Study On The Extraction And Isolation Process Of Flavonoids And Alkaloids Active Ingredients From Tephroseris Kirilowii

Tephroseris kirilowii belongs to Asteraceae,perennial herb,it is widely distributed in the north,north-east,east and south-west of China.The whole plant can be applied to as medicine,which is helpful for relieving heat and detoxification,quicking the blood and dispersing swelling,resolving tetany and ulcer,et al.Recent study showed that 60 percent ethanol extract of Tephroseris kirilowii(mainly containing water alcohol compatibility alkaloids and flavonoids) had a strong inhibitory effect on mouse lymphocytic leukemia L1210 cells.It has been recorded in Chinese Medicinal Dictionary "Tephroseris kirilowii at dose of 3g/ml had inhibitory effect on leukemia cells".At home and abroad,studies about Tephroseris kirilowii's anti-leukemia effect are few.Reports about extraction and purification research of its active ingredients are few too. In this study,we used Tephroseris kirilowii as raw material,study the extraction,separation and purification process of Tephroseris kirilowii at the basis of preliminary pharmacological experiments,and its chemical composition(flavonoids,alkaloids) was analysed by the qualitative and quantitative assay.All these results maybe provide a theory basic for the further separation of flavonoids and alkaloids from Tephroseris kirilowii.PartⅠ:Pharmacological activity tests of Tephroseris kirilowii's ethanol extractStudy on the ethanol extract(30%,60%,95%) of Tephroseris kirilowii affecting on the weight,the average survival time and the impact of abdominal circumference of the L1210 tumor-bearing mice by intraperitoneal injection.The results showed that 60%and 95%ethanol extract of Tephroseris kirilowii had the better anti-leukemia effect.In consideration of saving solvent,60 percent ethanol was choosed as the extraction concentration.PartⅡ:To optimize the extraction process of Tephroseris kirilowii active ingredientsIn the method of one-way and orthogonal test,extraction time, extraction temperature,extraction times and the rate of solvent to material were studied,and make a qualitative and quantitative tests.The pararneters of the best total flavonoids extraction technology were as follows:extraction temperature at 80℃,liquid ratio at 1:10,extraction time for 3h,extract times for 2 times.Under the conditions of optimal extraction process,the average extraction rate of total flavonoids was 0.523 percent,the total flavonoids content was 6.897 percent measured by Colorimetry.The parameters of the best total alkaloids extraction technology were as follows:extraction temperature at 80℃,liquid ratio at 1:10,extraction time for 3h,extract times for 3 times.Under the conditions of optimal extraction process,the average total alkaloids extraction rate was 0.211%.PartⅢ.To optimize the isolation and purification process of total flavonoids from Tephroseris kirilowiiPurificated total flavonoids of Tephroseris kirilowii using D101 macroporous resin,the factors of affecting the rate of adsorption and desorption of macroporous resin D101 were researched in order to find out the optimum purification conditions,such as effluent speed,effluent concentration,effluent pH,sample flow rate,sample pH,eluant volume fraction,sample concentration,elution concentration and so on。A better adsorption-desorption conditions were as follows:static absorption time for 24h,sample concentration at 5mg/ml,sample pH from 6 to 8,sample flow rate at 2BV·h^-1;better elution conditions were as follows:eluant volume fraction of 70%ethanol,eluant pH from 6 to 8,eluant volume 2.5BV,elution flow rate for 2BV·h^-1.After the treatment by D101 macroporous resin,the purity of flavonoids was increased 2.3 times. PartⅣ:Initial isolation of Flavonoids from Tephroseris kirilowiiMade a initial isolation of the ethyl acetate part(total flavonoids part) from Tephroseris kirilowii using silica gel and Sephadex LH-20, and identificated the structural of isolated compounds using modern spectrum technology.