Isolation and purification of ester catechins by macroporous

Transcription

1 ZHANG Yan-lai et al. Isolation and purification of ester catechins by macroporous adsorption resin Isolation and purification of ester catechins by macroporous adsorption resin ZHANG Yan-lai, Li Nan (School of Chemistry and Engineering, Dalian University of Technology, DaLian,116023,P.R.China) Abstract: The single factor tests, thogonal experimental designs and variance analyses were applied to optimize the manipulation parameters of HPD-600 macroporous adsorption resin for purifying ester catechins. The results indicated that the ideal adsorption condition was feeding rate at 1.5BV/h. The optimum eluting conditions were A 1 B 3 C 2 D 1, i.e. eluting volume 4BV, eluting fasion water -10 %-20%-45%, eluting rate 1BV/h and the ratio between the diameter and height of chromatography column 3:30. Key words: ester catechin, EGCG, ECG, isolation and purification, macroporous adsorption resin 1. Introduction Leaves of the green tea plant camellia sinensis contain up to 36% polyphenols on a dry weight basis. However, their composition varies with climate, season, variety and state of maturity. Green tea catechins are the predorminant group of green tea polyphenols. Examples of catechins are (-)-epicaechin(ec), (-)-epigallocatechin gallate(egcg),epigallocatechin(egc), and epicatechin gallate(ecg) 1. In recent years, tea polyphenols have received more and more concerns in such new applied areas as medicine, food, chemical commodity, etc. Especially two kind of common ester components EGCG and ECG are the most noticeable in catechins. Ester catechins are superior to other catechins in terms of many pharmacology effection because they have more ortho- phenol hydroxyl(scheme 1). It has been demonstrated that EGCG, ECG or their mixture has many medical effect such as antimutagenic effect, antibacterial effect and also a beneficial effect on cholesterol level in blood, etc. On the other hand, the other catechins in tea are much less effective [2-4]. With the study on ester catechins being deepened and their pharmacology function verified further, it is very imperious to separate and prepare ester catechins for the sake of deeply study and HO O O O C HO O H Scheme1 Structure of EGCG and EC application. From the available literatures, the methods used in the separation and preparation process of ester catechins include High Performance Liquid Chromatogram(HPLC), Precipitation- Adsorption, Supercritical Fluid Extraction(SFE) and Column Chromatogram. The method HPLC does not permit an economical production of ester catechins in a technical scale because of the high cost of the instrument and its too little preparing amount. Furthermore, the process described in Precipitation-Adsorption does not permit the production of ester catichins, which can be added to food or medical products because the solvents mixture used are not food-approved. So far as SFE, its yield is too low. Moreover, silica gel column, 352

2 The Proceedings of the 3rd International Conference on Functional Molecules although, suitable for industrialization because of its large producing amount, is being substituted by emerging techmology of microporous adsorption resin column due to the latter s low price and better performance. So a great effort has been made toward to explore a feasible process that can be industrialized by using the microporous adsorption resin. 2. Experimental 2.1 Reagents ECG and EGCG were obtained from Sigma Chemical Co. Sodium nitrite, Alminium nitrite, Sodium hydroxide, ethanol(edible), chloroform, ethyl acetate, eather, formic acid, methanol, citric acid and silica gel were purchased from Shenlian Chemical Reagent and Glass Instrument Co., Ltd.(DaLian). HPD-600 microporous adsorption resin was supplied by Cangzhou Bon Chemical Co., Ltd.(Hebei). Tea polyphenols, deionized water and the silica gel thin layers were prepared in our own lab. 2.2 Instrumentation and Procedures 722 Visible Raster Spectrophotometer and JASCO High Performance Liquid Chromatogram were used. The adsorption column was a glass tube (12mm i.d., 400mm length) in which a small pad of cottonwool had been placed beforehand The quantitive determination of catechins by Visible Spectrophotometer Displaced 1mL sample unknown concentration into 10mL measuring flask, added 0.4mL 5% NaNO 2, 0.4mL 10 Al(NO 2 ) 3, 4mL Na into the flask in sequence. Determinated at the wavelength of 280nm The qualititive determination of catechins by Thin Layer Chromatogram(TLC) The chromatogram conditions: chloroform: ethyl acetate: formic acid: eather=9:7:3: The determination of the content of ester components by HPLC The chromatogram conditions: moble phases: CH 3 :H 2 O(1 citric acid)=25:75, eluting rate: 1mL/min, wave lenth: 280nm Dynamic adsorption procedure Filled 16ml HPD-600 microporous resin pretreated into the glass column. Subjected the tea polyphenols to the chromatography on a macroporous resin at ambient temperature. Keep the feeding rate at 3BV/h. Regarded the volume of flowing out liquid as the abscissa, and the concentration of catechins as the ordinate to gain the dymamic assorption curve of catechins on the HPD-600 microporous resin Adsorption conditions Filled 20ml wet resin pretreated into the glass column respectively. Fixing the feeding rate or the ratio between the diameter and height of the chromatography column to see the effect of every adsorption condition respectively on amount adsored. Collected the flowing out liquid subsectively and detected the concentration of the catechins. Stopped adsorbing when the concentration reached 0.05mg/ml. Calculated the dynamic adsorbed amount according to the volume of the flowing out liquid and the concentration difference of catechins before and after absorbing The single factor tests of eluting conditions Filled 20ml wet resin pretreated into the glass column respectively, subjected the tea polyphenols to the chromatography under the ideal adsorption condition gained from tests above. Eluted under fixed ethanol concentration, eluting rate, eluting volume and the ratio between the diameter and height of chromatography column separately. Collected the flowing out liquid subsecctively, detected the concentration of catechins and calculated the desorption percentage. Rrgarded the eluting manipulation parameter as the abscissa and the desorption percentage the ordinate to see the effect of each eluting manipulation on the eluting results Thogonal experiment of eluting manipulation parameter The experiment regarded influential ethanol concentration, eluting rate, eluting volume and the ratio between the diameter and height of the chromatography column as the facters investigated 353

3 ZHANG Yan-lai et al. Isolation and purification of ester catechins by macroporous adsorption resin with three levels each, and the content of ester catechins as investigated index. The experiments were disigned according to L 9 (3 4 ) The calculation formula of absorbed amount Q=( C 0 -C)V/W Thereinto, Q is the adsorbed amount(mg/ml), C 0 is the concentration of ester catechins before adsorbing(mg/ml), C is the concentration of ester after adsorbing(mg/ml), V is the total volume of the eluting liquid(ml), W is the volume of the resin(ml) The calculation formual of the desorption percentage DP(%)=VC%/W Thereinto, DP is the desorption percentage, W is the total adsorbed amount(mg/ml), V is the total eluting volume(ml), C is the concentration of the ester catechin in the eluting liquid(mg/ml). 3. Results and Discussion 3.1 Dynamic adsorption procedure From Fig.1 we know Feeding liquid begins to let out when the volume of the flowing out liquid reaches about 1.25BV(1BV=the total volume of the column fillings), then the rate of letting out bagins to increase till the flowing out volume gets to 5BV. The adsorption saturates when the volume gets to 6BV. So the feeding volume sould be about 1.25BV. Concentration(mg/mL) The volume of the flowing out liquid(ml) Fig.1 Dynamic adsorption on the HPD-600 microporous resin 3.2 The effect of adsoption condition on the adsorbed amount The effect of feeding rate on the adsorbed amount From Fig.2 we can see the adsorbed amount is mg/mL and mg/mL respectively when the feeding rate is 1BV/h and 1.5BV/h correspondingly. As the rate rises to 2BV/h the dynamic adsorbed amount drops to mg/mL because catechins are macromolecule compounds, the rate of diffusion is relatively slow, as the flowing velocity increases, a large amount of catechins flow out of the post before being adsorbed. Though the low speed is favorable to absorbing, operating time is prolonged when the flowing velocity is too low. So choosing the feeding rate should be considered synthetically in actual production. 1.5BV/h was suitable. Fig.2 Amount absorbed(mg/ml) Feeding rate(bv/h) The effect of feeding rate on the adsored amount The effect of the ratio between the diameter and height of the chromatography column Fig.3 shows the change of the ratio between the diameter and height of the column has a little effect on the absorbed amount. We didn t look at it as a facter in choosing adsorption conditions. Otherwise, It is generally acknowledged, ph value is influential on the absorbed amount and the acid material is easy to be absorbed under the environment of acidity. But in this experiment, the ph value of the raw material is up to 4 already, consequently, the influence of the ph value on the adsorption didn t be consided in this experiment. In sum, the feeding rate plays a decisive role in the course of absorbing, therefore, we considered that the ideal adsorption condition was feeding at the 354

4 The Proceedings of the 3rd International Conference on Functional Molecules rate of 1.5BV/h. Amount adsorbed(mg/ml) inverse proportionate relationship. When the velocity of eluting is 0.5BV/h, the percentage can be up to 97.8%, but down to 70.5% at the speed of 3BV/h. This may be because when the velocity of flowing is too fast, the mobile phase still fails to replace out catechins from the macroporous resin. It is obvious that the eluting rate should be confined to between 0.5BV/h and 2.5BV/h : 10 1: 20 1: 30 Diameter : height of chromatography column Fig. 3 The effect of diameter: height of chromatography column 3.3 The optimization of the eluting manipulation parameter The effect of the eluting volume on the desorption percentage It can be found from the Fig.4 that the eluting capability and consumption prent a direct proportionate relation, the greater consumption is, the bigger the desorption pencentage is, vice versa. The desorption pencentage increases 12 percentage when the eluting consumption varies from 2BV to 3.5BV, on this basis, the enhance of eluting volume has a little impact, so 4BV eluting liquid is suitable in view of saving the cost. Desorption percentage(%) Eluting rate(bv/h) Fig. 5 The effect of the eluting rate on the desorption percentage The effect of the ratio between the diameter and height of the column Just like the situation in the adsorption tests, Fig.6 indicates the change of the ratio between the diameter and height of the column has a little effect on the desorption percentage too. 100 Desorption percentage(%) Desroption percentage(%) :10 3:20 3:30 Diameter: height of the column Eluting volume(ml) Fig. 4 The effect of the eluting volume on the desorption percentage The effect of the eluting rate on the desorption percentage The eluting rate has obvious influence on the desorption pencentage in Fig.5, the two present Fig. 6 The effect of the ratio between the diameter and height of the column The effect of the concentration of ethanol on the separation of catechins We can see from the Fig.7 that there is eluting peak in every eluting part with different concentration of alcohol. Qualititively examined the component in each eluting peak with TLC and found 355

5 ZHANG Yan-lai et al. Isolation and purification of ester catechins by macroporous adsorption resin that ester catechins existed only in the 10%, 20%, 30%, 45% eluting share. So we decided to adopt the way of gradient elution with the alcohol concentration 10%, 20%, 30% and 45% respectively. Concentration of catechins(mg/ml) Eluting volume with diffrent ethanol concentration(ml) Fig. 7 The effect of the concentration of ethanol on the separation of catechins Thogonal experimental designs of eluting fasion We have investigated the relations between the single factor and the eluting results in the tests above. But, generally, the influence between every factor is interactive. The result investigated alone might not be in conformity with actual situation. So, in order to get the optimum conditions closer to actual conditions, on the basis of single factor tests we decided to design thogonal experiment further. The experiments were disigned according to Tab.1. The concentration of ester catechins in the eluting liquid was listed in the Tab.2. From the results we can conclude that the optimum eluting conditions is A 1 B 3 C 2 D 1 i.e. eluting rate: 1BV/h, the ratio between the diameter and height of column: 3:30, eluting volume: 4BV, eluting fasion: water-10%-20%-45%. Tab. 1 Facters and levels of eluting manipulation Level A B C D Eluting rate(bv/h) The ratio between ID and HEI Eluting volume(bv) Eluting fasion 1 1 3: Water-10%-20%-45% :20 4 Water-10%-30%-45% 3 2 3: Water-10%-45% Tab. 2 Thogonal experimental design Experiment No. A B C D The concentration of ester catechins K j K j K j k j k j k j R

6 The Proceedings of the 3rd International Conference on Functional Molecules Tab. 3 The analysis of variance of the thogonal expriment Resource of square deviation SUMSQ Deg-freedom Mean sauare deviatin F P A >0.1 B C >0.1 D <0.1 Tatal error F 0.1 (2,2)=9; F 0.05 (2,2)=19 The analysis of variance of the thogonal experiment is listed in the Tab.3. We can know from it that there is no obvious difference in three levels of every investigated factor(0.05), but there are certain significance differences in the way of eluting(<0.1)which indicates that the result is determined mainly from the way of eluting. Every level of other three factors can be chosen randomly. In addition, it can be found according to R value from Tab.2 that the influence sequence on purification efficiency of every factor is D>A>C>B. 4 Conclusion 4.1 The optimum eluting conditions are A 1 B 3 C 2 D 1 i.e. eluting rate: 1BV/h, the ratio between the diameter and height of column: 3:30, eluting volume: 4BV, eluting fasion: water-10%-20%-45%. In this process, the content of ester catechins was increased from 70% below to 85% above. 4.2 The present process has several advantages over previously described methods: First, it has reduced the cost of separating greatly for the use of cheap column fillings, which can be regenerated again and again. Second, the mobile phase used is food-approved which makes the products safer in the use of medicine and food. Third, this precess has increased greatly preparing amount so that can be easily industrialized. In addition, the gradient elution system is introduced into separation of different components in catechins for the first time, which improves the chromatographic efficiency. Acknowkedgements We gratefully acknowledge the analytical center at School of Chemical and Engineering for measurement of content of ester catechins. Special thanks go to Dr. Zhanming Gao at Department of Chemical Engineering for his help in the preparation of the manuscript. References [1] Yang X Q. Tea Polyphenols Chemistry [M]. 1st ed. Shanghai: Shanghai Science and Technology Publishing House, [2] [3] Qiong Guo. ESR study on the structure-antioxiant activity relationship of tea catechins and their epimers[j]. Biochimica et Biophysica Acta,1999,14: [4] Pan S J. Study on antioxidative activity and antimicrobial effect of different type of tea polyphenols[a].dissertation of Master Degree of Hunan Agriculture University [D], Hunan: