Study On The Extraction And Separation Of The Alkaloids From Radix Sophorae Tonkinensis And Its Anti-oxidative And Anti-tumor Activity

Radix sophorae tonkinensis is the dried root and rhizome of Sophora tonkinensisGagnep. belonging to Leguminosae, distributed mainly in the south of China. Itpossesses antipyretic, antiinflammatory, analgesic effects and is used to treat sorethroats, sore gums and tongue festered and so on. There are abundant alkaloids,flavonoids and triterpenoid glycosides in Radix sophorae tonkinensis, among them,alkaloids are the major active constituents. Antitumor activity, antioxidative activity andantiarrhythmic activity of extract of Radix sophorae tonkinensis were reported.Therefore, Radix sophorae tonkinensis plays an extremely important role in medicine,shall be researched and developmented.Firstly, the experiment studies the optimal extraction technology of alkaloids fromRadix sophorae tonkinensis. The content of the total alkaloids and matrine andoxymatrine is as the inspection index. Meanwhile, the total content of matrine andoxymatrine from Radix sophorae tonkinensis is determined using high performanceliquid chromatography （HPLC）. The extraction efficiency of warm immersionextraction, ultrasonic extraction, reflux extraction and accelerated solvent extraction(ASE) is compared. The results obtain that accelerated solvent extraction is superior tothe other three methods. The chromatographic condition is that Ascentis RP-Amide,Catalog#:565325-U is as the chromatographic column (250×4.6mm，5μm), the mobilephase A is acetonitrile and B is phosphoric acid with pH of3. The gradient elutionsequence is0~29min, from A:B=0:100to A:B=6:94;30~32min, A:B=100:0. Theultraviolet wavelength detection is210nm, the flow rate is1mL/min, the sample size is2μL, the column temperature is40℃. The extraction conditions of ultrasonic extraction, reflux extraction and accelerated solvent extraction are optimized throughsingle-factor test and orthogonal experimental design. The optimal technology ofaccelerated solvent extraction is that the ethanol concentration was40%, extractiontemperature is90℃,the time of extraction is13min and the times of extraction is twice.The total content of matrine and oxymatrine from Radix sophorae tonkinensis is15.5277mg/g. Compare to the other three methods, accelerated solvent extraction willgreatly shorten the time of extraction and obtain high extraction rate, which provides anew method for the extraction of alkaloids from Radix sophorae tonkinensis. Butbecause of the amount of the extract of crude drugs obtained from accelerated solventextraction is limited, consequently, it still requires ultrasonic extraction or refluxextraction for the extraction preparation of a large number of the total alkaloids.According to the results of orthogonal test, the optimal technology of ultrasonicextraction is that the ethanol concentration is40%, the ratio of material to liquid is1:20,the time of extraction is1.5h and the times of extraction is twice. The content of thetotal alkaloids from Radix sophorae tonkinensis is16.7137mg/g. While the optimaltechnology of reflux extraction is that the ethanol concentration is60%, the ratio ofmaterial to liquid is1:25, the time of extraction is1h and three times for the extraction.The content of the total alkaloids from Radix sophorae tonkinensis is16.7685mg/g.The results obtain that the difference of the content of the total alkaloids from Radixsophorae tonkinensis between ultrasonic extraction and reflux extraction in the optimalextraction conditions is not obvious, the consumables and the time of extraction in bothmethods are pretty close, you can choose either one of them for the extraction. Thisexperiment uses reflux extraction.Preparative high-performance counter-current chromatography (HPCCC) isapplied to separate and purify the alkaloids from Radix sophorae tonkinensis for thefirst time. The two-phase solvent system composed of dichloromethane-methanol-water (5:3:2, v/v/v) is selected for HPCCC, using the upper aqueous phase as stationary phaseand the lower organic phase as mobile phase; the flow rate is2mL/min; the revolutionspeed is1600rpm; the detection wavelength is210nm; the column temperature is30℃;the sample size is150mg; the retention of the stationary phase is70%. The collectedfractions are analyzed by high-performance liquid chromatography (HPLC) andHPLC-DAD-MS/MS. The results obtain that two kinds of alkaloids are obtained,matrine and oxymatrine with their purity of100%, respectively. It turned out thathigh-performance counter-current chromatography can separate a large of samples withhigh purity, and provides a new effective separation method for the preparationseparation of alkaloids from Radix sophorae tonkinensis.Finally, this experiment determines the ability of elimination hydroxyl radical ofcrude extract from Radix sophorae tonkinensis through DPPH method. Theexperimental results showes that its ability of elimination hydroxyl radical along withthe increase of the concentration enhanced, thus, it turned out that the crude extract ofRadix sophorae tonkinensis has better antioxidant activity. To detect the inhibitingcapacity of matrine and oxymatrine to MDCC-MSB cells’ reproductive activity throughthe MTT method. It turned out that all of matrine and oxymatrine can inhibitMDCC-MSB cells’ reproductive activity obviously, its inhibiting capacity show obviousdose-time dependent.