| Ginkgo Biloba L, ancient relict plant, had a long history in the application of Chinese herbal. Flavonoids and ginkgo lactones were effective ingredients of Ginkgo Biloba leaves. Flavonoids could scavenge free radicals, inhibite lipid peroxidation, expanse blood vessels and improve cerebral artery. They also could prevent alzheimer, hypertension, arteriosclerosis and coronary heart disease. Ginkgo lactones were antagonism of platelet activating factor. They had potential application in the treatment of asthma, endotoxin shock, organ transplant rejection, cardiovascular and cerebrovascular diseases.Quercetin, isorhamnetin and kaempferol were main constituents of ginkgo flavonoids, while ginkgo lactones mainly included ginkgolide A, B, C and bilobalide. These seven compounds were not only the effective ingredients of Ginkgo biloba extract, but also an important quality indicator of Ginkgo biloba preparation. Monomers of these seven components were used to control the quality of Ginkgo biloba preparation by detecting contents of flavonoids and ginkgo lactones. With the development of Chinese herbal quality control and the increase of studies on single component medicine, the requirement of monomers of these seven components was increasing.Three kinds of flavonoids and four kinds of ginkgo lactones were chemical analogues. Their chemical structures and polarity were similar to each other. It was difficult to obtain monomers of flavonoids and ginkgo lactones merely using traditional purification technology, such as solvent extraction and column chromatography. Traditional technology had the disadvantage of large lost of sample, complex process and high cost. High-speed countercurrent chromatography (HSCCC) was a new type of liquid-liquid technology with the advantage of high recovery rate, large injection volume, high separation ability and easy operation. HSCCC had applied to purified flavonoids and lactones at home and abroad. Quercetin could be isolated and purified by one HSCCC preparation. Isorhamnetin and kaempferol were unable to be separated from each other, because their chemical structures and polarity were more similar. They could be separated by multidimensional HSCCC using more than two countercurrent chromatography apparatus connected in series. But it was difficult to carry out in normal laboratory. Bilobalide was separated and obtained from Ginkgo biloba extract by HSCCC using chloroform-methanol-water as the solvent system, but not other three ginkgo lactones.On this study, crude extract flavonoids and crude extract of ginkgo lactones were prepared firstly by preparation technology we optimized. Monomer of quercetin was obtained from crude extract of flavonoids by HSCCC. Isorhamnetin and kaempferol were separated and purified originally by recycling HSCCC using only one countercurrent chromatography apparatus. Monomers of bilobalide and ginkgolide A, B, C were isolated and obtained from crude extract of lactones originally by HSCCC. The results were as follows:1. Preparation of quercetin monomer:Contents of flavonoids and quercetin in the Ginkgo biloba leaves we picked were 1.16% and 0.5% detected by high performance liquid chromatography (HPLC). Ginkgo biloba leaves were extracted by 70% alcohol, purified by chloroform extraction and D-101 macroporous resin column to prepare crude extract of flavonoids. Mass fraction of flavonoids was 24% in the crude extract. Monomer of quercetin was obtained form the crude extract by HSCCC with a solvent system composed of hexane-ethyl acetate-methanol-water at a volume ratio of 5.5:4.5:5:5 and the maximal purity achieved 98.6%2. Preparation of monomers of isorhamnetin and kaempferol:Isorhamnetin and kaempferol were unable to be separated from each other by HSCCC, because their chemical structures and polarity were more similar. But they could be separated by recycling HSCCC using only one countercurrent chromatography apparatus. The commixture of isorhamnetin and kaempferol was circularly separated and purified in the apparatus by connecting outlet of detector and inlet of infusion pump. Monomers of isorhamnetin and kaempferol were successfully prepared by recycling HSCCC with a solvent system composed of n-hexane-ethyl acetate-methanol-water (5:5:5:5, v/v/v/v) and the maximal purity were all over 97%.3. Preparation of monomers of bilobalide and ginkgolide A, B, C:Ginkgo biloba leaves were extracted by 25% alcohol, purified by acetic ether extraction, D-101 macroporous resin column and Al2O3 (pH4.0) column to prepare crude extract of lactones. Mass fraction of lactones was 44.98% in the crude extract. Monomers of bilobalide and ginkgolide A, B were obtained form the crude extract by HSCCC with a solvent system composed of hexane-ethyl acetate-methanol-water at a volume ratio of 4:5:3:5 and the maximal purity of bilobalide and ginkgolide A, B achieved 98.3%,98.9% and 98.8%. Ginkgolide C could not be separated in this solvent system. Ginkgolide C was isolated and purified by HSCCC using hexane-ethyl acetate-methanol-water at a volume ratio of 2:6:3:5 as the two-phase solvent system and the maximal purity achieved 98.4%. |
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