| Bioactive ingredients extracted from green tea such as catechins, theanine and caffeine have a lot of health benefits and have been used widely in various neutraceuticals, pharmaceuticals, and cosmetics. Thus, the demand for these bioactive ingredients is increasing. However, considering negative effects of caffeine in some cases coupled with the requirements of the purity for pharmaceutical use, related isolation and purification techniques should be improved to remove caffeine and to get high-purity catechins or theanine. Adsorption separation methods using adsorbents to enrich bioactive ingredients from plant materials are available. Focusing on preparing high-purity catechins and theanine from tea leaves or tea extracts, this paper investigated the adsorption properties of19tested adsorbents including macroporous adsorption resins (MAR), anion exchange resins (AER), cation exchange resins (CER) for use in preparing catechins, caffeine and theanine. Those which have good adsorption capacity and selectivity were selected as adsorbents for isolation of the target ingredients. The major results can be summarized as follows:(1) Screening of candidate adsorbentsPolyvinylpolypyrrolidone (PVPP) and polymide-6(PA-6) displayed strong and selective adsorption characteristics for catechins due to their capacity to form hydrogen bonds between their amide group and the phenolic hydroxyl group of catechins, and they adsorbed little caffeine and theanine, indicating PVPP and PA-6can be used to isolate catechins from caffeine or theanine.Among the tested MARs, HPD-400had good adsorption capacity for caffeine in solution, but had weak adsorption for theanine. It can be used for decaffeination of crude theanine product.The tested CER had an excellent adsorption capability to theanine, among which ZGSPC106Na had higher adsorption capacity than D001SD, while D001SD had better adsorption selectivity to theanine. Both ZGSPC106Na and D001SD can be used to enrich theanine from tea extract. (2) Adsorption behaviors of PVPP to catechins and caffeineThe adsorption behaviors of PVPP to catechins and caffeine were investigated by static adsorption testing. The results showed that catechins rather than caffeine were preferred to adsorb onto PVPP. The adsorption was driven by hydrogen bonds between the proton donor from the catechins and the carbonyl group from PVPP, together with π-bond overlap and hydrophobic interactions between the aromatic ring of the catechins and the pyrrolidone ring of PVPP, among which hydrogen bond appeared was the major driving force. The adsorption of catechins onto PVPP fitted the pseudo-second-order model. The results also showed that the adsorption capacity of catechins and caffeine decreased with an increase in temperature, and that both Freundlich and Langmuir models were suitable for describing the isothermal adsorption of catechins, but not suitable for caffeine. The predicted maximum monolayer adsorption capacity of total catechins by PVPP was671.77mg g-1at20℃. The thermodynamics analysis showed that the adsorption of catechins onto PVPP was a spontaneous and exothermic physisorption process, and decreasing temperature was favorable for the adsorption of catechins. Elution tests showed that the desorption rates of caffeine and catechins were higher than91%and99%by water eluting stage and dimethyl sulfoxide/ethanol (DMSO/EtOH) solution eluting stage, respectively, resulting in the final catechins product containing more than95%catechins and less than0.1%caffeine. PVPP was considered to be an excellent alternative adsorbent for preparing high-purity catechins from tea extracts.(3) Adsorption behavior of the theanine onto cation exchange resinsTwo cation exchange resins, i.e. D001SD and ZGSPC106Na, were used to test their adsorption capacity to theanine in static adsorption test using model solution of theanine and caffeine respectively. The effects of adsorption time, temperature and pH on resin exchange capacity were investigated. Between pH3.18and pH5.67, pH had little effect on the adsorption of the tested resins to theanine. However, as the solution acidity was below pH3.18or above pH5.67, the adsorption capacity decreased. It was proposed that the adsorption process should be carried out at natural pH level of tea extract. The adsorption equilibrium of theanine onto the two CER was obtained in30min of adsorption time, among which equilibrium time of ZGSPC106Na was shorter than that of D001SD. The adsorption process was fitted to the pseudo-second-order kinetics model, and the equilibrium parameters could be calculated using both the Langmuir model and the Freundlich model. As temperature ranging293K to323K, the adsorption capacity of the resins to theanine decreased with increase in temperature, suggesting that lowering temperature favored the adsorption capacity. However, resin ZGSPC106Na was less sensitive to temperature than resin D001SD.Effects of NaCl, NaH2PO4and Na2CO3and ammonia at various pH levels and ionic strength levels on the elution of theanine were investigated. Ammonia solution was confirmed to be good eluant to elute theanine from the cation exchange resins, which avoided the salt-leaching process.(4) Isolation of catechins and caffeine from crude theanineAn adsorption separation method was developed to isolate catechins and caffeine from crude theanine extract (theanine content being30%) using PA-6or HPD-400as adsorbents. It showed that PA-6adsorbed catechins specifically but HPD-400adsorbed both catechins and caffeine. As the extract was passed through the PA-6or HPD-400column, most of catechins and caffeine were retained in the column. When the the theanine extract solution was sequentially loaded onto the PA-6or HPD-400column, catechins and caffeine were retained in the columns. As the effluent which was passed through the columns was concentrated and freeze dried, the product contained theanine more than50%, withy a recovery rate>97%. The catechins and caffeine retained in the PA-6or HPD-400columns could be recovered by eluting using80%(v/v) aqueous ethanol. The above abtained crude theanine was further purified by CER column (packed with ZGSPC106Na or D001SD) chromatography using stepwise elution process, i.e.1) sequentially elution using water and80%(v/v) aqueous ethanol to wash away catechins and caffeine;2) elution using0.5M ammonia solution. The ammonia solution eluate which contained theanine was collected, concentrated and freeze dried, by which a refined theanine powder was obtained. This theanine level in the product was more than92%, and the theanine recovery rate was upto98%.(5) Simultaneous preparation of catechins and theanine from tea leavesBased on above study, a method for simultaneous preparing catechins and theanine from tea extract was developed. Green tea extracts was loaded on a PA-6column and washed sequentially with water and80%aqueous ethanol. The ethanol eluate was collected, concentrated and freeze dried, resulting in a product with total catechins concentration being854.08mg g-1and caffeine less than2.53mg g-1. The effluent passed through the PA-6column and the previous water eluate fraction was combined and further passed through a HPD-400resin column and then washed using water and80%ethanol. The ethanol eluate was collected, concentrated and freeze dried, by which a product with total catechins being352.14mg g-1and caffeine399.76mg g-1was obtained. The effluent was then loaded on D001SD cation exchange resin column and eluted using water and ammonia solution. The ammonia solution eluate was concentrated and freeze dried, by which a product containing more than37%theanine was obtained. By this process, three products were separated from a material of tea leaf. |
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