Separation Of Epigallocatechin Gallate Research Articles

DEVELOPMENT OF THE TECHNOLOGY FOR THE SEPARATION OF THE COMPONENTS FROM GREEN TEA USING THE PREPARATIVE LIQUID CHROMATOGRAPHY BY DESIGN OF PERIODICAL PROCESS

Leaves of the green tea plant camellis sinensis contain up to 36 % polyphenols. Epigallocatechin gallate (EGCG) is the most interesting polyphenols because it exhibits a strong antioxidant effect. Furthermore, it has been demonstrated that EGCG has an antimutagenic and an anticancer effects, an antibacterial effect and a beneficial effect on cholesterol level in blood. Therefore, is needed to isolate EGCG in a pure form in high yield by simple and commercial process. This method for isolation of EGCG contents the following steps: a) selection of macroporous polar resin for the preparative liquid chromatography; b) selection of polar elution solvent; c) determination the temperature and pressure for adsorbtion and desorbtion of the polyphenols of the green tea. Therefore, it was searched the interconnection between yield of EGCG and the range of temperature and amount of eluent. Finally, the process for the separation of Epigallocatechin gallate from green tea extract was developed in the laboratory and EGCG was isolated with the concentration 92% by the yield 43%. Accordingly, the technology of EGCG production based on the preparative liquid chromatography was launched and introduced on the market.

Separation of epigallocatechin gallate and epicatechin gallate from tea polyphenols by macroporous resin and crystallization.

Epigallocatechin gallate (EGCG) and epicatechin gallate (ECG) are the most abundant ester catechins of green tea polyphenols (GTPs) with numerous potential bioactivities, which have wide application prospects in the fields of medicine and functional foods. In this study, a new method using macroporous resin and crystallization was established to separate and purify EGCG and ECG. Two resins with high adsorption and desorption capacities for EGCG and ECG were screened through static adsorption/desorption tests, and the LX-20B resin was selected through column chromatography due to its best separation effect. Moreover, the column separation parameters of LX-20B resin (sample amount, ethanol elution concentration, elution volume, and elution flow rate) were optimized. After resin purification, the EGCG and ECG purity were 70.08 ± 2.55% and 74.97 ± 2.66%, respectively, and the recovery rates were 68.07 ± 2.43% and 74.28 ± 2.24%, respectively. After crystallization, the EGCG purity reached 95.87 ± 0.89%, with a total recovery rate of 58.66%, and the ECG purity reached 95.55 ± 1.30%, with a total recovery rate of 62.45%. The separation efficiency of the resin showed no significant change after 6 cycles. These results show the proposed method to be a simple, eco-friendly, and cost-effective separation method for the industrial separation and purification of EGCG and ECG from GTPs.

Separation of Epigallocatechin Gallate from Natural Plant Extracts Using Crowding Agents—Assisted Imprinted Polymers

A methodology-based molecularly imprinted polymer (MIP) was proposed to separate epigallocatechin gallate (EGCG) from natural plant extracts. A molecular crowding agent was introduced to improve the affinity and recognizing ability of EGCG in the preparation of an MIP monolith. Acrylamide was used as the functional monomer, and ethylene glycol dimethacrylate was the crosslinking monomer, using a solution of polystyrene in tetrahydrofuran as a molecular crowding agent with isooctane as a coporogen. In addition, it was found that the ratio of the macromolecule agent, the amount of templates and crosslinking degree, and the composition of the mobile phase greatly affected the retention of the template and performance of molecular recognition. The imprinting factor of the resulting MIP monolith obtained was up to 9.67. The resulting MIP can be used to purify EGCG from crude tea polyphenol efficiently with mean recoveries of 87.42 %.

Two-step chromatographic procedure for the preparative separation and purification of epigallocatechin gallate from green tea extracts

The preparative separation and purification of epigallocatechin gallate (EGCG) from green tea extracts was achieved by a two-step chromatographic procedure. In the first step, a preparative β-cyclodextrin (β-CD) bonded silica chromatography operated in the polar organic mode using methanol/acetonitrile/acetic acid as the mobile phase was applied. Due to a high selectivity for EGCG over epicatechin (EC) and caffeine (CA), an efficient separation of EGCG from EC and CA was achieved and the system could be scaled-up with high recovery of EGCG (>90%). The EGCG-rich fraction obtained from the first step was subjected to the second step where a preparative C-18 chromatography operated in the reversed-phase mode using water/methanol/acetic acid as the mobile phase was applied. The resolution of EGCG was significantly enhanced as the sample contained no EC and CA. The system could also be scaled-up and both the purity and recovery of EGCG reached about 90%. In summary, the chromatographic procedure consisting of two such complementary steps gave a satisfactory purification results: the purity and recovery of EGCG were >90% and >80%, respectively. Most significantly, the purification method could be more easily scaled-up, showing good potential for industrial applications.

Separation of epigallocatechin gallate from tea polyphenol by simulated moving bed chromatography

(−)-Epigallocatechin gallate (EGCG) was purified from tea polyphenol by a two-step simulated moving bed (SMB) chromatographic process with C18-bonded silica gel as stationary phase and a mixture of methanol and water as mobile phase, removing the more retained components firstly and the less retained components secondly. An open-loop 3-zone SMB approach was used, a gradient of solvent strength in zones I and II was formed by different methanol dosage in purge and desorbent solutions and pre-equilibration was reached by the elution at the end of zone III. Under the approximation including linear adsorption and ideal chromatographic model, the operating parameters of the simulated moving bed were selected according to the “triangle theory”. In the first SMB separation, a solution of tea polyphenol with 55.5% purity of EGCG was used for feeding and a raffinate solution with 92.2% purity and 99.7% recovery of EGCG was obtained. The raffinate solution was concentrated for feeding in the second SMB separation and an extract solution with 97.8% purity and 99.8% recovery of EGCG was obtained.

Preparative separation of EGCG from Korean green tea by high performance liquid chromatography

Among catechin compounds abundant mainly in green tea, (−)epigallocatechin gallate (EGCG) has antioxidative and anticarcinogenic properties. Korean green tea (Bosung, Chonnam) was used as a feed sample. The separation was done by a C18 reversed-phase preparative column (22×250 mm) with mobile phases of 0.1% acetic acid in water, acetonitrile, methanol, and ethyl acetate considered. The injection volume and the flow rate of mobile phase were fixed at 400 μL and 16 mL/min, respectively. To purify EGCG preparatively, the effluent was collected from the column outlet and concentrated, and analyzed by analytical column (3.9×300 mm). From the experimental results, the optimum mobile phase condition for separating EGCG from Korean green tea was 0.1% acetic acid in water/acetonitrile/methanol/ethyl acetate (80/16/1/3 vol.%). In the experimental condition, 0.6 mg of EGCG was obtained purely.

Separation of Epigallocatechin Gallate and Gallocatechin Gallate Using Multiple Instruments Connected in Series

A synthetic mixture of epigallocatechin gallate and gallocatechin gallate was separated using multiple countercurrent chromatographic instruments by connecting the separation columns in series. Peak resolution increased according to the formula Rs-n = n1/2Rs-1, where n is the number of columns connected; Rs-n and Rs-1, the peak resolution obtained from n columns and a single column, respectively. Various sample sizes and concentrations were applied to four columns connected in series. The results indicated that the sample loading capacity is increased 11 times that of the single instrument for the same peak resolution.