| Canner is a detriment disease which death rate is second only than cardiovascular disease. So long time, searching anticancer biotic resources and naturally occurring drugs, is a common wash of every country medical, ph, chemistry, and biologist. In recent, it is more and more thinked highly by people to extract active component from natural drugs. Taxus is as a natural broad-spectrum anticancer resource that curing more much cancer. But its resource is lack and utility content lower; presently, extraction technology and preparation technology exist much problems, so it is necessary to improve and solove them.The main purpose of this study is wash to multiple utilization anticancer active component. The same time, to production more correspond actuality requestion, more convenient, economy, safe, and environmental protection; the other part of the study also on flavonoids in branches and leaves of Taxus yunnanensis, including riching total flavonoids, cell- pharmaco experiment, and a method for the determination of flavonoids, so profit more development and utilize in flavonoids of Taxus yunnanensis in afterward This thesis includes six parts, which methods and results describe as follows:1. Review articles. Using computer or handworked searehes, summarize and analyse the international and domestic literatures, and reasonable analyse their study results. Finally propose derection and purpose on this study.2. Extraction technology of anticancer active component in branches and leaves of Taxus yunnanensis.Methods: from prepation of crude drug as start, study the technology of extract and defat with L9 (34) orthogonal test. With EtOH extraction and water sed, alkali washing, EtOAC extraction as main methods of eliminate dopant. Finally with alky AlzO3 chromatographic column, rich and gain anticancer active site. The maximum response was achieved at a 40 min extracting time with 10 times volume 90% EtOH added to the sample for the first time and 30 min for the second time. Adding 10 times water will produce precipitation; Place the precipitation into 6 times petroleum benzin to de-fat at 60℃. Use 1 mol/L NaOH solution to wash the sample to remove the acid impurity. Al2O3 chromatography eluting with cyclohexane/acetone, collect the cyclohexane/acetone 6:4 fraction.Result: The anticancer active component includes: taxol 9.45%,cephalomanine: 9.63%,10- deacetylbaccatinⅢ: 22.64%,total flavonoids: 19.15%.3. The preparation of Seiadopitysin controlMethods: Take dry Ginkgo leaves and extracted three times by sonication with 95% EtOH, solutions were concentrated under vacuum, after defatting by petroleum benzin, the sample was extracted with EtOAc and the solutions were concentrated under vacuum. Take silica gel chromatography eluting with EtOAc / petroleum ether(1:9～5:5). The production was washed several times by MeOH, and dissolved in dimethyl formamide(DMF) to recrystallize, we got yellow power crystal.Result: (1) The purity of the production was over 95% detected by RP-HPLC. (2) General qualitative reactions consistent with the refererices. (3)The spectrums data of UV,IR,MS and NMR consistent with the references. So the product was sciadopitysin.4. An Investigation of the method for determinating the contents of total flavonoids and sciadopitysin in branches and leaves of Taxus yunnanensisMethods: Using HPLC and UV-Spectro-photometry method to determinate the contents of total flavonoids and sciadopitysin in branches and leaves of Taxus yunnanensisResult: We take the experiments at 272 nm, total flavonoids show a good linear relationship (r=0.9992) at a range of 12～28μg/ml. The average addition recovery is 96.04% and RSD is 2.7%. The average content of total flavonoids is 5.01%. Sciadopitysin shows a good linear relationship (r=0.9999) at a range of 0.88～4.4μg/ml. The average recover rate is 95.4% and RSD is 3.5%. The average content of sciadopitysin is0.147% (RSD is 2.86%).5.The technology to rich the total flavonoids in branches and leaves of Taxus yunnanensis.Methods: (1) To extract the sample by sonication; (2) Study the technology of extract and defat with L9 (34) orthogonal test; (3)Study the process of purification by macroporous resin: containing resin type, liquor concentration, the category and quantity of eluant.Result: Make sure the best condition to obtain total flavonoids was extraction by sonication and defatting 3 times with an 15 times volume of petroleum benzin. To extract the crude drugs for 2 hours for the first time with 6 times volume 80% EtOH added to the sample and 1.5 hours for the second time. We pack the column with resin of AB-8, the density of liquor is 6mg·mL-1, then the resin is eluated orderly with water, 20% and 40% alcohol, and the dosage in order is 1.5～2BV,1.5～2BV and 1.5～2BV. The content of total flavonoids was 51.26%.6. Preliminary Study on Pharmacological Activities of total flavonoids and sciadopitysinMethods: Named the sciadopitysin,total flavonoids and total extraction were 1,2 and 3, and dissolved in little DMSO, adding N. S. to some density. To study the influence of all these samples on the growth of tumor cells HePG2,A549 and K562 in vivo by MTT assay. The densities of the samples were 50,100,150 (μg/well). According the absorbance, calculate the inhibition rate of the different drug. Then make the inhibition rate curve with inhibition rate as ordinateand and density as abocissa.Result: All the samples exhibited markedly antitumor activities in vivo in all densities, and the inhibitory rate order was: sample 2＞sample 1＞sample 3; The inhibitory rate against K562 of all the three samples in middle and large densities were all over 50%.Conclusion: Made sure the extraction method from branches and leaves of Taxus yunnanensis. All methods were simple,ecoenmical and environmental protection. Study the flavonoids in branches and leaves of Taxus yunnanensis, including the preparation of control, the determination of the flavonoids content, the rich of component and preliminary pharmacological Activities experiments. In one word, the result will be a good foundation for the next research. |
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