| With high attack rate and death rate, malignancy threats mankind's healthiness badly. And colon carcinoma is one of the most rapid onset rate of common malignant tumor. Because of side effects such as immunity-restraining,marrow-restraining etc,it is hard to improve survival quality of cancer-patients by chemotherapeutics medicine only.In recent years,studies of TCM on anti-tumor are very prolific.Natural drugs have distinctive superiority in anti-tumor because of the fewer poison, side effects and expedient resourses.Among these drugs, taxol extracted from Taxus has influential effects on curing tumor. Taxol has been found favourable therapeutic actions not only in ovarian cancer but also in colon carcinoma although the price is expensive and the side effects are visible. But the researches of anti-tumor components in Taxus are limited by the mode of western medicine,many other anti-cancer active components in Taxus are refused in taxol injection because of the special preparation.With the guide of Chinese medical science, anti-cancer components were extracted from needles and twings of Taxus Yunnanensis.The projects are to overcome a series of problems caused by taxol solitary. And other components such as total flavonoids, total lignanoids,alkaloids, and so on also have anti-tumor effects to some extent. Compatible with taxol, they can improve curative effects and degrade the poison and side effects of taxol. Extract of Taxus is indissolvable in water and can't be absorbed well for oral use. Solid dispersion technology can enhance the dissolution rate in vitro. Colon targeted preparations can deliver drugs to colon directly,which can improve the bioavailability in vivo.Purposes:1. To summarize the international literature of Taxus,taxol—as the most important active component,and other related chemical compounds. To summarize literatures about colon carcinoma. To summarize literatures about solid dispersion technology and colonic targeted technology. To analyse researches of Taxus preparations.2. To establish the method of quantification of anti-tumor active components.3. To study the origin of Taxus. To study the main factors influencing the quality of needles and twigs of Taxus Yunnanensis.4. To study the extraction procedure,initially impurity refused procedure and refined procedure on needles and twigs of Taxus Yunnanensis for reforming traditional extraction and seperation procedure.5. To study the antitumor effects of Extract of Taxus on human colorectal carcinoma cell lines in vitro.To study the quality standard of extract of taxus6. To prepare the solid dispersion and colon targeted micro pellets.7. To evaluate the release of colon targeted pellets in vitro and to establish the method of quality standard of colon targeted pellets.Methods:1. Using computer or handworked searches, CA,BP,MEDLARS,CBM-Disc,CNKI,CMCC and so on,plentiful and all-sided literatures of research advancements of Taxus, structure and character of major chemical compositions, methods of extraction and separation, pharmaco-research and clinical application were got. Literatures of colon carcinoma and preparation of colon targed were consulted.2. Comparing different methods of sample processing. A HPLC method was developed for the determination of the contents of axanes compounds in needles and twigs of Taxus Yunnanensis. An ultraviolet spectrophotometry method was developed for the determination of the contents of total flavonoids.3. Contents of taxanes compounds were used as the judgement criteria. The origins, the different medication parts, the growth time and the stored time of Taxus were studied. Three batches of medical materials were inspected.4. Total terpene yield and contents of taxanes compounds in extract were used as the judgement criteria to compare different extraction methods. The effect on the impurity refusing and the losing rate of the active components contents were investigated by extraction with NaHCO3, defatting with petroleum ether, and precipitation with lime cream. To refine extract, different column chromatography such as Silica gel column, macroporous resin column and alumina column were considered.5. Colorectal carcinoma cell lines SW480 and HCT-8 were chosen to study the antitumor effects of the extract of taxus. Five groups were compared and every group had five concentrations. Methyl thiazolyl tetrazolium analysis was used to detect the inhibitory effets of extract. The concents of taxanes compounds in the extract were determined by HPLC and the quality standard of the extract was assured.6. Prescription of solid dispersion was chosen by in vitro dissolution test of artificial colon juice. Micro-pellets were made by coat method.Different diluents, diffetent ratio of diluent and diffetent binder solution were chosen with the judgement criteria of slick and sly condition, the size distribution of pellets and bulk density.7. The release of the micro-pellets differently in artificial gastric juice, artificial small intestinal juice and artificial colon juice was evaluated in vitro,and the quality standard of micro-pellets was assured..Results:1. Literature summarization: About 90 literatures were collected by the above tools,Among them, overseas literatures were focusing on the studies of chemical constituents,domestic literatures were focusing on the studies of production places,assaying,pharmaco-researching, drug action and clinical application. The quality of Taxus cuspidate Sieb.et.Zucc had been published by Finger printing. It had been studied on the extraction,identification and anti-tumor action of Taxus Yunnanensis.The experiments of taxol immunoliposome and targeting human colorectal carcinoma cell had also been found.2. Studies on the determination of anti—cancer active components contents2.1 Preparation of sampleSolid phase extraction was used to separate the product,and different fraction were collected. The recovery rate was high.2.2 Determination of Taxanes CompoundsThe contents of three kinds of Taxanes compounds were determined by HPLC. The linear range were 0.04μg～3.0μg. The content of total flavonoids was determined by UV and the linear range was 8～48μg/ml.3. Studies on the quality of medical material3.1 Studies on the origin of TaxusContents of taxanes compounds in Taxus Yunnanensis were the highest. Needles and twigs of Taxus Yunnanensis could be used as medical material.3.2 Studies on the different medication parts of TaxusAlthough the contents of taxanes compounds in Needles and twigs were lower than that in barks, collecting needles and twigs would not hurt the plants, and they could also regenerate.So it had great significance to get taxanes compounds from needles and twigs.3.3 Influence of growth time and stored time on the quality of Taxus.The contents of Taxanes compounds were higher and higher with growth time increasing.The contents were lower and lower with stored time increasing.So the needles and twigs of Taxus Yunnanensi should be collected with more than five years growth time and stored less than half a year.4. Studies on the preparation of extrac of Taxus4.1 Study on extraction technologyThe optimum extraction condition was to infuse the herb with 7 and 5 times volume of 85% alcohol in turn.SFE extraction was better than the traditional extraction.4.2 Studies on the initially impurity refused procedureThe emulsification and the loss of taxanes compounds were serious when extracting with NaHCO3. Although most impurity could be eliminated by lime cream preparation,terminal point was difficult to control. Petroleum ether showed good effect on defatting, Physic liquor after defatting should be extracted for four times with chloroform. And then decoloured with 0.25% (W/V) activated carbon at 35℃.4.3 Studies on the refined technologySilica gel column, macroporous resin column and alumina column were used to refine the extract. The extract was dissolved in chloroform and added to the top of Silica gel column, then washed with chloroform until the eluents were achromic,finally washed with chloroform-methanol (97:3),and collected three times dosage of eluent. Eluent was concentrated in vacuum. And then the concrete was dissolved in sufficient acetic ether-acetone (1:1), and filtered out impurities, The filterate was mixed with macroporous resin column and added to the top of macroporous resin column,The column was eluted with three times column volume of 30% alcohol and 6 times column volume of 85% alcohol in turn,The 85% alcohol eluent was concentrated in vacuum. The residue was dissolved in sufficient chloroform,and added to the top of alumina column.Firstly, chloroform was used to wash the column until eluent was achromic. Secondly, 7 times column volume of chloroform-methanol (97:3) was used. Thirdly, the chloroform-methanol (97:3) eluent was evaporated in vacuum and the extract of Taxus was got.5. Studies on the antitumor effects on hunman colorectal carcinoma cell and the quality standard of the extract of Taxus.In the selected limits, the extract of Taxus, taxol, mixture of taxol and cephalomannine,mixture of taxol and cephalomannine and taxinninⅣall exhibited antitumor effects on human colorectal carcinoma cell lines SW480 and HCT-8 in vitro after 72 hours, the effects of mixtures were more stronger, It was showed that cephalomannine and taxinninⅣhad synergistic inhibitory effects.Drily calculated, The content of taxol in the extract should not be less than 16% and the total amount of cephalomannine and taxinninⅣshould not be less than 35%.6. Preparation of solid dispersion: the solid dispersion was prepared with acrylic resin L100—55 as carrier materials. One portion of extrac of Taxus dissolved in dehydrated alcohol was mixed with two portions of acrylic resin L100—55 dissolved in dehydrated alcohol. The mixture was evaporated to dryness on aqueous bath, and dried under reduced pressure and at last comminuted to 80 mesh.Preparation of micro-pellets: Solid dispersion-MCC-amylon-talc powder were mixed at the ratio of 3:0.6:0.3:0.1, and 5%(w)PVP used as the adhesive, The mixture was made to soft wood and then rooled in the coating pan to preparate pellets, The pellets of 14～30 mesh were collected after dried on 40℃.7. Studies on the evaluation in vitro and quality standard of colon targeted pellets.Discerning from the stripping curve, in artificial gastric juice there was no drug release within two hours. In artificial small intestinal juice taxanes compounds had a dissolution of 9～14% and within 9 hours in artificial colon juice the pellets released completely.Drily calculated, the content of taxol in pellets should not be lower than 4% and total amount of cephalomannine and taxinninⅣshould not be lower than 9%.Conclusion:1. Literatures and investigation report had showed that the same researches as ours were not found.2. The methods are accurate, feasible, reproducible, and can be used for the determination of taxol, cephalomannine,taxinninⅣand total flavonoids.3. Medical material was confined to needles and twigs of Taxus yunnanensis Cheng et L.K.Fu,and they should be collected with more than five years growth time and stored less than half a year.4. The key of the study is the extraction technique,the initially impurity refused procedure and the refined technology.The opimum technique could extract active priciple efficiently from Taxus. Through collecting main category components of Taxanes compounds, total flavonoids and total lignanoids, the method changed the traditional method which collected components with systematic separation. The method reduced the dosage of poisonous reagents on some extent,and decurtated the cycle time of passing column. Separation technique of main category components was in line with the theory of TCM. It was also expected to educe synergistic effects and reduce poison and side effects after compatibility.5. The experiment of cell inhibition of colorectal carcinoma cell lines SW480 and HCT-8 in vitro showed that cephalomannine and taxinninⅣhad synergistic inhibitory effects, which is coincident to Literatures.6. Appling appropriate conditions on adjuvant and preparation, micro-pellets of solid dispersion of the extract could be made successfully. Micro-pellets could elevate the dissolution rate of Taxanes compounds.Quality standard can control the quality of micro-pellets effectly.The antitumor effects on human colorectal carcinoma cell lines in vitro has proved that anti-cancer active components extracted from Taxus Yunnanensis has antineoplasmic activity, The results offer theory-gist for quality control of needles and twigs of Taxus Yunnanensi, Extraction, seperation and refinement technology of anti-cancer active components, Antitumor effects of Extract of Taxus on human colorectal carcinoma cell lines in vitro, Quality standard of extract, Preparation of Colon targeted solid dispersion and micro pellets. Quality standard of pellets.But partly some researches havn't been finished, it is required to do more deeply in pharmacodynamics and process of releasing in vivo.
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