| “Prescription embodied in Preparation”, Chinese medicine preparation, aims to study the specific form of Chinese medicine from raw materials to preparation for acting on patients directly. With the development of new technologies and new theories,Traditional Chinese Medicine(TCM) preparation emerged in a new stage: preparation of component structural TCM, with the characteristics of definited material basis, clear mechanisms, determined ADME/T properties, reasonable drug release system designs and scientific productions quality controls. Multiple drug release system which was made by component is the basic form of preparation of component structural TCM. It emphasizes on different components by refining and purification to represent the whole TCM. Then technology of modern pharmaceutics was used to prepare different drug release unit for the purpose of a procedure to release. At last, it can play the role of multiple ingredients and targets.Zexie, Alisma orientalis(Sam.) Juzep., one of Traditional Chinese Medicine in clinic, was recorded in Su wen because of containing a variety of chemical composition,such as terpenoids, polysaccharide, volatile oil and so on, which was effective or toxic have been isolated. However, the chemical composition of nephrotoxicity is still unclear,and toxicology mechanism is unknown. In this study, we studied relevancy of efficacy-toxicity-chromatogram and its mechanism of total terpenoids as preparation units basing on “component structure” theory to direct against preparation of component structural TCM. It provides an ideal for illuminating the safety and effectiveness of praperation unit and also promots the development of preparation of component structural TCM. The main content of this study was as follows:1. Efficacy and evaluation of safety of ZexieIn this part, distilled water was used to fill the stomach to make water load model in rats for studying the effect “clearing damp and promoting diuresis” of Zexie from seven different places. The results indicated that Alisma orientalis from different placehad diuretic efficacy by significantly increasing the volume of rat urine and the concentration of Na+, K+ and Cl- in urine and significantly decreasing the level of AQP-2 in rat renal medulla and HK-2 cells. Female SD rats were used for evaluation of safety after administrating of its ethanol extraction in different concentrations for about6 months. The results indicated that the protein expression of Ceruloplasmin, Clusterin,Kim-1, LCN2, Osteopontin and TIMP1 investigated in rat kidney. Good preparation unit needs consideing excellent efficiency and safety, so we need further study about how to control the quantity relationship of preparation basic unit for reducing its toxicity.2. Identification of relationship between efficacy and chromatogram and total terpenoids from ZexieThe basic unit of preparation of component structural TCM needs to be clarified material base. In this section, we analyzed the components of salted Zexie from different places to represent the best effect of clearing damp and promoting diuresis using relationship between efficacy and chromatogram basing on its optimal efficiency of Zexie from Fujian. The results indicated that the chromatographic peak with retention time was 72.53, Alisol A 24-acetate, Alisol B and Alisol B 23-acetate were the main components in Zexie. Due to many components in Zexie, we usually think that components transported into blood were effective component. Thus we used UPLC/Q-TOF-MS to differentiate components as terpenoids in the blood which was Alisol F, Alisol A, Alisol B 23-acetate, Alisol A 24-acetate and Alisol B. Overall consideration, we chose Alisol A 24-acetate, Alisol B and Alisol B 23-acetate as main components whose ratio of structural features was 1:0.4:0.13. So we chose total terpenoids as basic unit of components praperation.3. Preparation and determination of total terpenoids from ZexieResponse surface method was used to investigate the optimum technological condition of total terpenoids extracting by ethanol. The optimum technological condition of total terpenoids extracting by ethanol was extracted by 80% ethanol for 2times with 30 min each time. Orthogonal design was used to investigate the optimum technological condition of macroporous resin purifying total terpenoids. The optimum technological condition of macroporous resin purifying total terpenoids was purified by AB-8 macroporous resin with 70% ethanol at the speed of 2BV/h and elution volume was 4BV. The best technology was used to prepare total terpenoids and simultaneous finish content determination. 8 terpenoids including Alismoxide, Alisol F, Alisol A,Alisol F 24-acetate, Alisol A 24-acetate, 25-Methoxyalisol A, Alisol B and Alisol B23-acetate, whose content was 61.74% calculated by peak area while the content of Alisol A 24-acetate, Alisol B and Alisol B 23-acetate was 38.13%. Whether total terpenoids prepared by us was good preparation unit, we need further study.4. Evaluation for safety of total terpenoidsThis section we respectively investigated whether total terpenoids have renal toxicity in vivo and in vitro. And its possible mechanism of toxicity and relationship among efficacy, tocxity and chromatogram were also explored with Alisol A 24-acetate,Alisol B and Alisol B 23-acetate. The results of HK-2 cells in vitro indicated that total terpenoids could significantly increase the protein and m RNA level of Kim-1, Clusterin and TFF-3. Rats fed with total terpenoids for 4 months, BUN in urine increased while the content of BUN could significantly increase. In vivo, pathological changes of kidneys from rats were identified. Interstitial inflammation, renal tubular epithelial cell exfoliation and morphological changes were found in rat kidneys. Meanwhile the protein level of Clusterin and Kim-1 significantly increased. 3-MA was used for the further study of its mechanism. The results indicated that Alisol A 24-acetate, Alisol B and Alisol B 23-acetate induced autophagy possibly resulted in apoptosis and nephrotoxicity through PI3K/Akt/m TOR signaling pathway. Then we chose the ratio of Alisol A 24-acetate, Alisol B and Alisol B 23-acetate for further study. At last we identified the relationship of efficacy-toxicity-chromatogram with the ratio of Alisol A24-acetate, Alisol B and Alisol B 23-acetate was 5.38:14.34:11.31. This ratio was related to the ratio of structural features from Fujian Zexie. This ratio of total terpenoidscould ensure the efficacy of Traditional Chinese Medicine preparation, and guarantee the safety of its basic unit of preparation of component structural TCM.5. SummaryBasing on “component structure” theory, the main topic of this study was focused on the study of total terpenoids before prapration, mainly includes discrimination and preparation of total terpenoids, evaluation of safety for total terpenoids, analysis of relationship among toxicity, efficacy and content and so on. The results indicated that toxicity and efficacy of Zexie was related to the content of components in total terpenoids which created conditions for the preparation of component structural Traditional Chinese medicine and provided a research approach for the study of preparation of component structural Traditional Chinese medicine. |
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