| This dissertation includes three parts:summary of the related literatures; studies on the extraction and purification technology of the alkaloid fraction of Caulis Sinomenii; establishment of methods to control the quality of alkaloid fraction and sinomenine; the HPLC fingerprint of Tinospora sinensis; establishment of methods to control the quality of alkaloid fraction and Jatrorrhizine hydrochloride, Palmatine chloride, Berberine.Part one:summary of the related literaturesThis account is a review on the development of the genus Tinospora’s (family Menispermaceae) phytochemicals and biological activities during1995to2012. A total of132compounds were found reported in this genus, consisting of100terpenoids,24alkaloids,8lignans and some others. Part two:studies on the extraction and purification technology of the alkaloid fraction of Caulis Sinomenii; establishment of methods to control the quality of alkaloid fraction and sinomenine.The extraction technology of alkaloid fraction of Caulis Sinomenii has been studied. L9(34) orthogonal test has been used to evaluate the optimum extraction technology, including the best extraction solvent, amount of the solvent, hour and number of times. The optimum extraction technology was evaluated by the extraction amount of total alkaloid as well as sinomenine. Finally, the optimum technology is that using70%ethanol, the amount of which is10times of crude drugs, to extract Caulis Sinomenii2times,1.5hour each time.The purification technology of alkaloid fraction of Caulis Sinomenii has been established. After investigating the absorptive property of5kinds of ion-exchange resins, D001-CC ion-exchange resin was selected due to its preeminent absorptive and separate property of total alkaloids as well as sinomenine. Then the purification technology of purifying the alkaloid fraction of Caulis Sinomenii with D001-CC ion-exchange resin was optimized, such as concentration of the solution, the pH, velocity of absorption, the ratio of diameter and height, loading capacity, purifying solvent, eluting solvent, the amount of purifying and eluting solvent, ammonia and ethanol concentration of eluting solvent and et al. Finally, the best purification technology of alkaloid fraction of Caulis Sinomenii with D001-CC ion-exchange resin is as follow:First, crude drugs of Caulis Sinomenii were extracted by70%ethanol under reflux, the amount of which is lOtimes of crude drugs, to extract2times,1.5hour each time. The EtOH was evaporated under reduced pressure, the residue was ultrasonic dispersed (30min,100kHz) with10times0.1%HCl of crude drugs, then centrifugalize (3000rpm,30min) it to get leavings and the water solution, the leavings was treated with the same method, mixing two supernatant (the concentration of the supernatant is0.05g/mL, calculated by crude drugs). Second, the supernatant was absorbed by D001-CC ion-exchange resin (diameter/height1:8of resin column) with the velocity of flow of3BV/h and the proportion between the crude drugs and the resin column was1.61g/mL,9times volume of resins of70%ethanol to wipe off impurity, then30times volume of resins of0.5%alcohol ammonia (concentration of ethanol is90%) to elute with10BV/h, collecting eluent of alcohol ammonia and evaporating alcohol ammonia to dryness under reduced pressure.The extraction and purification technology described above was tested and verified through3batches of samples of alkaloid fraction of Caulis Sinomenii. The yield ratio of alkaloid fraction of Caulis Sinomenii is6.20-6.76%. In the alkaloid fraction, the content of total alkaloid was64.31-71.55%and the transferring ratio was beyond71.11%(71.11-74.96%); the content of sinomenine was23.16-24.88%and the transferring ratio was beyond70.14%(70.14-71.12%). The results were steady and satisfied.The acid dye colorimetry was established to determine the total alkaloids in the alkaloid fraction of Caulis Sinomenii. The content of total alkaloids was determined with acid dye colorimetry, and selected sinomenine as reference substance. Sinomenine had a linear relationship with the absorbance in the range of1.69-10.16ug/mL and the regression equation was as follows:Y=0.0841X+0.0283(r=0.9996). The average recovery for sinomenine was101.12%, RSD=1.89%(n=6).HPLC methods were established for the determination of sinomenine in the alkaloid fraction of Caulis Sinomenii. The linear ranges of sinomenine was0.02064-0.1032mg/mL, respectively. The regression equation of sinomenine was:Y=1.7513×107X-67660(r=0.9996). The average recovery for sinomenine was99.33%. The results manifest that the method was steady and reliable and can be used as the determination methods.Part three:the UPLC fingerprint of Tinospora sinensis; establishment of methods to control the quality of alkaloid fraction and Jatrorrhizine hydrochloride, Palmatine chloride and Berberine.The extraction technology of alkaloid fraction of Tinospora sinensis has been studied. Lc>(34) orthogonal test has been used to evaluate the optimum extraction technology, including the best extraction solvent, amount of the solvent, hour and number of times. The optimum extraction technology was evaluated by the extraction amount of total alkaloid. Finally, the optimum technology is that using Hydrochloric acid-70%ethanol (1:100), the amount of which is8times of crude drugs, to extract Tinospora sinensis3times,1.0hour each time.The uv method to determine the content of total alkaloids was established, which was performed with Jatrorrhizine hydrochloride as the reference substance. The optimum extraction technology was evaluated including the best extraction solvent, amount of the solvent, hour and so on. The optimum extraction technology was evaluated by the extraction amount of total alkaloid. And the content of total alkaloids in different place was between0.26%and0.76%.To establish the chromatographic fingerprints of Tinospora sinensis by UPLC which potentially benefit to the quality control in production. Under the above chromatographic conditions, sixteen peaks were identified as the characteristic fingerprints of Tinospora sinensis. The UPLC fingerprint method is found to have satisfactory accuracy, stability and reproducibility.HPLC methods were established for the determination of Jatrorrhizine hydrochloride, Palmatine chloride and Berberine. The optimum extraction technology was evaluated including the best extraction solvent, amount of the solvent, hour and so on. The optimum extraction technology was evaluated by the extraction amount of indicative constituents. And the content of indicative constituents in different place were0.00015%-0.00094%,0.000086%-0.00048%,0.000040%-0.00028%respectively. |
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