| Inception rate of Alzheimer disease(AD) is increasing rapidly year by year with population's aging of our country. Now, the number suffering from AD is about 50 million all over the world and more and more patients become younger than before when they are to fall ill. It was not know for etiopathogenisis of AD accurately, having several hypothesizes was only suggested. AD is not only a difficult problem at medicine, but also a serious social problem."Dingzhixiaowan"(DX-9386) came from Tang Dynasty《Beijiqianjinfang》which was written by Sun Simiao called"medicine king".This prescription composed of ginseng , hoelen, pologalae and acori graminei have the functions of invigorating the spleen and supplying the heart, transforming phlegm,opening the orifices, quieting the spirit and sharpening the wits, and it is a basic formula of the Traditional Chinese Medicine to treat AD. The traditional preparation was honeyed pill which was not convenient to take medicine. DX-9386 was studied by advanced technologies and methods and a new dosage form was obtained.Modern pharmacology studes have shown that effective compositions of DX-9386 were ginsenoside, polygalic acid, pachymaran and essential oil which come from acori graminei. According to traditional Chinese medicine theory additionally, total polysaccharide, total saponin and pharmacology's result was used as evaluation index, by L9(3)4 orthogonal experiment of alcohol-precipitation and dynamic absorption test of macroporous resin, the extraction and purification technology were established Finally. Amount of effective compositions extracted were maxlmum, purity of effective compositions was elevated , a stable feasible technology of extraction and purification of DX-9386 were formulated.As the same time, Four compounds have been separated from hydrolyzates of polygalae.One is polygalacic acid, the others are its analogs. Polygalacic acid can be used as reference substance.at deteremination of Radix PolygalaeObjective: To study extraction and purification technology, to establish evaluation index of technology, to make initial pharmacological experiments , to obtain polygalacic acid used as Chinese medicine reference substance.Methods:The first part:①Study on extraction method of DX-9386: According to PRC pharmacopeia(2005) to choose qualified four kinds of herbs,and herb mixtures were extracted with two mixed solvent (water-extraction, 50%alcohol-extraction) to decide a solvent. Dedermined absorption rate , soak time , amount of water-adding, extraction time, to decide extraction technology of essential oil; Optimization for extraction condition: to decide times of extraction by one factor control test and to optimize the extraction condition of DX-9386 by L9(3)4 orthogonal experiment(three factors: amount of solvent, extraction time, concentration of alcohol ).②Study on purification technology of DX-9386: The first purification was carried out by alcohol-precipitation which was optimized by L9(3)4 orthogonal experiment. Macroporous resin adsorption technology was decided by dynamic absorption test of macroporous resin, including resin models,adsorptive capacity,elution solvent and its amount , Then the second time purification was carried out.③Evaluation index of technology: Total polysaccharide and total saponin were determinated by colorimetric method.④Preliminary pharmacology test: The effect of learning and memory ability of administration group DX-9386 was compared with blank grounp and positive control grounp through step-down test and active of SOD,MDA,AchE.The second part:Separation and structure elucidation of compounds: Polygalae was extracted by 50%-alcohol, then enriched by n-butanol and macroporous resin. The total saponin was hydrolyzed by hydrochloric acid. The separation was performed on silica gel colume and prepared HPLC, the TLC was used to determine the separating condition and to monitor the proceeding of separation. Structure of compounds were elucidation by UV,NMR,MS technology.Results:The first part:①Technical condition of extraction: Through contrast test, the best extraction condition of acori graminei essential oil was: soaking for12 hours with 8 times volume water, reflux extracting for 6 hours and essential oil was extracted by petroleum erther,According to orthogonal experiment and LSD-t test, the best project was: reflux extracting with 8 times volume 50%-ethanol for 2 times, each time for 1.5 hours.②Technical condition of purification: adding 95-ethanol to extraction liquor (1g herb/mL) until ethanol concentration 75%. Through macroporous resin dynamic test , D101 macroporous resin was the best choice, the best technology parameter: extraction liquor 0.4g herb /mL which has been precipitated was used as sample of macroporous resin column, to collect water fraction for 3BV, to abandon 30%-ethanol fraction for 3BV, to collect 60%-ethanol fraction again.③Evaluation index of technology: Total polysaccharide and total saponin determined by colourimetry was reasonable and suitable to industrial manufacture. Studies on analysis techonolygy showed that these method were simple,accurate and can be used to quality control.④Preliminary pharmacology test: Extract of DX-9386 could reduce errors and relong latency of mice's memory acquisition in step-down test; at the same time, effect for SOD,AChE,MDA had statistical significance.The second part:Four compounds from hydrolyzate of polygalae were polygalacic acid,senegenin,polygalacic acid-3-O-β-D- gluco -side and senegenin -3-O-β-D-glucoside.Conclusions: The extract technology was make extraction to kept all kinds of effective compounds maxlmum; alcohol-precipitation and macroporous resin decreased the amound of extract, together, avoided loss of effective constituent, reduced oral dosage. Among four compounds obtained, polygalacic acid was reference reference substance for determination of polygalae.
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