin exhibited conspicuous cytotoxic activity.Tricin and isojacareubin had significant cytotoxic activities against Bel-7402 cells,Hela cells,Colo cells,A549 cells, SGC-7901 cells and Heps cells in a concentration-dependent manner,with IC₅₀ 7.61-20.4μg.mL^-1and 3.86-8.52μg·mL^-1,respectively. We established a method for the determination of isojacareubin in Hypericum japonicum Thunb.by RP-HPLC.The separation was performed on an Agilent Zorbax SB-C₁₈column(250mm×4.6mm,5μm)with the mobile phase consisting of acetonitrile-0.04%aqueous phosphonic acid(47:53,v/v)at the flow rate of 1.0 mL·min^-1.The detection wavelength was set at 254 nm and the column temperature was set at 30℃.The calibration curve was linear over the range of 1.68-56 mg·L^-1(r =0.9997).The average recoveries were 101.5%(RSD=2.5%,n=9).The method is simple,accurate and reproducible,and suitable for the quality control of Hypericum japonicum Thunb.A reversed-phase high performance liquid chromatographic method was developed for the simultaneous determination of four flavonoids,namely isoquercitrin, quercitrin,quercetin-7-O-rhamnoside,quercetin in Hypericum japonicum Thunb.The separation was performed on an Agilent Zorbax SB-C₁₈column(250mm×4.6mm,5μm)with a gradient of acetontrile and 0.5%aqueous acetic acid(v/v)at a flow rate of 1.0 mL·min^-1.The detection wavelength was set at 360 nm and the column temperature was set at 35℃.All calibration curves showed good linearity(r²ï¼ž0.999) within the test ranges and the recoveries were 97.5%-102.6%.Satisfactory precisions were obtained with relative standard deviations less than 3%.The method is simple,accurate and reproducible,it can be used for the quality control of Hypericum japonicum Thunb.