| This dissertation includes five chapters. Chapter one is the review of Rhizoma arisaematis; Chapter two is the preparation and anticancer activity screening of different fractions from Rhizoma arisaematis; Chapter three is the studies on the technological process of the anticancer effective fractions of Rhizoma arisaematis; Chapter four is the establishment of the quantitative analysis methods for the anticancer effective fractions of Rhizoma arisaematis; Chapter five is the summary and discussion about this dissertation.Chapter One The progress of Rhizoma arisaematisThe review summarized the progress of sources, chemical constituents, pharmacological activities, clinical applications, and quality control methods of Rhizoma arisaematis.78pieces of references are cited.Chapter Two The preparation and anticancer activity screening of different fractions from Rhizoma arisaematisScreen and affirm the anti-cancer activity of Rhizoma arisaematis. By the organization crushing method, crushing the fresh Rhizoma arisaematis to get its homogenate. Fresh herbs soaking with70%ethanol1:2, soak3days, get filtration, immerse3times, combine the filtrate, get the total extract. Concentration of the total extract is gradient eluted with water-ethanol by AB-8macroporous resin, then in the vitro response test the clearing end, get four polarity parts (D-1, D-2, D-3, D-4). In the anti-cancer cell experiment, test the inhibition of the total extract and4polarity parts to SK-BR3, NCI-H460, HepG2, SW620and normal cells with PrestoblueTM method. Screening and affirm the D-3and D-4is the main anti-cancer active parts in Rhizoma arisaematis.Chapter Three The studies on the technological process of the anticancer effective fraction of Rhizoma arisaematisTo study the extraction technology of the effective fractions of Rhizoma arisaematis.Using L9(43) orthogonal test, which were inspected by the extraction efficiency of total steroid terpene, total alkaloid and trigonelline. To analyze the extraction quantity of total steroid terpenoid and total alkaloid, factor A, factor B, factor C, factor D have significant difference, the best extraction technology is A2B3C3D2; to analyze the extraction quantity of trigonelline, factor A, factor B, factor C, have significant difference, factor D has no significant difference, the best extraction technology is A2B2C3D1. To analyse all the experiments results comprehensively, the best extraction technology is A2B3C3D2,70%ethanol extract3times,5days for one time,and the quantity of solvent is4times of the medical material.To study the purification technology of the effective fraction of Rhizoma arisaematis. After investigated the adsorptive property of4kind of macroporous resin, A resin was selected due to its best adsorption and separative property of total steroid terpene and total alkaloid effective fraction.We investigated the loading capacity, the ratio of diameter and height, concentration of aborsorptive solution, absorptive rate, purified solvent, velocity of flow, eluting solvent and at all. The optimized purification technology of A resin is the extraction of Rhizoma arisaematis with concentration of C2mg/mL was absorpted to resin coloumn (diameter/height H2) with velocity of flow of V3BV/h, the retio of loading capacity and resin volume was Y, Q1BV40%EtOH to wipe off impurity with I3BV/h velocity of flow, then N BV90%EtOH to elute with the flow of I3BV/h, reclaim the solvent, vacuum drying, weighting, the effective fraction of Rhizoma arisaematis was obtained.The extraction and purification technology descried above was tested and verified through3batches of samples. The yield ratio of the effective fraction was1.11%～1.18%, the content of total steroid terpenes is5.86%～6.55%, the content of total alkaloids is9.48%～11.42%. Chapter Four The establishment of the quantitative analysis methods for the anticancer effective fractions of Rhizoma arisaematisThe quantitative method of total steroid terpene by UV-Vis which was carried out Vanillin-perchloric acid reaction was destablished. The linear regression was Y=53.147X+0.0201, r=0.9990(n=8), using (3-sitosterol as indicative constituent, that the concentration of β-sitosterol and absorptance had a favourable linear relationship in the range of0.0049～0.0147mg/mL, the average recovery was100.54%, RSD=1.22%(n=6). The methods were steady and reliable and can be used as the determination methods.The quantitative method of total alkaloid by UV-Vis which was carried out bromorthymol blue reaction was destablished. The linear regression was Y=31.73X-0.0156, r=0.9966(n=8), using trigonelline as indicative constituent, that the concentration of trigonelline and absorptance had a favourable linear relationship in the range of0.0083～0.0249mg/mL, the average recovery was100.34%, RSD=2.15%(n=6). The methods were steady and reliable and can be used as the determination methods.The quantitative methods of indicative constituents trigonelline by HPLC were established The linear regression was Y=30988X-430.36, r=0.9995(n=7), using trigonelline as indicative constituent, that the concentration of trigonelline and absorptance had a favourable linear relationship in the range of0.190～3.792μg/mL, the average recovery was100.74%, RSD=0.97%. The methods were steady and reliable and can be used as the determination methods.Chapter Five Summary and DiscussionThe polarity parts is obtained by preparation of Rhizoma arisaematis separation screening of anticancer activity. With four kinds of cancer cell inhibition rate as index, the anticancer effective part of Rhizoma arisaematis is determined.Preparation of Rhizoma arisaematis anticancer effective parts and purification process conditions are optimized, and the process of optimization method is verified experiment.Established the quality control methods of medicinal materials, the total extract and the effective parts of Rhizoma arisaematis, including the content determination method of total terpene steroid terpene, total alkaloid and trigonelline in medicinal materials, the total extract and the effective parts of Rhizoma arisaematis. For Rhizoma arisaematis anticancer effective part provides the optimum seeking method of the preparation process, and lay the foundations for the further quality control of new drugs research. |
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