| With medlar leaf as the research object in this study, taking the extraction rate of medlar leaf alcohol extra and the viability of microglial as index,the extraction process of effective composition in medlar leaf alcohol extra is studied.The alcohol extra of medlar leaf is systematic isolated, the chemical components of four fractions are tested by preliminary chemical analysis, and MTT method is used to determine the effective part can protect microglial which is injuried by methymercury chloride(MMC). The me-thods such as silica gel column chromatography, Sephadex LH-20, preparative HPLC, are used in the isolation and purification of the components. The structure of compoun-ds are identified by technique of 1H-NMR,13C-NMR.The effective compounds are dete-rmined through activity test. The main findings are as follows:1. Determine the optimal extraction condition of effective compositionTaking the extraction rate of medlar leaf alcohol extra and the viability of microglial as index,the influencing factors of extraction efficiency are studied by single factor experiment, including ethanol concentration, extraction time, ratio of solid to liquid and extraction temperature. The orthogonal experiment was conducted on the base of the single factor experiment.Finally,the optimal extraction condition were obtained as follows: ethanol concentration 70%, extraction temperature 80℃,ratio of solid to liquid 1:10,extraction time 2.5h,repeated for twice.And through the verification test indicate the extraction process is reasonable, reliable, and reproducible.2. Extracting effective compositions and preliminary chemical analysis8500g meal of medlar leaf are extracted in accordance with the optimal extraction condition,the weight of medlar leaf alcohol extra is 2192 g. 2141 g alcohol extra use different polarity solvent for extraction and separation,in turn by petroleum ether,ethyl acetate, n-butanol, respectively,have to each part for dry paste 84.27 g(3.94%), 71.60 g(3.48%),120.77 g(5.64%),1864.36g(86.94%).This result indicate the main components of medlar leaf alcohol extra is water soluble constituents.The test tube method and filter paper method are used for chemical components test of four parts.Researches on chemical components indicate that petroleum ether pa-rt must contains axunge.Ethyl acetate part must contains flaconoids,steroids,terpenoids, phenols,organic acids.N-butanol part must contains flaconoids,alkaloids, phenols.Water part must contains polysaccharide,amino acids.3. Determine the effective partMTT method is used to determine the effective part can protect microglial which is injuried by MMC.The result indicate that main effective parts of medlar leaf alcohol extra are Ethyl acetate part and N-butanol part.4. Isolation and structure identification of compounds from effective partsThe methods such as silica gel column chromatography, Sephadex LH-20,preparati-ve HPLC, are used in the isolation and purification of the compounds.Fourteen comp-ounds are isolated,and ten compounds are identified.They are 4-methoxybenzoic acid(3), 4-methoxycinnamic acid(5), scopoletin(6), 4-hydroxybenzaldehyde(7), 4’-hydroxyacet ophenone(8),(Z)-2-ethylidene-3-methylsuccinic acid(9),quercetin(10), adenosine(11),scop olin(13),rutinum(14).Compounds 3,5,7,9,11 are firstly isolated from the plants in genus of Lycium L,and compounds 6,8,13 are firstly isolated from medlar leaf.5. Determine the effective compoundsQuercetin and Rutin are determined as effective compounds by MTT and Griess methods.Quercetin and Rutin in medlar leaf alcohol extra can improve the viability of microglial,and inhibit release of inflammatory factor NO, provides a theoretical basis for finding the drug for treatment of Alzheimer’s Disease. |
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