Separation Of Major Active Flavonoid In Ginkgo Biloba L. And Pueraria Lobata(Wild.) Ohwi By Coordination Chromatography

Active components in natural products possess various pharmacologic effects and receive considerable attention in the literature. Isolation, purfication and analysis were essential in their routine application. Over 4000 species of flavonoids was discovered up to now, which are structure related compounds and great efforts are needed to conquer the complexation of separation. This assay was focus on the bottleneck of R&D procedure of flavonoid. Separation method of three diverse model compoments was achieved by coordination chromatography technology.Interaction of target compound and coordination agents was studied by chemical calculation. Coordination chromatography experimental research was carried out accordingly and mathmatical model of coordination chromatography was established. Different flavonoid aglycone and flavonoid glycoside was tested to investigate coorperative effectiveness of coordination agents to chromatographic separation. Separation mechanism of coordination chromatography was resolved preliminarily. Main research finding was following:1. Isolation and purifcation method of flavonol and isoflavone was accomplished by coordination chromatography using transitional metal ion(Zn²⁺, Ni²⁺) as coordination agents. Deduced by he rusults of MM and QM computation and experimental investigation, the complexation strength of transitional metal ion with flavonoid was: solitory hydroxyl group 3-OH,5-OH>3'-OH,4'-OH,5'-OH>7-OH, ortho- group C=O,C-OH>C-OH,C-OH>C-OH, C-OCH₃, different domain of target compounds ring A> ring B, inferior part of ring A> superior part of ring A, -OCH₃ attenuate the coordination affinity of its neighboring group.2. Isolation and purifcation method of flavone, flavonol and isoflavone was accomplished by coordination chromatography using cyclodextrin as coordination agents. Deduced by he rusults of MM computation and experimental investigation, the complexation strength of cyclodextrin with flavonoid was: solitory group -OCH₃>-H>-OH, different domain of target compounds ring B> ring A, superior part of ring A> inferior part of ring A, -OCH3 facilitate the coordination affinity of its neighboring group.3. Retention behavior of isoflavonoid in Radix puerariae was different in various mobile phase of HPLC. Reversed elution order was observed in tetrahydrofuran and methanol modified mobile phase. Resolution was decreased as polority of tetrahydrofuran modified mobile phase increaseing, while resolution was decreased as polority of methanol modified mobile phase decreaseing. Hydrogen bonding played a significient role in this phenomenon by the computation results. Mobile phase should be choosing carefully according to the nature of coordination agents.4.β-Cyclodextrin adjusted chromatographic resolution of puerarin and 3 '-methoxy puerarin in coordination chromatography. When the mixture of tetrahydrofuran and water was used as mobile phase, low concentration ofβ-cyclodextrin elevated the resolution of puerarin and 3'-methoxy puerarin, yet high concentration of cyclodextrin depressed the resolution.5. Experimental condition for isoflavonoid in Radix puerariae of coordination chromatography was optimized to satify the resolution, analysis time and tailing factors simultaneous. Box-Behnken method was used to design of experiment for simulation the chromatographic response, back-propagation artificial neural networks was employed to model isocratic HPLC separation of puerarin and 3'-methoxypuerarin, multi decision Multicriteria optimisation of separation of puerarin and 3'-methoxypuerarin was obtained by differential evolution, a global optimization method. The optimized condition for separation was concentrations ofβ-cyclodextrin, triethylamine and acetic acid was 3.7 mmol·L-1,0.02% and 0.8% respectively, which was testified by experimental test to qualify the research objective.