WO2007041891A1

WO2007041891A1 - Methods of separating catechins from green tea leaves

Info

Publication number: WO2007041891A1
Application number: PCT/CN2005/001644
Authority: WO
Inventors: Tak-Hang Chan; Wai-Har Lam
Original assignee: The Hong Kong Polytechnic University; Mcgill University
Priority date: 2005-10-08
Filing date: 2005-10-08
Publication date: 2007-04-19
Also published as: CN101316830B; CN101316830A

Abstract

There have been a number of reports on the isolation of (-)-EGCG from green tea. However, the presence of many components with their similar water solubility and the structural similarities of the catechins make the isolation of pure (-)-EGCG difficult. The absence of a simple, efficient and inexpensive method of obtaining pure (-)-EGCG has hampered the evaluations of (-)-EGCG in animal and human clinical studies. This invention provides a method of isolating catechins, including (-)-EGCG, by first converting the catechins to their respective ester forms. The esterified catechins are then separated by chromatography, and converted back to catechins. The method of this invention is relatively simple, and a substantial amount of catechins may be obtained.

Description

Methods of Separating Catechins from Green Tea Leaves

Field of the Invention

This invention relates to methods of separating catechins from green tea leaves.

Background of the Invention

Green tea contains many constituents, including polyphenols which are commonly known as catechins. Catechins are thought to be responsible for many of the biological effects of tea. Some major components of green tea polyphenols are (-)-epzgallocatechin-3- gallate (EGCG), (-)-ep/gallocatechin (EGC), e/?/catechin-3-gallate (ECG) and (-)-epZcatechin (EC). In particular, (-)-EGCG, the most abundant catechin, has been found to have anticancer, antibacterial, antiviral, and a beneficial effect on cholesterol level in blood. Although the other catechins in green tea were found to be less effective in the above applications compared to EGCG, they may have other biological activities as well, and increasing their availability may assist in discovering such activities. Green tea also contains other components such as caffeine, proanthocyanidins, carbohydrates, amino acids and soluble minerals which are soluble in water together with the catechins in green tea infusions.

There have been a number of reports on the isolation of (-)-EGCG from green tea. However, the presence of many components with similar water solubility and the structural similarities of the catechins make the isolation of pure (-)-EGCG difficult. US patent no. 6,210,679 describes a four-step process involving three column chromatographic separations to give eventually (-)-EGCG of 95-98% purity. Because of the need of using expensive reverse phase column fillings, the method is time consuming and not economical. US patent publication no. 20030083270 describes a process of providing (-)-EGCG by subjecting a green tea extract to chromatography on a macroporous polar resin with a polar elution solvent under pressure. The method provides (-)-EGCG of 75% to 97% purity. However, the recovery of (-)-EGCG from the green tea extract was only about 73%, and the process is considered to be not efficient. The absence of a simple, efficient and inexpensive method of obtaining pure (-)-EGCG has hampered the evaluations of (-)-EGCG in animal and human clinical studies. Objects of the Invention

Therefore, it is an object of this invention to provide methods for separating catechins from a green tea extract that involve relatively simple steps, and are preferably more efficient.

It is also an object of this invention to resolve at least one or more of the problems as set forth in the prior art. As a minimum, it is an object of this invention to provide the public with a useful choice.

Summary of the Invention

Accordingly, this invention provides a method of separating catechins from green tea leaves. The catechins are first converted to corresponding ester forms to form a ester mixture, and then separated by suitable methods.

Preferably, the catechins are converted to their respective peracetate forms. More preferably, the catechins are converted to corresponding peracetate forms by acetic anhydride.

The converted catechins may be separated by column chromatorgraphy, preferably eluted on silica gel by a mixture comprising hexane and ethyl acetate. More preferably, the hexane and the ethyl acetate are in a ratio of 1 :2 v/v.

Optionally, the ester mixture may first be filtered to obtain a filtrate before the converted catechins are separated. More preferably, the filtrate may then be washed by water, dried and then concentrated before the converted catechins are separated.

After the converted catechins are separated, they may be converted back to the catechins, preferably by a methanol solution of ammonium acetate.

The catechins that may be separated by the method of this invention may include of (- )-e/?z^'gallocatechin-3-gallate, (-)-epzgallocatechin, e/7/catechin-3-gallate and (-)-epZcatechin. Brief description of the drawings

Preferred embodiments of the present invention will now be explained by way of example and with reference to the accompanying drawings in which:

Figure 1 shows a scheme of a preferred embodiment of the separation method of this invention; and

Figure 2 shows the X-ray diffraction crystal structure of (-)-EGC hexaacetate (2).

Detailed Description of the Preferred Embodiment

This invention is now described by way of example with reference to the figures in the following paragraphs.

Objects, features, and aspects of the present invention are disclosed in or are obvious from the following description. It is to be understood by one of ordinary skilled in the art that the present discussion is a description of exemplary embodiments only, and is not intended as limiting the broader aspects of the present invention, which broader aspects are embodied in the exemplary constructions.

According to this invention, it was surprisingly found that the catechins in crude green tea extracts may be separated relatively easily after the catechins are converted to their respective ester forms. The converted catechins may then be separated. Column chromatography is one of the widely used separation methods. Without any proof at this stage, it is believed that the conversion of the catechins into their respective ester forms may enhance the differentiation of the physical properties of the catechins towards, for example, the stationary support used during chromatographic separation.

The catechins may be converted to various different ester forms. Although it is possible to only convert a portion of the hydroxide groups on the catechins into ester forms, it is preferable to have all of the hydroxide groups converted to a single type of ester group. Firstly, this reduces the complexity of the later separation. Additionally, it is more economical. In the following examples, the catechins are converted to their respective peracetate forms by excess amount of acetic anhydride. It should be noted that other ester forms, for example perbenzoate form, may be used. Of course, the catechins may be converted to their respective peracetate forms using any suitable methods, for example using acetyl chloride instead of acetic anhydride. Although the catechins may also be converted to ether forms, this is not desirable as the vigorous reaction conditions involved, either converting the catechins into the ester forms or re-converting the esters back into the catechins, may destroy the catechins themselves.

Figure 1 shows a scheme of a preferred embodiment of the separation method of this invention. As shown in Figure 1, the catechins in the green tea leaves are first converted to their peracetate forms. Direct application of esterification agent, for example acetic anhydride, to the green tea leaves may achieve this purpose. Alternatively, crude green tea extracts may first be obtained by putting green tea leaves in hot water, and the hot water extract may be dried then react with the esterification agent. However, this may be less desirable as this extra extraction step may reduce the overall extraction efficiency. The converted catechins are then separated by traditional column chromatography. The eluent may be a mixture comprising hexane and ethyl acetate, preferably in a ration of 1 :2 v/v.

To further enhance the efficiency of the separation method of this invention, the peracetated mixture may first be filtered before the converted catechins are separated by chromatography. The filtrate may then be washed by water, dried and then concentrated by evaporation.

After the catechins are separated in their respective ester forms, they may then be converting catechins by, for example, using a methanol solution of ammonium acetate.

According to the method of this invention, catechins including (-)-e/>zgallocatechin-3- gallate, (-)-epz^'gallocatechin, e/?/catechin-3-gallate and (-)-epzcatechin may be separated from green tea extracts.

Examples General Green tea packs were purchased from SEIYU with OSK trademark. Alternately, Shou

Mee green tea leaves were purchased from Ying Kee Tea House, Hong Kong. All reagents and solvents from purchased from commercial suppliers and were used without further purification unless otherwise specified. Ammonium acetate was dried under reduced pressure before use. The water used in HPLC was doubly de-ionized water. ¹H and '³C NM R spectra were determined with a Varian-500 NMR spectrometer. Low and high resolution mass spectra were recorded on a Finnigan Model Mat 95 ST mass spectrometer, using positive ion electrospray ionization techniques. Melting point determinations were recorded on a Bϋchi Melting Point B-545 instrument and were uncorrected. Thin layer chromatography was performed on Merck pre-coated silica gel 60 F254 plates and was viewed under a 254 nm lamp. Silica gel (Merck, 230-400 mesh) was used for column chromatography. HPLC was conducted on a HP 1100 liquid chromatograph, equipped with a C- 18 reverse phase column (CAPCELL PAK C18 UG 120, 4.6 mm i.d. x 250 mm).

Isolation of individual catechins from green tea leaves from SEIYU

Pyridine (12 mL) and acetic anhydride (8 mL) were added to green tea leaves (4.55 g, purchased from SEIYU) in a round bottom flask. The mixture was stirred at room temperature overnight. Then it was dried under vacuum. The reaction mixture was washed with ethyl acetate (20 mL x 4) and filtered. The filtrate was washed with water (20 mL x 4). The filtrate was dried over sodium sulfate and evaporated. The crude product was purified by column chromatography over silica gel using «-hexane:ethyl acetate (v:v=l :2) as eluent to afford the crude (-)-EGCG peracetate 1 (Rf value was 0.3, green color). The crude 1 was dissolved in ethyl acetate and filtered through a layer of activated charcoal (1 cm width x 3 cm long) to give pure (-)-EGCG peracetate (1) as a white solid (0.19 g, 4.3% yield) after evaporation. Besides 1, (-)-EGC hexaacetate (2) was also isolated from column chromatography (hexane: ethyl acetate 1 :2, R_f value is 0.6) and was filtered through a layer of activated charcoal. The crude compound 2 was recrystallized from dichloromethane: diethyl ether: hexane (1 :2:0.5) to give pure EGC hexaacetate 2 as white solid (0.07 g, 1.5% yield).

(-)-EGCG peracetate (1): Mp 1 12.4-1 13.8 °C (compared with the one synthesized from commercially available (-)-EGCG, 1 15 ⁰C); [α]D -67.7 (c 1.0, CHCl₃); ¹H NMR (CDCl₃, 500MHz): 67.62 (s, 2H), 7.23 (s, 2H), 6.73 (d, J=2.5 Hz, IH), 6.61 (d, J=2.5 Hz, IH), 5.63 (br s, IH), 5.18 (s, IH), 3.02 (m, 2H), 2.28 (s, 3H), 2.27 (s, 9H), 2.26 (s, 3H), 2.24 (s, 3H), 2.22 (s, 6H); ¹³C NMR (125 MHz, CDCl₃): δ 168.89, 168.40, 167.59, 167.43, 166.72, 166.20, 163.51, 154.71, 149.72, 149.64, 143.38, 143.29, 138.93, 135.06, 134.34, 127.41 , 122.34, 1 18.79, 109.42, 109.00, 108.06, 76.46, 67.98, 25.85, 21.06, 20.75, 20.54, 20.1 1 ; LRMS m/z (ESI) 817 [M+Na]⁺; HRMS: Calcd for C₃₈H₃₄Oi₉Na, 817.1592; found, 817.1586. (-)-EGC hexaacetate (2): Mp 189.5-191.2 °C; [α]D -14.6 (c=1.0, CHCl₃); ¹H NMR (CDCl₃, 500MHz): 67.22 (s, 2H), 6.67 (d, J=2.0 Hz, IH), 6.57 (d, J=2.0 Hz, IH), 5.38 (br s, IH), 5.08 (s, IH), 2.93 (m, 2H), 2.29 (s, 6H), 2.28 (s, 6H), 2.28 (s, 3H), 1.94 (s, 3H); ¹³C NMR (CDCl₃, 120MHz): δ 170.58, 169.06, 168.50, 167.72, 166.90, 154.80, 149.73, 149.69, 143.26, 135.48, 134.28, 119.01, 109.56, 108.86, 108.08, 76.49, 66.44, 25.95, 21.10, 20.80, 20.77, 20.69, 20.19: LRMS m/z (ESI):581 [M+Na]⁺; HRMS: Calcd for C₂₇H₂₆O₁₃Na, 581.1271 ; found, 581.1252.

Larger scale isolation ofcatechin acetates from green tea leaves from SEIYU

Green tea leaves (83.2 g, purchased from SElYU) was added to a mixture of pyridine (160 mL) and acetic anhydride (80 niL). The resulting mixture was stirred at room temperature for overnight. The solvent was removed under reduced pressure. Then the mixture was filtered with a sintered glass funnel and washed with ethyl acetate (100 mL x 5). The filtrate was washed with water (100 mL x 5) and brine solution (20 mL). The organic phase was dried over MgSO₄, filtered and evaporated by rotary evaporator. The crude product was purified by column chromatography over silica gel with w-hexane/ethyl acetate (v:v=l:2) as eluent. Three columns (25 x 6 cm i. d., 25 x 6 cm i.d. and 24 x 3.5 cm i.d. respectively, with 3 cm of activated charcoal loaded on the top of the third column) were used. The crude products were chromatographed over these columns and fractions collected with TLC analysis using n-hexane/ethyl acetate (v:v=l :2) as eluent. The fractions containing the compound with R_f value of 0.32 were combined and evaporated to give (-)-EGCG peracetate (1) as a white solid (2.83 g, 3.4% yield). The fractions containing the compound with R_f value of 0.46 were combined and evaporated to give (-)-ECG heptaacetate (4). This was followed by the fractions containing the compound with Rf value of 0.58 which was evaporated to give (-)-EGC hexaacetate (2) (0.92 g, 1.1% yield). Finally, the fractions containing the compound with Rf value of 0.68 were also combined and evaporated to give the (-)-EC pentaacetate (3). The yields of compounds 3 and 4 were less than 0.01%.

EC pentaacetate (3): ¹H NMR (CDCl₃, 500MHz): 57.22 (m, 3H ), 6.67 (s, IH), 6.57 (s, IH), 5.38 (br s. I H), 5.08 (s, IH), 2.94 (m, 2H), 2.29 (m, 15H); HRMS (ESI): Calcd for C₂₅H₂₄On Na (M+Na)⁺ 523,1216, found, 523.1198. ECG heptaacetate (4): ¹H NMR (CDCl₃, 500MHz): 67.62 (s, 2H), 7.25 (m, 3H), 6.73 (s, IH), 6.60 (s, IH), 5.63 (br s, I H), 5.20 (s, IH), 3.06 (m, 2H), 2.27 (m, 21H); HRMS (ESI): Calcd for C₃₆H₃₂O₁₇Na (M+Na)⁺ 759.1537, found, 759.1515.

Isolation of individual catechins from Shou Mee green tea leaves

Green tea leaves (26.9g) (Shou Mee) were ground into powder. The powder was added to acetic anhydride (3OmL), and pyridine (4OmL) in an ice-water bath. The resulting mixture was stirred for 12 hours at room temperature. Excess pyridine and acetic anhydride were then removed under reduced pressure (20 mmHg). The mixture was filtered with a layer of silica gel and the filter cake was washed with ethyl acetate (5 x 10OmL). The filtrate was washed with 0.1M HCl (5 x 10OmL), saturated sodium hydrogencarbonate solution (3 x 10OmL) and brine solution (200 niL). The organic phase was dried over MgSO₄ and filtered. The filtrate (~40mL) was evaporated by rotary evaporator. The crude product was in foam- like form with dark green colour. The crude product was purified by column chromatography (silica gel, 10 g) with n-hexane/ethyl acetate (1 :2 v/v) as the eluent.

The fractions containing the (-)-EGCG peracetate (1) was collected and the solvents were evaporated to obtain the (-)-EGCG peracetate. (1.25g, 4.6%) with physical and spectroscopic properties identical to authentic sample.

The fractions containing the (-)-ECG heptaacetate (4) was collected and the solvents

120 were evaporated to obtain the (-)-ECG heptaacetate. (0.24g, 0.88%): Mp 104 - HOO ; [af_L

57.6 (c = 0.60, CHCl₃); ¹H NMR (CDCl₃, 400MHz) δ 7.62 (s, 2H), 7.29-7.33 (m, 2H), 7.21 (s, IH), 6.73 (d, J= 2.2Hz, IH), 6.60 (d, J= 2.1Hz, IH), 5.63 (s, IH), 5.20 (s, IH), 3.08 (dd, J = 17.9, 4.6Hz, IH), 2.98 (dd, J = 17.9, 2.0Hz, IH), 2.14-2.29 (m, 21H); ¹³C NMR (CDCl₃, 100MHz) δ 168.9, 168.4, 168.0, 167.9, 167.4, 166.2, 163.5, 154.9, 149.7, 143.3, 142.1 , 142.0, 138.9, 135.4, 127.4, 124.4, 123.5, 122.3, 121.8, 118.7, 109.5, 108.9, 108.0, 76.7, 68.1, 25.9, 21.1, 20.7, 20.5, 20.1; LRMS (ESI) m/∑ 759 ([M+Na]⁺, 66), 737 ([M+H]⁺, 42); HRMS calcd for C₃₆H₃₂O_nNa 759.1537, found 759.1508.

The fractions containing the (-)-EGC hexaacetate (2) was collected and the solvents were evaporated to obtain the (-)-EGC hexaacetate with physical properties identical to an authentic sample. The fractions containing the (-)-EC pentaacetate (3) was collected and the solvents were evaporated to obtain the (-)-EC pentaacetate with physical properties identical to an authentic sample. The combined isolated yield of 2 and 3 was less than 0.5 %.

Procedure for the synthesis of (-)-EGCG from (-)-EGCG per acetate (1)

NH₄OAc (4.3 g, 56 mmol) was added to (-)-EGCG peracetate 1 (350 mg, 0.42 mmol) and vitamin C (35 mg) in MeOHiH₂O (4:1) (20 mL) at room temperature. The resulting mixture was stirred at 40 °C until 1 disappeared and EGCG was formed (reaction progress was monitored with HPLC: C-18 reverse phase column; flow rate, 1 mL/min; detection, UV 280 nm; mobile phase, 0-8 min (20% aqueous acetonitrile and 0.016% TFA), 8-13 min (varying from 20% aqueous acetonitrile with 0.016% TFA to 60% aqueous acetonitrile with 0.008% TFA). The reaction mixture was dried under vacuum. Water was added and the mixture was extracted with ethyl acetate (8 mL x 3). The filtrate was then dried over sodium sulfate and evaporated. The crude product was purified by column chromatography over silica gel (ethyl acetate, R_f value was 0.5) to afford the EGCG (180 mg, 90% yield) as off white solid. Its identity with an authentic sample of (-)-EGCG was confirmed by NMR spectroscopy and other physical data.

Determination of Crystal structure

The crystal structure of compound 2 was determined by the direct method on a Bruker CCD area detector diffractometer using MoK (λ radiation = 0.71073 A) from generator operating at 50KV, 30mA condition. The positions of part of non-hydrogen atoms and subsequent difference Fourier syntheses were employed to locate all of the remaining non- hydrogen atoms which did not show up in the initial structure. All of non-hydrogen atoms were refined anisotropically. Hydrogen atoms were located basing on difference Frouier Syntheses connecting geometrical analysis. All experiment and computation were performed on a pc computer with program of Bruker Smart and Bruker Shelxtl package. The crystal structure of 2 is shown in Figure 2.

Results and Discussion

Green tea leaves were treated with acetic anhydride and pyridine directly at room temperature overnight. All the catechins, including (-)-EGCG, were presumably converted to the corresponding acetates (Figure 1 ) as excess amount of acetic anhydride was used. The reaction mixture was then extracted with ethyl acetate and filtered. The catechin acetates were then separated from each other by simple column chromatography on silica gel with hexane:ethyl acetate (1 :2 v/v) to give pure crystalline EGCG peracetate (1) as the major product in 3.4 - 4.6% dry weight of the green tea leaves. The EGC hexaacetate (2) was also obtained as crystals in 1.1 -1.5% dry weight. Compound 1 thus isolated was identical in all respects with the sample synthesized previously. The structure of compound 2, in addition to being consistent with the spectroscopic data, was confirmed by crystallographic determination with X-ray diffraction (Figure 2). In addition to the isolation of 1 and 2, small amounts of (-)-EC pentaacetate (3) and (-)-ECG heptaacetate (4) were also isolated. There is marked contrast between the ease of separation of the different catechin acetates 1-4 and the difficulty in the separation of the catechins with their free phenolic groups by normal phase silica gel chromatography.

The ready available compound 1 allows its conversion to the precursor (-)-EGCG ammonium acetate in aqueous methanol to give (-)-EGCG in 90% yield as white crystals with identical NMR spectra as authentic sample:

The use of (-)-EGCG peracetate (1) and other catechin acetates as prodrugs for proteasome inhibition and apoptosis inducers of human cancer cells is described. [D. Kuhn, W. H. Lam, A. Kazi, K. G. Daniel, S. Song, L. M. C. Chow, T. H. Chan, Q. P. Dou, Synthetic peracetate polyphenols as potent proteasome inhibitors and apoptosis inducers in human cancer cells, Frontiers in Bioscience, 10, 1010-1023 (2005); W. H. Lam, A. Kazi, D. J. Kuhn, L. M. C. Chow, A. S. C. Chan, Q. P. Dou, T. H. Chan, A potential prodrug for a green tea polyphenol proteasome inhibitor: evaluation of the peracetate ester of (-)-epigallocatechin gallatc [(-)-EGCG], Bioorg. Med. Chem., 12, 5587-5593 (2004).] These ester derivatives are usually prepared from the esterifϊcation of the pure (-)-EGCG or other catechins. The absence of a simple, efficient and inexpensive method of obtaining pure (-)-EGCG or other catechins has also hampered the preparation of (-)-EGCG peracetate (1) and the evaluations of 1 or other catechin ester derivatives in animal and human clinical studies. Using the method of this invention, catechins in green tea extracts can become more readily available. This may facilitate the biological viability studies of this compounds .

While the preferred embodiment of the present invention has been described in detail by the examples, it is apparent that modifications and adaptations of the present invention will occur to those skilled in the art. Furthermore, the embodiments of the present invention shall not be interpreted to be restricted by the examples or figures only. It is to be expressly understood, however, that such modifications and adaptations are within the scope of the present invention, as set forth in the following claims. For instance, features illustrated or described as part of one embodiment can be used on another embodiment to yield a still further embodiment. Thus, it is intended that the present invention cover such modifications and variations as come within the scope of the claims and their equivalents.

Claims

CLAIMS:

1. A method of separating catechins from green tea leaves, including the steps of: converting the catechins to corresponding ester forms to form a ester mixture; separating the converted catechins in the ester mixture.

2. The method of claim 1, wherein the catechins are converted to their respective peracetate forms.

3. The method of claim 2, wherein the catechins are converted to corresponding peracetate forms by acetic anhydride.

4. The method of claim 1, wherein the converted catechins are separated by column chromatorgraphy.

5. The method of claim 4, wherein the converted catechins are eluted on silica gel by a mixture comprising hexane and ethyl acetate.

6. The method of claim 5, wherein the hexane and the ethyl acetate are in a ratio of 1 :2 v/v.

7. The method of claim 1 further including the step of filtering the ester mixture to obtain a filtrate before the converted catechins are separated.

8. The method of claim 7 further including the step of washing the filtrate by water, drying the filtrate and then concentrating the filtrate before the converted catechins are separated.

9. The method of claim 1 further including the step of converting the converted catechins back to the catechins after being separated.

10. The method of claim 9, wherein the converted catechins back to the catechins by a methanol solution of ammonium acetate.

11. The method of claim 1 , wherein the catechins is selected from the group consisting of (-)-ep/gallocatechin-3-gallate, (-)-e/?/gallocatechin, epj^'catechin-3-gallate and (-)- e/7/catechin.