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Studies On Isolation And Purification Of Ginkgolic Acids And Their Bioactivities

Posted on:2005-05-25Degree:DoctorType:Dissertation
Country:ChinaCandidate:X W NiFull Text:PDF
GTID:1101360125469106Subject:Agricultural Products Processing and Storage
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Ginkgolic acids are claimed to be harmful compounds and strictly limited in the extract of Ginkgo biloba leaves (EGb). Contents of Ginkgolic acids are the highest in Ginkgo biloba exopleuras and their special pharmacological and poisonous activities have increasingly caused the extensive concern of people. But compared with Ginkgo biloba leaves, the research of Ginkgolic acids is still few. This paper is aimed to establish an efficiently quantitative and analytical method of Ginkgolic acids by using modern chromatographic separation theory and identification technique. The technological conditions of isolation and purification Ginkgolic acids from Ginkgo biloba exopleuras were studied systematically and their structures were identified. The antibacterial activity of Ginkgolic acids was also discussed and their antitumor mechanism was studied by immune adjustment and apoptosis. The main results are as follows:1. Determination of Ginkgolic acids by reversed-phase high performance liquid chromatography (RP-HPLC)According to the difference of Ginkgolic acids in structure and character, an RP-HPLC analytical method was developed for separation of Ginkgolic acids. Effects of mobile phase constitute, pH and temperature on retention behaviors of Ginkgolic acids in column were discussed. The optimal chromatographic conditions of RP-HPLC were as follows: chromatographic column was SUPELCOSIL?LC-18(15cm X4.6mm,5um); mobile phase was MethanoI/H2O(90:10,V/V), adding acetic acid and making pH become 3.15; UV detection wavelength was at 310nm; flow rate was 0.7ml/min and AUFS was 0.02; column temperature was 30*C. Under the above conditions, the peaking high resolutions (Rj,) between different Ginkgolic acids were all over 1, and the capacity factors (k) of Ginkgolic acids were between 3 and 8.The quantitative determination method of Ginkgolic acids was established based on RP-HPLC separation theory. The UV spectrums of Ginkgolic acids were identified by HPLC-DAD, the results showed that the spectrums of standard Ginkgolic acids were identical to that of Ginkgolic acids from samples. The sample could be analyzed directly by HPLC after extracting with methanol and filtration. The average recoveries of the method were 97.02% and the coefficients of variation were 1.28%(n=5). The linear range was from 0.498ug to 8.879g, correlation coefficient was 0.9998. The minimum thatcould be determined was 7.2ng. This simple, rapid, sensitive and accurate method could be used in quantitative determination of Ginkgolic acids in ginkgo products.2. Extracting technology, purification and structure identification of Ginkgolic acidsThe technological conditions of extracting Ginkgolic acids from Ginkgo biloba exopleuras were optimized, and the adsorbing and desorbing properties of Ginkgolic acids on different macroreticular adsorbents were studied. The optimal purification conditions were as follows: Ginkgo biloba exopleuras was extracted with 7 volumes of 80% methanol by refluxing at 60 for 3 times and 4h for each time. The extracts were filtrated, concentrated and purified on D4020 resin (HaO, 65%, 90% ethanol grads desorbing). The desorbing solution was concentrated and extracted with solvent S|. Then, Ginkgolic acids were further purified on AB-8 resin (75% and 95% ethanol grads desorbing). The obtained Ginkgolic acids product had 95% purity and 0.89% yield.UV, IR, HPLC-DAD and LC/ESI/MS were applied to identify the structure of Ginkgolic acids from Ginkgo biloba exopleuras. Five Ginkgolic acids C13:0, C15:l, C17:2, C15:0 and C17:l were confirmed, their proportion relative were!2.0: 41.77: 4.89: 3.52: 37.83.3. Antibacterial activity of Ginkgolic acidsThe antibacterial activity of Ginkgolic acids was discussed by the dilution broth method. Water extraction and ethanol extraction of Ginkgo biloba exopleuras, purified Ginkgolic acids all showed strong antibacterial and antifungal activities. As contents of Ginkgolic acids increasing, the antibacterial activity became stronger. Ginkgolic acids were the main inhibitor. Ginkgolic acid...
Keywords/Search Tags:Ginkgolic acids, RP-HPLC, determination, extracting technology, structure identification, antibacterial activity, antitumor, immune adjustment, apoptosis, gene expression
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