Preparative Separation and Purification ofEpigallocatechin Gallate from Green Tea Extracts UsingSilica Adsorbents Containing Beta-Cyclodextrin

Autor: Jhe-Yu Gu, 顧哲瑜
Rok vydání: 2011
Druh dokumentu: 學位論文 ; thesis
Popis: 99
Green tea is recognized for its high content of tea polyphenols, in particular tea catechins. The four represented catechins in green tea are, epigallocatechin (EGC), epicatechin (EC), epigallocatechin gallate (EGCG), and epicatechin gallate (ECG). Among these four major compounds, EGCG is the most abundant and biologically active compound in green tea. Many researches in the past found that green tea polyphenols have physiological effects in anti-oxidant, anti-tumorgenic, anti- mutagenic, anti-pathogenic, anti-inflammation, and anti-fungus, etc. Many health foods and products containing green tea polyphenols are already in markets, such as toiletries, cosmetics, foods, and beverages. In this study, the optimal techniques for preparation of silica adsorbents containing β-cyclodextrin (β-CD) and their use in column chromatography were investigated. The objective is using the self-prepared silica adsorbents containing β-CD to enhance the adsorptivity and selectivity of EGCG and then to effectively separate and purify caffeine (CA) and each of the four catechins from green tea extracts. The preparation of silica adsorbents containing β-CD includes the following two steps: (1) modification of 3-glycidoxypropyl-trimethoxysilane (GOPS) on silica particles to produce epoxy terminated GOPS_silica particles, (2) chemical grafting of Na-β-CD, obtained from the reaction of β-CD with sodium hydride, on GOPS_silica particles to produce β-CD_GOPS_silica adsorbents. Three kinds of silica gels with pore sizes of 60?? 100?? and 150? were used as the solid matrix. The best preparation results show that, the amount of GOPS modified on GOPS_silica and the amount of β-CD grafted on β-CD_GOPS_silica, analyzed by elemental analysis (EA) and thermo-gravimetric analysis (TGA), were about 0.19~0.25 mmol GOPS/g Silica using 100 ? silica gel and about 0.04~0.07 mmol β-CD/g Silica using 100? and 150? silica gels, respectively. First, the crude green tea extracts were concentrated by liquid-liquid extraction using ethyl acetate as the extractant and under the following conditions: concentration of 50 mg/mL, water/oil ratio of 1:1, extraction temperature of 30℃, extraction time of 2 hr, and without pH adjustment. The purity of the four catechins was raised from 14.0% to about 60.0% (a concentration ratio of about 4.3) and the purity of EGCG was raised from 6.5% to about 35.3% (a concentration ratio of about 5.4). The green tea concentrates were then used as the feed materials in the following batch adsorption and column chromatographic experiments. In the batch adsorption experiments, when the bare silica and the GOPS_silica were used as the adsorbents, the adsorption ratios for caffeine and the four catechins were found to be very low (95%) was obtained. The great improvement in adsorption capacity proved that the β-CD_GOPS_silica adsorbents were successfully prepared in this study. In the column chromatographic experiments operated in the frontal (step-injection) mode, the adsorption results show that, a high adsorption capacity (＞30 mg/g adsorbent) and a good selectivity with respect to EGCG were obtained using (60?? β-CD_GOPS_silica as the adsorbent. However, the desorption results show that, due to the very strong interaction between β-CD_GOPS_silica and EGCG, EGCG was very difficult to be desorbed, even tried various kinds of solvents with different degrees of polarity. In the column chromatographic experiments operated in the step-gradient elution mode, the green tea concentrates were dissolved in acetonitrile, injected into the glass column packed with (60?? β-CD_GOPS_silica, eluted using acetonitrile: methanol: acetic acid (%) as the eluant and operated under the optimal elution chromatographic conditions for achieving the best separation resolution for EGCG. The purity and recovery of EGCG reached about 75% and 89%, respectively. However, due to the limitation on the EGCG resolution, the system could not be further scaled-up. Finally, a two-stage column elution chromatographic system was adopted to enhance the EGCG purification. In the first stage, the glass column packed with (60?? β-CD_GOPS_silica was used. The chromatographic system, eluted using acetonitrile: methanol: acetic acid (%) as the eluant, could be scaled-up under the optimal elution chromatographic conditions for achieving the best separation between the groups of CA+EC and EGCG+ECG+EGC. In the second stage, a preparative C-18 stainless steel column was used to purify EGCG from the mixture of EGCG+ECG+EGC. The chromatographic system was eluted using water (1% acetic acid)/methanol as the eluant and operated under the optimal elution chromatographic conditions for achieving the best separation resolution for EGCG. The purity and recovery of EGCG reached about 89% and 91%, respectively.
Databáze: Networked Digital Library of Theses & Dissertations
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