TWI293628B - - Google Patents

Description

Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printing 1293628 A7 _B7_ V. Description of the invention (1) The present invention relates to the production method of _ _ · 掊 catechin catechin (EGCG). The present invention specifically describes a method for separating and preparing EGCG from tea catechus by treatment with a coarse pore resin. The leaf of the green tea plant camellia sinensis contains up to 36% of the dry weight, but the composition varies with climate, season, species and maturity. Green tea catechu The acid is the main body of green tea polyphenols. Examples of catechins are - epicatechin (EC), (-)-epiphylline catechin (EGCG), epigallocatechin (EGC) and epicatechin (ECG). EGCG is the most advantageous compound of the above catechins because it exerts strong antioxidant activity. It has been confirmed that EGCG has an anti-mutation effect, a bactericidal effect and a positive effect on the level of blood cholesterol. Other catechins in tea are far less effective. Green tea also contains other ingredients such as caffeine, protein, pectin and/or metal ions that may not be suitable. Therefore, it is necessary to separate the pure EGCG in a high yield by a simple and economical method. However, the similar structure of various tea catechins makes it difficult to separate individual catechins. Moreover, tea catechins are usually accompanied by caffeine content of green tea leaves to a dry mass of 4%. It is known that caffeine is combined with catechin and is not easily removed. The manufacture of tea extracts is well known in the art. U.S. Patent No. 5,879,733 describes the preparation of green tea extracts which improve clarity and color. Effectively remove the metal cation-containing green tea extract from the metal cation exchange resin in the extract at a temperature ranging from 25 ° C to 60 ° C with a quantity of food grade cation exchange resin. The treated extract is then contacted with a nanofiltration membrane. However, the method described in U.S. Patent 5,879,733 is not suitable for the separation of EGCG from a tea catechin mixture. U.S. Patent No. 4,613,672 describes the preparation of pure EGCG with the following steps: Applying the Chinese National Standard (CNS) A4 specification (210 X 297 mm) to -4- this paper scale --------- U-- -----t — order--------Aw (please read the notes on the back and fill out this page) 1293628 A7 B7 V. Description of invention (2) Hot water or 40- 75% methanol, The tea leaves are extracted from 40_75% ethanol or 30-80 〇/〇 acetone. The resulting extract was washed with a gas imitation, and the washed extract was dissolved in an organic solvent. The organic solvent is distilled off, and the concentrated extract component is subjected to high-speed liquid chromatography using a reverse phase distribution column for propanol/tetrahydrofuran/water (〇_ 25 : 〇- 35 : 65-85, vol%). (1) Epicatechin, (1) epigallocatechin, (_) epicatechin citrate and (1) epigallocatechin catechin. The process described in U.S. Patent No. 4,613,672 is not economically capable of producing EGCG in an industrial scale due to the use of the ruthenium packing. Moreover, the method described in U.S. Patent No. 4,613,672 is not approved for use in the use of a solvent mixture (acetone/tetrahydrofuran/chloroform) to produce EGCG which can be added to food products. Technical Note The production of catechins is a mixture, and there is still a need for simple, safe and economical production of pure EGCG as a component for incorporation of additives and foods. It has been found that EGCG can be isolated from tea catechins and/or coffee face mixtures, and a coarse-pored polar resin and a suitable polar eluting solvent are used for the separation to obtain improved selectivity. Therefore, the present invention directs a method for producing epigallocatechin citrate (E〇CG), including the steps of: Ministry of Economic Affairs, Intellectual Property Bureau, Staff Consumption Cooperative, Printing ---------------- ------* 丨定· (Please read the phonetic transcription on the back? Please fill out this page again) a) Make a green tea extract; b) Place the green tea extract at a temperature of about 〇 °C to about The color layer separation is carried out on the coarse pore polar resin at 80 ° C; c) the solvent is eluted with a polarity of from about 1 〇X: to about 80. (: FFCG in the temperature of the park and the pressure in the range of about 0.1 bar to about 50 bar; d) arbitrarily concentrate c) the eluate of the step; e) freely desorb the remaining catechin to regenerate the coarse hole Polar resin, ··-5- This paper scale applies to China National Standard (CNS) A4 specification (210 X 297 mm) 1293628 A7 B7 Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printing 5, invention description (3) f) random Concentrate e) to desorb catechins. Green tea extracts for the manufacture of starting materials are well known in the art. For example, green tea leaves are typically extracted from hot or cold water as a solution containing tea catechins and caffeine. The green tea solution can be re-concentrated to form a concentrated concentrate or dry powder. The extract or powder can contain stabilizers such as food-accepted acids such as citric acid, ascorbic acid, isoascorbic acid and the like. The tea extract powder may also be purchased from a product such as Guizhou Highyin Biological Product Co., Guiyang, PR China, or Zhejang Zhongke Plant Technical Co. Ltd., Hangzhou, Zhejang, PR China 0 EGCG, and the green tea extract is filled. The rough hole polarity tree is reached. As used herein, "coarse polar resin" refers to acrylic resins such as polyacrylates such as AMBERLITE® XAD-7 or polymethacrylate available from Rohm & Haas, Philadelphia, Pa_, such as AMBERCHROM available from Toso Haas. ® CG-71 or DIAION HP 2MG from Mitsubishi Chemical, Pennsylvania; or Polyamide from Merck, Darmstadt, Germany, such as Polyamide 11; Polyamide 6 and Nylon 6, 6 from Fluka, Buchs, Switzerland (in 1J) 02395 and 74712); (Grilamid L 25 natur) Polyamide 12 from EMS Chemie, Domat, Switzerland; or polyethylidene phloprofenone 6755 from Sigma; or aromatic polyamine and polyester. The resin operation is preferably degassed and equilibrated with the elution solvent. The process according to the invention is carried out at a temperature of from about 10 ° C to about 80 ° C, preferably from about 40 ° C to about 60 ° C. The tower can be thermostatically controlled in a thermostatic control zone such as a heating jacket. -6- This paper scale applies to China National Standard (CNS) A4 specification (210 X 297 mm) ------------Installation-------I------- (Please read the notes on the back and fill out this page) I293628

V. Description of invention (4) Printed by the Consumers' Cooperative of the Intellectual Property Office of the Ministry of Economic Affairs The hydraulic energy of the mobile phase passing through the tower varies within wide limits. The flow rate is preferably from about 1 to about 50 bar, preferably from about 〇 to about 2 mbar, and particularly preferably from 〇 to about 1 Torr. The mobile phase comprises a polar eluting solvent which is a mixture of water and an organic solvent. The term "organic solvent" as used herein refers to alcohols such as methanol, ethanol and isopropanol, ketones such as acetone or esters such as ethyl acetate or mixtures thereof, etc. The use of food grade alcohols with ethanol and isopropanol is used. Preferably, the flow of a mixture of organic solvents containing from about 7 Torr to about 95 v 〇 1%, preferably about 90 vol%, and from about 5 to about 3 vol%, preferably about 1 〇v 〇 1%, is used. The results obtained are particularly good. The activity phase is degassed and maintained in an inert atmosphere such as nitrogen or in the bottom. The column is regulated by the mobile phase. The flow rate of the mobile phase through the column can vary within wide limits. The flow rate is about 0.5 to Between about 20 bed volume / h range, preferably from about 55 to about 1 〇 bed volume / h, more preferably from about 〇 8 to about 5 bed volume / h. The volume of the trampoline is equivalent to i m3 solution or per m3 resin Solvent). After the equilibrium between the stationary phase and the mobile phase is established, the tea extract is introduced into the mobile phase, and then the green tea extract is subjected to chromatography on the crude pore polar resin. If the green tea extract is used as the starting material, the powder is dissolved in the mobile phase. If the aqueous green tea extract is added, the organic solvent is added to conveniently adjust the water in the extract to the organic solvent to the mobile phase. An important feature of the present invention is that the green tea extract is treated with a coarse pore polar resin at a temperature ranging from about 10 ° C to about 80 ° C, preferably from about 40 ° C to about 60 ° C, and washed with a polar eluting solvent. The special interaction of the three characteristics of the resin, the eluent and the temperature forms an important scene of the invention, guiding the clear separation of the EGCG from the tea catechin and/or the coffee test mixture, and extracting an EGCG part after the elution. This paper scale applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) ------r — l! Pack·! — I f I order ί------- (please read the back first) Note: Please fill out this page again) 1293628 A7 B7 V. Inventive Note (5) According to the total amount of all catechins in the extract or concentrate, at least 75%, preferably more than 85%, especially about 90% to about 97% of EGCG. (Please read the notes on the back and fill out this page.) The ability of coarse pore polar resin to absorb caffeine, EGCG and remaining catechins varies depending on the eluent used and temperature. The affinity for caffeine is inferior to that of caffeine. For EGCG, if there is a coffee test, it will be washed out first and can be separated. If it is convenient, it can be recycled. Morphine can also be an economic advantage. Secondly, it contains EGCG. The other tea catechins have stronger affinity than EGCG, so they are regenerated with a solvent that can desorb the remaining catechins until the resin absorbs the rest. For example, The remaining catechins can be eluted with a pure organic solvent or the ratio of water to organic solvent in the mobile phase. Suitable regenerating solvent is, for example, a pure organic solvent or a solvent of about 10-60 vo 1% water and about 40_90 vol% organic. A mixture of solvents, preferably about 40 vol% water and about 60 vol% organic solvent. The concentration of EGCG in the eluate can be accomplished by methods well known in the art, such as evaporation. The EGCG eluate can be evaporated to a powder containing high purity EGCG or concentrated to crystallize. Concentration can add a stabilizer such as a food-recognized acid such as citric acid, ascorbic acid, and isoascorbic acid to the eluate. The amount of acid added is preferably from about 0.1 to about 2.5% relative to EGCG. Printed by the Intellectual Property Office of the Ministry of Economic Affairs, the Consumer Cooperatives, the pre-parts containing caffeine and the e) step containing the remaining catechins can be concentrated as described above. The process can be carried out using a single column or multiple chromatography columns. The procedure can also be accomplished using techniques known in the art as "simulated moving bed chromatography" or "ring tomography". The method of the present invention can be realized in a simple and economical operation, and thus can be practically used for mass production in terms of production and processing. The above-mentioned EGCG has strong antioxidant activity, so it can be used as a variety of food--8-paper scales. It is applicable to China National Standard (CNS) A4 specification (210 X 297 mm). 1293628 A7 _B7_ V. Invention description (6) / , Antioxidants such as cosmetics and oils. In addition, EGCG has an anti-mutagenic effect, a bactericidal effect, and a beneficial effect on the level of cholesterol in the blood. Therefore, concentrated or pure EGCG is suitable for use in health care preparations and the like. The invention is illustrated in more detail with reference to the following examples.

Example 1 Separation of EGCG A green tea extract containing different catechins and caffeine (made by Highyin Bioproducts Co., Ltd., Guiyang, China), green tea extract, minimum 95% polyphenols π was used as the starting material. The tea component concentration was determined by UV absorbance HPLC and expressed in wt.%. The contents of EGCG, caffeine, other catechins and citric acid are shown in Table 1. Table 1 Concentration of tea components in the starting materials (please read the notes on the back and then fill out this page) Installed by the Ministry of Economic Affairs, Intellectual Property Bureau, Staff and Consumers Cooperatives, printed compound tea extracts. Example 1 HPLC/wt.-% Tea extract example 1 Relative percentage % decanoic acid 0.01 0.02 catechin 2.3 3.11 Coffee test 11.0 15.10 EGCG 38.1 52.34 Epicatechin 5.2 7.11 GCG 6.6 8.99 ECG 9.7 13.35 Total 72· 8 100.00 AMBERLITE® XAD-7 resin with particle size 0.3 - 1.2 mm 33.5 € (26 kg) is filled into an intermediate specification tower with an inner diameter of 150 mm, a length of 2 m and a volume of 35.4 €. The tower is provided with a heating jacket. The resin was thoroughly washed with water and equilibrated with a mixture of water/isopropanol (volume ratio: 9:1). The device and solvent used are degassed and kept under an inert nitrogen atmosphere before use. -9- This paper scale applies to China National Standard (CNS) A4 specification (210 X 297 mm) 1293628 A7 B7 V. Invention description (7) Filled tower insulation 60 ° C, 0.4 kg of the above green tea essence (Table 1) The 152.5 g EGCG was dissolved in a 1.8 kg water/isopropanol mixture (9:1 by volume) and added to the top of the column. The EGCG was eluted from the column by a pump at a pressure of 0.5 bar and at 60X: with a water/isopropanol (volume ratio of 9:1) at a strange flow rate of 50 kg/h. After the initial washout (pre-part) 144 kg, the main eluate was collected and 174 kg of the main polyphenol component containing 112 g of EGCG. The concentration of EGCG in the main eluate was 0.064%. The EGCG yield isolated from 152.5 g EGCG in tea spirit was 73.5%. The remaining catechins were eluted with 78.3 kg of water/isopropanol (volume ratio 4:6) for the regenerated resin. Prior to the second separation, the column was conditioned at a flow rate of 120 kg/h with a mixture of 86 kg of water/isopropanol (9:1 by volume) in a backwashing manner. Table 2 illustrates that the separation effect is seen by the relative percentage of EGCG. The concentration of the inner tea component in the main eluate is determined by HPLC using UV absorbance, expressed in wt.-% or ppm. --------*1 ♦装· II (Please read the notes on the back and fill out this page) 卜丨定· Ministry of Economic Affairs Intellectual Property Bureau Staff Consumer Cooperatives Printed Table 2 Main Washing Liquid Tea Group Part of the concentration of the compound main part Example 1 HPLC / ppm relative percentage rate % citric acid 0 0.0 catechin 21 3.1 coffee test 1 0.2 EGCG 644 92.1 epicatechin 29 4.2 GCG 3 0.4 ECG 1 0.2 total 699 100.0 Example 2 10 - This paper size applies to China National Standard (CNS) A4 specification (210 X 297 mm) 1293628 A7 B7 V. Invention description (8) (Please read the back note first and then fill in this page) Use another batch of Guizhou Highyin creatures The product company's ''Green tea essence, at least 95% polyphenols' repeated Example 1, the composition of which is shown in Table 3. Table 3 Tea composition concentration in the starting material Compound tea extract Example 2 HPLC/wt.-% Example 2 tea Example 2 Relative Percentage/% Tannic Acid <0.1 0.05 Catechin 1.4 1.94 Coffee Test 13.8 18.89 EGCG 35.1 47.95 Table Free Tea 3.3 4.47 GCG 8.2 11.20 ECG 11.4 15.49 Total 73.2 100.00 Ministry of Economic Affairs Intellectual Property Bureau Staff Consumer Cooperative Printed Example 1 wash The tower is kept at a constant temperature of 60 ° C for separation. 0.4 kg of the above green tea extract (Table 3) containing 140.5 g of pure EGCG dissolved in 1.8 kg of water / isopropanol (9:1 by volume) mixture, added to the tower Top. The elution was then carried out as described in Example 1. After initial elution of 200 kg (pre-part), 117 kg of main eluate was collected containing 72.8 g of EGCG as the main polyphenol component. The concentration of EGCG in the main eluate was 0.062%. The yield of EGCG isolated from the initial extraction of 140.5 g EGCG from the tea extract was 51.8%. For resin regeneration, stripping with 100 kg of water/isopropanol (volume ratio 4:6). Desorption before the next separation step 100 kg of water/isopropanol (9:1 by volume) mixture was conditioned at a flow rate of 120 kg/h in a backwashing manner. Table 4 illustrates the separation effect as a function of the relative percentage of EGCG. The main component of the tea in the eluate The concentration is measured by HPLC using UV absorption, expressed in ppm. -11 - This paper scale applies Chinese National Standard (CNS) A4 specification (210 X 297 mm) 1293628 A7 B7 Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printed five , Invention Description (9) Example 1 ratio, obtaining a higher percentage of EGCG. Table 4 Main tea extract concentration in the main eluate. Example 2 HPLC/ppm Relative percentage/% decanoic acid <0.1 0.01 catechin 10 1.49 Coffee test 1 0.17 EGCG 622 96.53 Epicatechin 2 0.27 GCG 6 0.95 ECG 4 0.57 Total 645 100.00 Example 3 Eluate Concentration The 9008 kg absorption/desorption column eluate prepared by the repeated operation described in Example 2 was stabilized by adding 2% citric acid as calculated by the amount of EGCG. The eluate was concentrated at a temperature of 40 ° C and a pressure of 55 mbar using a stainless steel falling film evaporator with a heat exchange surface area of 1.1 m2. The catechin and coffee samples of the feed solution receiving the evaporation unit are listed in Table 5. Table 5 Concentration of Tea Components in Evaporated Purified EGCG Liquid Compound Feed Falling Film Evaporator HPLC/ppm Relative Percentage/% Tannic Acid 0.1 0.01 Catechin 10 1.30 Coffee Test 1 0.14 EGCG 712 96.20 Epicatechin 7 0.96 GCG 7 0.97 ECG 3 0.41 Total 740 100.00 -12- ---------?----- (Please read the notes on the back and fill out this page) This paper scale applies to Chinese national standards ( CNS)A4 size (210 X 297 mm) . 1293628 A7 B7 V. Description of invention (10) (Please read the note on the back and fill out this page) Adjust the evaporator feed flow to the range of 120-130 kg/h Flow at a circulating flow rate of 300 kg/h. The remaining flow rate is 123.5 kg/h and the bottom product removal rate is 0.52 kg/h. The first part of the concentration process was sampled and analyzed, followed by the second part. The total amount of the two parts of EGCG concentrate is 63.5 kg. Table 6 shows the concentration of the tea component in the bottom product. The EGCG recovery was 95.9%. The results of the analysis clearly indicate that the high purity of the separated EGCG can be maintained during concentration of the solution. EGCG can be spray dried or concentrated by solid state from the dope. Table 6 Concentration of Tea Components in EGCG Concentrate in Falling Film Evaporator Compound Falling Film Evaporator: Composition Ist Part I HPLC/wt.-% Relative Percentage t Residual Product Composition 2nd Part II HPLC/wt··% Relative Percentage /% 掊酸0.00 0.00 0.00 0.00 catechin 0.17 1.55 0.14 1.59 咖嗔0.01 0.12 0.02 0.28 EGCG 10.70 95.36 8.07 94.93 Epicatechin 0.16 1.39 0.11 1.24 GCG 0.13 1.14 0.13 1.52 ECG 0.05 0.44 0.04 0.44 Total 11.23 100.00 8.50 100.00 The total mass of the solution is 39.0 kg 24.5 kg. Example 4 Ministry of Economic Affairs Intellectual Property Office Staff Consumer Cooperatives Printed 450 AMBERCHROM® CG-71c with an average particle diameter of 120 μm Filled into a stainless steel laboratory chromatography column with an inner diameter of 2.2 cm It is 103 cm long and is equipped with a heating jacket. The resin was washed and equilibrated with water/ethanol (9:1 by volume). -13 - This paper size is applicable to China National Standard (CNS) A4 specification (210 X 297 mm) 1293628 A7 B7_ V. Invention description (11) Dissolving 20 g concentrated catechin powder from Guizhou Highyin Bioproducts Co., Ltd. , a mixture of at least 95% polyphenols (starting material) in 20 water/ethanol (9:1 by volume). This solution was then added to the top of the column with 14 g (equivalent to 2.99 g EGCG). The EGCG was eluted by a toluene pump at a pressure of 2_3 bar and at a temperature of 60 ° C with a water/ethanol (9:1 by volume) mixture at a constant flow rate of 16 /min. The eluent is degassed and kept in a nitrogen atmosphere for use. Initially, 2.48 € (pre-part) was added and the flow rate was changed to 25.5 m€ /min, and 5.40 was collected. The eluate had an EGCG concentration of 0.627 g/€. The purity of EGCG in the main eluate was determined by HPLC as a relative percentage of 97.13% relative to other catechins and caffeine. During the experiment, the pressure variation in the system was determined by the flow rate from 2 to 3 Bar. Table 7 compares the concentration of the eluate with the tea component in the starting material, which is a representation of the relative percentage of separation effect such as EGCG. The concentration of the tea component in the starting material and the main portion was determined by HPLC using UV absorption and expressed in wt.% or ppm. Table 7 MBERCHROM CG-71c separation, 60 ° C, solvent system: water / --------- ΓΙΛ - installed - (Please read the back of the note before you fill out this page) Intellectual Property Bureau Staff Consumer Cooperative Printed Ethanol Compound Tea Concentrate (Starting Material) Example 4 Main Section Example 4 HPLC wt.-% Relative Percentage/% HPLC/ppm Relative Percentage/% Niobic Acid 0.08 0.10 0 0.00 Catechin 0.50 0.61 1 0.22 咖嗓9.29 11.31 5 1.29 EGCG 42.23 51.41 407 97.13 Epicatechin 4.24 5.17 3 0.78 GCG 8.09 9.85 1 0.20 ECG 17.70 21.55 2 0.38 Total 82.15 100.00 419 100.00 -14- This paper scale applies to Chinese National Standard (CNS) A4 size (210 X 297 mm) 1293628 A7 B7_ V. Description of invention (12) Example 5 (Please read the note on the back and fill out this page) In a glass laboratory layer with an inner diameter of 2.4 cm and a length of 100 cm The column was filled with Amberlite® XAD-7 450 ml with a particle diameter of 0.3 to 1.2 mm. The tower body is provided with a heating jacket, and a porous frit P3 is arranged on the bottom. The resin was thoroughly washed with deionized water and equilibrated with a water/ethanol (volume ratio of 9:1) mixture. Dissolve 20 g of concentrated catechin powder '' green tea extract of Guizhou Highyin Bioproducts Co., Ltd., at least 95% of the mixture (starting material) in a mixture of 20 mf water/ethanol (9:1 by volume). This solution was then added to 14 g (equivalent to 2.91 g of EGCG) at the top of the column. The EGCG was eluted with a water/ethanol (volume ratio of 9:1) mixture at a constant flow rate of 16.9 / min at a temperature of 60 ° C and a pressure of 0.5 _ 1 bar. The eluent is degassed and kept under nitrogen before use. After the initial elution of 2.48 € (pre-part), the flow rate was changed to 23.6 minutes, and the main eluate was collected at 4.95 £ with an EGCG concentration of 0.470 g/. For other major catechins and caffeine, the purity of EGCG in the main part was determined by HPLC to be 86.22% (see Table 8). The yield based on the EGCG basis was 79.8 %. The pressure variation in the system during the experiment was determined by the flow rate used from 0.5 to 1 bar. Printed by the Intellectual Property Office of the Ministry of Economic Affairs, the Consumer Cooperatives. For the purpose of recycled resin, the remaining catechins were desorbed at a flow rate of 22.5 / min at a water/ethanol (volume ratio of 4:6) of 1.35. This part can also be used to further purify or isolate desorbed catechins. Table 8 compares the concentration of the tea component in the eluate with the starting material, thus exemplifying the separation effect as indicated by the relative percentage of EGCG. The concentration of the tea component in the starting material and in the main portion was determined by HPLC using UV absorbance and expressed in wt.% or ppm. -15- This paper size is applicable to China National Standard (CNS) A4 specification (210 X 297 mm) " 1293628 A7 B7 V. Inventive Note (13) Table 8 is separated on Amberlite® XAD-7, 60 ° C, solvent System: water/ethanol compound tea concentrate (starting material) Example 5 HPLC/wt.-% relative percentage/% Main part Example 5 HPLC/ppm Relative percentage/% decanoic acid 0.09 0.11 0 0.00 catechin 0.50 0.62 2 0.32 Coffee #验9.17 11.47 7 1.22 Please read the note on the back first

EGCG 41.16 51.46 470 86.22 Epicatechin 4.16 5.20 5 0.98

GCG 7.75 9.69 22 3.97

ECG 17.16 21.46 40 7.28 Total 79.99 100.00 545 100.00 Wins and then W Fills I. Example 6 Raw resin for Example 5 Pumped in a laboratory tower as described in Example 5 Water/Ethanol (body ratio 9:1) The mixture is equilibrated by a resin. Dissolve 20 g of high-quality catechin powder from Guizhou Highyin Bioproducts Co., Ltd., green tea extract, a mixture of at least 95% polyphenol π (starting material) in 20 water/ethanol (9:1 by volume). 14 g of this solution (equivalent to 3.04 g EGCG) was then added to the top of the column. The eluted EGCG water/ethanol mixture (volume ratio 9:1) was carried out at a constant flow rate of 22.5 / min at a column temperature of 40 ° C and a pressure of 1-2 bar. The eluent is degassed and kept under nitrogen before use. After the initial 3.60 € elution (pre-part), change the flow rate to 26.3 / min flow and collect the main eluate 4.73. The concentration of EGCG in the main wash was 0.278 g/f. Regarding other main catechins and caffeine, the purity of the catechin catechol in the main eluate was determined by HPLC to be 93.2%. The yield based on EGCG was 42.8%. The pressure change in the system during the experiment depends on the flow rate of 1 to 2 Bar. For regenerated resin, 1.98, water/ethanol (volume ratio 4:6) at a flow rate of 26.3 -16- This paper scale applies Chinese National Standard (CNS) A4 specification (210 X 297 mm). Co-operative printing 1293628 A7 B7 V. Description of invention (14) / min and 40 ° C temperature elution and desorption. This section can also be used to further purify or separate desorbed catechins. Table 9 compares the concentrations of the tea components in the eluate and in the starting material, thus indicating that the separation effect is as shown by the relative EMIG. The concentration of the tea component in the starting material and in the main portion was determined by HPLC using UV absorbance and expressed in wt.% or ppm. Table 9 Separated on Amberlite® XAD-7, 40 ° C, Solvent System: Water/Ethanol Tea Concentrate (Starting Material) Example 6 Please read the notes on the back ▲ then fill in the main part of the sample of Example 6 HPLC/ppm HPLC/wt.-% Relative Percentage/% Relative Percentage/% Niobic Acid 0.08 0.10 0 0.00 Catechin 0.51 0.61 1.00 9.48 11.33 1.43

EGCG 43.01 51.40 276 93.24 Epicatechin 4.34 5.18 9 3.15

GCG 8.23 9.83 2 0.51

ECG 18.03 21.54 2 0.66 Total 83.68 100.00 296 100.00 Ministry of Economic Affairs Intellectual Property Office Staff Consumer Cooperative Printed Example 7 The regenerated resin of Example 6 was equilibrated with a mixture of water/isopropanol (9:1 by volume). Dissolve 20 g of high-concentration catechin powder from Guizhou Highyin Bioproducts, a blend of green tea extract, at least 95% polyphenol (starting material) in 20 water/isopropanol (9:1 by volume). Subsequently, 14 g of this solution (corresponding to 3.21 g of EGCG) was added to the top of the column, and the mixture with water/isopropanol (volume ratio of 9:1) was eluted at a constant flow rate of 18 /min at 60 ° C. The eluent was degassed and kept under nitrogen before use. Start the eluate 1.35, (pre-part) and change the flow rate to 16.5 m / / min, -17- This paper scale applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) 1293628 A7 B7 V. Description of the invention (15) Collect the main eluate for 2.30 €. The concentration of EGCG in the main eluate was 0.998. Related to other major catechins and caffeine, the purity of the epicatechin in the main eluate was determined by HPLC to be 85.7%. The yield based on EGCG is 62.8%. The pressure variation in the system during the experiment was determined by the flow rate used by 1 to 2 Bax. For the regenerated resin, the remaining catechins were desorbed with a mixture of 2.03 € water/isopropanol (volume ratio 4:6) at a flow rate of 16.5 /min and a temperature of 40 °C. This part can also be used for further purification or separation of desorbed catechins. Table 10 compares the tea component concentrations in the eluate and in the starting material, thus indicating the separation effect as indicated by the relative EGCG percentage. The concentration of the tea component in the starting material and in the main portion was determined by HPLC using UV absorbance and expressed in wt·-% or ppm. Table 10 Separated on Amberlite® XAD-7, 60 ° C, Solvent System: Water / Isopropyl Alcohol - - * * - # - Pack! (Please read the notes on the back and fill out this page) Ministry of Economic Affairs, Intellectual Property Office, Staff and Consumer Cooperatives, Printing Compound Tea Concentrate i (Initial Phase HPLC/wt.-% German-Material) V Percentage/% Real-time 丨 Major HPLC/ppm 17% relative percentage/% Capric acid 0.05 0.06 0 0.01 catechin 0.38 0.44 8 0.69 Coffee test 9.48 10.82 28 2.39 EGCG 45.42 51.83 998 85.68 Epicatechin 4.38 5.00 16 1.38 GCG 8.80 10.04 35 3.02 ECG 19.12 21.81 80 6.84 Total 87.63 100.00 1165 100.00 痈-18 - This paper scale applies to China National Standard (CNS) A4 specification (210 X 297 mm) 1293628 Ministry of Economic Affairs Intellectual Property Office Staff Consumer Cooperative Printed A7 B7_ V. Invention Description (16) Example 8 Recycled resin of Example 7 in water/ The mixture of isopropanol (9:1 by volume) was equilibrated. Dissolve 20 g of concentrated catechin powder from Guizhou Highyin Bioproducts Co., Ltd. "Green tea essence, at least 95% multiple test" (starting material) in a mixture of 20 water / isopropanol (9:1 by volume). This solution was then added to the top of the column at 14 g (equivalent to 3.10 g EGCG). The water/isopropyl alcohol (volume ratio of 9:1) mixture was eluted at a temperature of 40 ° C in a strange flow of 16.9 m € / min. The eluent was degassed and kept under nitrogen before use. After the initial eluate 2.48 / (pre-part), change the flow rate to 23.66 m € / min and collect the main eluate 4.95. The concentration of EGCG in the main eluate was 0.370 g/. Related to other major catechins and caffeine, the purity of EGCG in the main part was determined by HPLC to be 86.6%. The yield based on the EGCG basis was 59.2%. The system pressure variation during the experiment was determined by the flow rate used from 1 to 2 Bar. For the regenerated resin, the remaining catechin was desorbed with a mixture of 2.03 mmol of water/isopropanol (volume ratio of 4:6) at a flow rate of 16.5 /min and a temperature of 40 °C. This part can also be used for further purification or separation of desorbed catechins. Table 11 compares the tea component concentrations in the eluate with the starting material, thus indicating the separation effect as indicated by the relative EGCG percentage. The concentration of the tea component in the starting material and in the main portion is determined by HPLC using UV absorbance and expressed in wt.-% or ppm. -19- This paper size is applicable to China National Standard (CNS) A4 specification (210 X 297 mm) ---------*—I·Install·II (Please read the notes on the back and fill out this page. )·1293628 A7 B7 V. INSTRUCTIONS (17) Table 11 Separation on Amberlite XAD-7, 40 ° C, Solvent System: Water/Isopropanol Compound Tea Concentrate Main Part (Starting Material) Example 8 Example 8 HPLC/wt.-% Relative Percentage/% HPLC/ppm Relative Percentage Citrate 0.20 0.23 0 0.00 Catechin 0.49 0.57 4 0.83 Curry Verification 9.21 10.85 6 1.50 EGCG 43.74 51.52 370 86.62 Epicatechin 4.23 4.99 14 3.21 GCG 8.50 10.02 11 2.49 ECG 18.52 21.82 23 5.35 Total 84.91 100.00 428 100.00 Please read the back note first

Example 9 Separation of EGCG from Polyamide with Organic Solvent 11 Commercial Green Tea Extract (''Green Tea Extract, Minimum 95% Polyphenols') Guiyang Guizhou Highyin Bioproducts Co., Ltd. Batch No. 960328) Contains catechins and caffeine The starting materials are as shown in Table 12. The concentration of the tea components is determined by the UV absorption rate of HPLC. Ministry of Economic Affairs, Intellectual Property Bureau, Staff Consumer Cooperatives, Table 12, Concentration of tea components in the starting materials, compound tea extract HPLC/ Wt.-% relative percentage of tea extract /% decanoic acid 0.1 0.01 EGC 2.02 2.99 catechin 0.78 1.15 coffee test 8.48 12.54 EGCG 36.87 54.52 epicatechin 4.48 6.62 GCG 4.77 7.05 ECG 10.22 15.11 total 67.63 100.00 -20- paper scale Applicable to China National Standard (CNS) A4 specification (210 X 297 mm) 1293628 A7 B7 V. Description of invention (18) 250 g of commercially available polyamido 11 (source German Dermstadt Merck, catalogue 1.07435.0100) particles The size of 5 - 40 microns is suspended in 300 acetic acid, transferred into a tower with an inner diameter of 5 cm and a length of 3 6 cm. The tower has a heating device and a temperature of 40 ° C. Dissolve 3 g of starting green tea. Table 12 characteristics, containing 1.11 g of pure EGCG 153 ethyl acetate, added to the top of the column. Ethyl acetate / ethanol gradient elution [500 ethyl acetate, 1000 mi ethyl acetate / ethanol (8.5: 1 · 5 v / v ), 1000 m 彳 ethyl acetate / ethanol (7:3 v / v), 2000 m 彳 ethyl acetate / ethanol (1 : 1 v / v)] produced a major part of 550 at a pressure of 0.3 bar, after evaporation of the solvent 1.12 g of solids containing 0.87 g of EGCG as the main catechin component. The concentration of EGCG in the main eluate was 0.186%. The calculation of the separation of £000 from the initial catechin was 1.106§£0€0. Desorb the remaining catechins with 500 ethanol for the resin regeneration meter. Adjust the column with 500 ethyl acetate before the next separation. Table 13 shows the separation effect. The concentration of the components in the main eluate is the UV absorption rate by HPLC. ------- -. - Installation - (Please read the notes on the back and fill out this page) 纛 Ministry of Economic Affairs Intellectual Property Bureau Staff Consumer Cooperatives Printed Table 13 Main Eluates (after evaporation of solvents) The main part of the tea component concentration in the residue remains HPLC/wt.-% The remaining part of the main part is relative percentage /% 掊酸0 0 EGC 0 0 Tea 0 0 咖# 验 0 0 EGCG 77.4 96.50 Epicatechin 0.05 0.06 GCG 0.74 0.92 ECG 2.07 2.58 Total 80.21 100.00 -21 - This paper scale applies to Chinese National Standard (CNS) A4 specification (210 X 297 mm) 8 2 6 3 912 A7B7 V. Description of invention (19)

ί^Μ_0 Separation of EGCG from a polyamine 11 using an aqueous mixed solvent The starting amount of the catechin-containing and green tea aqueous solution shown in Table 14 was used. The concentration of the tea component was determined by HPLC using UV absorption rate, and the concentration of the tea component in the residue of the tea semen (after evaporation of the solvent) was determined by wt.-% Table 7JT 〇 J J soil compound tea extract HPLC/wt.- % Percentage of tea extract /% Citrate 1.36 4.77 EGC 3.61 12.65 Catechin 1.45 5.08 Coffee test 6.89 24.14 EGCG 10.14 35.53 Epicatechin 1.59 5.57 GCG 0.99 3.47 ECG 2.51 8.79 Total 28.54 100.00 Suspended particle size 5 40 μm polyp Amine 11 (sourced from Dernstadt, Germany, catalogue 1.07435.0100) was adjusted to pH 6.5 in 100 water. Transfer the suspension into a tower with an inner diameter of 3 cm and a length of 8 cm. Add the above (Table 14) green tea extract containing 0.078 g of pure EGCG to the top of the column. One water / ethanol gradient elution [500 water, 600 water / ethanol (7: 3 v / v), 350 water / ethanol (6: 4 v / v), 500 water / ethanol (1: 1 v / v)] A major fraction of 110 (0.072 g) was produced at a flow rate of 5 m/min with 0.046 g EGCG. The concentration of EGCG in the main eluate was 0.06%. The yield of EGCG from the start of 0.078 g of EGCG in the tea extract was 59%. For the regenerated resin, the remaining catechins were desorbed by washing with 500 m of ethanol.下-22- -------- — ΙΓΙ·-装- (Please read the notes on the back and fill out this page). 镰Ministry of Economic Affairs, Intellectual Property Bureau, Staff and Consumer Cooperatives, Print a Moderate Ruler Paper 21 /V Grid A4 S) N (C standard standard PCT 97 1293628 A7 B7 V. Invention description (2Q) 500 water conditioning tower before separation. Table 15 shows the separation effect. The concentration of tea component in the main washing liquid It is determined by the absorption rate of 玎v. Table 11 The main part (after evaporating the solvent) The concentration of the tea component in the residue The remainder of the main part of the compound remains HPLC/wt.-% The remaining percentage of the main part is %/% 掊酸1.10 1.59 EGC 0.00 0.00 catechin 1.29 1.86 Coffee test 0.00 0.00 EGCG 63.53 91.70 Epicatechin 0.00 0.00 GCG 0.16 0.23 ECG 3.20 4.62 Total 69.28 100.00 11 Separate EGCG from Amberlite XAD-7, solvent system · water / ethanol will Amberlite XAD-7 resin 416 with a particle diameter between 〇·3 and 1.2 mm is packed into a laboratory chromatography column, made of glass (ECO 25/999 M3V-K, manufactured by Wallisellen, Switzerland, Stagroma AG). 2.5 cm in diameter and the Intellectual Property Officer of the Ministry of Economic Affairs Printed by consumer cooperatives ------Package ------ 订 · · (Please read the note on the back and fill in this page) Length 100 cm. Tower heating set, constant temperature 60 ° C. Resin The water/ethanol (9:1 by volume) mixture was washed and balanced. The commercially available green tea extract (Chinese Phyto-Zhejiang-Zhejiang-Zhejiang-Zhejiang Chinese Plant Technology Co., Ltd. π-tea multi-test TP-80π-containing catechin and coffee test as shown in Table 16 The materials shown are the starting materials. The tea component concentration is determined by UV absorption rate by HPLC and expressed in wt··%. -23- The paper scale is applicable to China National Standard (CNS) A4 specification (210 X 297 mm) 1293628 Economy Ministry of Intellectual Property Bureau Staff Consumer Cooperative Printed A7 B7 V. Invention Description (21) Table 16 Tea Composition Concentration Compound Example 13 Tea Extract HPLC/wt.-% Example 13 Tea Extract Relative Percentage /% Tannin 0.1 0.1 EGC 8.6 10.1 catechin 1.9 2.2 Coffee 6.2 7.3 EGCG 40.3 47.4 Epicatechin 10.4 12.2 GCG 0.9 1.1 ECG 16.6 19.5 Total 85.0 100.00 Dissolved 11.2 g The characteristics shown in Table 16 contain 4.5 g of pure EGCG starting green tea Finer than 112.5, from the water, join the top of the tower. The catechin water/ethanol mixture (volume ratio 9:1) was eluted at a constant flow rate of 0.6 ^ / hr at 60 ° C. The eluent is degassed and kept under nitrogen before use. After the initial stripping of 1.2 €, the composition of the eluent was changed to a water/ethanol volume ratio of 8:2. The total amount of this elution was 1.5, resulting in a major portion of 900 containing 2.15 g of EGCG. The concentration of EGCG in the main part was 0.245%. The EGCG yield starting from 4.5 g of EGCG in tea spirit was 47%. The pressure in the system varied from 0.8 to 1.5 bar during the experiment. The mixture was again eluted with a water/ethanol 4:6 volume ratio mixture for the regenerated resin, and the ethanol was then desorbed from the remaining catechins. The tower was treated with a water/ethanol volume ratio of 9:1 before the next separation. Table 17 illustrates the separation effect. The concentration of the tea component in the remainder of the main portion (after evaporation of the solvent) was determined by HPLC using UV absorbance and expressed as wt.-%. -24- This paper size is applicable to China National Standard (CNS) A4 specification (210 X 297 mm) --------.--I--------^------ -- (Please read the notes on the back and fill out this page) 1293628 Ministry of Economic Affairs Intellectual Property Office Staff Consumer Cooperatives Print A7 B7 V. Inventions (22) Table 17 Main part (after evaporation of solvent) Residual tea group Part of the concentration of the compound Example 13 Remaining HPLC/wt.-% The main part of Example 13 Residual relative percentage /% Citrate 0 0 EGC 0 0 Catechin 0.6 0.7 Coffee test 0.3 0.3 EGCG 81.4 94.5 Epicatechin 1.7 2.0 GCG 0.2 0.2 ECG 1.9 2.2 Total 86.1 100.00 -25- ---------,---装-------- -定------- (Please read the back first Note on this page. This paper size applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm).

Claims (1)

Road No. 16495 Patent Application Year of September) VI. Application for Special ρτFan Group A8 B8 C8 D8 Reporting|Revised 丨 Supplementary Year" A method for manufacturing epigallocatechin citrate (EGCG), including steps : "% a) Preparation of a green tea extract containing tea catechins and caffeine; b) Green tea extract in the range of (7) art to rib art temperature in the absorption or decanting common coarse pore polar resin Accepting chromatographic separation, wherein the coarse pore polar resin is selected from the group consisting of polyethylidene pyrrolidone, polyacrylate, polyisobutyl vinegar, polyamide and polyester; and C) using a polar eluting solvent EGCG is eluted from the crude pore polar resin in a temperature range of 1 〇r to 8 〇〇c and a pressure range of 〇·丨巴 to 5 〇, wherein the polar eluting solvent is water and one selected from the group consisting of alcohols, ketones and esters And a mixture of organic and granules of the mixture thereof. 2 The eluate according to the method of claim 1 of the patent application. 3. Regenerating the crude pore polar resin according to the catechin remaining in the method of claim i. According to the method of item 3 of the scope of patent application _ Please refer to item 3 of the patent scope The dehydrated catechin. 5. According to the method of claim 1, wherein the polyacrylate resin is AMBERLITE® XAD-7. 6 according to the method of the scope of the patent application, wherein the polymethacrylate tree month The invention is directed to AMBERCHROM® CG·71. 7. The method according to the scope of the patent application, wherein the polyamine resin is Polyamide 1 卜 1. further comprises concentration c) the step further comprises desorption and further comprising a concentration such as a binding line Wherein b) & the method according to any one of claims 6 to 6 and the hydrazine step are carried out at a temperature ranging from 20 ° C to 60 ° C. Where c) 9) According to the method of any of items 1 to 7 of the scope of the patent application, the steps are carried out at a pressure of from 0. 1 bar to 20 bar. Where c) 10. According to the method of any one of the scopes of the patent application, from step VII to step 7, the step is carried out at a pressure of from 0.1 bar to 1 bar. U: The method according to claim 9, wherein the eluting solvent is a mixture comprising 70% of water of 1% to 95% by weight and 5v of 1% of W5v〇i% of organic solvent. According to the method of claim 9, wherein the eluting solvent is a mixture containing 90% of water and 10% by volume of an organic solvent. According to the party of the patent scope, the organic solvent is ethanol, isopropanol, ethyl acetate or propylene. η. The method according to any of the preceding claims, wherein the flow rate of the washing solvent is in the range of 0.5 to 20 bed capacity/h. The method according to any one of claims 1 to 7, wherein the flow rate of the eluting solvent is in the range of 〇5_10 bed capacity/h. Dip according to the scope of the patent application! The method of any one of the items 7, wherein the flow rate of the eluting solvent is in the range of 〇.8_5 bed capacity. π. The method of claim 2, wherein the concentration of the eluate of the hydrazine step is accomplished by the addition of citric acid, ascorbic acid or isoascorbic acid. According to the application for patent ribs 7 7 stone, and target 罘 17 items, the amount of acid added is relative to EGCG 0.1 -2·5 % 〇_0:\65\65463^hm 1 .ΡΟΓ\ This paper scale is suitable Guan Jia Xian (CNS) AB c D 1293628 VI. Scope of Application - ~"~ 19· According to the method of claim 3 or 4, the regeneration of the crude pore polar resin is a pure organic solvent or a 1〇_ 6〇v〇l% water is mixed with 4〇_ 9〇vol% organic solvent. 2. The method according to claim 3, wherein the regeneration of the crude pore polar resin is carried out by using a pure organic solvent or a mixture of 4 〇v〇1〇/〇 water and 6 〇v〇1% organic solvent. carry out. A green tea component comprising at least 91.7 wt% iEGCG and up to j 43 wt-% coffee. 22. The green scorpion component according to item 21 of the patent application, which contains at most 〇.2 w t - % of the coffee test. 23. A green tea component according to claim 21, which comprises up to 315 wt-% of epicatechin. 24. A green tea component according to claim 21, which comprises up to 2% wt-% of epicatechin. 25. A green tea component according to claim 21, which comprises up to 139 wt-% of epicatechin. 26. The green tea component according to item 21 of the patent scope includes at most 3, except for egcg, epicatechin, catechin citrate (GCG) and epicatechin citrate (ecg). · 1 wt-0 / catechin catechin. 27. The green tea component according to item 21 of the patent application, in addition to egcg, epicatechin, GCG and ECG, contains at most the tea acid. 28. According to the green tea component of claim 21, it contains at most 152 _Γ) ΠΓ\ ^ ^ This paper scale applies to Chinese national standards (CN^T^See (210Χ 297 公|): ---- 8 2 6 9 2 8 8 8 8 ABCD VI. Apply for the GCG of the patent model park wt-%. 29. According to the green tea component of claim 21, it contains up to 0.97 wt-% of GCG 0 30. According to the patent application The green glutinous component of the range 21, which contains up to 4.62 wt-% of ECG 0 31. The green tea component according to claim 21, which contains up to 0.66 wt-% of ECG. The green tea component of the range of item 21, which comprises from 7,000 liters of from 01.7 to 1% of £0 to from 0. From 0.2 wt-% to 1.52 wt-% of GCG, from 0.38 wt-% to 4.62 wt-% of ECG and from 0 to 1.43 wt-% of caffeine. 33. Green tea component according to claim 21 It comprises 96.53 ^ 1-% EGCG, 0.27 wt-% EC, 1.49 wt-% catechin, 0.95 ^-% GCG, 0.57 wt-% ECG and 0.17 wt-% caffeine. . Green tea component according to item 21 of the patent application scope It comprises 96.2 wt-% EGCG, 0.96 wt-% EC, 1.3 wt-% catechin, 0.97 wt-% GCG, 0.41 wt-% ECG, and 0.14 wt-% juvenile mouth test. The green tea component according to claim 21, which comprises 95.36 to 1-% of EGCG, 1.39 wt-% of EC, 1.55 wt-% of catechin, 1.14 wt-% of GCG, and 0.44 wt-%. ECG and 0.12 wt-% of caffeine. 36. The green tea component according to claim 21 of the patent application, comprising 94.93 ^ 1-% of EGCG, 1.24 wt-% of EC, 1.59 wt-% of catechin, 1.52 \¥^% of GCG, 0.44 wt-% of ECG and 0.28 wt-% of caffeine. This paper scale applies to China National Standard (CNS) Α4 specification (210Χ 297 mm). ABCD 1293628 6. Patent application scope 37. The green tea component of claim 21, which comprises 97.13\¥1-% of EGCG, 0.78 wt-% of EC, 0.22 wt-% of catechin, 0. 2 wt-% of GCG, 0.38 wt-% ECG and 1.29 wt-% caffeine. 38. The green tea component according to claim 21, which comprises 93.24 wt-% of EGCG, 3.15 wt-% of EC, 1.0 wt-% of catechin, 0.5 1 wt-% of GCG, 0.66 wt- % ECG and 1.43wt-% coffee test. 39. A green tea component according to claim 21, which comprises 91.7 wt-% EGCG, 1.86 wt-% catechin, 0.23 wt-% GCG and 4.62 wt-% ECG. 40. The green tea component according to claim 21, comprising 94.5 to 1-% of EGCG, 2.0 wt-% of EC, 0.7 wt-% of catechin, 0.2 wt-% of GCG, 2.2 wt- % ECG and 0.3 wt-% coffee _ test. 41. A health care preparation comprising EGCG, such that the EGCG is a component of the green carp according to item 21 of the scope of the patent application. 42. According to the health care preparation of claim 41, the green peony component contains up to 0.2 wt-% of coffee. 43. The green tea component contains up to 3.15 wt-% EC according to the health care preparation of claim 41. 44. The green tea component contains up to 2.0 wt-% of EC according to the health care preparation of claim 41. 45. The green tea component contains up to 1.39 wt-% EC according to the health care preparation of claim 41. 46. According to the health care preparations in the scope of patent application No. 41, except EGCG, Table _nnr\S_一 A -__ This paper scale applies Chinese National Standard (CNS) A4 specification (210 297 297 mm). 1293628 Patent application range In addition to catechol, GCG and ECG, the green tea component contains up to 3.1 wt-% catechin. 47. The green preparation according to claim 41 of the patent application, in addition to egcg, excipient acid, GCG and ECG, the green tea component contains up to 0.59 w t -% of catechin. 48. The green tea component contains up to 1.52 wt-% of GCG according to the health care preparation of claim 4 of the patent application. 49. The green tea composition according to claim 41, wherein the green tea component contains up to 0.97 wt-% of GCG. 50. The green tea component contains up to 4.62 wt% ECG according to the health care preparation of claim 41. 51. The green peony component contains up to 0.66 wt-% ECG according to the health care preparation of claim 41. 52. The health care preparation according to claim 4, wherein the green tea component contains from 91.7 wt-% to 97.13 wt-% of EGCG, from 0 to 3.15 wt-0/〇 of EC, from 〇 to 3.1 wt-% Tea yoghurt, from wt 2 wt 〇 / o to! 52 correction _% of GCG, from 0.38 wt-% to 4.62 wt% ECG and caffeine from 〇 to 1.43 wt-〇/0. ΰ_:\6$\6546.ν960921 ΡΟΓ\ 5_____ 6 _ This paper scale applies to the Chinese National Standard (CNS) Α4 specification (210X 297 mm).