| Hydroxytyrosol(HT), which is also called3,4-Dihydroxyphenylethanol, is one of secoiridoids hydroxyaromatic compounds. It is an ortho-diphenol and holds lipid and water solubility. It showed that anti-oxidizability, aiti-platelet aggregation, anti-atherosclerosis, antibiosis and anticancer and so on bioactivities. It plays an important role in the mediterranean diet because it is a main composition of olive oil. Glioma is a common disease and occurred in central nervous system(CNS), its treatment is a difficult problem, which is accepted in the world medical. Although the microscopy techniques were used extensivly, it could still not reach to the expected effect that the glioma would be removed completely after operation because it holds infiltrative growth in pathology. At present, there are many chenotherapeutics which utilized to cure this disease, Temozolomide is one of more authentic drugs, but its effect is still difficult to be satisfied. So that, a new medicine which can cure glioma we expected, and holds these features of efficient and harmfulless and so on.This study aims to gain the HT with high purity by separating and purifing from hydrolyzed oleuropein crude extract or olive leaves powder with hydrochloric acid, and then research that it can whether to induce the normal brain HEB cells or glioma U251cells apoptosis so that we will go deep into discussing its anti-glioma bioactives, with the result that it will afford the experimental and theoretical basis for the fully apply of the olive leaves or the resources which are rich in the olive saponins and expand this field that hydroxytyrosol will be applied to cancer prevention and treatment and so on. The processs and conclusions summarized at experimental stages belows:(1) Hydrolyzed oleuropein crude extract with0.5M HC1, the condition consisits of15min,105℃, solid-liquid ratio(1/30). The hydrolyzate was centrifuged to remove precipitation, and designed the orthogonal experiment to screen out the best clarification of the ZTC1+1-Ⅱ clarifying agents, this result showed the best fining condition that the final concentration of the B component was0.2g·L-1and that of A component was0.1g·L-1, pH5.0and the optimal clarified temperature was100℃ with2hours. We found that the effect was better after clarifying three times by clarifying this hydrolyzate with the best clarify condition judging by the weight of dry deposit which came from the clarified liquer, and selected the best extractive solvent(ethyl acetate) for the clarified liquer. At last, the purity of HT could reach43.38%after clarfing, extracting and concentrating, the recovery rate also reached to82.35%.(2) Hydrolyzed olive leaves powder with0.5M HC1, the condition of hydrolyzing, fining and time of clarfing was the same as the above. Elected the best macroporous resin by the clarified liquer was absorbed with13macroporous resins respectively, and used this macroporous resin to screen out the best absorption capacity, the result showed that1g LX-18C could absorbed5mL maximally and the absorption rate could also reach90.14%. At last, we would go on separating the sample which have eluted from the LX-18C with ODS fillers by AKTATMpurifier instrumentation. Finally, the purity of HT was47.82%and the recovery rate was only1.11%which we got.(3) We took the resveratrol(Res) as the positive control drug when we researched the hydroxytyrosol(HT) whether or not could induce growth arrest and apoptosis in the normal brain HEB cells or glioma U251cells. The impact of HT on the HEB or U251cells viability was evaluated by MTT test in the same time that the half inhibit concentration(IC5o) was obtained at48h. The change of the shape and structure could been observed with transmission electron microscope(TEM). DNA agarose gel electrophoresis was used to observe the cell apoptosis. The alteration of cell cycle and apoptosis rate was defected by flow cytometry. Western blotting was adopted to study the levels of expressions of the Bcl-2or Caspase-3proteins on the HEB or U251cells trested with different concentrations of HT. The results showed that the IC50of HT effected on the HEB or U251cells was0.846and0.410mM at48h, respectively. Microvilli reduction, euchromatin gather, nuclear condensation and nuclear-cytoplasm ratio descending would be detected after the HEB and U251cells were treared with different concentrations of HT. The typical DNA ladder on agarose gel electrophoresis for analysis of cellular apoptosis was significantly appeared in the treated HEB and U251cells. The apoptosis rate of HEB or U251cells was increased gradually and their cell cycles were arrested at S stage obviously with the concentrations increased of HT. The expression of antiapoptosis Bcl-2protein was down-regulated while expression of related proapoptosis Caspase-3protein was up-regulated significantly along with the concentration of HT increases.In summary, it was practicable that separating and purifying HT from the solution of hydrolyzed oleuropein crude extract or olive leaves powder with hydrochloric acid. At present, we could only get the quality score of HT which was more43%. But it couldn't reach this purity of HT which was used in the followup cell experiments. We showed that HT could induce the apoptosis of HEB or U251cells through some apoptosis test methods. The lower concentration of HT could reach the same apoptosis effect of the U251cells than it impacted on the HEB cells, but the induce strength of HT was weaker than Res as the same concentration, so that we didn't think about that HT was took as one of anti-glioma drugs. |
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