CN102432577B - Method for separating and purifying epicatechin gallate (ECG) monomer - Google Patents

Abstract

The invention discloses a method for separating and purifying epicatechin gallate monomer from green tea. The method takes Yunnan big-leaf green tea as raw material and directly extracts it with ethyl acetate, and the crude extract is subjected to two different methods respectively. Adsorption resin column chromatography adsorption and elution, collection of desorption fractions rich in epicatechin gallate (ECG), high-efficiency external circulation concentration and recovery of ethanol, and then low-temperature concentration with reverse osmosis membrane, 95% ethanol cryogenic refrigeration crystallization, and finally freeze-drying to obtain 98% epicatechin gallate (ECG) monomer. The method of the invention is simple, short in preparation period, low in cost, meets the requirements of environmental protection and safety, and is suitable for large-scale industrial production.

Description

Separation and purification method of epicatechin gallate (ECG) monomer

technical field

The invention relates to a method for separating and purifying epicatechin gallate (ECG) monomer from green tea raw materials, which belongs to the technical field of food.

Background technique

Catechins have obvious pharmacological functions such as anti-aging, anti-cancer and anti-mutation, fat loss and weight loss, lowering blood sugar, blood lipid and cholesterol, and preventing cardiovascular diseases. They have been widely used in food processing, medical care, and daily chemical industries. ^[1-3] . In October 2006, the U.S. Food and Drug Administration (FDA) approved tea polyphenols as a new prescription drug for topical (external) treatment of genital warts caused by human papillomavirus. The first plant (herbal) drug approved for marketing ^[4] . In the past two decades, due to breakthroughs in the research on the health care and pharmacological functions of tea polyphenols/catechins, not only the share of green tea in the international market has been expanded, but also the development of the tea extract industry has been greatly promoted. The research and development of functional ingredients has laid a good industrial and market foundation. At present, my country has formed an industrial scale with an annual output of about 5,000 tons of tea polyphenols/catechins. The traditional extraction and separation processes of crude catechin mainly include ion precipitation, organic solvent extraction and adsorption column chromatography ^[5-7] . The most notable ones in the pharmacological health function research and industrial application of catechin are epigallocatechin gallate (EGCG), epigallocatechin (EGC) and epicatechin gallate (ECG), so It has become the focus of catechin monomer isolation, purification and clinical application research. However, due to the complex composition of tea leaves, it is quite difficult to prepare high-purity catechin monomers from tea leaves. The technical difficulty is the fine separation. At the same time, the effective substances and the natural flavor of the tea should be prevented from being damaged and lost. The processing process should be as low temperature as possible. At present, the processes successfully applied to the separation and preparation of EGCG monomer at home and abroad mainly include high-speed countercurrent chromatographic extraction, simulated moving bed chromatographic separation and other technologies ^[8,9] . Research results at home and abroad show that ECG plays a significant role in anti-oxidation, scavenging lipid free radicals, and inhibiting human virus infection; it has strong antibacterial activity and is expected to be developed into a natural antibiotic-like substance; it can also significantly improve Trauma wound healing and scar formation; at the same time, ECG also has obvious neuroprotective effect on astrocyte injury ^[10~13] . Zhang Ying et al. used a self-made preparative countercurrent chromatograph to separate and purify ECG monomer from catechin with petroleum ether-ethyl acetate-water (0.2:1:2, v/v) as the solvent system, with a purity of 98%. % ^[8] ; Huang Yongdong et al. used the B-type three-zone simulated moving bed technology, using C ₁₈ bonded silica gel as the stationary phase, ethanol and water (20:80, v/v) as the mobile phase, and using the Varicol process to obtain a purity of 91.33% ECG, the recovery rate is 91.41% ^[9] , but the above two methods have disadvantages such as expensive C ₁₈ bonded silica gel, large amount of organic solvent, and small loading capacity of the separation platform (countercurrent chromatography, moving bed), etc. Not suitable for scale-up. So far, there is no report on the industrial preparation of EGC monomer at home and abroad.

The references are as follows:

[1] Wang Jing, Gao Yutang. Epidemiological research on anti-cancer effect of tea polyphenols [J]. Tumor, 2011, 31(6): 553-556

[2] Liu Xueming, Liang Shizhong. The health care and pharmacological effects and application prospects of tea polyphenols [J]. Food and Fermentation Industry, 1998, 24(5):47-51,71

[3] Yokozawa Tx, Dong Ex, Nakagawa Tx, el al. In vitro and in vivo studies on the radical scavenging activity of tea [J]. J Agric Food Chem, 1998,46(6):2143-2150

[4] Huang Fanghua. Discussion on the pharmacokinetic research and evaluation strategy of traditional Chinese medicine from the first approved herbal medicine in the United States [J]. Chinese Journal of Traditional Chinese Medicine, 2010, 35(7):932-935

[5] Huang Siyong, Jin Xianping, Kang Zhenxing, Gan Guoping. Study on extraction and purification process of tea polyphenols in tea leftovers [J]. Journal of Hubei University of Traditional Chinese Medicine, 2011, 13(4): 30-32

[6] Zhang Sheng, Liu Zhonghua, Huang Jian'an, Liu Ailing, Shi Zhaopeng. Research on the preparation of high-purity catechin by adsorption resin [J]. Tea Science, 2002, 22 (2): 125-130

[7] Hu Lijuan. Study on Extraction Technology of Tea Polyphenols in Green Tea [J]. Journal of Yunnan Agricultural University, 2011, 26(3):426-429

[8] Zhang Ying, Shi Zhao-peng, Nie Hong-yong, et al . Preparative Isolation and Purification of Catechins from Green Tea Extract by Preparative Counter-current Chromatography [J]. Journal of Hunan Agricultural University (Natural Science), 2003 , 29(5): 408-411, 417

[9] Huang Yongdong, Jiang Heyuan, Jiang Yongwen, etc. Comparative study on the separation and purification of ECG and EGCG by traditional SMB, Varicol and Partial-discard processes [J]. Tea Science, 2011, 31 (3): 201-210

[10] Guo Q, Zhao BL, Li MF, et al. Studies on protective mechanisms of four components of green tea polyphenols against lipid peroxidation in synaptosomes biochim[J]. Biophys Acta,1996,13-32.21

[11] Yoko Yanagawa, Yoshimasa Yamamoto, Yukihiko Hara, Tadakatsu Shimamura. A Combination Effect of Epigallocatechin Gallate, a Major Compound of Green Tea Catechins, with Antibiotics on Helicobacter pylori Growth In Vitro [J]. Current Microbiology, 2003, 47:24 -249.

[12] Renata T. Abib, Andre′ Quincozes-Santos, Patricia Nardin, et al. Epicatechin gallate increases glutamate uptake and S100B secretion in C6 cell lineage [J]. Mol Cell Biochem, 2008, 310:153-158.

[13] Kapoor M, Howard R, Hall I et al. Effects of epicatechin gallate on wound healing and scar formation in a full thickness incisional wound healing model in rats [J]. Am J Pathol, 2004, 165:299-307.

Contents of the invention

The technical problem to be solved by the present invention is to provide a method for separating and purifying epicatechin gallate monomer from green tea. The method is simple, does not use highly toxic organic solvents, has short preparation period, low cost, and conforms to environmental protection and safety requirements, the extraction rate of the epicatechin gallate monomer and the product purity are high, and it is suitable for large-scale industrial production.

The technical scheme provided by the invention is: a method for separating and purifying epicatechin gallate monomer from green tea, comprising the steps of:

(1) Extraction of total catechins in green tea raw materials: Weigh green tea raw materials, add 4~7 BV ethyl acetate, heat, stir intermittently, recover ethyl acetate, concentrate the extract, and then spray dry to obtain total catechin products ;

(2) Separation of ester-type catechins and non-ester-type catechins: add pure water to dissolve total catechin products to prepare a column feed solution with a concentration of 3% (w/v), and use an adsorption resin column to separate and purify. The desorption solvent is ethanol, the ethanol desorption fraction is collected, the ethanol is concentrated and recovered by high-efficiency external circulation, the concentrated desorption distillation is centrifuged, spray-dried, and a dry product is obtained;

(3) Separate and purify the dry product in the above step (2) with an adsorption resin column: add pure water to dissolve the dry product and configure it as a column feed solution with a concentration of 3% (w/v), and use an adsorption resin column Separation and purification, the desorption solvent is ethanol, the ethanol desorption fraction is collected, the ethanol is first concentrated and recovered by a high-efficiency external circulation to an alcohol content <5, and then further concentrated by a reverse osmosis membrane and then centrifugally spray-dried;

(4) Dissolve the dried product obtained in step (3) above with 3 to 4 times the amount (w/v) of 95% ethanol, place it, crystallize it, filter it, and freeze-dry the crystal to obtain epicatechin gall ester monomer.

In the method described above, the green tea raw material described in step (1) is Yunnan big-leaf green tea, the temperature of the heated water bath is 45°C-50°C, and the stirring is performed intermittently for 30 minutes for 5 minutes; after the green tea raw material is weighed, it is extracted three times with ethyl acetate , each extraction time is 1h, the first, second, third added ethyl acetate is divided into 7 BV, 6BV, 4BV, all the extraction solutions are combined, the ethyl acetate is recovered under reduced pressure, and concentrated to 6-7 pome degrees Afterwards, spray and dry with a centrifugal spray tower, control the air inlet temperature at 160°C to 180°C, and the exhaust air temperature at 70°C to 80°C. The first-stage cyclone separator takes out the spray dry powder every 30 minutes, and the product is the total catechin product of large-leaf species. .

The method described in (2) is separated and purified with an adsorption resin column, and the adsorption resin is one of D101, HP20, polyamide (Polyamide) or DM130; the amount of resin is: 40 times the weight of the total catechin product ;Flow rate of upper column: 1.0 BV/h; Desorption: first use 2BV 25% ethanol to desorb the fraction rich in non-ester catechin, then use 2BV 60% ethanol to desorb the fraction rich in ester catechin, the desorption flow rate is 1.0 BV/h.

The method described in (3) is separated and purified with an adsorption resin column, the adsorption resin is one of HP ₂ MGL, NKA-9, AB-8 or S-8, and the amount of resin is 40 times the weight of the dry product; Flow rate of upper column: 3.0 BV/h; after adsorption is completed, use 3BV of distilled water to rinse and remove impurities at the same flow rate, desorption solvent: 2BV 80% ethanol; desorption flow rate: 1.0BV/h.

The catechins in tea are divided into two categories: ester type catechins (mainly EGCG and ECG) and non-ester type catechins (mainly EGC). Chromatography separates the ester type/non-ester type two major types of catechins, and the resin used in this step has a strong adsorption force for the gallic acid ester group; in step (3), another type of resin is used to separate the ECG and EGCG are separated, and the resin used in this step of the present invention has selective adsorption for the difference in molecular weight and polarity of ECG and EGCG .

In the method, the temperature is controlled below 45°C when ethanol is recovered in steps (2) and (3), the vacuum degree is controlled below -0.08Mpa, and the operation temperature is controlled when the reverse osmosis membrane is concentrated in step (3). Below 40°C, the membrane inlet pressure is controlled below 15 Bar.

The present invention has the following beneficial effects:

The method of the present invention is combined with ethyl acetate extraction, two different adsorption resin column chromatography adsorption separation, reverse osmosis membrane concentration and freeze-drying technology to separate and purify epicatechin gallate (ECG purity> 98%) monomer, optimized product separation and purification process, high monomer extraction rate and product purity. The product has antibacterial and antiviral effects, and can significantly improve wound healing and scar formation. It is also an important raw material for the development of many functional foods. It can be used alone or in combination with vitamins and natural plant ingredients in many fields such as health food, cosmetics and pharmaceuticals. The method of the invention is simple, short in preparation period, low in cost, meets the requirements of environmental protection and safety, and is suitable for large-scale industrial production.

Detailed ways

The present invention will be further clarified through the detailed description of specific embodiments below, but it is not intended to limit the present invention, but only for illustration.

The method for separating and purifying epicatechin gallate (ECG) monomer from raw materials of Yunnan big-leaf green tea comprises the following steps:

1. Ethyl acetate extraction of total catechins in green tea raw materials:

Weigh the raw material of Yunnan big-leaf green tea (purchased from Shuangjiang County, Yunnan, with an ECG content of 4.10%), add 7 BV/6BV/4BV ethyl acetate and heat and extract three times (1h/time, keep the water bath temperature at 45°C-50°C , intermittent 30min and stirring for 5min), combine all the extraction solutions, recover ethyl acetate under reduced pressure, add pure water to fully replace the ethyl acetate in the later stage, concentrate to 6-7 Pomi degrees, and then spray dry with a centrifugal spray tower to control the The wind temperature is 160°C-180°C, the exhaust air temperature is 70°C-80°C, and the first-stage cyclone separator takes out the sprayed dry powder every 30 minutes. The product is the total catechin product of the large-leaved species (the ECG content of HPLC is 21.08%). For the method of detecting ECG content by HPLC, please refer to: Liu Ailing, Zhang Sheng, Yang Weili. A Preliminary Study on the Dynamic Research of Green Tea Extraction [J]. Tea Communication, 1999, (3): 32-35; He Chunlei, Luo Xueping, Chen Qiang, etc. Effect of acetic acid Study on the extraction effect of ethyl acetate on tea polyphenols and catechins[J]. Food Research and Development, 2007, 28 (1): 29-33; Deng Yanli, Gong Zhihua, Hu Yabei, etc. Extraction of tea polyphenols with ethyl acetate Dose-effect relationship research [J]. Journal of Analytical Testing, 2009, 28(10): 1115-1120.

2. Separation of ester catechins (ECG, EGCG) and non-ester catechins (EGC) by Type-A adsorption resin column chromatography:

Add pure water to dissolve the total catechin products of large-leaved species to prepare a column feed solution with a concentration of 3% (w/v), and then separate and purify with an adsorption resin column (resin dosage: 40 times the weight of the total catechin products; Flow rate: 1.0 BV/h; Desorption: first use 2BV 25% ethanol to desorb the fraction rich in non-ester catechins (mainly containing EGC), and then use 2BV 60% ethanol to desorb the fraction rich in ester catechins (mainly containing ECG and EGCG), the desorption flow rate is 1.0BV/h). Collect 60% ethanol desorption fraction, use high-efficiency external circulation to concentrate and recover ethanol (control the temperature below 45°C, and control the vacuum degree below -0.08Mpa), concentrate to 6-7 degrees Pomi, and then centrifugal spray-dry to obtain epicatechin Crude gallate (ECG content detected by HPLC is 40.52%, the detection method can refer to: Gong Yushun, Liu Zhonghua, Huang Jianan, etc. Study on separation of tea catechin and caffeine with macroporous adsorption resin[J]. Journal of Hunan Agricultural University, 2005, 31(1): 50-52; Zhang Sheng, Liu Zhonghua, Huang Jianan, et al. Preparation of high-purity catechins by adsorption resin [J]. Tea Science, 2002, 22(2): 125-130） .

Type-A adsorption resin is one of D101, HP20, Polyamide or DM130.

3. Separation and purification of the above crude epicatechin gallate (ECG) by Type-B adsorption resin column chromatography:

Add pure water to dissolve the crude ECG product and configure it as a column feed solution with a concentration of 3% (w/v), and then separate and purify it on the adsorption resin column (resin dosage: 40 times the weight of the ECG crude product; column flow rate: 3.0 BV/h; after the adsorption is completed, Use 3BV of distilled water to rinse and remove impurities at the same flow rate. Desorption solvent: 2BV 80% ethanol; desorption flow rate: 1.0BV/h). Collect the desorption fraction of 80% ethanol, concentrate and recover the ethanol with high-efficiency external circulation until the alcohol content is less than 5 (the temperature is controlled below 45°C, and the vacuum degree is controlled below -0.08Mpa), and then further concentrated to 6-7 bar with reverse osmosis membrane Mido (the operating temperature is controlled below 40°C, and the membrane inlet pressure is controlled below 15 Bar), centrifugal spray drying to obtain a product with high content of epicatechin gallate (ECG content detected by HPLC is 80.25%), the detection method can refer to: Zhang Sheng, Liu Zhonghua, Huang Jianan, et al. Study on Purification Process of High ECG Type Catechin[J]. Journal of Hunan Agricultural University, 2003, 29(2): 144-146; Sun Yanjuan, Zhu Yuejin, Li Dawei, et al. Membrane Separation Research on the application of technology in the development of mushroom instant tea [J]. China Tea Processing, 2010, (4): 42-45.

Type-B adsorption resin is one of HP ₂ MGL, NKA-9, AB-8 or S-8.

4. Dissolve the above-mentioned high-content epicatechin gallate (ECG >80%) product with 3 to 4 times the amount (w/v) of 95% ethanol, place it at 4°C for crystallization treatment, filter the crystal under normal pressure with filter paper, and freeze the crystal For drying, the product is first cooled to -40°C for pre-freezing, and then vacuumized. When the vacuum reaches 30Pa, heating and sublimation drying is started. The temperature of the product is controlled not to exceed 20°C, and the drying time is 12~16h (for specific operations, please refer to: Zhu Chuanjiang. Freeze Drying Process principle and related equipment [J]. Qilu Pharmaceutical Affairs, 2006, 25(8): 503-504; Xu Chenghai, Zhang Shiwei, Guan Kuizhi, etc. Freeze-drying experiments of several materials [J]. Vacuum and low temperature , 1998, 4(3): 161-164; Shen Shanming. Drying of heat-sensitive drugs [J]. Pharmaceutical Engineering Design, 2010, 31(6): 1-4), a light yellow powdery solid was obtained.

The above light yellow powder product was mixed with a standard product of epicatechin gallate (ECG) [purchased from Sigma-Aldrich, USA, C ₂₂ H ₁₈ O ₁₀ , MW 442.37, number: E3893, purity ≥ 98% ( HPLC)] Control identification and analysis and detection by high performance liquid chromatography (for specific identification and detection methods, please refer to: Ding Pingping, Li Yanfang, Song Hang, etc. Comparison of analysis methods for catechin monomers[J].Sichuan Chemical Industry, 2005 , 8 (4): 38-41; Wu Di, Yang Lizhu, Qiu Peihong, et al. Research progress on separation and analysis methods of catechins in tea [J]. Journal of Analytical Science, 2008, 24(4): 468-472; Liu Ting , Yang Hongmei, Guo Qilei, et al. Determination of catechins in tea beverages by high performance liquid chromatography[J]. Analytical Laboratory, 2009, 28: 277-280; Gu Xungang, Cai Jibao, Zhang Zhengzhu, et al. Analysis of catechins in black tea by HPLC-DAD Method research on ketones and caffeine[J].Journal of Anhui Agricultural University, 2010, 37 (1): 5-10), the results showed that the light yellow powder solid had a molecular weight of 442, (-) ESI-MS m/z : 441 [M－H] ^- , EGC ≥98%, molecular formula is C ₂₂ H ₁₈ O ₁₀ .

Claims (8)

1. the method for a separation and purification L-Epicatechin gallate monomer from green tea, is characterized in that comprising the steps:

(1) extract total catechins in green tea materials: take green tea materials, add the ethyl acetate post-heating of 4 ~ 7 times of volumes, intermittent stirring, reclaim ethyl acetate, concentrated extracting solution, and then spraying drying, obtain the total catechins goods;

(2) separate ester catechin and non-ester catechin: add pure water to dissolve the total catechins article configurations and become the upper prop feed liquid that concentration is 3% w/v, use the polymeric adsorbent column separating purification, desorption solvent is ethanol, collect ethanol desorb cut, with efficient outer circulation concentration and recovery ethanol, concentrated desorb cut is centrifugal, spraying drying, obtain drying products;

(3) by the drying products polymeric adsorbent column separating purification in above-mentioned (2) step: add pure water to dissolve described drying products and be configured to the upper prop feed liquid that concentration is 3% w/v, use the polymeric adsorbent column separating purification, desorption solvent is ethanol, collect ethanol desorb cut, with the first concentration and recovery ethanol of efficient outer circulation to alcoholic strength<5, then centrifugal spray drying after further concentrated with reverse osmosis membrane;

(4) 95% dissolve with ethanol of 3 ~ 4 times of amount w/v for the product above-mentioned (3) step drying obtained, place, and crystallization is filtered, and the product that the crystal lyophilize is obtained is the L-Epicatechin gallate monomer.

2. in accordance with the method for claim 1, it is characterized in that: the green tea materials described in (1) step is big-leaf species in yunnan green tea, 45 ℃～50 ℃ of the bath temperatures of heating, and intermittently 30min stirs 5min.

3. in accordance with the method for claim 1, it is characterized in that: in (1) step, after taking green tea materials, by ethyl acetate, extract three times, each extraction time is 1h, first, two, the amount of the ethyl acetate added for three times is divided into other 7 times of volumes, 6 times of volumes, 4 times of volumes, merge all extraction solution, the reclaim under reduced pressure ethyl acetate, use the centrifugal spray tower spraying drying after being concentrated into 6～7 mother-in-law U.S. degree, control 160 ℃～180 ℃ of inlet temperature, 70 ℃～80 ℃ of temperature of outgoing airs, primary cyclone takes out spraying dry powder every 30min, product is large leaf total catechins goods.

4. in accordance with the method for claim 1, it is characterized in that: use the polymeric adsorbent column separating purification described in (2), resin demand is: the weight of 40 times of total catechins goods; Upper column flow rate: 1.0 BV/h; Desorb: first with 2BV 25% ethanol desorb, be rich in the non-ester catechin cut, then be rich in the ester catechin cut with 2BV 60% ethanol desorb, the desorb flow velocity is 1.0BV/h.

5. in accordance with the method for claim 4, it is characterized in that: use the polymeric adsorbent column separating purification described in (2), polymeric adsorbent is D101, one of HP20, polymeric amide or DM130.

6. in accordance with the method for claim 1, it is characterized in that: described in (3), use polymeric adsorbent column separating purification, resin demand: 40 times of drying products weight; Upper column flow rate: 3.0 BV/h; Adsorb complete, with the distilled water of 3BV with same flow velocity drip washing removal of impurities, desorption solvent: 2BV 80% ethanol; Desorb flow velocity: 1.0BV/h.

7. in accordance with the method for claim 1, it is characterized in that: use the polymeric adsorbent column separating purification described in (3), polymeric adsorbent is HP ₂one of MGL, NKA-9, AB-8 or S-8.

8. in accordance with the method for claim 1, it is characterized in that: while in (2) step and (3) step, reclaiming ethanol, temperature is controlled at below 45 ℃, vacuum degree control-below 0.08Mpa, when in (3) step, reverse osmosis membrane is concentrated, service temperature is controlled at below 40 ℃, advances film pressure and is controlled at below 15 Bar.