Chemical Studies Of The Effective Fractions Of Kushen

This dissertation includes four parts: summary of the related literatures; studies on the extraction and purification technology of the flavonoid fraction and alkaloid fraction; studies on the chemical constituents of flavonoid fraction; establishment of methods to control the quality of flavonoid fraction and alkaloid fraction.Part one: summary of the related literaturesStudies on chemical constituents and pharmacological activities of Sophora flavescens Ait. in recent years were reviewed. 81 references were cited.Part two: studies on the extraction and purification technology of the flavonoid fraction and alkaloid fractionThe extraction technology of flavonoid fraction and alkaloid fraction of Kushen has been studied. L9(34) orthogonal test has been used to evaluate the best extraction technology, including the best extraction solvent, amount of the solvent, hour and number of times, of flavonoid fraction and alkaloid fraction which was evaluated by the extraction efficiency of total flavonoids, total alkaloid as well as the indicative constituents including nor-kurarinone, kuraridin, trifolirhizin, oxymatrine, N-oxysophocarpine, matrine and sophocarpine. Finally, the best technology is that using A₂% ethanol, the amount of which is D₂ times of crude drugs, to extract Kushen three times, C₂ hour for B₃ time.The purification technology of flavonoid fraction of Kushen has been established. The best purification method of flavonoid fraction is as follows: firstly, disperse the extraction of Kushen by water (with the concentration of 30mg/mL) and then centrifugalize it to get residue; secondly, the residue was resolved by J5% ethanol, the amount of which is r5 times of the residue, and then the solution was centrifugalized to have transparent ethanol; thirdly, j5% ethanol was recycled to obtain the other residue that was resolved by x%HCl with an amount of s times of the residue and then the solution was centrifugalized to acquire the residue; at last, the residue was watered in order to obtain pH=7, dried with a reduced pressure, and then the purified flavonoid fraction was got.The purification technology of alkaloid fraction of Kushen has been established. After investigating the absorptive property of 5 kinds of macroporous absorptive resins, E resin was selected due to its preeminent absorptive and separate property of total alkaloids as well as the indicative constituents like oxymatrine, N-oxysophocarpine, matrine and sophocarpine. Then the purification technology of purifying the alkaloid fraction of Kushen with E resin was optimized, such as the pH, concentration of the solution, velocity of absorption, the ratio of diameter and height, loading capacity, purifying solvent, eluting solvent, the amount of purifying and eluting solvent, and the repeated using times of resins and et al. Finally, the best purification technology of alkaloid fraction of Kushen with E resin is as follow: in the first place, disperse the extraction of Kushen by water (with the concentration of 30mg/mL) and then centrifugalize it to get residue and the water solution; in the second place, the residue was resolved by J5% ethanol, the amount of which is r5 times of the residue, and then the solution was centrifugalized to have transparent ethanol; in the third place, J5% ethanol was recycled to obtain the other residue that was resolved by x%HCl with an amount of s times of the residue and then the solution was centrifugalized to acquire the HCl solution; in the fourth place, amalgamate the water solution and HCl solution, using 2mol/L NaOH to basify the merged solution to obtain pH=7; finally, the basified solution(pH=7) was concentrated with the concentration of c3g/mL, alkalinized to pH=a2, absorbed by E resin(diameter/height d2 of resin column) with the velocity of flow of s2BV/h and the proportion between the total amount of the solution and the resin column was p mg/mL, x2 times volume of resins of water to wipe off impurity, then 5 times volume of resins of Q% ethanol to elute with e2BV/h, the resin can be repeatedly used for7 times after refining with 95% ethanol and 5%NaOH.The extraction and purification technology described above was tested and verified through 3 batches of samples of flavonoid fraction and alkaloid fraction of Kushen. The preparation and determination results showed that the established extraction and purification technologies were steady and repeated, the yield ratio of extraction, flavonoid fraction and alkaloid fraction of Kushen is 24²6%,4.2⁴.6%, 7.0⁷.5%, respectively. In the flavonoid fraction, the content of total flavonoids was 58⁶1% and the transferring ratio was beyond 68% (68⁷2%); the content of nor-kurarinone was 4.0⁴.5% and the transferring ratio was beyond 46% (46⁵1%); the content of xanthohumol was 0.21⁰.26% and the transferring ratio was beyond 225% (225²80%); the content of kuraridin was 15.0¹6.0% and the transferring ratio was beyond 526%(526⁶21%); the content of trifolirhizin was 1.7².0% and the transferring ratio was beyond 34%(34³6%). In the alkaloid fraction, the content of total alkaloid was 60⁶2% and the transferring ratio was beyond 78% (78⁸2%); the content of oxymatrine was 14¹5% and the transferring ratio was beyond 45% (45⁴8%); the content of N-oxysophocarpine was 2.5³.0% and the transferring ratio was beyond 39% (39⁴3%); the content of matrine was 15¹7% and the transferring ratio was beyond 565% (565⁷02%); the content of sophocarpine was 8.0⁸.3% and the transferring ratio was beyond 90% (90⁹5%). The results were steady and satisfied. Part three: studies on the chemical constituents of flavonoid fractionSeveral separation and purification methods and determination technology(such as UV,IR,EI-MS,ESI-MS,1HNMR,13CNMR) were used to study the chemical constituents of flavonoid fraction. 12 flavonoid compounds were separated and identified, including, 1 flavonol, named Nor-anhydroicaritin; 4 flavanones, named kushenol A, nor-kurarinone, kurarinone, isokurarinone, respectively; 3 pterocarpans, named trifolirhizin, maackiain, trifolirhizin 6'-monoacetate, respectively; 2 chalcones, named kuraridin, xanthohumol, respectively; 1 coumaroyl phenothrin, named medicagol and 1 isoflavone named formononetin. Among them, formononetin was firstly separated from Kushen.Part four: establishment of methods to control the quality of flavonoid fraction and alkaloid fractionThe Mg-HCl colorimetry was established to determine the total flavonoids in the flavonoid fraction of Kushen. The content of total flavonoids was determined with HCl-Mg reaction colorimetry, and selected nor-kurarinone as reference substance. Nor-kurarinone had a liner relationship with the absorbance in the range of 8.27～41.36μg/mL and the regression equation was as follows: Y = 0.0154 X + 0.0001(r = 0.9992). The average recovery for nor-kurarinone was 99.91%, RSD=2.21% (n=6). The contents of total flavonoids in three batches of the flavonoid fraction were 61.32%, 60.56%, 60.02%, respectively. The contents of total flavonoids in three batches of the extraction of Kushen were 12.32%, 13.56%, 13.02%, respectively; the contents of total flavonoids in three batches of the raw drug were 3.82%, 3.56%, 3.71%, respectively.The acid dye colorimetry was established to determine the total alkaloids in the alkaloid fraction of Kushen. The content of total alkaloids was determined with acid dye colorimetry, and selected matrine as reference substance. Matrine had a liner relationship with the absorbance in the range of 1.60⁸.0μg/mL and the regression equation was as follows: Y = 0.0786X + 0.005 (r = 0.9999). The average recovery for matrine was 99.61%, RSD=0.74% (n=6). The contents of total alkaloids in three batches of the alkaloid fraction were 60.48%, 59.39%, 59.94%, respectively. The contents of total alkaloids in three batches of the extraction of Kushen were 19.18%, 19.39%, 18.94%, respectively; the contents of total alkaloids in three batches of the raw drug were 5.42%, 5.26%, 5.18%, respectively.HPLC methods were established for the determination of four flavonoids, i.e. nor-kurarinone, xanthonumal and kuraridin, trifolirhizin in the flavonoid fraction of Kushen. The linear ranges of nor-kurarinone, xanthonumal, kuraridin and trifolirhizin were 0.12μg⁰.60μg, 0.004μg ⁰.020μg, 0.20μg¹.02μg, 0.05616μg～0.3370μg, respectively. The regression equations of these four flavonoids were as follows: Y=1810432X-2838.7 (r=0.9999), Y=735164X +4543.1 (r=0.9996), Y=938204X +3373.6 (r=0.9997), Y=735164X+4543.1 (r=0.9991). The average recoveries for these four flavonoids were 102.45% with RSD=2.90% (n=6), 101.48% with RSD=2.53% (n=6), 99.83% with RSD=1.64% (n=6) and 102.04% with RSD=1.12% (n=6), respectively. The results of the contents of these four flavonoids in three batches of the flavonoid fraction, extraction and raw drug showed the methods were steady and reliable and can be used as the determination methods.HPLC methods were established for the determination of four alkaloids, i.e. nor-kurarinone, xanthonumal and kuraridin, trifolirhizin in the flavonoid fraction of Kushen. The linear ranges of oxymatrine, N-oxysophocarpine, matrine and sophocarpine were 0.202μg 1.213μg, 0.220μg 1.320μg, 0.207μg 1.240μg, 0.124μg 0.746μg, respectively. The regression equations of these four alkaloids were as follows: Y = 1019192X + 1630.3 (r = 0.9999),Y = 1599822X + 19713 (r = 0.9999),Y = 1407124X + 20031 (r = 0.9995),Y = 3377452X– 15677 (r = 0.9994). The average recoveries for these four alkaloids were 101.43% with RSD=1.30% (n=6), 99.08% with RSD=0.94% (n=6), 102.59% with RSD=1.69% (n=6) and 102.06 with RSD=1.10% (n=6), respectively. The results of the contents of these four alkaloids in three batches of the alkaloid fraction, extraction and raw drug showed the methods were steady and reliable and can be used as the determination methods.The HPLC fingerprint of flavonoid fraction of Kushen was established under the detective wavelength 320nm. 31 peaks (or peak groups) were found. Meanwhile, the chemical constituents which these peaks stood for were analyzed by adding reference substances to the sample. 11 peaks were concerned, which are all flavonoids. The 11 flavnoids are as follows: peak 1 (trifolirhizin), peak5 (trifolirhizin 6'-monoacetate), peak 10 (formononetin), peak 11 (maackiain), peak 14 (kurarinone), peak 17(nor-anhydroicaritin), peak 22(nor-kurarinone), peak 25 (xanthohumol), peak 28 (isokurarinone), peak 30 (kuraridin) and peak 31 (kushenol A).The contents of four flavonoids (trifolirhizin, nor-kurarinone, xanthonumal and kuraridin) in 25 market samples of Kushen were determined by HPLC methods, in order to discuss the necessity and feasibility to use these four constituents to evaluate the quality of raw drug, decoction piece and preparation of Kushen. The content range of trifolirhizin was 0.05%～0.32%, nor-kurarinone was 0.05%～0.51%, xanthonumal was 0.0005%～0.0045% and kuraridin was 0.06%～0.23%. The results of contents of four flavonoids in 25 market samples of Kushen showed that the contents of these four flavonoids from different districts were changed dramatically between 4 times and 10 times. Besides, the contents of kuraridin from different districts had the lowest variation while nor-kurarinone had the largest distinction.