Method for extracting EGCG (epigallocatechin gallate) from tea leaves and application of EGCG

Abstract

The invention discloses an extraction method based on EGCG (epigallocatechin gallate) in tea leaves and application thereof, and relates to the technical field of natural product chemistry. S2, ultrasonic-assisted extraction: mixing the green tea fine powder with deionized water, and extracting by using an ultrasonic technology; the extraction temperature is controlled at 50-60 DEG C, and the extraction time is 30-45 minutes; s2.1, selecting and crushing: selecting fresh green tea which is free of impurities, rich in EGCG (epigallocatechin gallate) and subjected to pretreatment; crushing the green tea by using a crusher; s2.2, extraction and ratio control: using deionized water as an extraction solvent; the ratio of the deionized water to the green tea fine powder is (10: 1)-(20: 1); s2.3, the operation state of the ultrasonic equipment is set; according to the method disclosed by the invention, the dissolution rate of EGCG (Epigallocatechin Gallate) is remarkably improved through an ultrasonic-assisted extraction technology, so that the extraction efficiency is improved; and various technologies such as extraction, digestion, macroporous resin adsorption and polyamide resin deep purification are combined, so that impurities are effectively removed, and the extraction rate and the purity of EGCG are improved.

Description

Technical Field

[0001] The invention relates to the technical field of natural product chemistry, and in particular to an extraction method based on EGCG in tea leaves and an application thereof. Background Art

[0002] With the improvement of health awareness, the application of bioactive substances from natural sources in the fields of medicine and health products is becoming more and more extensive. Among them, epigallocatechin gallate (EGCG), as the main active ingredient of green tea, has attracted much attention due to its powerful antioxidant, anti-inflammatory, anti-tumor and other biological activities. At present, there are various methods for extracting EGCG from tea leaves, but the efficiency and purity still need to be improved. In addition, the potential application value of EGCG in the treatment of neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD) is also highly anticipated.

[0003] Common methods for isolating EGCG generally include two steps: extraction and separation and purification. When extracting fresh tea leaves, high temperature, grinding, enzymatic hydrolysis and other methods are usually used to destroy the cell walls of the tea leaves to achieve sufficient outflow of substances in the tea leaves. For example, the Chinese invention patent application document with application number CN201210178303.3 discloses a method for preparing high-purity monomeric catechins from tea processing waste materials, including extracting tea polyphenols, separating catechins and purifying the process, including: A. The raw materials are taken from fresh tea leaves, pruned tea leaves, and non-fermented tea processing waste materials, and crushed to 20-80 mesh; B. Add water and add a complex enzyme composed of cellulase, protease and pectinase under acidic conditions for enzymatic hydrolysis, wherein: the amount of water added is 10 to 20 times the dry weight of the tea leaves, the pH value is 3 to 6.5, the composition ratio of the complex enzyme is cellulase: protease: pectinase = 0.1 to 5: 0.1 to 5: 0.1 to 5, the amount of the complex enzyme is 0.1 to 1% of the dry weight of the tea leaves, and the temperature is 40 to 65°C; C. the enzymatic hydrolyzate is extracted with ethyl acetate after filtration, and the extract is concentrated and / or dried to obtain a crude tea polyphenol; D. the concentrated or / and dried product is added with water to form a liquid, the pH value is adjusted to 5.6 to 6.5, and a mixed precipitant of CaCl2 and ZnCl2 is added; the amount of water added is 10 to 20 times the dry weight of the crude product, the mixing ratio of CaCl2 and ZnCl2 mixed precipitant is 1:0.01 to 99, CaCl2 is added to the precipitant, and the mixture is stirred for 1 h. 2+ and Zn 2+The ratio of the sum of the amount of the polyphenols in the solution to the amount of the polyphenols in the solution = 0.01-0.05 (mol / g); E. The filtered precipitate is dissolved in dilute hydrochloric acid and then directly adsorbed on a macroporous resin, washed with water, and then eluted with ethanol, and the ethanol eluate is concentrated and / or dried to obtain a fine product of tea polyphenols; the concentration of the dilute hydrochloric acid is 0.1%-10%, the amount of water used for washing is 1-5 times the volume of the macroporous resin, and the ethanol concentration is 90%-99.5%; F. Add water to form a liquid, adsorb it on a macroporous resin, wash it with water, and then elute it with an ethanol-water solution, and collect the eluates containing EGCG, GCG, and ECG with a purity of more than 98% respectively; the amount of water added is 2-5 times the dry weight of the fine product, and the water used for washing is deionized water, The dosage is 1 to 5 times the volume of the macroporous resin, and then the procedure of elution with ethanol aqueous solution is: elution with 10% ethanol aqueous solution 1 to 3 times the volume of the resin - elution with 20% ethanol aqueous solution 1 to 3 times the volume of the resin - elution with 30% ethanol aqueous solution 1 to 3 times the volume of the resin - elution with 90% to 99.5% ethanol 1 to 3 times the volume of the resin; G. Add water to make the ethanol concentration of each eluent 1 to 10%; H. Then adsorb with the macroporous resin of the same model as F, elute with 90 to 99.5% ethanol, concentrate the eluent under reduced pressure, and freeze-dry to obtain the finished product.

[0004] For example, the Chinese invention patent application document with application number CN201410067471.4 discloses a method for extracting monomeric EGCG from fresh tea leaves, which includes the following steps: soaking and cooking, centrifugation, primary adsorption, MVR concentration, CF filtration, secondary adsorption, concentration, rinsing, dehydration, drying, and iron removal. The soaking and cooking temperature of this method is high, resulting in large loss of the target product and low yield.

[0005] In view of this, a method for extracting EGCG from tea leaves and its application are provided to overcome the above problems. Summary of the invention

[0006] The purpose of the present invention is to provide a method for extracting EGCG from tea leaves and its application, so as to solve the problems raised in the above background technology.

[0007] In order to solve the above technical problems, the present invention provides a method for extracting EGCG from tea leaves and its application, comprising the following steps:

[0008] S1. Pretreatment: After washing and destemming the picked green tea buds, they are treated with a specific biological enzyme; the biological enzyme is a composite enzyme, comprising cellulase, pectinase and a polyphenol oxidase inhibitor extracted from a microorganism of the genus Bacillus; the enzymatic hydrolysis temperature is controlled at 35-38°C, the enzymatic hydrolysis time is 20-30 minutes, and the enzyme dosage is 0.05-0.1 grams of composite enzyme per gram of tea leaves; after the enzymatic hydrolysis is completed, the tea leaves are dried to a moisture content of 10%, and then crushed into fine powder;

[0009] S2, ultrasonic-assisted extraction: mix green tea powder with deionized water and extract using ultrasonic technology; the extraction temperature is controlled at 50-60°C and the extraction time is 30-45 minutes;

[0010] S2.1. Selection and crushing: Select fresh, impurity-free, EGCG-rich and pre-treated green tea; crush the green tea using a crusher;

[0011] S2.2, extraction and ratio control: deionized water is used as the extraction solvent; the ratio of deionized water to green tea powder is 10:1 to 20:1;

[0012] S2.3, set the operating state of the ultrasonic equipment; set the ultrasonic power to 200W; control the temperature between 53-57℃;

[0013] S2.4, mixing and time control: mix the green tea powder and deionized water evenly, put them into the container of the ultrasonic device; turn on the ultrasonic device to make the ultrasonic wave act evenly on the mixture; the extraction time is controlled within 30-45 minutes;

[0014] S3, extraction: add citrate aqueous solution to the extract, adjust the pH value to 5.0-6.0, heat to 60-70°C, and maintain for 2-3 hours for extraction;

[0015] S4. Macroporous resin adsorption: The extract is centrifuged to remove solid residues to obtain an extract; the extract is then adsorbed through a macroporous resin column to remove most impurities; the pore size and polarity of the resin are carefully selected for efficient adsorption of EGCG;

[0016] S5, deep purification: the effluent after macroporous resin adsorption is further passed through a polyamide resin column for deep purification to obtain a high-purity EGCG solution;

[0017] S5.1: Prepare a polyamide resin column: select a polyamide resin column with a diameter of 2.5 cm and a height of 30 cm; use 50 g of polyamide resin;

[0018] S5.2: Sample loading: After filtering or clarifying the effluent from the macroporous resin adsorption, use a pump to load it into the polyamide resin column; and control the loading flow rate to maintain at 1-2 mL / min;

[0019] S5.3: Elution: Use ethanol-water mixture for elution; control the elution flow rate to maintain at 2-4 mL / min, and collect the eluate at the same time; set multiple elution gradients;

[0020] S5.4: Collection and detection: Collect the eluates of each elution gradient and mark them clearly; use high performance liquid chromatography detection method to quantitatively analyze the EGCG content in the eluate; according to the test results, combine the eluates containing higher EGCG concentrations to obtain a high-purity EGCG solution;

[0021] S5.5: Resin regeneration: After deep purification is completed, the polyamide resin column is regenerated with an acid solution to restore its adsorption performance; after the regeneration is completed, the resin column is rinsed with clean water and stored in a suitable environment for next use;

[0022] S6, concentration and crystallization;

[0023] S7. Drying and packaging.

[0024] Furthermore, in S1, the pretreatment includes: washing, destemming, drying the picked green tea buds to a moisture content of 10%, and then crushing them into fine powder.

[0025] Furthermore, in S2.2, the ratio of deionized water to green tea powder is 10:1 to 20:1.

[0026] Furthermore, in S2.3, the following steps are included: setting the frequency of the ultrasonic wave to 40 kHz; and selecting an ultrasonic probe with a diameter of 10 cm.

[0027] Furthermore, in S2.3, the following steps are also included: Device parameter verification: adjusting the position and angle of the probe.

[0028] Furthermore, in S5.3, the elution gradient is set:

[0029] Gradient 1: pure water (0% ethanol) - 100 mL;

[0030] Gradient 2: ethanol-water (20:80, v / v) - 200 mL;

[0031] Gradient 3: ethanol-water (50:50, v / v) - 300 mL;

[0032] Gradient 4: ethanol-water (80:20, v / v) - 200 mL;

[0033] Gradient 5: pure ethanol (100% ethanol) - 100 mL.

[0034] Further, in S6, the concentration and crystallization include: concentrating the high-purity EGCG solution, and then crystallizing it at a low temperature to obtain EGCG crystals.

[0035] The invention also provides epigallocatechin gallate extracted by the extraction method.

[0036] The present invention also provides an application of the epigallocatechin gallate in the preparation of drugs for treating Alzheimer's disease and Parkinson's disease, including the preparation of EGCG tablets and EGCG capsules. The EGCG tablets are prepared as follows: 50% (calculated by weight, i.e., the weight proportion of EGCG in each tablet) of EGCG and EGCG are uniformly mixed with 48% (filler and thickener) of microcrystalline cellulose and 2% (lubricant); the mixture is made into granules by using wet granulation technology; the granules are dried and the particle size is adjusted by a granulator; the granules are quality inspected, including content determination and appearance inspection; and the qualified granules are pressed into tablets, each containing 50 mg of EGCG.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] Extraction of EGCG from Longjing tea; Type of green tea: Longjing; Mass of green tea fine powder: 10 g; Deionized water: 150 ml (mass ratio 15:1); Ultrasonic conditions: power 200 W, frequency 40 kHz; Extraction temperature: 55°C; Extraction time: 40 minutes; The best embodiment; EGCG dissolution rate (%) is 93.2; High EGCG content and less impurities are ensured; The ultrasonic-assisted extraction technology significantly improves the dissolution rate of EGCG, thereby improving the extraction efficiency.

[0039] Combining multiple technologies such as ultrasonic-assisted extraction, leaching, macroporous resin adsorption and deep purification of polyamide resin, impurities are effectively removed, the extraction rate and purity of EGCG are improved, and the operation is simple and environmentally friendly.

[0040] EGCG: 50%, microcrystalline cellulose: 48%, magnesium stearate: 2% are the optimal components for the preparation of EGCG tablets. The preparation process of EGCG tablets is simple and low-cost, and it has broad application prospects in the field of neurodegenerative disease treatment, namely the treatment of Alzheimer's disease and Parkinson's disease.

[0041] In the pretreatment stage, a composite enzyme containing specific ingredients is used to treat the cleaned and destemmed green tea buds under precisely controlled temperature, time and dosage conditions, and then dried and crushed. This can not only inhibit the oxidation of other easily oxidized polyphenols in tea to build a solid and stable raw material foundation, ensure the smooth progress of subsequent steps, but also facilitate full contact with the solvent during subsequent extraction. It has a positive impact on each step, providing high-quality raw materials for ultrasonic-assisted extraction to improve the dissolution rate, making the extraction more sufficient, helping macroporous resin adsorption and deep purification to be carried out efficiently, ensuring that high-quality crystals are obtained through concentrated crystallization, facilitating drying and packaging to ensure product quality, and enabling EGCG tablets to better exert their efficacy. On the whole, the synergistic cooperation of various technologies has improved the extraction rate and purity of EGCG, and the operation is simple and environmentally friendly. The obtained high-purity EGCG products have broad application prospects. The preparation process of EGCG tablets is simple and low-cost, and has obvious advantages in the treatment of Alzheimer's disease and Parkinson's disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 The present invention is a schematic diagram of an extraction method of EGCG in tea leaves and its application. DETAILED DESCRIPTION

[0043] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0044] See also Figure 1 , the present invention provides a technical solution:

[0045] See also Figure 1 As shown, a method for extracting EGCG from tea leaves and its application, comprising the following steps:

[0046] Extraction method of EGCG;

[0047] S1. Preprocessing:

[0048] In the entire extraction process, the pretreatment stage plays a key role in the foundation. The specific steps are as follows:

[0049] Specific biological enzyme treatment: First, the picked green tea buds are washed and destemmed, and then introduced into a specific biological enzyme treatment. This biological enzyme is a composite enzyme that carefully contains cellulase, pectinase and polyphenol oxidase inhibitors extracted from Bacillus microorganisms. Bacillus microorganisms are widely distributed in the natural environment, which makes them extremely easy to obtain. For example, the industry has a relatively mature cultivation technology for Bacillus subtilis, which provides great convenience for the large-scale production of polyphenol oxidase inhibitors for tea pretreatment, while effectively reducing costs. The Bacillus microorganisms produce proteins or polypeptides, which cleverly change the spatial structure of the enzyme by binding to polyphenol oxidase, thereby providing a relatively stable inhibitory effect. This unique inhibition method has significant beneficial effects. Without affecting the activity of EGCG, it can strongly inhibit the oxidation of other easily oxidized polyphenols in tea during the pretreatment process, laying a solid and stable raw material foundation for the subsequent extraction steps, ensuring that the subsequent links can be carried out smoothly based on high-quality raw materials, and minimizing the loss of components caused by raw material oxidation and the impact on extraction purity. During this process, the enzymatic hydrolysis temperature needs to be precisely controlled at 35-38°C, the enzymatic hydrolysis time is set to 20-30 minutes, and the enzyme dosage is strictly controlled to 0.05-0.1 grams of compound enzyme per gram of tea to ensure that the biological enzyme has the best treatment effect.

[0050] Drying and crushing: After enzymatic hydrolysis, the tea leaves are dried to a moisture content of 10% and then crushed into fine powder so that the tea leaves can fully contact with the solvent in the subsequent extraction step, which is conducive to the dissolution of the active ingredients.

[0051] S2, Ultrasonic assisted extraction:

[0052] S2.1. Raw material selection and crushing: Select fresh green tea (such as Longjing, Maofeng and other well-known varieties) that is free of impurities and rich in EGCG after the above pretreatment, and use a grinder to crush it to an appropriate particle size (for example, 100-200 mesh). This treatment makes the tea cell walls easier to break, thereby increasing the dissolution rate of EGCG. The stable foundation provided for the raw materials during the pretreatment further ensures the supply of high-quality raw materials for EGCG dissolution here, indirectly helping to improve the dissolution rate.

[0053] S2.2, solvent ratio: Deionized water is used as the extraction solvent in the extraction process. Because of its pure and impurity-free characteristics, it can ensure that there are no impurities interfering with the extraction effect of EGCG. And deionized water and green tea powder are mixed in a ratio of 10:1 to 20:1 (mass ratio). For example, when 10 grams of green tea powder is used, 100 ml to 200 ml of deionized water is added accordingly. The reasonable solvent ratio is combined with the raw material stability guaranteed by the pretreatment, which is more conducive to the full dissolution of EGCG.

[0054] S2.3, Ultrasonic equipment settings:

[0055] Power and frequency settings: According to the experimental needs, through the control panel, the ultrasonic power can be set to 200W (it can also be flexibly adjusted within the range of 100-500W according to the actual experimental needs), and the frequency can be set to 40kHz (or appropriately adjusted within the range of 20-100kHz according to the specific characteristics of green tea and experimental requirements).

[0056] Probe and container selection: Select an ultrasonic probe with a diameter of 10 cm and properly install it on the bracket of the extractor. Make sure that the probe is at an appropriate distance from the bottom of the extraction container so that the ultrasonic wave can evenly act on the mixture of green tea powder and deionized water.

[0057] Equipment parameter verification: Carefully adjust the position and angle of the probe to ensure that the ultrasound can act evenly on the entire mixture. During the extraction process, continuously monitor the temperature of the extract, and adjust the power of the ultrasonic equipment or the heating equipment accordingly according to the actual situation to strictly control the temperature between 53-57°C. If you want to further increase the dissolution rate of EGCG, you can also use the stirring function of the equipment or stir it manually. The good state of the raw materials guaranteed in the pretreatment stage can allow ultrasound to better promote dissolution under such conditions and improve the extraction efficiency.

[0058] S2.4, mixing and extraction control: put the green tea powder and deionized water mixed in proportion into the container of the ultrasonic equipment. After turning on the ultrasonic equipment, accurately adjust the power and frequency to the appropriate values ​​to ensure that the ultrasonic wave acts evenly on the mixture. During the entire extraction process, the temperature of the extract is monitored at all times. By reasonably adjusting the power of the ultrasonic equipment or the heating equipment, the temperature is stabilized between 50-60°C, and the extraction time is controlled at 30-45 minutes. During this period, the mixture can also be stirred appropriately as needed to further increase the dissolution rate of EGCG. The quality of the raw materials guaranteed in the pretreatment stage enables EGCG to be dissolved more efficiently and stably during this extraction process.

[0059] S2.5, post-extraction processing: After the extraction is completed, turn off the ultrasonic equipment, take out the extract, and preliminarily judge the dissolution of EGCG by observing the color, transparency and other appearance characteristics of the extract. After that, the extract will enter the subsequent processing process, including centrifugal separation, macroporous resin adsorption, deep purification and other steps, aiming to further improve the purity and concentration of EGCG. The high-quality raw material foundation laid by the pretreatment reduces the interference of impurities in the subsequent purification links and improves the overall purification effect.

[0060] Example display:

[0061] Example 1: Extraction of EGCG from Longjing tea:

[0062] Materials and methods: The type of green tea selected was Longjing. 10 g of green tea fine powder was mixed with 150 ml of deionized water (mass ratio 15:1). The ultrasonic conditions were set to 200 W power and 40 kHz frequency. The extraction temperature was controlled at 55 °C and the extraction time was 40 min.

[0063] Results: Under this specific ultrasonic condition, the EGCG dissolution rate of up to 93.2% was achieved, which fully demonstrated that this method is extremely effective for the extraction of EGCG in Longjing tea. This achievement was achieved thanks to the optimization of raw materials in the early pretreatment stage and the reasonable setting of various extraction parameters, which worked together to achieve a high dissolution rate and also proved the importance of the coordination of various links. The appropriate solvent ratio, ultrasonic power and frequency, extraction temperature and time and other factors all have a significant impact on the dissolution rate of EGCG that cannot be ignored.

[0064] Example 2: Extraction of EGCG from Biluochun:

[0065] Materials and methods: Biluochun was used as the green tea raw material. 10 g of its fine powder was taken and 180 ml of deionized water was added (mass ratio 18:1). The ultrasonic conditions were set to 300 W power, 30 kHz frequency, 58 °C extraction temperature and 35 min extraction time.

[0066] Results: For Biluochun, a higher EGCG dissolution rate was also achieved by specifically adjusting the ultrasonic conditions, which once again verified that the extraction method is universal for different varieties of green tea. The consistency and stability of the raw materials ensured in the pretreatment step is one of the foundations for achieving this universality.

[0067] Example 3: Extraction of EGCG from Huangshan Maofeng:

[0068] Materials and methods: Huangshan Maofeng was used as the green tea sample, with a fine powder mass of 10 g and 120 ml of deionized water (mass ratio 12:1). The ultrasonic conditions were set as power 250 W, frequency 50 kHz, extraction temperature 52 °C, and extraction time 45 min.

[0069] Results: For Huangshan Maofeng, after reasonable adjustment of ultrasonic conditions, the EGCG dissolution rate was similar to that of Longjing and Biluochun, which strongly proved that the extraction method is not only universal, but also can maintain its effectiveness under different conditions. The pretreatment step played an important role in providing a stable state for different varieties of green tea raw materials.

[0070]

[0071] From the data analysis of the above table, it can be seen that Example 1 (i.e., EGCG extraction from Longjing tea, specific parameters are: green tea type is Longjing, green tea fine powder mass is 10 g, deionized water is 150 ml, mass ratio is 15:1, ultrasonic condition power is 200 W, frequency is 40 kHz, extraction temperature is 55 ° C, extraction time is 40 minutes) is the best embodiment, and its EGCG dissolution rate can reach 93.2%. The achievement of this optimal effect is inseparable from the fine treatment of the raw materials in the pretreatment link and the precise coordination of the subsequent steps.

[0072] S3, extraction:

[0073] Add citrate aqueous solution to the extract, adjust the pH value precisely to 5.0-6.0, then heat to 60-70°C and keep it for 2-3 hours for extraction. The raw material stability guaranteed by pretreatment allows EGCG to be further dissolved from the raw material more fully under suitable acid-base environment and temperature conditions during the extraction process, reducing the impact of extraction efficiency and effect caused by early changes in raw materials, which helps to increase the overall extraction volume.

[0074] S4, macroporous resin adsorption:

[0075] The extract is first centrifuged to remove the solid residue, so as to obtain a relatively pure extract. Then the extract is adsorbed by a macroporous resin column with carefully selected pore size and polarity, and most of the impurities are removed by the specific adsorption of EGCG by the resin. The effective control of impurities in the raw materials and the stability of the components guaranteed by the pretreatment stage enable the macroporous resin adsorption process to more efficiently adsorb the target component EGCG, reduce the problem of poor adsorption effect caused by factors such as excessive impurities, and ensure the basis for subsequent further purification.

[0076] S5, deep purification:

[0077] S5.1. Preparation of polyamide resin column: Select a polyamide resin column with a diameter of 2.5 cm and a height of 30 cm, use 50 g of polyamide resin, and carefully load the polyamide resin into the resin column according to the standard operating instructions to ensure that the resin bed is uniform and free of bubbles, creating good conditions for subsequent adsorption and purification. The quality control of raw materials and intermediates in the early steps, especially the pretreatment stage, will help improve the deep purification effect of the polyamide resin column here, so that higher purity EGCG can be obtained by subsequent elution.

[0078] S5.2. Sample loading: After filtering or clarifying the effluent from the macroporous resin adsorption, use a pump to transport it to the polyamide resin column. At the same time, strictly control the loading flow rate to keep it at 1-2mL / min to ensure that the sample can fully contact with the resin. On the basis of high-quality raw materials and good treatment of each link in the early stage, the effect of deep purification is further guaranteed, and the purity of EGCG is improved and the efficiency is improved.

[0079] S5.3, elution: ethanol-water mixture was used for elution operation, and the elution flow rate was precisely controlled at 2-4 mL / min. The eluate was carefully collected at the same time. The recovery rate and purity of EGCG were optimized by reasonably setting multiple elution gradients. The effects of each elution gradient are as follows:

[0080] Gradient 1: 100 mL pure water (0% ethanol): mainly used to remove nonspecific impurities adsorbed on the resin, clearing the way for more accurate elution of EGCG in the subsequent steps. Thanks to the effective control of impurities in raw materials and intermediates in the previous steps, this gradient can better play the role of removing impurities.

[0081] Gradient 2: 200 mL ethanol-water (20:80, v / v): As the ethanol concentration gradually increases, a portion of impurities that are relatively weakly bound to the resin and a small amount of EGCG can be eluted, and the preliminary separation of impurities and components can be achieved in an orderly manner based on overall good raw materials and processes.

[0082] Gradient three: 300 mL ethanol-water (50:50, v / v): This is the key gradient after a large number of experimental optimizations. According to past experimental data, the ethanol-water mixture at this concentration can elute EGCG very effectively, while minimizing the elution of other impurities. It is an important step in obtaining a high-purity EGCG solution. The early stages, especially the pretreatment, ensure the quality of the raw materials and intermediates, so that EGCG can be eluted more efficiently and maintain a high purity under this gradient.

[0083] Gradient 4: 200 mL ethanol-water (80:20, v / v): further increase the ethanol concentration to elute impurities that are tightly bound to the resin and residual EGCG, and further improve the effect of impurity removal and EGCG recovery.

[0084] Gradient 5: 100 mL pure ethanol (100% ethanol): Use pure ethanol to ensure that all substances on the resin can be eluted, to ensure that EGCG and other related components are recovered as completely as possible, and to provide a comprehensive sample basis for subsequent further analysis and processing.

[0085] Operation and detection: During each gradient elution, always maintain the same elution flow rate (such as 2-4mL / min), and carefully collect the eluate of each gradient, and then use high performance liquid chromatography (HPLC) to quantitatively analyze the eluate of each gradient, accurately detect the content of EGCG and other impurities therein, and carry out scientific and reasonable subsequent treatment based on the test results.

[0086] Results analysis: HPLC detection found that the EGCG content in the eluent of gradient 3 (ethanol-water 50:50, v / v) was the highest, and the impurity content was relatively low. Therefore, by combining the eluents of gradient 3, a high-purity EGCG solution can be obtained. After further HPLC detection, the purity of EGCG in the solution can reach more than 95%, and the recovery rate is also at a high level. This series of good results is inseparable from the coordination of various links in the entire extraction process, especially the solid foundation laid by the pretreatment link.

[0087] S5.4. Collection and detection: Collect the eluates from each elution gradient and mark them clearly. Continue to use high-performance liquid chromatography to perform accurate quantitative analysis of the EGCG content in the eluate. Based on the test results, rigorously combine the eluates containing higher EGCG concentrations to obtain a high-purity EGCG solution. The entire process relies on the effective treatment and quality control of the samples in the previous steps to ensure that a high-purity target product is finally obtained.

[0088] S5.5, Resin regeneration: After the deep purification work is completed, the polyamide resin column is regenerated with an acid solution to restore its adsorption performance, so that it can be recycled and reduce costs. After the regeneration treatment is completed, the resin column is carefully rinsed with clean water and then properly stored in a suitable environment for the next use, ensuring the sustainability and economy of the entire extraction process. The stable and high-quality raw material supply and the good operation of each link will help to extend the service life of the resin and maintain good performance after regeneration.

[0089] S6, Concentration and Crystallization

[0090] The high-purity EGCG solution obtained after deep purification is reasonably concentrated and then placed in a low-temperature environment for crystallization to obtain EGCG crystals. The high-purity EGCG solution guaranteed by the previous steps provides a high-quality raw material basis for the crystallization process, which helps to obtain high-quality, high-purity crystal products.

[0091] S7. Drying and packaging

[0092] The generated EGCG crystals are vacuum dried to remove residual moisture and other impurities to ensure the stability and quality of the product. They are then packaged and stored in a standardized manner for subsequent storage, transportation and application. The close coordination and high-quality operation of each link in the entire extraction process ultimately ensure that the product enters the application link in good condition.

[0093] Summary of the advantages of extraction methods:

[0094] Advantages of the best embodiment:

[0095] Taking the extraction of EGCG from Longjing tea as the best example, it fully ensures the high content of EGCG and the low level of impurities. This advantage is achieved thanks to the fine treatment of raw materials in the pretreatment stage and the reasonable coordination of a series of technical links such as subsequent ultrasonic-assisted extraction. The ultrasonic-assisted extraction technology significantly improves the dissolution rate of EGCG, thereby effectively improving the overall extraction efficiency and laying the foundation for obtaining high-quality EGCG products.

[0096] Comprehensive technical advantages:

[0097] This extraction method organically integrates a variety of advanced and effective technical means such as ultrasonic-assisted extraction, leaching, macroporous resin adsorption and polyamide resin deep purification. Each link complements each other. The pretreatment link provides a stable and high-quality raw material basis for the entire process. The subsequent steps extract and purify EGCG from different angles. The synergistic effect can effectively remove impurities, greatly improving the extraction rate and purity of EGCG. The entire operation process is relatively simple, green and environmentally friendly, in line with the current high standards for chemical processes, and has good promotion and application value.

[0098] Product application advantages:

[0099] The high-purity EGCG products extracted by this method have broad application prospects in many fields such as food, health products, and cosmetics, and can provide high-quality raw material support for related industries. At the same time, the extraction method itself is easy to operate, has low energy consumption, and is pollution-free. It is highly consistent with the concepts of green chemistry and sustainable development, and shows significant advantages in terms of product quality and process characteristics.

[0100] EGCG product related applications:

[0101] Products that provide extracted EGCG:

[0102] The present invention successfully provides epigallocatechin gallate (EGCG) obtained by the above-mentioned rigorous and efficient extraction method, which has the characteristics of high purity and high quality, and creates good conditions for subsequent applications in different fields, especially the medical field.

[0103] Application in drug preparation:

[0104] The present invention further explores the application of the above-mentioned EGCG in the preparation of drugs for treating Alzheimer's disease and Parkinson's disease, specifically covering two aspects of the preparation of EGCG tablets and the preparation of EGCG capsules.

[0105] Drug preparation:

[0106] Material preparation: EGCG: The high-purity EGCG obtained by the extraction method described in detail above has quality and purity advantages that lay the foundation for the efficacy and stability of the drug.

[0107] Excipients: Select commonly used pharmaceutical preparation excipients, such as microcrystalline cellulose, magnesium stearate, etc., which play a key role in drug molding and performance optimization.

[0108] Preparation method:

[0109] Example 1: Preparation of EGCG tablets:

[0110] Optimization of auxiliary material ratio: EGCG is mixed evenly with auxiliary materials such as microcrystalline cellulose and magnesium stearate in a specific ratio. Among them, the optimal composition verified by a large number of experiments is 50% EGCG (calculated by weight, that is, the weight proportion of EGCG in each tablet), 48% microcrystalline cellulose (as a filler and thickener, it helps to maintain the shape and texture of the tablet), and 2% magnesium stearate (acts as a lubricant to ensure the smooth progress of the tableting process). This reasonable ratio not only takes into account the effective content of the active ingredients of the drug, but also takes into account the feasibility of the preparation process and the quality of the final tablets. The high purity of EGCG itself is the key prerequisite to ensure the effectiveness of the drug. The quality of EGCG guaranteed by the pretreatment and extraction process enables it to better exert its efficacy in tablets.

[0111] Granulation and subsequent processing: The wet granulation technology is used to make the above-mentioned uniform mixture into granules, and then the granules are dried, and then the particle size is adjusted by a granulator to reach an appropriate range to meet the subsequent tableting requirements. Then, strict quality inspection work is carried out on the granules, covering content determination, appearance inspection and other aspects to ensure that each batch of granules meets the high-quality drug production standards. Finally, the qualified granules are pressed into tablets by professional equipment. Each tablet contains a precise dose of EGCG (50mg), which provides a guarantee for the accurate use of the drug.

[0112] Example 2: Preparation of EGCG capsules:

[0113] Mixing and filling: EGCG is fully and evenly mixed with auxiliary materials such as microcrystalline cellulose in a reasonable proportion, and then the mixture is accurately filled into empty capsules using a filling machine. During this process, the high-quality characteristics of EGCG ensure the stability of the drug in the capsule and the effectiveness of subsequent release. The entire filling operation must be strictly controlled to ensure that the filling amount of each capsule is accurate and uniform.

[0114] Quality inspection and packaging: The filled capsules are also subject to comprehensive quality inspection, including key items such as content determination and appearance inspection. Only capsules that pass the inspection can enter the packaging stage. Capsule products that have been standardized and packaged are easy to store, transport and use by patients, and are then put on the market to serve the treatment needs of related diseases.

[0115] Experimental design and data analysis

[0116] Experimental design:

[0117] In order to scientifically evaluate the efficacy of EGCG tablets and capsules in the treatment of Alzheimer's disease (AD) and Parkinson's disease (PD), the following animal experiments were carefully designed: two groups of professionally modeled mouse models were selected, namely AD model and PD model. For each group of mice, EGCG tablets, EGCG capsules and control group drugs (drugs available on the market and commonly used for AD or PD treatment were selected as controls), and the dosing cycle was uniformly set to 4 weeks. Through such a rigorous and standardized experimental design, it aims to objectively compare the differences in the effects of different dosage forms of EGCG drugs and existing therapeutic drugs, and provide a strong basis for the subsequent judgment of the application value of EGCG in the treatment of related diseases.

[0118] Comparative data presentation and analysis:

[0119] AD Model:

[0120]

[0121]

[0122] It can be clearly seen from the experimental data of the above AD model that under the same dosage (50 mg / kg), the therapeutic effect of EGCG tablets is significantly better than that of EGCG capsules, and its behavioral score (improvement rate) reached 40%, compared with 35% for capsules, and both were significantly better than the control group (improvement rate was only 20%). This result shows that the high-purity EGCG obtained by the extraction method described above has different efficacy in different dosage forms when preparing drugs for the treatment of AD, and the tablet dosage form shows more prominent advantages. It also confirms that the extracted EGCG itself has good medicinal value, which is due to the quality assurance of EGCG in the entire extraction process.

[0123] PD Model:

[0124]

[0125] Similar results were also presented in the PD model, which once again verified that EGCG is a key active ingredient in drugs, and its content change has a crucial impact on the therapeutic effect of the drug. This also indirectly reflects the scientificity and necessity of the extraction method adopted in the present invention to obtain high-purity EGCG and determine a reasonable proportion of excipients. The combined effect of the two ensures the application advantages of the drug in the field of neurodegenerative disease treatment.

[0126] It is necessary to add that:

[0127] In the entire extraction process, the pretreatment link plays a key role in the foundation. First, the picked green tea buds are washed and destemmed, and then treated with a specific biological enzyme. This biological enzyme is a composite enzyme, including cellulase, pectinase and polyphenol oxidase inhibitor extracted from Bacillus microorganisms. Bacillus microorganisms are widely distributed in the natural environment and are easy to obtain. For example, Bacillus subtilis has a relatively mature cultivation technology in industry, which makes it convenient to mass-produce polyphenol oxidase inhibitors for tea pretreatment, and the cost is relatively low. The Bacillus microorganism produces proteins or polypeptides to inhibit polyphenol oxidase, which changes the spatial structure of the enzyme by binding to polyphenol oxidase, thereby providing a relatively stable inhibitory effect. This inhibition method has a significant effect. Without affecting the activity of EGCG, it can strongly inhibit the oxidation of other easily oxidized polyphenols in tea during the pretreatment process, minimize the loss of components caused by raw material oxidation and the impact on the extraction purity, and lay a solid foundation for the subsequent extraction steps. Stable raw material foundation, ensuring that the subsequent links can be carried out smoothly based on high-quality raw materials. In addition, the enzymatic hydrolysis temperature is strictly controlled at 35-38°C, the enzymatic hydrolysis time is set to 20-30 minutes, and the enzyme dosage is precisely controlled to 0.05-0.1 grams of compound enzyme per gram of tea, so as to ensure that the biological enzyme has the best treatment effect. After the enzymatic hydrolysis is completed, the tea leaves are dried to a moisture content of 10% and then crushed into fine powder. This treatment allows the tea leaves to fully contact with the solvent in the subsequent extraction step, which is conducive to the dissolution of the active ingredients.

[0128] In the ultrasonic-assisted extraction step, fresh, impurity-free green tea rich in EGCG after the above-mentioned pretreatment is selected for crushing and other operations. The stable foundation provided for the raw materials in the pretreatment further guarantees the supply of high-quality raw materials for the dissolution of EGCG here, and indirectly helps to improve the dissolution rate. For example, in the embodiment, when EGCG is extracted from Longjing tea, the pretreated raw materials enable an EGCG dissolution rate of up to 93.2% under specific ultrasonic conditions (power 200W, frequency 40kHz, extraction temperature 55°C, extraction time 40 minutes), which fully demonstrates that the pretreatment provides a high-quality raw material prerequisite for ultrasonic-assisted extraction, so that the extraction process can be carried out more efficiently, allowing ultrasound to better play a role in promoting dissolution and improve extraction efficiency.

[0129] During the extraction process, the raw material stability ensured by the pretreatment allows EGCG to be further dissolved from the raw material more fully under suitable acid-base environment (adding citrate aqueous solution to adjust the pH value to 5.0-6.0) and temperature conditions (heating to 60-70°C for 2-3 hours), reducing the impact of the extraction efficiency and effect caused by the previous changes in the raw materials, helping to increase the overall extraction volume and ensure the continuity and efficiency of the entire extraction process.

[0130] The effective control of impurities in the raw materials and the stability of the components guaranteed in the pretreatment stage enable the macroporous resin adsorption process to more efficiently adsorb the target component EGCG, reducing the problem of poor adsorption effect caused by factors such as excessive impurities, and ensuring the basis for subsequent further purification. The macroporous resin can remove most impurities more smoothly by virtue of its specific adsorption effect on EGCG, laying a good foundation for the subsequent deep purification stage.

[0131] In the deep purification stage, the quality control of raw materials and intermediates in the early steps, especially the pretreatment stage, helps to improve the deep purification effect of the polyamide resin column here. For example, in the elution process of the polyamide resin column, the quality of the raw materials guaranteed by the pretreatment enables each elution gradient to play a better role. For example, gradient three (ethanol-water 50:50, v / v) can elute EGCG very effectively, while minimizing the elution of other impurities, and finally obtain a high-purity EGCG solution after deep purification (purity reaches more than 95%, and the recovery rate is also at a high level). This series of good results is inseparable from the solid foundation laid by the pretreatment stage, which helps the entire deep purification process achieve high-quality purification goals.

[0132] The high quality of raw materials and intermediate products ensured in the previous steps, especially the pretreatment stage, ensures that the high-purity EGCG solution obtained after deep purification provides a high-quality raw material basis for the crystallization stage, which helps to obtain high-quality, high-purity EGCG crystals and ensure the quality of the final product.

[0133] The close coordination and high-quality operation of the previous links of the entire extraction process, including the pretreatment link, ultimately ensure the high quality of the generated EGCG crystals, so that during the drying and packaging steps, when the crystals are vacuum dried, residual moisture and other impurities can be better removed, ensuring the stability and quality of the product, so that it can be packaged and stored in a standardized manner later, allowing the product to enter the application link in good condition.

[0134] In the preparation of EGCG tablets, high-purity EGCG obtained by the extraction method described above is used, and the quality of EGCG guaranteed by the pretreatment and the entire extraction process enables it to better exert its efficacy in tablets. Especially in the optimization of the ratio of excipients, high-quality EGCG raw materials combined with a reasonable ratio of excipients (EGCG accounts for 50%, microcrystalline cellulose accounts for 48%, and magnesium stearate accounts for 2%) not only take into account the effective content of the active ingredients of the drug, but also take into account the feasibility of the preparation process and the quality of the final tablets, so that the prepared tablets can show better efficacy in the treatment of neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease, and ensure the smooth progress of the tablet preparation process, realizing the transformation from high-quality raw materials to high-quality drug products.

[0135] In general, the present invention combines various technical means such as ultrasonic assisted extraction, leaching, macroporous resin adsorption and polyamide resin deep purification through such pretreatment steps, and each link complements each other. Pretreatment provides a stable and high-quality raw material basis for the entire process. The subsequent steps extract and purify EGCG from different angles respectively. Under the synergistic effect, impurities can be removed efficiently, which greatly improves the extraction rate and purity of EGCG, so that the EGCG product finally extracted has the characteristics of high purity and high quality. This not only makes EGCG have broad application prospects in many fields such as food, health products, and cosmetics, but also shows significant advantages in the preparation of drugs for treating neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. For example, the preparation process of EGCG tablets is simple and low in cost. It has good efficacy in the treatment of related diseases and has broad application prospects. The overall extraction method is simple to operate, has low energy consumption, and is pollution-free. It is highly consistent with the concepts of green chemistry and sustainable development. It reflects significant advantages and important values ​​in every link from raw material pretreatment to final product application.

[0136] Extraction of EGCG from Longjing tea; Type of green tea: Longjing; Mass of green tea fine powder: 10 g; Deionized water: 150 ml (mass ratio 15:1); Ultrasonic conditions: power 200 W, frequency 40 kHz; Extraction temperature: 55°C; Extraction time: 40 minutes; The best embodiment; EGCG dissolution rate (%) is 93.2; High EGCG content and less impurities are ensured; The ultrasonic-assisted extraction technology significantly improves the dissolution rate of EGCG, thereby improving the extraction efficiency.

[0137] Combining multiple technologies such as ultrasonic-assisted extraction, leaching, macroporous resin adsorption and deep purification of polyamide resin, impurities are effectively removed, the extraction rate and purity of EGCG are improved, and the operation is simple and environmentally friendly.

[0138] EGCG: 50%, microcrystalline cellulose: 48%, magnesium stearate: 2% are the optimal components for the preparation of EGCG tablets. The preparation process of EGCG tablets is simple and low-cost, and it has broad application prospects in the field of neurodegenerative disease treatment, namely the treatment of Alzheimer's disease and Parkinson's disease.

[0139] Summarize:

[0140] Advantages of optimal components:

[0141] Through the above rigorous comparative experiments (comparison between experimental group 1 (optimal component) and experimental group 2 (control group component)), it is clearly observed that a reasonable increase in EGCG content (from 40% to 50%) can significantly improve the therapeutic effect of the drug in AD and PD models. This result intuitively and powerfully proves that EGCG is the core active ingredient of the drug to exert its therapeutic effect, and the precise control of its content plays a decisive role in improving the efficacy of the drug. At the same time, it also re-emphasized the importance of ensuring the high purity and high quality of EGCG throughout the extraction process. Only high-quality EGCG raw materials can give full play to its efficacy advantages under a reasonable formula and provide strong support for the treatment of related diseases.

[0142] Simple preparation method advantages:

[0143] In the preparation process of EGCG tablets, the wet granulation and tableting technology used has significant advantages. The application of these two technologies makes the entire tablet preparation process simple and efficient, and easy to carry out production operations on an industrial scale. It can not only ensure the quality stability of the drug, but also effectively control production costs and improve production efficiency, laying a solid technological foundation for large-scale production and market promotion of drugs. In addition, high-quality EGCG raw materials can be better converted into drug products with good therapeutic effects under such a preparation process, realizing efficient conversion from raw materials to finished drugs.

[0144] Good application prospects advantages:

[0145] Taking into account the significant therapeutic effects of EGCG in AD and PD models, as well as the simplicity of the tablet preparation method and the characteristics of industrial production, it can be clearly seen that this drug has broad application prospects in the treatment of neurodegenerative diseases. In the future, with further research and development, it is expected that more effective treatment plans will be developed based on this high-quality EGCG raw material and mature preparation process, which will bring good news to the vast number of patients suffering from such diseases, and also provide new directions and impetus for the development of the pharmaceutical industry in related fields, and promote the entire industry to move towards a more efficient and innovative direction.

[0146] In summary, the extraction method based on EGCG in tea leaves and its application in the preparation of drugs for the treatment of Alzheimer's disease and Parkinson's disease proposed in the present invention have significant advantages and important values ​​from the perspectives of the innovation and scientificity of the extraction process, the rationality of drug preparation and the broad application prospects, and are expected to play a positive and far-reaching role in related fields.

Claims

1. A method for extracting EGCG from tea leaves, characterized in that: The following steps are involved: S1. Pretreatment: After washing and destemming the picked green tea buds, they are treated with a specific biological enzyme; the biological enzyme is a composite enzyme, comprising cellulase, pectinase and a polyphenol oxidase inhibitor extracted from a microorganism of the genus Bacillus; the enzymatic hydrolysis temperature is controlled at 35-38°C, the enzymatic hydrolysis time is 20-30 minutes, and the enzyme dosage is 0.05-0.1 grams of composite enzyme per gram of tea leaves; after the enzymatic hydrolysis is completed, the tea leaves are dried to a moisture content of 10%, and then crushed into fine powder; S2, ultrasonic-assisted extraction: mix green tea powder with deionized water and extract using ultrasonic technology; the extraction temperature is controlled at 50-60°C and the extraction time is 30-45 minutes; S2.

1. Selection and crushing: Select fresh, impurity-free, EGCG-rich and pre-treated green tea; crush the green tea using a crusher; S2.2, extraction and ratio control: deionized water is used as the extraction solvent; the ratio of deionized water to green tea powder is 10:1 to 20:1; S2.3, set the operating state of the ultrasonic equipment; set the ultrasonic power to 200W; control the temperature between 53-57℃; S2.4, mixing and time control: mix the green tea powder and deionized water evenly, put them into the container of the ultrasonic device; turn on the ultrasonic device to make the ultrasonic wave act evenly on the mixture; the extraction time is controlled within 30-45 minutes; S3, extraction: add citrate aqueous solution to the extract, adjust the pH value to 5.0-6.0, heat to 60-70°C, and maintain for 2-3 hours for extraction; S4, macroporous resin adsorption: centrifuging the extract to remove solid residues to obtain an extract; The extract is then passed through a macroporous resin column for adsorption to remove most of the impurities; The pore size and polarity of the resin were carefully selected for efficient adsorption of EGCG; S5, deep purification: the effluent after macroporous resin adsorption is further passed through a polyamide resin column for deep purification to obtain a high-purity EGCG solution; S5.1: Prepare a polyamide resin column: select a polyamide resin column with a diameter of 2.5 cm and a height of 30 cm; use 50 g of polyamide resin; S5.2: Sample loading: After filtering or clarifying the effluent from the macroporous resin adsorption, use a pump to load it into the polyamide resin column; and control the loading flow rate to maintain at 1-2 mL / min; S5.3: Elution: Use ethanol-water mixture for elution; control the elution flow rate to maintain at 2-4 mL / min, and collect the eluate at the same time; set multiple elution gradients; S5.4: Collection and detection: Collect the eluates of each elution gradient and mark them clearly; use high performance liquid chromatography detection method to quantitatively analyze the EGCG content in the eluate; according to the test results, combine the eluates containing higher EGCG concentrations to obtain a high-purity EGCG solution; S5.5: Resin regeneration: After deep purification is completed, the polyamide resin column is regenerated with an acid solution to restore its adsorption performance; after the regeneration is completed, the resin column is rinsed with clean water and stored in a suitable environment for next use; S6, concentration and crystallization; S7. Drying and packaging.

2. A method for extracting EGCG from tea leaves as claimed in claim 1, characterized in that: In S1, the pretreatment includes: washing, destemming, drying the picked green tea buds to a moisture content of 10%, and then crushing them into fine powder.

3. A method for extracting EGCG from tea leaves as claimed in claim 1, characterized in that: In S2.2, the ratio of deionized water to green tea fine powder is 10:1 to 20:

1.

4. A method for extracting EGCG from tea leaves as claimed in claim 1, characterized in that: In S2.3, the following steps are included: setting the frequency of the ultrasonic wave to 40 kHz; and selecting an ultrasonic probe with a diameter of 10 cm.

5. A method for extracting EGCG from tea leaves as claimed in claim 1, characterized in that: In S2.3, the following steps are also included: Equipment parameter verification: adjusting the position and angle of the probe.

6. A method for extracting EGCG from tea leaves as claimed in claim 1, characterized in that: In S5.3, the elution gradient is set as follows: Gradient 1: pure water (0% ethanol) - 100 mL; Gradient 2: ethanol-water (20:80, v / v) - 200 mL; Gradient 3: ethanol-water (50:50, v / v) - 300 mL; Gradient 4: ethanol-water (80:20, v / v) - 200 mL; Gradient 5: pure ethanol (100% ethanol) - 100 mL.

7. A method for extracting EGCG from tea leaves as claimed in claim 1, characterized in that: In S6, the concentration and crystallization include: concentrating the high-purity EGCG solution, and then crystallizing it at a low temperature to obtain EGCG crystals.

8. Epigallocatechin gallate extracted by the extraction method according to any one of claims 1 to 7.

9. The use of epigallocatechin gallate as claimed in claim 8 in the preparation of drugs for treating Alzheimer's disease and Parkinson's disease, characterized in that: The method includes the preparation of EGCG tablets and the preparation of EGCG capsules. The preparation of EGCG tablets is as follows: 50% of EGCG and 48% of microcrystalline cellulose and 2% of magnesium stearate are uniformly mixed; the mixture is made into granules by using wet granulation technology; the granules are dried and the particle size is adjusted by a granulator; the granules are quality inspected, including content determination and appearance inspection; and the qualified granules are pressed into tablets, each tablet containing 50 mg of EGCG.

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