Extraction, Determination And Analysis Of Flavonoids In Leaf And Pollen Of Male Plant In Ginkgo Biloba L.

Gingko( Ginkgo boliba L.), a precious plant with multi-purpose, is native to China in which its resources are rich.The leaf and pollen of male plant in gingko have been developed and utilized as the important resources with various active ingredients such as flavonoids etc. According to the requirements of development and utilizationon on the active ingredients in gingko and their research advances at home and abroad at present, the research on extraction and determination of the flavonoids in leaf and pollen of gingko's male plant and selection of male plants with higher active ingredients have an important theoretical significance and application value. In this study, the leaf and microstrobilus of 87 male plants distributing in Yangzhou city and Xuzhou city of main producing areas of gingko in our country were randomly taken during blossom and July to September in running two years, then, were used to extract the flavonoids from the leaf and pollen by the optimized system based on orthogonal design and to detect their contents by RP-HPLC (Reverse-phase high performance Liquid Chromatography), and to take a cluster analysis on them, respectively. The above research results are described as follows:1. In the male plants of gingko in this experiment, the content of quercetin,kaempferol,isorhamnetin in leaf was 0.595 %,0.566 % and 0.44 %,which in pollen was 0.081 %,2.196 % and 0.060 %,total of which in leaf and pollen was 1.601 % and 2.187 %, respectively. In which there was a bigger difference between both of leaf and pollen. The content of flavonids in leaf was much significantly positive correlative with leaf thickness and specific leaf weight(SLW), respectively, so the leaf thickness and SLW could be used to evaluate the content of flavonoids in leaf as an important index.2. The content of flavonids, especialy, kaempferol in pollen was higher than that of leaf. In which there was a significant and positive correlation between both of them ( r=0.9270*).3. Four factors of solvent concentration, ratio of sample to liquid, temperature and time, 3 treatments each factor, were selected to make a system as the main effective factors for extracting flavonoids from leaf and pollen of male plant in gingko. After experiment by orthogonal design, an optimized extracting conbination including alcohol of 70 %, ratio of 1 sample to 15 liquids, temperature of 80℃and supersonic extracting time of 40 min was decided.4. Water was used as a solvent to extract the flavonoids from leaf and pollen of male plant in gingko and to compare with alcohol-water solution, the optimized treatments of which based on orthogonal design should consist of ratio of 1 sample to 15 liquids, temperature of 80℃and supersonic extracting time of 60 min. The percentage of flavonoids extracted by alcohol-water solution from leaf was 2.32 %, and 0.396 % by water solvent, the former was much more than the latter.5. The flavonoids in pollen of male plant in gingko could be effectively extracted by breaking wall of liquefied nitrogen-boiling water thermotreatment.6. The flavonoids in samples of leaf and pollen of male plant in gingko could be effectively isolated by supersonic wave extracting, petroleum ether and n-hexane extraction, and centrifugation etc.7. The flavonoids in leaf and pollen of male plant in gingko could be effectively purified by adsorption of ADS—17 wide-aperture resin for 30 min, then by elution of alcohol of 70 % which was three times as more as resin bed at volume at flow velocity of 1 ml/min.8. Analysis by RP-HPLC showed that there were significant differences at contens of each flavone aglycone and the sum of them among the male plants of gingko in this experiment, the finger-print maps of flavone aglycones made up by HPLC could be applied as a main basis for evaluating the flavonoids levels in leaf and pollen of male plants in gingko. 9.In cluster analysis, when the threshold value of 15 was taken the male plants of gingko based on the flavonids contents in leaves could be divided into 3 groups which consisting of 67 trees of groupⅠ,15 trees of groupⅡand 5 trees of groupⅢ,the flavonids contents of which was 1.542%,2.082% and 0.951%;when the threshold value of 13 was taken for that of pollen,above ones could also be divided into 3 groups which consisting of 48 trees of groupⅠ,38 trees of groupⅡand 1 trees of groupⅢ,the flavonids contents of which was 1.658%,2.845% and 4.161%,respectively.10.In this experiment, 15 male plants of gingko including No49,10,85,2,5,39,11,4,12,80,74,69,59in order of flavonids contents from higher to lower could be selected in as the recommended ones with the flavone aglycone contents of more than 2.00% in leaf and pollen.The above results provides an important theoretical basis and technological support for development and utilization of active ingredients in leaf and pollen of male plant in gingko.