Journal of Bioscience and Bioengineering VOL. 111 No. 2, 232 236, 2011 www.elsevier.

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Preparation of tea catechins using polyamide

Jian-Hui Ye,1 Liu-Xiang Wang,1 Hao Chen,1 Jun-Jie Dong,1 Jian-Liang Lu,2 Xin-Qiang Zheng,2 Ming-Yan Wu,1 and Yue-Rong Liang1,
Zhejiang University Tea Research Institute, 268 Kaixuan Road, Hangzhou 310029, China 1 and Key Laboratory of Horticultural Plants Growth, Development and Biotechnology, Agricultural Ministry of China, Hangzhou 310058, China 2 Received 27 May 2010; accepted 30 September 2010 Available online 9 November 2010

An adsorption separation method using Polyamide-6 (PA) as an adsorbent was developed to separate catechins from green B tea extract. The adsorption capacity of total catechins for PA was 193.128 mg g1 with an adsorption selectivity coefficient KA of total catechins over caffeine 21.717, which was better than macroporous resin model HPD 600. The Langmuir model and the pseudo-second order mode were primely fitted to describe its equilibrium data and adsorption kinetics, respectively. PA column separation by two-step elution using water and 80% (v/v) aqueous ethanol was established to prepare catechins complex which contained 670.808 mg g1 total catechins and 1.828 mg g1 caffeine. It is considered that PA was a promising adsorbent for selective isolation of catechins. 2010, The Society for Biotechnology, Japan. All rights reserved.
[Key words: Camellia sinensis; Caprolactam; Caffeine-free; Two-step elution]

Tea catechins are a group of bioactive components in tea leaf which are confirmed to have healthy benefits including antimutagenic, anticarcinogenic, anticlastogenic and antioxidant effects (13), and are increasingly used as additives in the functional food stuff (4,5). The usual methods to isolate a mixture of tea catechins are mainly liquidliquid extraction and adsorption separation. The advantages of liquidliquid extraction are less energy consumption and large capacity. However, large quantities of organic solvents such as ethyl acetate, propyl acetate, chloroform and dichloromethane are used in liquidliquid extraction, resulting in a trace of undesirable solvents remained in the final products, making the food safety problems hang over. The adsorption separation method may be advantageous because of its simple process and solvent control. The adsorbents adopted in adsorption separation of tea catechins include C-18 bonded silica and resins Amberlite XAD-7HP and Amberlite XAD-4 (6). Polyamide (PA) contains functional groups including acylamino, terminal amino and carboxyl groups which can reversibly form strong hydrogen bonding with substrates and eluents. It is used in the chromatography of phenols and carboxylic acids (7,8). A thin layer chromatography of theaflavins on PA is reported (9). However, there is no report on the preparative isolation of tea catechins using PA as an adsorbent. It is reported that the cyclic and linear oligomers from the manufacturer of PA have an adverse impact on the properties of final polymer, such as chemical resistance, thermal stability and mechanical intensity (10,11), indicating a pretreatment of PA is required before use.

In the present study, PA was used to isolate catechins from tea solution, and the adsorption characteristics, including kinetics, isotherm and column chromatography, were studied. Mixtures of tea catechins from green tea extract were prepared in a fixed bed column packed with PA.
MATERIALS AND METHODS Materials Green tea extract powder and green tea leaf used in the present study were supplied by CinoTea CO., Ltd. (Hangzhou, China). The green tea extract powder was prepared by extracting the green tea leaf at 90C in water, filtering through 0.25 m cellulose acetate ultrafiltration membrane, concentrated by reverse osmosis and finally spray drying. The tea powder was completely dissolved in water without suspended matter. Polyamide-6 (PA, 0.250.60 mm in diameter) was purchased from Luqiao SI-JIA Biochemical Plastic Company (Taizhou City, China). Chemical purity grade ethanol was from Sinopharm Chemical Reagent CO., Ltd. (Beijing, China). HPLC reference compounds of catechins and caffeine was Sigma products (Sigma-Aldrich, St. Louis, MO, USA). The other chemical reagents used were of HPLC grade (Jinmei Biotech Corporation, Tianjin, China). The water used was prepared by an EASYPure II UVUltraPure Water System (Barnstead International, Dubuque, IA, USA). Pretreatment of PA To remove the oligomers and ethanol-dissolved substances from the PA adsorbent, 1 kg PA was drenched in 8 L 95% (v/v) ethanol on 70C water bath for 40 min, decanted the ethanol and replaced with fresh ethanol as before. The PA was then washed using 8 L water for three times. The pretreated PA was naturally dried at room temperature. Test of adsorption selectivity Tea solution was prepared by dissolving 13.5 g green tea powder in 1 L pure water. Six g of the pretreated PA was placed in 500-mL glass flasks containing 200 mL of the tea solution. The flasks were shaken at 150 rpm and 30C for 12 h in a H2S-H water bath shaker (Donglian Electronic Technology CO., Ltd., Harbin, China). The solution was separated from the PA by filtrating through a Double-Ring no.101 filter paper (Xinhua Paper Co. Ltd., Hangzhou, China) and centrifuged at 5000 g for 25 min. The supernatant was injected into HPLC. The adsorption capacity was calculated by the following Eq. 1: q= V C0 C G 1

Corresponding author. Tel.: + 86 571 86971260; fax: + 86 571 86971704. E-mail address: yrliang@zju.edu.cn (Y.-R. Liang).

1389-1723/$ - see front matter 2010, The Society for Biotechnology, Japan. All rights reserved. doi:10.1016/j.jbiosc.2010.09.019

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where q is adsorption capacity per gram adsorbent (mg g-1), C0 and C are the initial and final solute concentrations of the solution (mg mL-1), respectively. V is the solution volume (mL) and G is the weight of adsorbent used (g). Adsorption selectivity refers to the extent to which different adsorbates bound onto an adsorbent. Selectivity coefficient KB is the equilibrium constant for the reaction A of displacement of one adsorbate by another adsorbate in a complex with the substrate. The tea solution contains tea catechins and caffeine. Decaffeination is necessary during isolation of catechins complex. The KB was used to evaluate the selective adsorption A capacity of PA to catechins over caffeine and was calculated by the following Eq. 2:
B KA =

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RB = RA C RB = A CB = CA CB RA

B where KA is selectivity coefficient in which A and B represent caffeine and total catechins respectively. CA and CB are concentrations (mg mL1) of caffeine and total catechins in the solution at equilibrium; RA and RB are adsorption capacities (mg g1) of caffeine and total catechins in the adsorbent phase at equilibrium, respectively. Test of adsorption kinetics Ten grams of the pretreated PA was placed in a 500 mL glass flask with 360 mL of tea solution (15 g L1) and shaken at 150 rpm in water bath at thermodynamic temperatures 288 K, 303 K and 318 K, respectively. The thermodynamic temperature is calculated by +273C. The tea solutions sampled at different time intervals (0360 min) were filtered and centrifuged as above before HPLC. Test of adsorption isotherm PA (1.5 g) was shaken with 50 mL of tea solution at concentrations ranging from 4.5 g L-1 to 31.5 g L1 in H2S-H water bath shaker at 150 rpm for 12 h at temperatures of 288 K, 303 K and 318 K, respectively. The solutions were filtered and centrifuged as above before HPLC. Gradient elution One milliliter of the tea solution (180 g L1) was loaded on the PA column (10 mm i.d. 120 mm). The column was washed with 30 mL pure water and then eluted with 20 mL ethanol solutions at increasing concentrations (10, 20, 40, 60 and 80%, v/v) at flow rate 1 mL min1. The eluates were collected in 5 mL fractions for HPLC analysis. Preparation of catechins complex from green tea leaf One kilogram of green tea leaf was extracted with 15 L pure water at 90C for 40 min. The tea solution was filtered through two layers of gauze and then loaded onto a column (66 mm i. d. 550 mm) packed with PA at a flow rate 100 mL min1. The column was rinsed with 6 L water and sequentially eluted with 6 L of 80% (v/v) ethanol solution at 35 mL min1. The ethanol eluate was collected and concentrated to 1 L in an evaporator at reduced pressure and 45C, and finally dried using an OPD-8 laboratory spray dryer (Ohkawara Dryers CO., Ltd., Shanghai, China) at inlet temperature 190C and outlet temperature 105C. The obtained tea catechins complex was kept in a desiccator containing silica gel at room temperature. The column was washed with pure water to remove ethanol, and the adsorption process was repeated again. The regeneration of PA column was carried out in the fixed bed by using 6 L of 1 N NaOH to drench the used PA for 12 h, washing to pH 7 with pure water. Then the bed was soaked in 6 L of 0.2 N HCl overnight, and finally rinsed with pure water to pH 7 for recycle test. HPLC analysis Concentrations of catechins and caffeine were analyzed by a modified method described in previous paper (12): injection volume 10 L, phenomenex C18 column (4.6 mm 150 mm), column temperature 28C, mobile phase A = acetonitrile/acetic acid/water (6:1:193, v), mobile phase B = acetonitrile/ acetic acid/ water(60:1:139, v), linear gradient elution: from 70%(v) A/30%(v) B to 15% (v) A / 85%(v) B during early 33 min and then 15% (v) A/85% (v) B till 38 min at flow rate 1 ml min1, monitored by a Shimadzu SPD ultraviolet detector at 280 nm. Data analysis All the above tests were carried out in triplicate and the mean values of the triplicate tests were presented.

B equilibrium (Table 1), with a selectivity coefficient KA of total catechins over caffeine 21.717. Previous research showed that the adsorption capacities of model macroporous resin HPD 600 to total catechins and caffeine were 156.3 mg g1 and 18.8 mg g1 respecB tively, with a selectivity coefficient KA of 0.54 (13). Both adsorption B capacity and selectivity coefficientKA at equilibrium are important parameters to evaluate the performance of adsorbents. Here catechins are the primary adsorbates and caffeine is the interfering adsorbate. The higher the selectivity coefficient is, the less the effect of the interference comes from caffeine. These suggest that the PA adsorption capacity to catechins and selectivity are better than those of macroporous resin HPD 600, showing PA is a promising adsorbent for separation of tea catechins from green tea. PA, a polycondensate of caprolactam, has functional acylamino groups which can reversibly form strong hydrogen bonding with phenolic compounds (14). Tea catechins are a group of plant polyphenols with plentiful hydroxyl groups. For example, the molecule of ()-epigallocatechin gallate (EGCg), the most abundant polyphenols in tea, has eight hydroxyl groups. PA aggregates catechins from tea solution through hydrogen bonding as shown in Fig. 1. The molecular structure of caffeine without any hydroxyl group is greatly different from that of tea catechins, and this resulted in a good adsorption selectivity of PA to total catechins. The previously reported macroporous resin HPD 600, polymerized by divinylbenzene and with a three-dimension structure full of pores, mainly interacts with tea catechins through Van der Waals force due to its tremendous surface area. Van der Waals force is regarded as the main action for macroporous resin to isolate natural compounds but it is a weaker interaction between molecules than that of hydrogen bonding. As a result, caffeine, the interfering adsorbate in the tea solution, is more adsorbed by macroporous resin HPD 600. These explain why PA had a better selectivity adsorption than macroporous resin HPD 600. Adsorption kinetics Parameters of adsorption kinetics are usually used to evaluate the reaction pathways and the mechanism of adsorption reactions. The fits to the pseudo-first order and the pseudo-second order models of present data were investigated. The pseudo-first order model of Lagergren (15) is expressed as Eq. 3:

dqt = k1 qe q dt Integrating and rearranging the Eq. 3 to a linear form as Eq. 4: logqe qt = log qe k1 t 2:303

RESULTS AND DISCUSSION Adsorption selectivity The PA adsorption capacities were 193.128 mg g1 to total catechins and 2.189 mg g1 to caffeine at

where qe is the solute concentration in the solid phase (mg g1) at equilibrium, qt is the solute concentration in the solid phase at any given time t (mg g1) and k1 is the rate constant.

TABLE 1. Concentrations of tea catechins and caffeine in tested samples (mean SD) a. Samples Tea powder b (mg g1) PA adsorption c (mg g1) Eq Conc d (g L1) Ini Conc e (g L1)
a b c d e

GC

EGC

EC

EGCg

GCg

ECg

Cg

Total catechins

Caffeine

8.444 0.074 64.815 0.963 1.778 0.074 15.111 0.222 119.333 0.667 5.407 0.074 29.333 0.074 1.037 0.000 245.259 1.407 22.000 0.074 3.152 0.000 16.546 0.963 0.788 0.088 0.078 0.001 0.114 0.001 0.686 0.002 0.015 0.000 0.875 0.013 0.024 0.001 4.815 0.175 127.993 0.875 6.041 0.088 32.830 0.088 0.963 0.000 193.128 1.596 0.149 0.001 0.204 0.003 0.149 0.001 0.004 0.000 1.611 0.009 0.073 0.001 0.021 0.000 0.003 0.000 0.395 0.002 0.014 0.000 1.105 0.001 3.310 0.019 2.189 0.088 0.272 0.002 0.296 0.002

SD, standard deviation. Tea powder used to prepare tea solution. Adsorption capacity of PA at equilibrium. Eq Conc, concentration at equilibrium. Ini Conc, initial concentration.

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TABLE 3. Langmuir parameters of total catechins onto PA at different temperatures a. Temperature Kb 288 K 303 K 318 K
a

Langmuir qm 331.126 326.797 271.003 R2 0.961 0.981 0.982

2.904 2.661 2.097

Initial concentrations 2.25-31.50 g L1.

FIG. 1. Scheme of hydrogen bonding interaction between PA and tea catechins.

to the pseudo-first order model, suggesting that chemisorption played a dominant role in the adsorption process of tea catechins onto PA, accompanying physical diffusion transport phenomena experimentally inseparable (17). The increase in temperature accelerates chemical reaction and diffusion of tea catechins in solution, resulting in an advanced equilibrium process. Adsorption isotherm Adsorption isotherm reveals the relationship between the adsorption capacity of adsorbates and concentration of adsorbates in solution at a given temperature (18). Langmuir model was usually used to describe the equilibrium data and it is expressed by Eq. 8: 1 1 1 = + qe qm Kb Ce qm 8

The pseudo-second order model (16) was described by Eq. 5: dqt = k2 qe q2 dt Integrating and rearranging the Eq. 5 to a linear form as t 1 1 = + t qt qe k2 q2 e 6 5

where k2 (g mg 1 min 1) is the second-order rate constant determined from the plot of t/qt vs t. The second-order rate constants were used to calculate the initial adsorption rate (h), given by Eq. 7: h = k2 q2 e 7

The kinetic parameters of total catechins adsorbed onto PA showed good fits to both pseudo-first order and pseudo-second order models (Table 2). However, based on the correlation coefficients R2, it was more fitted to the pseudo-second order model than the pseudo-first order model. The adsorption capacities at equilibrium calculated by the pseudo-second order model (qe (cal)) were close to the experimental data qe (exp) (Table 2). With an increase in temperature, the qe (cal) and qe (exp) decreased while k1, k2 and h increased (Table 2), implying that the increase in temperature accelerated the adsorption process but had a negative effect on adsorption capacity. Diffusion was assumed to be the rate- controlling step for the pseudo-first order model, while chemisorption was supposed to be the rate-limiting step for the pseudo-second order model. The chemisorption was the macroscopic concept of reaction relative to diffusion, involving valency forces through sharing or exchanging of electrons between adsorbents and adsorbates (16). The present kinetics data were more fitted to the pseudo-second order model than

where qe and Ce are the amount of adsorbates adsorbed by adsorbent (mg g1) and the adsorbate concentration in solution (mg mL1) at equilibrium respectively. Kb is the association constant and qm the adsorption capacity, which are the slope and intercept of plot of 1/qe versus 1/Ce, respectively (19). Table 3 shows the Langmuir parameters for adsorption of total catechins by PA at different temperatures, with correlation coefficients R2 above 0.961. Since the Langmuir isotherm is established on the assumption that the configuration of adsorbent is homogenous and each active site on adsorbent interacts with monolayer of adsorbate molecule. The results showed good fit to Langmuir model, indicating that prevalent surface of PA is homogeneous and the adsorption of catechins onto PA mostly occurs in a monolayer. With an increase in temperature from 288 K to 318 K, the qm for total catechins decreased from 331.126 mg g1 to 271.003 mg g1, with a decrease in Kb from 2.904 to 2.097, suggesting high temperature was not propitious to the adsorption capacity. Thermodynamic parameters Thermodynamic parameters, including standard Gibbs free energy change (G 0), enthalpy change (H 0) and entropy change (S 0), are usually used to describe temperature dependent adsorption properties. The G 0 was calculated based by Eq. 9: G0 = RT ln Kc 9

where R is the gas constant, Kc is equilibrium constant, i.e., the ratio of adsorbate concentration on solid adsorbent phase (CBe) to adsorbate concentration in the solution phase (CAe) at equilibrium, T is the thermodynamic temperature (20). The G 0 for physisorption ranges from 20 kJ mol1 to 0 kJ mol1 and for chemisorption from 80 kJ mol1 to 400 kJ mol1 (21). The G 0 values in the study ranged

TABLE 2. The pseudo-first order and pseudo-second order kinetic parameters of total catechins onto PA at different temperatures. a Temperature Pseudo-first-order model k1 (min1) 288 K 303 K 318 K
a

Pseudo-second-order mode (g mg1 min1) k2 h (mg g1min1) 10.902 12.498 12.593 R2 0.995 0.999 0.996 qe(exp) (mg g1) 221.163 213.966 204.058 qe(cal) (mg g1) 224.215 219.780 205.761

R2 0.982 0.913 0.979

0.01004 0.01064 0.01340

0.00022 0.00026 0.00030

Initial concentrations 15 g L1. qe(exp): experimental data; qe(cal): Theoretical value were calculated by Eq. 6.

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TABLE 4. Equilibrium constant (KC) and Gibbs free energy change (G 0) of total catechins onto PA. 288 K KC G 0(kJ mol1) 1210.384 16.998 303 K 636.871 16.226 318 K 305.897 15.132

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FIG. 2. Van't Hoff plot for the adsorption of total catechins onto PA.

from 16.998 kJ mol1 to 15.132 kJ mol1 (Table 4), suggesting the adsorption processes is spontaneous physisorption. Enthalpy change (H 0) and entropy change (S 0) were calculated from Kc through Van't Hoff Eq. 10: ln Kc = S0 H 0 R RT 10

Enthalpy change (H 0) is the amount of heat released or absorbed as the reaction occurs at constant pressure. Entropy change (S 0) is a

measure of freedom degree for the adsorbed species or the disorder of a system. The H 0 and S 0 which were obtained from the slope and intercept of the Van't Hoff plot of lnKc vs 1/T (Fig. 2) were 34.846 kJ mol1 and 61.760 J mol1 K1 respectively. The negative value of H 0 indicates that the adsorption of catechins onto PA was exothermic. Therefore, the adsorption equilibrium moved toward adverse orientation as the temperature rose. These are the reasons that a reduction in the adsorption capacity of total catechins occurred with the increase in temperature (Tables 2 and 3). The H 0 for Van der Waals force ranges from 10 kJ mol1 to 4 kJ mol1, for hydrogen bonding from 40 kJ mol1 to 2 kJ mol1 and for chemical bonding below 60 kJ mol1 (22). The H 0 value calculated by Eq. 10 in the present study was 34.846 kJ mol1, suggesting hydrogen bonding is the predominant interaction between tea catechins and PA. The S 0 calculated by Eq. 10 in the present study was 61.760 J mol1 K1, indicating that the uniformity of the adsorption system is increased, which is in accordance with the monolayer adsorption of catechins onto PA inferred above. Gradient elution Ethanol, a kind of non-toxic and efficient organic solvent weakens the hydrogen bonding between adsorbates and adsorbents and increases the solubility of adsorbates. Aqueous ethanol is generally used as an eluent in the isolation of natural compounds. Fig. 3 showed the chromatogram of catechins and caffeine (CAFF) eluted from PA column by gradient aqueous ethanol. Caffeine was eluted by water (Fig. 3A). Catechins were gradually eluted with an increase in ethanol concentration up to 60% (v/v). ()-Epigallocatechin gallate (EGCg) was partially eluted by 80% (v/v) ethanol. The results suggest that caffeine is perfectly separated from tea catechins during the gradient elution. This implies that two-step elution might be a good process to prepare caffeine-free catechins, i.e. using water to remove caffeine and then 80% (v/v) aqueous ethanol to elute catechins. Preparation of catechins complex from tea leaf Tea leaf extract was loaded onto the PA column and eluted sequentially using water and then 80% (v/v) ethanol. A complex powder of tea catechins was obtained as the ethanol phase was concentrated and spray dried. The yield of catechins complex ranged from 39.4 g to 42.8 g per kilogram tea leaf (Table 5). Composition of the catechins changed with the sorption desorption cycles (Table 5). Total catechins level was 670.808 mg g1 in

FIG. 3. Chromatogram of catechins and caffeine on PA with aqueous ethanol by gradient elution. Column, 10 mm i.d. 120 mm flow rate, 1 ml/min. The adsorbates were eluted with aqueous ethanol of increasing concentrations. The part marked by different alphabetic letters represented ethanol solution of various concentrations(v/v), respectively: A = 0 (30 ml); B = 10%(20 ml); C = 20%(20 ml); D = 40%(20 ml); E = 60%(20 ml); F = 80%(20 ml).

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TABLE 5. Chemical compositions of used tea leaves and catechins complex prepared by PA and regenerated PA columns (mean SD, mg g1) a. Sample TL b Ac Bd Ce
a b c d e

Weight (g) 1000.0 42.8 1.5 39.4 1.0 42.3 1.1

GC 17.5210.682 47.1330.815 46.4120.932 49.2150.973

EGC

EC

EGCg

GCg

ECg

Cg

Total catechins

Caffeine

53.0100.952 7.913 0.091 7.9140.097 57.1830.125 21.2640.153 12.5820.077 6.3180.203 83.9211.988 39.4260.592 35.9270.593 244.0123.137 131.2221.646 58.0120.719 31.1550.456 83.6372.103 35.7110.679 33.8470.475 238.6763.431 123.9341.593 55.4720.812 30.2340.348 84.5272.159 36.4200.443 35.2280.731 242.7382.912 127.9261.727 56.6140.629 34.9110.328

183.7051.598 49.5320.022 670.8085.742 1.8280.337 647.9235.541 13.3880.563 667.5794.330 2.1170.401

SD, standard deviation. TL, tea leaves used to prepare tea catechins complex. A, catechins complex powder prepared from the 1st sorptiondesorption cycle. B, tea catechins complex powder prepared from the 2nd sorptiondesorption cycle. C, tea catechins complex powder prepared by regenerated PA column.

the first sorptiondesorption cycle, with a caffeine level of 1.828 mg g1. Caffeine level was significantly increased in product obtained from the second sorptiondesorption cycle. However, the product isolated from the regenerated column after two sorptiondesorption cycles had a similar concentration of caffeine with that of the first cycle (Table 5). These suggest that column regeneration should be carried out after each sorptiondesorption cycle. The yield of catechins complex varied with the sorptiondesorption cycle and it was increased by column regeneration (Table 5). PA showed substantial advantages for preparing tea catechins compared to the reported results (13,23). The adsorption capacity of catechins for PA was higher than those of macroporous resin HPD 600 (13) and ion exchange fiber (23). The ratio of catechins to caffeine in the product of present study was higher than that prepared by macroporous resin HPD 600 (13), suggesting that PA had a better selectivity adsorption to catechins than the later. It is considered that PA is an ideal adsorbent for preparing catechins from tea solution. The adsorption process of catechins onto PA was an exothermic one mostly occurred in a monolayer, during which physisorption take place spontaneously with hydrogen bonding as the predominant interaction between tea catechins and PA. Caffeine in tea extract loaded onto PA column can be mostly removed by washing with water and catechins complex with low caffeine level can be obtained by elution with 80% (v/v) aqueous ethanol. The sorptiondesorption cycle had great influence on the caffeine concentration of product and regeneration is necessary after each sorptiondesorption cycle. ACKNOWLEDGMENTS This work was financially supported by the Agricultural Ministry of China (Project No. nyhyzx 2007-03-35-1). References
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