Separation Of Active Components Of Natural Products By Coupling Oligo-β-cyclodextrin On Polymeric Stationary Phases

β-cyclodextron (β-CD) is torus-shaped oligosacchrides made up of seven cyclic-arranged α-1,4-linked D-glycopyranose units. The outside of the cyclic molecule is hydrophilic and the central cavity is hydrophobic. β-CD and its derivatives have the ability to form specific inclusion complexes with some compounds, especially aromatics, the stability of inclusion complex depending on how the guest molecule fits into the cavity of the P-CD. This property has been widely employed in many separation techniques including classical liquid chromatography. In order to develop new media with improved selectivity, β-CD and its derivatives are used as a ligand in liquid chromatography. Compared to monomeric β-CD, immobilization of polymeric β-CD (oligo-β-CD) may be obtained the considerably higher local concentration of the β-CD group because the large soluble molecular aggregates were formed via the polymerization of the β-CD monomer before coupling. If oligo-β-CD is anchored on supports for target product, the selectivity of the media will be enhanced. In previous reports, oligo-P-CD immobilized agarose gel particles such as brominated allyl-group substituted Sepharose HP have been synthesized in our laboratory, and successfully employed for the purification of puerarin and epigallocatechin gallate from crude extracts. But the agarose support matrix is limited by its lack of mechanical rigidity, even in heavily cross-linked varieties, thus restricting its application in preparative scale operations. In this thesis, oligo-β-CD was coupled on polyacrylate beads media and polystyrene-based media. The novel stationary phases were used for separation puerarin from crude extraction. The experimental results showed that the coupled stationary phases had high selectivity for flavonoids of natural products and the separation efficiency was enhanced. This thesis optimized the process of separation and purification puerarin on the novel oligo-β-CD coupled stationary phases and investigated the adsorption mechanism. Processes utilized oligo-β-cyclodextrin substituted agarose gel medium for efficient purification of dadzin and paclitaxel were also studied. The main studies are as following:Polyacrylate resins have certain favorable characteristics such as biocompatibility and hydrophilicity. In this study, three types of homebred polyacrylic media were chosen as supports for coupling oligo-β-CD ligants in order to isolate target product puerarin. The present report shows the chemical immobilization methods of oligo-β-CD onto these polyacerylate beads. The preparation conditions were investigated in detail. The optimum coupled times were12h for PGMA,6h for PHEMA and D152; coupled reaction temperatures were50℃for PHEMA and D152matrix, and60℃for PGMA respectively; aqueous solution containing NaOH was determined as reaction solvent; the optimum values of molar ratio of reactants were1:1(PGMA: oligo-β-CD),1:1(PHEMA:oligo-β-CD), and1:1.5(D152:oligo-β-CD). The chromatographic separation characteristics of puerarin on the three novel coupled media were evaluated. By controlling the mobile phase composition and the packing particle sizes, D152-CDP stationary phase can provide efficient separation of isoflavonoids of the Radix puerariae crude extract. The optimal mobile phase was7%acetic acid in isocratic elution at a flow velocity of1.0ml/min on250×4.6mm column. In a single column pass on D152-CDP, the purity and recovery of puerarin can reach76.5%and96.8%respectively.The isolation selectivity of puerarin was studied when twelve native and coupled polystyrene-based macroporous resins were used as adsorbents by static adsorption, and an enhancement in selectivity was obtained when using coupled media. In term of representing the best adsorption and desorption capacity for puerarin than others, and its equilibrium adsorption data well fitting to the Freundlich isotherm which was used to describe the interactions between solutes and resin at different temperatures, the coupled resin HPD-100-CDP was selected as the most appropriate medium for the separation of puerarin. The performance for isolation of puerarin on HPD-100-CDP column in one step was evaluated sequentially. In order to improve the one-step isolation efficiency, uniform porous microsphere PS which was polymerized by combining microporous glass membrane emulsification technique and suspension polymerization process was chosen as support for coupled by oligo-β-CD based on the above experimental data. The optimal mobile phase was methanol/acetic acid/water=5.0/6.6/88.4(v/v/v) in isocratic elution at a flow velocity of1.0mL/min. The PS-CDP stationary phase can provide efficient separation of isoflavonoids from the Radix puerariae crude extract under optimum conditions. The purity was of95.3%with the recovery of86.7%for obtained puerarin in a single PS-CDP column. In addition, the PS-CDP column was used to separate the extract of soybean isoflavones. When the mobile phase was methanol/0.1mM NH4AC=65.0/35.0, dadzin can be separated with other isoflavones on base line.The sorption mechanism on oligo-β-cyclodextrin coupled polystyrene-based matrix (PS-CDP) has been investigated using the isosteric heat approach, Adsorptions of puerarin on the native resin PS and the coupled matrix PS-CDP at temperature from288to318K were studied for the interactions between the adsorbents and the adsorbates. Isotherms of Freundlich equation with characteristic parameters for the two media were well fitted to the equilibrium adsorption data. The isosteric enthalpy changes (△H) and the free energy changes (△G) were negative values in all cases, which indicates a favorable and exothermic process. The surface heterogeneity of the two polymeric adsorbents were revealed from the results of the sorption isosteric enthalpy decrease as the fractional surface coverages increase on the two tested media. The more heterogeneous surface of PS-CDP mostly resulted from the inclusion complexation between puerarin and β-CD. The formation of the inclusion complex puerarin/β-CD can be evidenced using NMR spectroscopy. The thermodynamic studies confirmed that multi-interaction cooperatively governed the isolation of puerarin from aqueous solution on PS-CDP matrix.In order to mimic the multiple weak interactions on D152-CDP in the process of adsorption puerarin from aqueous solution, the poly(glycidyl methacrylate) adsorbents P(GMA-EDMA) and P(GMA-DVB) were synthesized by the radical suspension-polymerization method and farther coupled by oligo-β-cyclodextrin (CDP) to obtain P(GMA-EDMA)-CDP and P(GMA-DVB)-CDP. The adsorption of puerarin from aqueous solution onto the four media i.e. P(GMA-EDMA), P(GMA-DVB), P(GMA-EDMA)-CDP and P(GMA-DVB)-CDP was studied for illuminating weak interactions between the puerarin molecules and the polymeric media. The interaction between the polymeric media and the puerarin was studied by FTIR. The result shows the adsorption efficiency on P(GMA-DVB)-CDP driven by multiple weak interactions is much higher than that on P(GMA-EDMA) driven by hydrogen bonding interaction only and on P(GMA-DVB) or P(GMA-EDMA)-CDP driven by two interactions. The synergistic effect of multiple weak interactions would contribute more to the adsorption process as acting simultaneously than that of acting individually.Oligo-β-cyclodextrin substituted agarose gel medium was utilized for efficient purification of dadzin and paclitaxel. Compared with the gel medium allyl-Sepharose HP, the separating ability of the oligo-β-cyclodextrin coupled medium was increased so much that dadzin can be purified thoroughly. The dadzin with purity more than97%and the recovery more than95%can be obtained on the oligo-β-CD-Sepharose HP column in one step. A process integrates extraction, Al2O3normal-phase chromatography, and oligo-β-cyclodextrin-Sepharose HP column chromatography for efficient purification of paclitaxel. The highest selectivity for paclitaxel was obtained on the oligo-β-cyclodextrin-Sepharose HP column compared with other β-cyclodextrin coupled gel media columns β-cyclodextrin-Superose12pg and β-cyclodextrin-Sepharose6B. The initial separation extract on Al2O3column was purified by the subsequent oligo-β-cyclodextrin-Sepharose HP column chromatography with a purity of about90.3%and a recovery of85.1%under the optimum mobile phase composed of methanol/acetonitrile/water=32/20/48(v/v/v). HPLC analysis and ESI-MS/MS spectrum were used for the detection and characterization of paclitaxel in isolated fraction.