Process for extracting and purifying tea polyphenol in large yellow tea

Abstract

The invention relates to the technical field of natural product extraction, in particular to an extraction and purification process of tea polyphenol in large yellow tea, the extraction process comprises the following steps: S1, raw material screening: selecting high-quality large yellow tea leaf raw materials, and treating and screening through an ultrafine grinder to obtain large yellow tea powder; s2, extracting: adding an ethanol solution with a certain concentration into the large yellow tea powder, extracting in a constant-temperature water bath, collecting primary filtrate after suction filtration, centrifuging, taking supernate, and performing low-pressure rotary evaporation to remove ethanol, so as to obtain a tea polyphenol concentrated solution; s3, extraction: transferring the concentrated solution into a Buchner funnel, respectively adding trichloromethane and ethyl acetate, carrying out organic solvent extraction at normal temperature, separating an organic phase, and retaining a tea polyphenol water layer. The process has the advantages of simple operation, high extraction efficiency, high product purity and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of natural product extraction, and particularly to an extraction and purification process of tea polyphenols from Huangdacha tea. Background Art

[0002] Tea polyphenols are the general term for polyphenolic substances in tea, including flavanols, anthocyanins, flavonoids, flavonols, phenolic acids, etc. Its main component is catechins (flavanols) and their derivatives, and catechins account for 60 - 80%. Catechins are mainly composed of several monomers such as EGC, DLC, EC, EGCG, GCG, ECG, etc. Research shows that tea polyphenols have various physiological activities such as antioxidant, radiation protection, anti - aging, lipid - lowering, blood - sugar - lowering, antibacterial, etc. In addition, tea polyphenols have been widely used in the fields of medicine, daily chemical industry, food, etc. as raw materials for health foods, natural cosmetics, or natural antioxidants.

[0003] There has been quite a lot of research on the extraction and purification process of tea polyphenols. Among them, solvent extraction and metal ion precipitation are the extraction and purification methods that have been studied and reported more. In recent years, there have also been some new methods reported, such as resin adsorption method, supercritical fluid extraction method, ultrasonic extraction method, microwave extraction method, etc. The organic solvent extraction method purifies tea polyphenols by utilizing the different solubilities of tea polyphenols and impurities in organic solvents. Commonly used extraction solvents include chloroform, petroleum ether, ethyl acetate, etc., and ethyl acetate is the most commonly used solvent in extraction. Column chromatography is also a commonly used technology for purifying tea polyphenols. The principle is to separate substances with different physical and chemical properties through the process of adsorption - desorption by utilizing the different distribution coefficients of the components of the tea polyphenol sample between two phases, so as to achieve the purpose of purification. According to the different fillers selected, it is divided into adsorption column separation method, ion - exchange column separation method, gel column separation method. Silica gel, polyamide, and macroporous resin are the most commonly used fillers at present.

[0004] Since a large number of studies have shown that the extraction efficiency, purity of tea polyphenols, and product yield of a single extraction method are not ideal, or there are various defects such as incomplete extraction, or more residues and impurities. For example, the solvent extraction method will dissolve impurities such as caffeine, which will reduce the purity of tea polyphenols, while the resin adsorption separation method has strict requirements for the quality and performance of the resin, and frequent resin replacement causes high costs. Moreover, in the existing tea polyphenol extraction methods, due to the use of heating or high - temperature methods in some processes, it is easy to cause the oxidation of tea polyphenols, the gelatinization of tea leaves or tea residues, and the co - extraction of extracts, resulting in the technical problem of low purity of tea polyphenols. Therefore, a co - extraction method that combines multiple extraction methods to extract tea polyphenols has emerged to overcome various defects existing in the single extraction method. Summary of the Invention

[0005] The object of the present invention is to solve the disadvantages existing in the prior art, and to propose an extraction and purification process of tea polyphenols from Huangda tea, so as to solve the problems of low extraction efficiency, low purity and complex operation in the prior art. The present invention realizes the efficient extraction and purification of tea polyphenols through the combination of ethanol extraction method, organic solvent extraction and macroporous resin chromatography, and has the advantages of simple operation, high extraction efficiency and high product purity.

[0006] In order to achieve the above object, the present invention adopts the following technical scheme:

[0007] An extraction and purification process of tea polyphenols from Huangda tea, the extraction process comprising the following steps:

[0008] S1. Raw material screening: Select high-quality Huangda tea leaf raw materials, process and sieve them through an ultrafine pulverizer to obtain Huangda tea powder;

[0009] S2. Extraction: Add an ethanol solution with a certain concentration to the Huangda tea powder, extract it in a constant temperature water bath, filter by suction and collect the primary filtrate, centrifuge to take the supernatant, and then perform low-pressure rotary evaporation to remove ethanol to obtain a tea polyphenol concentrate;

[0010] S3. Extraction: Transfer the concentrate to a Buchner funnel, add chloroform and ethyl acetate respectively, perform organic solvent extraction at room temperature, separate the organic phase and retain the tea polyphenol aqueous layer;

[0011] S4. Chromatographic purification: Pass the tea polyphenol aqueous layer through a macroporous adsorption resin column at a certain flow rate, elute with an ethanol solution, collect the ethanol eluate, rotary evaporate the eluate until the solid content ≥ 85%, transfer it to a vacuum freeze dryer for sublimation drying to obtain a light yellow powder of tea polyphenols.

[0012] Preferably, in the raw material screening of step S1, the following steps are included: Screen the purchased Huoshan Huangda tea leaves, select high-quality leaves, remove the leaves and branches with poor quality to ensure no mildew and no impurities, and perform ultrafine pulverization treatment on the Huangda tea leaves through a 40-mesh sieve.

[0013] Preferably, in step S2, the process is controlled by the following conditions: the material-liquid ratio of the Huangda tea coarse powder to the ethanol solution is 1:10 (g / mL) - 1:30 (g / mL), the time for extracting tea polyphenols in a constant temperature water bath with the ethanol solution is 20 - 60 min, the mass percentage of the ethanol solution used for extracting tea polyphenols is 30% - 70%, and the constant temperature water bath temperature for extracting tea polyphenols with the ethanol solution is 30 - 70 °C.

[0014] Preferably, in step S2, the primary tea polyphenol filtrate is centrifuged at 5000 r / min in a refrigerated centrifuge for 10 min, and the supernatant after centrifugation is concentrated to one-tenth of the original volume on a rotary evaporator, and the rotary evaporation temperature is 55 °C.

[0015] Preferably, in the step S3, chloroform with the same volume as the tea polyphenol concentrate is added for extraction to obtain a decaffeinated tea polyphenol extract. Then, ethyl acetate is added to the extract according to a volume ratio of 1:1 for extraction, and the extraction is carried out 2 - 3 times, 15 - 30 minutes each time. The extraction liquids are combined, and rotary evaporation is used for concentration to remove the residual ethyl acetate, and then drying on a freeze dryer can obtain the crude tea polyphenol powder.

[0016] Preferably, in the step S4, the macroporous resin chromatography column is a glass chromatography column with a specification of 16 mm × 500 mm, and the filler is macroporous resin. The chromatography column is vertically fixed on an iron stand, and the bottom of the chromatography column is plugged with absorbent cotton. The piston at the bottom of the chromatography column is closed, and ultrapure water with a height of three - quarters of the pre - installed column height is poured into the column. After the treated macroporous resin is ultrasonically degassed, it is poured into the chromatography column, and the macroporous resin is allowed to settle naturally. At the same time, the chromatography column is gently tapped by hand continuously to make the resin packed tightly in the column. After complete settlement, a layer of glass wool is covered on the adsorbent. The piston at the lower end of the chromatography column is opened to release the excess ultrapure water in the column, and the water is allowed to flow out slowly. When the liquid level is about 2 - 3 cm higher than the packing layer, the piston is closed.

[0017] Preferably, the macroporous resin filler is one of AB - 8, D101, NKA - 9, X - 5, and HPD - 600.

[0018] Preferably, in the step S4, during the macroporous resin purification process, the crude tea polyphenol powder is accurately weighed, dissolved in ultrapure water to prepare a tea polyphenol solution with a concentration of 0.5 - 3 mg / mL, and loaded onto the column at a flow rate of 1 - 4 mL / min. It is eluted isocratically with a 50% - 90% ethanol solution at a flow rate of 1 - 4 mL / min, and the elution fractions are collected, 20 mL each.

[0019] Preferably, in the step S4, the collected elution fractions are subjected to concentration determination, the fractions containing tea polyphenols are enriched and combined, concentrated by vacuum distillation on a rotary evaporator, and finally vacuum freeze - dried to obtain a light - yellow Huangda tea tea polyphenol powder.

[0020] Preferably, the Huangdacha tea powder prepared in step S1 is subjected to ethanol extraction, and single-factor experiments are carried out respectively to obtain the influence curves of extraction time, extraction temperature, ethanol concentration, and solid-liquid ratio on the extraction rate of tea polyphenols. According to the influence curves of extraction time, extraction temperature, ethanol concentration, and solid-liquid ratio on the extraction rate of tea polyphenols, the optimal extraction time, optimal extraction temperature, optimal ethanol concentration, and optimal solid-liquid ratio are obtained; Subsequently, according to the obtained optimal extraction time, optimal extraction temperature, optimal ethanol concentration, and optimal solid-liquid ratio, in accordance with the Box-Behnken experimental principle, with extraction time, extraction temperature, ethanol concentration, and solid-liquid ratio as independent variables and the extraction rate of tea polyphenols as the response value, a response surface experiment is designed to obtain the results of the response surface experiment; Finally, the obtained response surface experiment results are subjected to regression fitting analysis using the statistical software Design-Expert 12.0 software to obtain a quadratic multiple regression equation for predicting the fitting of the extraction rate Y of tea polyphenols with ethanol concentration A, solid-liquid ratio B, extraction time C, and extraction temperature D. According to the model fitting, the extraction process is obtained, and the extraction process includes the optimal extraction time, optimal extraction temperature, optimal ethanol concentration, and optimal solid-liquid ratio.

[0021] The present invention has the following beneficial effects:

[0022] Through the combination of ethanol extraction method, organic solvent extraction, and macroporous resin chromatography, the present invention realizes the efficient extraction and purification of tea polyphenols, and has the advantages of simple operation, high extraction efficiency, and high product purity. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required to be used in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0024] Figure 1 is the standard curve of gallic acid;

[0025] Figure 2 is the influence diagram of ethanol concentration on the extraction rate of tea polyphenols in yellow tea;

[0026] Figure 3 is the influence diagram of solid-liquid ratio on the extraction rate of tea polyphenols in yellow tea;

[0027] Figure 4 is the influence diagram of extraction temperature on the extraction rate of tea polyphenols in yellow tea;

[0028] Figure 5 is the influence diagram of extraction time on the extraction rate of tea polyphenols in yellow tea. DETAILED DESCRIPTION OF THE INVENTION

[0029] In order to make the above objects, features and advantages of the present invention more obvious and understandable, the following provides a detailed description of the specific embodiments of the present invention. Many specific details are set forth in the following description in order to fully understand the present invention. However, the present invention can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without departing from the connotation of the present invention. Therefore, the present invention is not limited by the specific embodiments disclosed below.

[0030] Refer to Figures 1-5 , an extraction and purification process of tea polyphenols from Huangda tea, and the extraction process includes the following steps:

[0031] S1. Raw material screening: Select high-quality Huangda tea leaves as raw materials, process them through an ultrafine pulverizer and sieve to obtain Huangda tea powder;

[0032] S2. Extraction: Add an ethanol solution with a certain concentration to the Huangda tea powder, extract it in a constant temperature water bath, filter by suction, collect the primary filtrate, centrifuge to take the supernatant, and then perform low-pressure rotary evaporation to remove ethanol to obtain a tea polyphenol concentrate;

[0033] S3. Extraction: Transfer the concentrate to a Buchner funnel, add chloroform and ethyl acetate respectively, perform organic solvent extraction at room temperature, and retain the tea polyphenol aqueous layer after separating the organic phase;

[0034] S4. Chromatographic purification: Pass the tea polyphenol aqueous layer through a macroporous adsorption resin column at a certain flow rate, elute with an ethanol solution, collect the ethanol eluate, rotate and evaporate the eluate until the solid content ≥ 85%, transfer it to a vacuum freeze dryer for sublimation drying to obtain a light yellow powder of tea polyphenols.

[0035] In order to make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the following clearly and completely describes the technical solutions in the embodiments of the present invention in conjunction with the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present invention.

[0036] Example 1

[0037] Determination of the standard curve, the specific steps are as follows:

[0038] (1) Prepare 5% sodium carbonate solution: Weigh 12.5 g of sodium carbonate and dissolve it in a conical flask, then make up the volume to 250 mL in a volumetric flask and shake well;

[0039] (2) Accurately weigh 10 mg of gallic acid standard product, make up the volume to 100 mL in a volumetric flask to prepare a 0.1 mg / mL gallic acid standard product mother liquor;

[0040] (3) Pipette 0 μL, 100 μL, 200 μL, 300 μL, 400 μL, 500 μL, 600 μL, 700 μL, 800 μL, and 900 μL of a 0.1 mg / mL gallic acid standard stock solution into 10 mL test tubes respectively;

[0041] Add 1 mL of Folin-Ciocalteu reagent respectively, shake well and react for 5 min, then add 4 mL of 5% Na2CO3 solution, make up the volume with ultrapure water, shake well to prepare a series of concentration standard solutions, react in the dark at room temperature for 40 min, and measure the absorbance of the samples at a wavelength of 765 nm;

[0042] Use the gallic acid sample concentration as the x-axis and the absorbance as the y-axis to plot the gallic acid standard curve. The plotted standard curve is as Figure 1 shown. The equation is y = 0.1529x + 0.0324, R2 = 0.9966, with good linearity. This equation can be used as the standard curve regression equation.

[0043] After obtaining the tea polyphenol extract, the total phenol content of the tea polyphenol extract is determined by the Folin-Ciocalteu method in the present invention.

[0044] In the present invention, the method for determining by the Folin-Ciocalteu colorimetric method preferably includes: mixing the tea polyphenol extract, the Folin-Ciocalteu dilution solution and the sodium carbonate solution, making up the volume with ultrapure water, then standing in the dark and filtering to obtain the filtrate; using distilled water as a blank control to measure the absorbance of the filtrate, calculating the polyphenol content of the upper phase using the standard curve, and then calculating the extraction rate of tea polyphenols according to the following formula:

[0045]

[0046] In the formula: Y is the extraction rate of tea polyphenols, %; A is the absorbance measured for the sample; V is the volume of the sample extraction solution measured, mL; a is the slope of the fitted curve; b is the intercept of the fitted curve; m is the mass of the sample, g.

[0047] Example 2:

[0048] A process optimization method for the extraction and purification of tea polyphenols from yellow tea, the steps are as follows:

[0049] (1) Single-factor experimental design: According to Figure 2 preliminarily determine that the optimal concentration of ethanol is 90%, and select 80%, 90%, and 100% as the optimizable values for ethanol concentration; According to Figure 3 determine that the optimal solid-liquid ratio of yellow large tea powder to ethanol solution is 1:25 g / mL, and select 1:20 g / mL, 1:25 g / mL, and 1:30 g / mL as the optimizable values for the solid-liquid ratio; According to Figure 4Select 30°C, 40°C, and 50°C as the optimizable values of temperature; according to Figure 5 Select 30 min, 40 min, and 50 min as the optimizable values of extraction time.

[0050] (2) According to the optimizable values of the optimal ethanol concentration, optimal solid-to-liquid ratio, optimal temperature, and optimal extraction time obtained in step (1) above, in accordance with the Box-Behnken experimental principle, using ethanol concentration, extraction time, temperature, and solid-to-liquid ratio as independent variables and the tea polyphenol extraction rate as the response value, design a response surface experiment, and the results of the response surface experiment are shown in the following table:

[0051]

[0052]

[0053] (3) Apply the statistical software Design-Expert 12.0 to perform regression fitting analysis on the results of the response surface experiment obtained in step (2), and the results are shown in the following table:

[0054]

[0055] Note: * indicates significant difference, P < 0.05; ** indicates extremely significant difference, P < 0.01.

[0056] Perform multiple regression fitting analysis on the experimental data in the above table, and the quadratic multiple regression equation for predicting the tea polyphenol extraction rate Y with alcohol addition amount A, temperature B, and solid-to-liquid ratio C is:

[0057] Y = 17.38 + 0.2808A - 0.65B - 0.2458C - 0.245D + 0.0325AB + 0.0375AC - 0.8825AD - 0.33BC - 0.3125BD + 0.39CD - 1.22A 2 -3.11B 2 -0.7992C 2 -0.9555D 2 .

[0058] For the significance analysis of the regression equation coefficients of the model, it can be seen from the table that taking the extraction rate of tea polyphenols as the index, the regression model is extremely significant (P < 0.0001), while the lack-of-fit term of the model is not significant (P > 0.05), indicating that the model can fit the experimental results well. The regression coefficient of the model R2 = 0.986, indicating that the experimental data is consistent with the model. The adjusted determination coefficient R2adj of the model is 0.972, indicating a high correlation between the measured values and the predicted values. The coefficient of variation (C.V. = 1.89%) is relatively low, indicating that the model has repeatability. Therefore, this regression equation can describe the influence of various factors on the extraction rate of tea polyphenols in Huangda tea and can be used to determine the optimal extraction process of tea polyphenols.

[0059] Under the optimal process, that is, the solid-liquid ratio is 1:25 (g / mL), the extraction temperature is 38 °C, the ethanol concentration is 52%, and the extraction time is 40 min, 3 parallel experiments are carried out for verification, and the actual value of the extraction rate of tea polyphenols is obtained as 18.42%. The difference between the model prediction and the actual value is less than 5%, proving that the response surface optimization extraction process method is reliable.

[0060] Example 3:

[0061] An extraction and purification process of tea polyphenols from yellow tea

[0062] (1) Raw material screening: Screen the purchased Huoshan Huangda tea leaves, select high-quality Huangda tea leaves, remove the leaves and branches with poor quality, ensure no mildew and no impurities, crush them with an ultra-fine grinder, and sieve them through a 40-mesh sieve to obtain the sieved Huangda tea powder;

[0063] (2) Extraction: Add 250 mL of 50% ethanol solution to 10 g of Huangda tea powder, extract it in a 30 °C constant temperature water bath for 30 min, perform vacuum filtration, collect the filtrate, centrifuge it at 5000 r / min in a refrigerated centrifuge for 10 min to obtain the Huangda tea polyphenol extract. The obtained tea polyphenol extract is concentrated under reduced pressure. The concentration temperature is 55 °C, the rotation speed is 100 rpm, and the pressure is 100 Kpa. Rotate and evaporate to remove ethanol to obtain the first extract for standby;

[0064] (3) Extraction: Transfer the extract to a separatory funnel, add an equal volume of chloroform, let it stand for layering and retain the aqueous phase to remove the fat-soluble impurities in the tea polyphenols. Then add an equal volume of ethyl acetate to the aqueous phase and repeat 2 - 3 times to enrich the tea polyphenols. Place the obtained tea polyphenol extract in a rotary evaporation flask, and concentrate and rotate and evaporate to remove ethyl acetate at 55 °C, 100 rpm, and 100 Kpa to obtain the second extract for standby;

[0065] (4) Chromatographic purification: The D101 macroporous resin was packed into a column by the wet method. The tea polyphenol injection solution with a concentration of 1.5 mg / mL was loaded for adsorption at a loading flow rate of 2 mL / min. A 50% ethanol solution by mass was used as the eluent for elution and desorption at an elution flow rate of 1 mL / min, and the eluate was collected. The collected eluate was concentrated under reduced pressure and then freeze-dried under vacuum to obtain the tea polyphenol extract. The extraction rate of tea polyphenols was 16.15%, and the purity was 82.5%.

[0066] Example 4:

[0067] A process for extracting and purifying tea polyphenols from yellow tea

[0068] (1) Raw material screening: The purchased Huoshan Huangda tea leaves were screened to select high-quality Huangda tea leaves, removing the leaves and branches of poor quality to ensure no mildew and no impurities. They were pulverized using an ultrafine grinder and sieved through a 40-mesh sieve to obtain the sieved Huangda tea powder.

[0069] (2) Extraction: 250 mL of 50% ethanol solution was added to 10 g of Huangda tea powder, and the mixture was extracted in a constant temperature water bath at 40 °C for 40 min. Vacuum filtration was used to collect the filtrate, which was centrifuged at 5000 r / min for 10 min in a refrigerated centrifuge to obtain the Huangda tea polyphenol extract. The obtained tea polyphenol extract was concentrated under reduced pressure at a concentration temperature of 55 °C, a rotation speed of 100 rpm, and a pressure of 100 Kpa to remove ethanol by rotary evaporation, obtaining the first extract for standby.

[0070] (3) Extraction: The extract was transferred to a separatory funnel, and an equal volume of chloroform was added. After standing for layer separation, the aqueous phase was retained to remove the fat-soluble impurities in the tea polyphenols. Then, an equal volume of ethyl acetate was added to the aqueous phase, and this was repeated 2 - 3 times to enrich the tea polyphenols. The obtained tea polyphenol extract was placed in a rotary evaporation flask and concentrated under reduced pressure at 55 °C, 100 rpm, and 100 Kpa to remove ethyl acetate by rotary evaporation, obtaining the second extract for standby.

[0071] (4) Chromatographic purification: The D101 macroporous resin was packed into a column by the wet method. The tea polyphenol injection solution with a concentration of 1.5 mg / mL was loaded for adsorption at a loading flow rate of 1 mL / min. A 50% ethanol solution by mass was used as the eluent for elution and desorption at an elution flow rate of 1 mL / min, and the eluate was collected. The collected eluate was concentrated under reduced pressure and then freeze-dried under vacuum to obtain the tea polyphenol extract. The extraction rate of tea polyphenols was 17.74%, and the purity was 87%.

[0072] Example 5:

[0073] A process for extracting and purifying tea polyphenols from yellow tea

[0074] (1) Raw material screening: Screen the purchased Huoshan Huangda tea leaves, select high-quality Huangda tea leaves, remove the leaves and branches with poor quality, ensure no mildew and no impurities, crush them with an ultrafine grinder, and sieve them through a 40-mesh sieve to obtain the sieved Huangda tea powder;

[0075] (2) Extraction: Add 10 g of Huangda tea powder to 250 mL of 60% ethanol solution, extract it in a constant temperature water bath at 30 °C for 40 min, perform vacuum filtration, collect the filtrate, centrifuge it at 5000 r / min in a refrigerated centrifuge for 10 min to obtain the Huangda tea polyphenol extract. The obtained polyphenol extract is concentrated under reduced pressure, the concentration temperature is 55 °C, the rotation speed is 100 rpm, and the pressure is 100 Kpa. Rotate and evaporate to remove ethanol to obtain the first extract for standby;

[0076] (3) Extraction: Transfer the extract to a separatory funnel, add an equal volume of chloroform, let it stand for layer separation, retain the aqueous phase to remove the fat-soluble impurities in the polyphenols, then add an equal volume of ethyl acetate to the aqueous phase, repeat 2 - 3 times to enrich the polyphenols. Place the obtained polyphenol extract in a rotary evaporation flask, concentrate it under reduced pressure at 55 °C, 100 rpm, and 100 Kpa, rotate and evaporate to remove ethyl acetate to obtain the second extract for standby;

[0077] (4) Chromatographic purification: Wet-pack the D101 macroporous resin column, load the 1.5 mg / mL polyphenol injection solution for adsorption, the loading flow rate is 2 mL / min, use a 60% ethanol solution by mass fraction as the eluent for elution and desorption, the elution flow rate is 1 mL / min, and collect the eluate; Concentrate the above-collected eluate under reduced pressure, and then perform vacuum freeze-drying to obtain the polyphenol extract. The polyphenol extraction rate is 16.99%, and the purity is 80%.

[0078] Example 6:

[0079] An extraction and purification process of polyphenols from yellow tea

[0080] (1) Raw material screening: Screen the purchased Huoshan Huangda tea leaves, select high-quality Huangda tea leaves, remove the leaves and branches with poor quality, ensure no mildew and no impurities, crush them with an ultrafine grinder, and sieve them through a 40-mesh sieve to obtain the sieved Huangda tea powder;

[0081] (2) Extraction: Add 300 mL of 40% ethanol solution to 10 g of yellow big tea powder, extract it in a constant temperature water bath at 40 °C for 40 min, perform vacuum filtration, collect the filtrate, centrifuge it at 5000 r / min in a refrigerated centrifuge for 10 min to obtain the yellow big tea tea polyphenol extract. Concentrate the obtained tea polyphenol extract under reduced pressure, with a concentration temperature of 55 °C, a rotation speed of 100 rpm, and a pressure of 100 Kpa, and rotate to evaporate ethanol to obtain the first extract for standby;

[0082] (3) Extraction: Transfer the extract to a separatory funnel, add an equal volume of chloroform, let it stand for layering and retain the aqueous phase to remove the fat-soluble impurities in the tea polyphenols. Then add an equal volume of ethyl acetate to the aqueous phase and repeat 2 - 3 times to enrich the tea polyphenols. Place the obtained tea polyphenol extract in a rotary evaporation flask, and concentrate it under reduced pressure at 55 °C, 100 rpm, and 100 Kpa, and rotate to evaporate ethyl acetate to obtain the second extract for standby;

[0083] (4) Chromatographic purification: Wet-pack a column with D101 macroporous resin, load the tea polyphenol injection solution with a concentration of 1 mg / mL, with a loading flow rate of 2 mL / min. Use an ethanol solution with a mass fraction of 50% as the eluent for elution and desorption, with an elution flow rate of 1 mL / min, and collect the eluate; Concentrate the above-collected eluate under reduced pressure, and then perform vacuum freeze-drying to obtain the tea polyphenol extract. The extraction rate of tea polyphenols is 12.05%, and the purity is 76.8%.

[0084] As described above, it is only a preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, according to the technical solution of the present invention and its inventive concept, makes equivalent substitutions or changes, and all should be covered within the protection scope of the present invention.

Claims

1. A process for extracting and purifying tea polyphenols from yellow tea, characterized in that: The extraction process comprises the following steps: S1. Raw material screening: Select high-quality yellow tea raw materials, process and sieve them through an ultra-fine grinder to obtain yellow tea powder; S2, extraction: adding a certain concentration of ethanol solution to the yellow tea powder, extracting in a constant temperature water bath, collecting the primary filtrate after suction filtration, centrifuging and taking the supernatant, and then performing low-pressure rotary evaporation to remove ethanol to obtain a tea polyphenols concentrate; S3, extraction: transfer the concentrated solution to a Buchner funnel, add chloroform and ethyl acetate respectively, perform organic solvent extraction at room temperature, separate the organic phase and retain the tea polyphenols aqueous layer; S4, chromatography purification: pass the tea polyphenol water layer through a macroporous adsorption resin column at a certain flow rate, elute with ethanol solution, collect the ethanol eluate, rotary evaporate the eluate to a solid content ≥ 85%, transfer to a vacuum freeze dryer for sublimation drying, and obtain a light yellow powder of tea polyphenol.

2. The process for extracting and purifying tea polyphenols from yellow tea according to claim 1, characterized in that: In the step S1 of raw material screening, the following steps are included: the purchased Huoshan Huangda tea leaves are screened to select high-quality leaves, remove leaves and branches of poor quality, ensure that there is no mildew and impurities, and the Huangda tea leaves are ultra-finely ground and passed through a 40-mesh sieve.

3. The process for extracting and purifying tea polyphenols from yellow tea according to claim 1, characterized in that: In step S2, the process is controlled by the following conditions: the solid-liquid ratio of yellow tea coarse powder to ethanol solution is 1:10 (g / mL)-1:30 (g / mL), the time for extracting tea polyphenols in a constant temperature water bath with ethanol solution is 20-60 minutes, the mass percentage of ethanol solution used for extracting tea polyphenols is 30%-70%, and the temperature of the constant temperature water bath for extracting tea polyphenols with ethanol solution is 30-70°C.

4. The process for extracting and purifying tea polyphenols from yellow tea according to claim 1, characterized in that: In step S2, the primary filtrate of tea polyphenols is centrifuged in a refrigerated centrifuge at 5000 r / min for 10 min, and the supernatant after centrifugation is concentrated to one tenth of the original volume on a rotary evaporator at a rotary evaporation temperature of 55°C.

5. The process for extracting and purifying tea polyphenols from yellow tea according to claim 1, characterized in that: In the step S3, chloroform is added in an amount equal to the volume of the tea polyphenols concentrated solution for extraction to obtain a decaffeinated tea polyphenols extract, and then ethyl acetate is added to the extract at a volume ratio of 1:1 for extraction, and the extraction is performed 2-3 times, each time for 15-30 minutes. The extracts are combined, concentrated by rotary evaporation to remove residual ethyl acetate, and dried on a freeze dryer to obtain a crude powder of tea polyphenols.

6. The process for extracting and purifying tea polyphenols from yellow tea according to claim 1, characterized in that: In the step S4, the macroporous resin chromatography column adopts a glass chromatography column with a specification of 16mm×500mm and a macroporous resin as a filler. The chromatography column is fixed vertically on an iron frame, the bottom of the chromatography column is plugged with absorbent cotton, the piston at the bottom of the chromatography column is closed, and ultrapure water pre-loaded to three-quarters of the column height is poured into the column. After the treated macroporous resin is ultrasonically removed of bubbles, it is poured into the chromatography column, and the macroporous resin is allowed to settle naturally. At the same time, the chromatography column is continuously and gently tapped by hand to ensure that the resin is tightly filled in the column; after complete sedimentation, a layer of glass wool is covered on the adsorbent; the piston at the lower end of the chromatography column is opened to release the excess ultrapure water in the column, allowing the water to flow out slowly until the liquid level is about 2-3cm higher than the filler layer, and then the piston is closed.

7. The process for extracting and purifying tea polyphenols from yellow tea according to claim 6, characterized in that: The macroporous resin filler is one of AB-8, D101, NKA-9, X-5, and HPD-600.

8. The process for extracting and purifying tea polyphenols from yellow tea according to claim 1, characterized in that: In step S4, the crude powder of tea polyphenols is accurately weighed during the macroporous resin purification process, dissolved in ultrapure water to prepare a 0.5-3 mg / mL tea polyphenol solution, loaded at a flow rate of 1-4 mL / min, and eluted isokinetically with 50%-90% ethanol solution at a flow rate of 1-4 mL / min, and the eluted fractions are collected, each 20 mL.

9. The process for extracting and purifying tea polyphenols from yellow tea according to claim 1, characterized in that: In step S4, the concentration of the collected eluted fractions is measured, the fractions containing tea polyphenols are enriched and combined, concentrated by reduced pressure distillation on a rotary evaporator, and vacuum freeze-dried to finally obtain light yellow yellow tea polyphenol powder.

10. The process for extracting and purifying tea polyphenols from yellow tea according to claim 1, characterized in that: The yellow tea powder prepared in step S1 is subjected to ethanol extraction, and single factor experiments are respectively carried out to obtain the influence curves of extraction time, extraction temperature, ethanol concentration, and solid-liquid ratio on the extraction rate of tea polyphenols, and the optimal extraction time, optimal extraction temperature, optimal ethanol concentration, and optimal solid-liquid ratio are obtained according to the influence curves of extraction time, extraction temperature, ethanol concentration, and solid-liquid ratio on the extraction rate of tea polyphenols; then, according to the obtained optimal extraction time, optimal extraction temperature, optimal ethanol concentration, and optimal solid-liquid ratio, the Box-Behnken test principle is used to determine the extraction time, extraction temperature, ethanol concentration, and solid-liquid ratio. Extraction temperature, ethanol concentration and solid-liquid ratio were used as independent variables, and tea polyphenols extraction rate was used as the response value to design a response surface experiment and obtain the response surface experiment results. Finally, the statistical software Design-Expert12.0 was used to perform regression fitting analysis on the response surface experiment results to obtain a quadratic polynomial regression equation for model prediction fitting of tea polyphenols extraction rate Y with ethanol concentration A, solid-liquid ratio B, extraction time C and extraction temperature D. According to the model fitting, the extraction process was obtained, which included the optimal extraction time, the optimal extraction temperature, the optimal ethanol concentration and the optimal solid-liquid ratio.

Images