Preparation method and application of phyllanthus emblica fruit juice polyphenol extract

Abstract

The invention relates to a preparation method of a phyllanthus emblica fruit juice polyphenol extract, in particular to a method for extracting polyphenol from phyllanthus emblica fruit juice by utilizing an ultrasonic-assisted aqueous two-phase extraction technology. The method relates to an aqueous two-phase reagent for preparing the phyllanthus emblica fruit juice polyphenol extract, the aqueous two-phase reagent comprises phyllanthus emblica fruit juice, ethanol and ammonium sulfate, the volume ratio of the phyllanthus emblica fruit juice to the ethanol is 1: (1.5-7), and the volume ratio of the ammonium sulfate to the phyllanthus emblica fruit juice is 0.5-2.5 g / mL. Placing the aqueous two-phase reagent in an ultrasonic treatment device, and carrying out sufficient ultrasonic treatment; and separating the aqueous two-phase reagent subjected to ultrasonic treatment, and taking an upper phase part which comprises total polyphenol, total polysaccharide, vitamin C, gallic acid, corilagin, chebulagic acid and ellagic acid. According to the extraction method provided by the invention, the problem that hydrolyzed tannin is reduced in a traditional method is avoided, so that relatively high polyphenol extraction rate and quality can be kept, and the extraction method has relatively high practical value and market prospect.

Description

Technical Field

[0001] The present invention relates to a method for preparing polyphenol extract from Phyllanthus emblica L. fruit juice, and particularly to a method for extracting polyphenols from Phyllanthus emblica L. fruit juice by using ultrasonic-assisted aqueous two-phase extraction technology. Background Art

[0002] Phyllanthus emblica L. is a traditional plant with both medicinal and edible uses, containing various chemical components, mainly including polyphenols, polysaccharides, fats, etc., as well as various vitamins, amino acids and trace elements. Among them, polyphenolic compounds are the main substances for Phyllanthus emblica L. to exert biological activities, having various biological activities such as antioxidant, anti-inflammatory, and antiviral effects. Modern research shows that Phyllanthus emblica L. polyphenolic compounds are used to treat liver injury and are traditional Asian medicines for treating liver diseases. Research also reports the hepatoprotective effects of Phyllanthus emblica L. against carbon tetrachloride and paracetamol.

[0003] Traditional methods for extracting Phyllanthus emblica L. polyphenols mainly include organic solvent extraction, ultrasonic extraction, enzymatic hydrolysis, supercritical extraction, ethanol reflux, ultrasonic extraction, enzymatic hydrolysis combined with ultrasonic extraction, etc. Among them, solvent extraction is one of the most commonly used methods, but this method has disadvantages such as cumbersome and complex processes, easy residue of organic solvents, and affecting the quality of polyphenol extracts. In addition, traditional extraction processes may also lead to a reduction in hydrolysable tannins, thereby reducing the biological activity of polyphenols. Therefore, it is of great significance to develop an efficient, environmentally friendly extraction method that can maintain the biological activity of polyphenols.

[0004] In view of the above problems, the present invention proposes a method for preparing polyphenol extract from Phyllanthus emblica L. fruit juice by using ultrasonic-assisted aqueous two-phase extraction technology. This method utilizes the mechanical fragmentation, cavitation effect and thermal effect of ultrasonic extraction to destroy plant cell tissues, increase the internal temperature of the plant, accelerate the diffusion rate of Phyllanthus emblica L. polyphenols into the solvent, and thus shorten the extraction time. Aqueous two-phase extraction technology (ATPS) is a new type of extraction and separation technology with mild conditions, fast mass transfer, low cost, easy operation, adjustable properties, and easy to scale up, and is particularly suitable for the separation and extraction of biological substances. Compared with traditional organic solvent extraction systems, ATPS has unique advantages in the separation of biological substances.

[0005] Traditional two-phase aqueous systems are mostly formed by polymers (such as polyethylene glycol PEG and dextran). Although the extraction efficiency is high, the cost is high and the system viscosity is large, which is not conducive to large-scale industrial production. In contrast, the two-phase aqueous system formed by low-level alcohols (such as ethanol) and salt aqueous solutions has low toxicity, simple recovery of organic solvents, low cost, and is suitable for large-scale industrial production. The two-phase aqueous system competes with ethanol for water molecules through the hydration of salt ions to form two phases of ethanol aqueous solution and salt water solution. The active ingredients in biological substances or natural products are selectively distributed between the two phases.

[0006] In summary, the existing extraction methods have many limitations, and it is urgent to develop a new method that is efficient, environmentally friendly and can maintain the biological activity of polyphenols to meet the needs of industrial extraction and processing. This study used an ultrasound-assisted aqueous two-phase extraction system to prepare the total polyphenol extract of emblica juice for the first time, aiming to solve the problems existing in the existing methods and provide a cutting-edge and effective green extraction method. Summary of the invention

[0007] The invention provides a two-aqueous phase reagent for preparing a polyphenol extract of emblica fruit juice. The reagent not only takes into account the efficient extraction of polyphenols, but also avoids the reduction of hydrolyzed tannins.

[0008] To achieve the above object, the technical solution of the present invention is:

[0009] The invention discloses a two-phase aqueous reagent for preparing a polyphenol extract of emblica fruit juice, comprising emblica fruit juice, ethanol and ammonium sulfate, wherein the volume ratio of the emblica fruit juice to the ethanol is 1:1.5-7, and the weight ratio of the ammonium sulfate to the volume of the emblica fruit juice is 0.5-2.5 g / mL.

[0010] As a preferred solution, in the aqueous two-phase reagent, the volume ratio of the emblica juice to ethanol is 1:4.8-5.5, and the weight ratio of the ammonium sulfate to the volume of the emblica juice is 1.5-2 g / mL.

[0011] As a preferred solution, the proportion of the two-phase aqueous reagent is as follows: when the volume of the emblica emblica juice is 3.1 mL, the volume of ethanol is 4.9 mL, and the weight of ammonium sulfate is 1.8 g.

[0012] As a preferred solution, the method for obtaining the emblica emblica juice is to remove the core of fresh emblica emblica, squeeze the juice and remove the pomace.

[0013] As a preferred embodiment, the mass concentration of gallic acid in the juice is 9.12 mg / mL.

[0014] The second object of the present invention is to provide a method for extracting polyphenols from emblica fruit juice, which is suitable for large-scale production.

[0015] To achieve the above object, the technical solution of the present invention is as follows:

[0016] A method for obtaining polyphenol extract from Phyllanthus emblica fruit juice using the aqueous two-phase reagent includes the following steps:

[0017] a) Place the aqueous two-phase reagent in an ultrasonic treatment device and perform sufficient ultrasonic treatment;

[0018] b) Separate the ultrasonic-treated aqueous two-phase reagent and take the upper phase part, which includes total polyphenols, total polysaccharides, vitamin C, gallic acid, corilagin, chebulic acid, and ellagic acid.

[0019] Total polyphenols are the main active components in Phyllanthus emblica fruit juice. Gallic acid, corilagin, chebulic acid, and ellagic acid are specific polyphenol components in Phyllanthus emblica, which have various biological activities.

[0020] The traditional aqueous two-phase system is composed of polymers (such as polyethylene glycol PEG and dextran). Although the extraction efficiency is high, the cost is high and the system viscosity is large, which is not conducive to large-scale industrial production. The novel aqueous two-phase system involved in this patent: consists of an aqueous solution of a lower alcohol (such as ethanol) and a salt (such as ammonium sulfate). This system has the following advantages: low cost: the cost of lower alcohol and salt is relatively low. Low toxicity: it has less impact on biological substances and the environment. Simple operation: the recovery operation of organic solvents is simple and easy to reuse. Suitable for large-scale production: low viscosity, easy to handle and operate, suitable for industrial production.

[0021] The principle of this method involves the hydration of salt ions, specifically: salt ions form a hydrated structure with water molecules, occupying a part of the water molecules. It also involves the competitive effect of ethanol, specifically: ethanol and salt ions compete for water molecules together, and finally form two phases: an ethanol aqueous solution and a salt aqueous solution. It also involves the principle of selective partitioning, specifically: the active components (such as polyphenols) in biological substances or natural products form a selective partition between the two phases, and usually, target components such as polyphenols will concentrate in one phase.

[0022] As a preferred solution, in the method, the conditions for ultrasonic treatment are: the power is 300W - 500W, and the ultrasonic time is 5 - 10 min.

[0023] As a preferred solution, in the method, the ultrasonic treatment step of the aqueous two-phase reagent further includes: when the volume of Phyllanthus emblica fruit juice is 3.1 mL, the volume of ethanol is 4.9 mL, and the weight of ammonium sulfate is 1.8 g, the aqueous two-phase reagent is placed in a 400W ultrasonic treatment device and ultrasonic-treated for 10 min.

[0024] As a preferred solution, the method further includes the following steps: recycling and reusing the lower phase portion of the separated aqueous two-phase reagent.

[0025] During the aqueous two-phase extraction process, after ultrasonic treatment, the reagent is divided into two phases: the upper phase and the lower phase. The upper phase is the main enrichment phase of the target component (such as polyphenols), while the lower phase contains other components and unextracted substances. The advantages of this are: reducing the consumption of reagents and lowering production costs. Reducing the generation of waste and alleviating environmental pressure. By recycling multiple times, the total extraction rate of the target component is increased.

[0026] As a preferred solution, the method further includes an evaluation and optimization module for the extraction method. Specifically: measuring the total polyphenol content in the upper phase portion, adding 1 mL of 10% Folin-Ciocalteu reagent and 0.8 mL of 7.5% sodium carbonate solution to the upper phase portion, using the corresponding reagent as a blank reference, and measuring the absorbance value at 765 nm; adjusting the ratio of each component of the aqueous two-phase reagent by the absorbance value, and / or adjusting the ultrasonic treatment parameters by the absorbance value.

[0027] Furthermore, measuring the gallic acid content in the upper phase portion, calculating the content according to the standard curve, and the regression equation of the standard curve is Y = 0.0367X + 0.2281, where X is the gallic acid concentration gradient and Y is the absorbance value; adjusting the ratio of each component of the aqueous two-phase reagent by the absorbance value, and / or adjusting the ultrasonic treatment parameters by the absorbance value.

[0028] The present invention also provides a method for measuring the total polyphenol content and gallic acid content in the upper phase portion to further optimize the extraction conditions. Specifically, adjusting the ratio of each component of the aqueous two-phase reagent by the absorbance value, and / or adjusting the ultrasonic treatment parameters by the absorbance value to ensure the extraction efficiency and quality.

[0029] The above evaluation and optimization module can optimize the extraction method and improve the extraction efficiency and purity of polyphenols. This optimization method helps to ensure the stability and high efficiency of the extraction process.

[0030] The third object of the present invention is to provide the application of the above extract, which provides a clinical treatment idea for the body damage caused by alcohol.

[0031] To achieve the above object, the technical solution of the present invention is:

[0032] The application of the polyphenol extract of Phyllanthus emblica fruit juice obtained by the method in the preparation of a drug for improving acute drunkenness state and / or alleviating acute drunken liver injury.

[0033] As a preferred solution, the polyphenol extract of Phyllanthus emblica fruit juice can reduce the serum ALT and AST contents.

[0034] Beneficial effects

[0035] By adopting the ultrasonic-assisted aqueous two-phase extraction technology, the extraction efficiency and quality are improved, and the extraction time is shortened. By strictly controlling the ratio of the aqueous two-phase reagents and the ultrasonic treatment conditions, the introduction of harmful impurities is avoided, and the biological activity of polyphenols is effectively maintained. The lower phase part of the separated aqueous two-phase reagents is recovered and reused, reducing the production cost and improving the resource utilization rate.

[0036] The extraction method provided by the present invention avoids the problem of reduced hydrolyzable tannins existing in the traditional method, so that a high polyphenol extraction rate and quality can be maintained, and it has high practical value and market prospects. Description of the drawings

[0037] Attached Figure 1 It is an analysis chart of the influence of the ethanol dosage on the polyphenol extraction rate.

[0038] Attached Figure 2 It is an analysis chart of the influence of the ammonium sulfate dosage on the polyphenol extraction rate.

[0039] Attached Figure 3 It is an analysis chart of the influence of the Phyllanthus emblica fruit juice dosage on the polyphenol extraction rate.

[0040] Attached Figure 4 It is a three-factor response surface diagram of the ethanol dosage, ammonium sulfate dosage and Phyllanthus emblica fruit juice dosage.

[0041] Attached Figure 5 It is the blood ethanol concentration-time curve of each group of rats.

[0042] Attached Figure 6 It is the influence of preventive administration of Phyllanthus emblica extract on gastric mucosal injury and bleeding in drunken rats.

[0043] Attached Figure 7 It is the influence of preventive administration of Phyllanthus emblica extract on the histopathology of gastric mucosa in drunken rats (100×).

[0044] Figure 8 It is the total phenol standard curve.

[0045] Figure 9 It is the total sugar standard curve.

[0046] Figure 10 It is the vitamin C standard curve. Detailed implementation manners

[0047] The technical solution of the present invention will be further described clearly and completely in conjunction with specific embodiments below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work belong to the protection scope of the present invention.

[0048] the term

[0049] Aqueous Two-Phase Reagent: A reagent for extracting polyphenols from emblica juice, consisting of emblica juice, ethanol and ammonium sulfate. Aqueous Two-Phase Reagent is an innovative extraction system that utilizes a specific ratio of emblica juice, ethanol and ammonium sulfate to form two aqueous phases to achieve selective extraction of specific components.

[0050] Ultrasonic treatment: A method of treating a two-phase aqueous reagent with an ultrasonic treatment device to accelerate the extraction process of polyphenols. Ultrasonic treatment uses the cavitation effect and mechanical fragmentation of ultrasound to promote the polyphenol components in the emblica juice to enter the solution phase and improve the extraction efficiency.

[0051] The experimental equipment and consumables involved in the present invention are as follows

[0052] 1. Instruments and Materials

[0053] JJ200B electronic balance (Changshu Shuangjie Testing Instrument Factory); electric constant temperature drying oven (Shanghai Shangdao Instrument Manufacturing Co., Ltd.); -80℃ ultra-low temperature refrigerator (Haier); Spark TM Multifunctional microplate reader (Tecan, Switzerland); IMS-20 flake ice maker (Changshu Xueke Electric Co., Ltd.); HWS-26 electric constant temperature water bath (Shanghai Shangdao Instrument Manufacturing Co., Ltd.); KQ-300DE CNC ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); VORTEX-5 vortex oscillator (Qilin Bell Instrument Manufacturing Co., Ltd., Haimen); BMBFree-17 desktop high-speed refrigerated centrifuge (Bomei Scientific Instrument (Guangzhou) Co., Ltd.); HNY-100B constant temperature culture oscillator (Tianjin Ounuo Instrument Co., Ltd.); pipette (Eppendorf); juicer (Shanghai Beihe Technology Co., Ltd.).

[0054] Phyllanthus emblica was purchased from Shantou City, Guangdong Province; gallic acid reference substance (batch number: C12211828), Folin-Ciocalteu phenol reagent (batch number: C16171888) were purchased from Shanghai Macklin Biochemical Co., Ltd.; ammonium sulfate (batch number: 20230801), absolute ethanol (batch number: 20230704), sodium carbonate (batch number: 20221201) were all purchased from Tianjin Zhiyuan Chemical Reagent Co., Ltd.; Gegenzhiju compound tablets (Herbsense Co., Ltd., batch number: 339419); 56% liquor was purchased from Beijing Red Star Co., Ltd. (batch number: 20230926081419); ALT test kit (batch number: 20240724), AST test kit (batch number: 20240726), ADH test kit (batch number: 20240727), ALDH (batch number: 20240305) test kits were all purchased from Nanjing Jiancheng Bioengineering Institute; ethanol content test kit (batch number: ADS20240730) was purchased from Jiangsu Edison Biotechnology Co., Ltd.; isoflurane (batch number: 20240905) was purchased from Shenzhen Rewod Life Science Co., Ltd.

[0055] Forty-two healthy SPF-grade male Sprague-Dawley (SD) rats, weighing 180 - 200 g, were provided by Zhuhai Bestong Biotechnology Co., Ltd., and the experimental animal certificate number was: SCXK (Guangdong) 2020 - 0051. The animals were housed in the Animal Experiment Center of Guangdong Pharmaceutical University. The housing environment: the temperature was 20℃ - 23℃, the relative humidity was 50% - 60%, the lighting time was from 8:00 to 20:00 every day, and they had free access to food and water. After adapting to the environment for 3 days, they were used for the experiment. This experiment was approved by the Animal Experiment Ethics Committee of Guangdong Pharmaceutical University.

[0056] Example 1

[0057] 1 Preparation of Phyllanthus emblica fruit juice sample: Fresh Phyllanthus emblica was washed, dried on the surface, pitted and juiced. After vacuum filtration to remove the fruit residue, yellowish-green Phyllanthus emblica fruit juice was obtained. (The mass concentration of gallic acid in the fruit juice was 9.12 mg·mL -1 )

[0058] 2 Ultrasonic-assisted aqueous two-phase extraction of polyphenols from Phyllanthus emblica fruit juice: Weighed a certain amount of ammonium sulfate, ethanol and a certain volume of Phyllanthus emblica fruit juice into a small beaker. After adding water to dissolve the ammonium sulfate, ultrasonicated for a period of time, transferred to a separatory funnel, separated the upper and lower phases, and measured the volume of the upper phase and the total polyphenol content.

[0059] 3 Determination method of total polyphenol content

[0060] 3.1 Preparation of reference substance solution: Accurately weighed 10.00 mg of gallic acid, dissolved it in water and fixed the volume to a 100 mL volumetric flask, and shook well to obtain.

[0061] 3.2 Preparation of test solution: Weigh a certain amount of ammonium sulfate, ethanol and a certain volume of Phyllanthus emblica fruit juice into a small beaker. After adding water to dissolve ammonium sulfate, sonicate for a period of time, transfer to a separatory funnel, separate the upper and lower phases. Accurately pipette 0.25 mL of the upper phase extract of Phyllanthus emblica fruit juice into a 25 mL volumetric flask and make up to the mark with distilled water to obtain the test solution.

[0062] 3.3 Determination of total polyphenol content: Pipette 0.2 mL of the test solution into a 5 mL EP tube, add 1 mL of 10% Folin-Ciocalteu reagent and 0.8 mL of 7.5% sodium carbonate solution. Place in the dark at room temperature for 60 min. Using the corresponding reagent as the blank reference, measure the absorbance at 765 nm. Perform parallel measurements three times, and conduct the experiment in the dark. Calculate the total polyphenol content (mg·mL -1 ) under different conditions according to the standard curve, and calculate the extraction rate according to the following formula (1).

[0063] Extraction rate (%) = m 1 / m 2 = (C 1 V 1 ) / (C 2 V 2 )(1)

[0064] Wherein, m 1 , m 2 are the masses of total polyphenols in the extract and Phyllanthus emblica fruit juice respectively; C 1 , C 2 are the concentrations of total polyphenols in the extract and Phyllanthus emblica fruit juice respectively; V 1 , V 2 are the volumes of the extract and Phyllanthus emblica fruit juice respectively;

[0065] 3.4 Methodological investigation

[0066] 3.4.1 Investigation of linear relationship: The Folin-Ciocalteu colorimetric method was slightly modified. Respectively take 0 μL, 20 μL, 40 μL, 60 μL, 80 μL, 100 μL, 120 μL, 140 μL, 160 μL, 180 μL, 200 μL of gallic acid reference solution into 5 mL centrifuge tubes, make up to 200 μL with distilled water, with 3 parallels for each. Add 1 mL of 10% Folin-Ciocalteu reagent respectively, shake well, react for 5 min, add 800 μL of 7.5% sodium carbonate solution and shake well. Place in the dark at room temperature for 60 min. Add 200 μL to each well in a 96-well plate, and measure the absorbance value at 765 nm with an enzyme-linked immunosorbent assay reader. At the same time, prepare a reagent blank. Perform linear regression with the concentration gradient (X) of gallic acid reference and the absorbance value (Y) to calculate the linear regression equation and correlation coefficient. The standard curve regression equation is Y = 0.0367X + 0.2281, R = 0.999, and the linearity is good in the range of 0 - 10 μg / mL.

[0067] 3.4.2 Precision Test

[0068] Take Phyllanthus emblica fruit juice, prepare the test solution according to the method under item "3.2", measure the absorbance value according to the method under item "3.3", and continuously measure 6 times. The RSD value of the gallic acid content is 0.12%, indicating that the precision of the instrument is good.

[0069] 3.4.3 Repeatability Test

[0070] Take 6 portions of Phyllanthus emblica fruit juice, prepare the test solution respectively according to the method under item "3.2", measure the absorbance value according to the method under item "3.3", and the RSD value of the gallic acid content is 0.96%, indicating that the repeatability of the preparation method of the test solution is good.

[0071] 3.4.4 Sample Addition Recovery Test

[0072] Precisely measure 1 mL of Phyllanthus emblica fruit juice with known gallic acid content, a total of 6 portions, precisely add an equal amount of reference substance solution, prepare the test solution according to the method under item "3.2", measure the absorbance value according to the method under item "3.3", and calculate the average recovery rate. As a result, the average recovery rate of gallic acid is 99.82%, and the RSD value is 1.59%, indicating that the established analytical method has good accuracy.

[0073] Example 2 Single-Factor Investigation Test of Extraction Process

[0074] 1 Ethanol Dosage

[0075] Precisely measure 2 mL of Phyllanthus emblica fruit juice, in parallel for 3 portions, fix the mass of ammonium sulfate at 2 g, add 4.5 mL, 5 mL, 5.5 mL, 6 mL, and 6.5 mL of ethanol respectively, make up the volume to 10 mL with water, place it in an ultrasonic cleaner with a power of 400 W, a temperature of 40 °C, and an ultrasonic time of 10 min. After phase separation, measure the polyphenol content in the upper phase. The results are shown in Figure 1 . It can be seen that as the volume of ethanol increases, the total polyphenol content in the upper-phase extract of Phyllanthus emblica fruit juice first increases and then decreases. When the volume of ethanol is 5 mL, the total polyphenol content reaches the maximum. Therefore, three levels of ethanol volume, namely 4.5 mL, 5 mL, and 5.5 mL, are selected for response surface test optimization.

[0076] 2 Ammonium Sulfate Dosage

[0077] Precisely measure 2 mL of Phyllanthus emblica fruit juice, in parallel for 3 portions, fix the ethanol volume at 5 mL, add 1.4 g, 1.6 g, 1.8 g, 2.0 g, and 2.2 g of ammonium sulfate respectively, make up the volume to 10 mL with water, place it in an ultrasonic cleaner with a power of 400 W, a temperature of 40 °C, and an ultrasonic time of 10 min. After phase separation, measure the polyphenol content in the upper phase. The results are shown in Figure 2It can be seen that with the increase of ammonium sulfate dosage, the total polyphenol content in the upper phase extract of Phyllanthus emblica juice first increased and then decreased. When the ammonium sulfate dosage was 1.8g, the total polyphenol content reached the maximum. Therefore, three levels of ammonium sulfate dosage of 1.6g, 1.8g, and 2.0g were selected for response surface experiment optimization.

[0078] 3. Dosage of Phyllanthus emblica juice

[0079] The amount of ammonium sulfate was fixed at 2 g and the volume of ethanol was 5 mL. Three parallel batches were added with 1.5 mL, 2 mL, 2.5 mL, 3 mL, and 3.5 mL of Phyllanthus emblica juice, respectively. Water was added to make the volume 10 mL. The mixture was placed in an ultrasonic cleaner with a power of 400 W, a temperature of 40 ° C, and an ultrasonic time of 10 min. After phase separation, the content of polyphenols in the upper phase was determined. The results are shown in the table. Figure 3 It can be seen that with the increase of the amount of emblica juice, the total polyphenol content in the upper phase extract of emblica juice first increased and then decreased. When the amount of emblica juice was 3mL, the total polyphenol content reached the maximum. Therefore, the response surface experiment was optimized with the amount of emblica juice at three levels: 2.5mL, 3mL, and 3.5mL.

[0080] 4Box-Behnken response surface experiment

[0081] Based on the results of the single factor experiment, the volume of ethanol (A), the amount of ammonium sulfate (B), and the amount of emblica juice (C) were selected as independent variables, and the extraction rate of total polyphenols in the above phases was selected as the dependent variable. The response surface method with three factors and three levels was used to optimize the extraction process of emblica juice polyphenols. The factors and levels are shown in Table 1, the experimental design is shown in Table 2, and the results are shown in Table 3.

[0082] Table 1 Factors and levels of Box-Behnken experimental design

[0083]

[0084] Table 2 Response surface experimental design

[0085]

[0086]

[0087] Table 3 Experimental results

[0088]

[0089] The Design-Expert10 software was used to perform a multiple regression analysis on the data in Table 3, and the equation was Y = 95.71-4.18A-2.92B+0.99C-0.60AB+0.011AC-0.52BC-7.68A. 2 -5.27B2 -4.81 °C 2 , the analysis of variance is shown in Table 4. It can be seen that the model has a high significance (P < 0.01); the lack-of-fit term (F = 4.53, P = 0.0893 > 0.05), indicating that the model is reliable; the correlation coefficient R 2 = 0.9877, indicating that the model can explain 98.77% of the variation in the response value; the P-value results show that factors A, B, C, A 2 , B 2 , C 2 have a significant influence (P < 0.001). The influence degree of each factor is: the amount of ethanol (B) > the amount of ammonium sulfate (A) > the amount of Phyllanthus emblica fruit juice (C).

[0090] Table 4 Analysis of variance of the multiple regression model

[0091]

[0092] Response surface analysis was carried out using Design-Expert 10.0 software, and the results are shown in Figure 4 . The three-dimensional response surface diagram can intuitively reflect the influence of the interaction of various factors on the response value, so as to determine the optimal process parameter range. In this experiment, in order to make the total polyphenol extraction rate reach the maximum value, the levels corresponding to the highest point of the surface were selected as the optimal experimental parameters. According to the analysis of Design-Expert 10 software, the optimal extraction process of Phyllanthus emblica fruit juice is 4.87 mL of ethanol, 1.75 g of ammonium sulfate, and 3.06 mL of Phyllanthus emblica fruit juice.

[0093] 6 Final determination and verification test

[0094] According to the analysis of Design-Expert 10 software, the optimal extraction process of Phyllanthus emblica fruit juice is 4.87 mL of ethanol, 1.75 g of ammonium sulfate, and 3.06 mL of Phyllanthus emblica fruit juice. Considering the practical feasibility, it was modified to 4.9 mL of ethanol, 1.8 g of ammonium sulfate, and 3.1 mL of Phyllanthus emblica fruit juice. Six portions of Phyllanthus emblica fruit juice were taken for verification tests with the optimized process. The results showed that the polyphenol extraction rates were 96.10%, 96.84%, 96.49%, and the average value was 96.48%, which was close to the predicted value of 96.70% (RSD was 0.37%), indicating that the process is reasonable and feasible.

[0095] Example 3 Improvement of alcoholic injury by the extract

[0096] 1 Administration and grouping

[0097] Take 1500 mL of Phyllanthus emblica fruit juice from Example 1. According to the optimal process parameters, perform ultrasonic-assisted aqueous two-phase extraction. After vacuum concentrating the upper-phase extract, freeze-dry it to obtain the polyphenol extract of Phyllanthus emblica fruit juice. Select 30 male SD rats with a body weight of 200 - 220 g. After 7 days of adaptive feeding, fast the rats for 12 h without water restriction, and randomly divide them into 5 groups with 6 rats in each group. The normal control group (control) and the model group (model) were given distilled water at 10 mL / kg (body weight). The high-dose group of Phyllanthus emblica fruit juice polyphenols (PEF-H) and the low-dose group of Phyllanthus emblica fruit juice polyphenols (PEF-L) were gavaged with the Phyllanthus emblica extract at a high dose of 500 mg / kg and a low dose of 250 mg / kg. The positive control group was given 250 mg / kg of Gegenzhijuzi compound tablets (GZF). 30 min after the first gavage, the normal control group was gavaged with 0.9% physiological saline, and the other groups were used to establish an acute alcohol intoxication model in rats by a single gavage of excessive alcohol, and were gavaged with 15 mL / kg of 56% edible alcohol solution.

[0098] 2 Observation indicators

[0099] 2.1 General condition observation

[0100] Observe whether there are deaths in each group of rats before and after drug gavage, and whether there are obvious abnormalities in appearance, behavior, feces, food intake, etc.; observe whether there are deaths, excitement and mania, ataxia, reduced activity, lethargy, etc. in each group of rats after alcohol gavage.

[0101] The results showed that there were no deaths in each group of rats before and after the first gavage, and there were no obvious abnormalities in appearance, behavior, feces, food intake, etc. After the second gavage, except for the normal control group, the other groups of rats showed obvious drunken manifestations, mostly manifested as excitement and mania, ataxia, and then reduced activity and lethargy. It shows that an acute alcohol intoxication model was successfully established, and no deaths occurred in all rats during the observation period. The mental state and activity ability of the rats in each administration group after drinking were significantly improved compared with the model group.

[0102] 2.2 Serum index determination

[0103] Blood ethanol concentration detection was performed on the rats in each group. The rats were bled from the orbital venous plexus under light anesthesia at 0.5, 1.0, 2.0, and 4.0 h after the second gavage. After standing for 15 min, centrifuge at 3500 r / min for 10 min at 0 - 4 °C, take the serum, and store it at -80 °C for later measurement. Use a kit to detect the ethanol content in the serum. Measure the blood ethanol concentration of the rats at different time points and obtain the blood ethanol kinetic parameter: the area under the blood ethanol concentration-time curve. At the same time, 5 mL of blood was taken from the abdominal aorta after anesthesia with isoflurane 8 h after the second gavage, and the serum was prepared in the same way, and the levels of ALT, AST, and ethanol content in the serum were detected using the corresponding kits.

[0104] The results were as Figure 5 shown. Compared with the normal control group, after intragastric administration of 56% edible alcohol, there were significant differences in the blood alcohol concentrations of rats in each group, indicating that the alcoholic intoxication model of rats was successfully established. At the same time, the blood alcohol concentration reached the peak at 0.5 h. Compared with the model group, the blood alcohol concentrations of rats in the positive control group and the high-dose fruit juice group were significantly lower than those in the model group (P < 0.001); the low-dose fruit juice group was significantly lower than the model group (P < 0.05). At 1.0, 2.0, and 4 h after intragastric administration of 56% edible alcohol, the blood ethanol concentrations of rats in the high-dose fruit juice group were significantly lower than those in the model group (P < 0.001), and the low-dose fruit juice group was significantly lower than the model group at the 4 h time point (P < 0.05); compared with the model group, the area under the blood ethanol concentration-time curve and the blood alcohol content after 8 h of rats in each administration group decreased (P < 0.001, P < 0.05), and the effect of the high-dose group was better than that of the low-dose group, showing a certain dose-dependence. See Table 5 for details.

[0105] Table 5 Comparison of blood ethanol concentrations of rats in each group at different times after intragastric administration of 56% edible alcohol ( n = 6)

[0106]

[0107] (Note: Compared with the model group, *P < 0.05, **P < 0.01, ***P < 0.001. Compared with the normal control, # P < 0.05, ## P < 0.01, ### P < 0.001.)

[0108] The contents of AST and ALT in serum are important indicators for measuring liver function. When the liver is affected by foreign poisons, hepatocytes will undergo inflammation, fibrosis, and necrosis, etc., the permeability of the cell membrane changes, and ALT and AST are released into the extracellular fluid, causing an increase in the enzyme activity in the blood. Compared with the normal control, the activities of serum ALT (P < 0.001) and AST (P < 0.001) in the model group were significantly increased, indicating that the intake of alcohol had caused damage to the liver cell membrane and mitochondria. Compared with the model group, the activities of ALT and AST in the serum of rats in the high-dose fruit juice polyphenol group were significantly decreased (P < 0.001), and those in the low-dose group also decreased, but there was no significant difference. See Table 6 for details.

[0109] Table 6 Comparison of the contents of AST, ALT, ADH, and ALDH in the serum of rats in each group ( n = 6)

[0110]

[0111] (Note: Compared with the model group, *P < 0.05, **P < 0.01, ***P < 0.001. Compared with the normal control, # P < 0.05, ## P < 0.01, ### P < 0.001.)

[0112] 2.3 Organ index

[0113] Eight hours after the end of modeling, after the rats were sacrificed by taking blood from the abdominal aorta, the organs (liver, kidney, spleen) were quickly removed, rinsed with pre-cooled normal saline and blotted dry with filter paper. Weigh them and calculate the organ index according to formula (2).

[0114] Organ index = organ weight (g) / body weight (g) x 100% (2)

[0115] By calculating the organ index, compared with the normal control group, the liver and kidney indices of the rats in the model group increased (P < 0.001), indicating that after a large amount of alcohol intake, the important organs of the body were damaged to a certain extent, and the liver and kidney were most significantly affected. The spleen index increased, but there was no significant difference compared with the normal group. Compared with the model group, the kidney indices of the rats in the high-dose and low-dose groups of Phyllanthus emblica fruit polyphenols decreased significantly (P < 0.05), indicating that Phyllanthus emblica extract has a certain protective effect on organ damage caused by acute alcoholism. See Table 7 for details

[0116] Table 7 Organ indices of rats in each group ( n = 6)

[0117]

[0118] (Note: Compared with the model group, *P < 0.05, **P < 0.01, ***P < 0.001. Compared with the normal control, # P < 0.05, ## P < 0.01, ### P < 0.001.)

[0119] 2.4 Determination of liver biochemical indexes

[0120] Determine the contents of ADH, ALDH, SOD, MDA, and GSH in liver tissues according to the method described in the kit instructions. The results are shown in Table 8. Low levels of SOD and high levels of MDA are important markers of oxidative stress in the liver. Compared with the normal group, the content of SOD in the liver of rats in the model group decreased significantly (P<0.001), and the content of MDA increased (P<0.001), indicating that the antioxidant capacity of the liver in ethanol-induced liver injury rats decreased and oxidative stress increased. The activity of ADH in the liver increased significantly, and the activity of ALDH decreased significantly (P<0.05, P<0.01); compared with the model group, the content of SOD in rats in each drug administration group increased significantly, the content of MDA decreased significantly, and the activities of ADH and ALDH in the liver increased significantly. Among them, the indexes in the high-dose group of Phyllanthus emblica fruit polyphenols were significantly lower than those in the low-dose group. The results show that Phyllanthus emblica extract can improve the oxidative stress state of the liver and accelerate ethanol metabolism by enhancing the activities of ADH and ALDH, thereby reducing the ethanol level in the blood, and there is a certain dose-effect relationship.

[0121] Table 8 Comparison of oxidative stress indexes and ADH and ALDH activities in the livers of rats in each group ( n = 6)

[0122]

[0123]

[0124] (Note: Compared with the model group, *P < 0.05, **P < 0.01, ***P < 0.001. Compared with the normal control, # P < 0.05, ## P < 0.01, ### P < 0.001.)

[0125] 2.5 Gastric mucosal injury evaluation

[0126] After sacrificing the rats by taking blood from the abdominal aorta and dissecting the stomach, ligate the cardiac and pyloric parts of the stomach. After fixing with 5 mL of 4% formalin for 5 minutes, cut open along the greater curvature of the stomach, wash with pre-cooled physiological saline and then dry with filter paper. Weigh it and spread it on a white bottom plate for photographing and recording. Use ImageJ image processing software to statistically analyze the total area of the glandular part of the gastric tissue and the area of gastric mucosal bleeding, and calculate the gastric mucosal injury index and gastric mucosal injury inhibition rate according to formula (3) and formula (4).

[0127] Gastric mucosal injury index (%) = area of gastric mucosal bleeding / total area of the corresponding glandular part of the gastric tissue x 100% (3)

[0128] Gastric mucosal injury inhibition rate (%) = (injury index of the model group - injury index of the drug administration group) / injury index of the model group x 100% (4)

[0129] Acute excessive alcohol consumption can directly damage the gastric mucosa and even cause gastric bleeding. Therefore, in the anti-alcoholism experiment, the protective effect of Phyllanthus emblica extract on the gastric mucosa was observed simultaneously. As Figure 6 shown, compared with the model group, after the rats were given ig white liquor for 8 h, the degree of gastric mucosa injury in the high-dose group of Phyllanthus emblica fruit juice polyphenols was significantly reduced, the gastric mucosa bleeding performance of the rats was alleviated, the number of bleeding points was significantly reduced, and the degree of gastric mucosa injury in the low-dose group of rats was also significantly reduced.

[0130] The total area of the glandular part of the gastric tissue and the bleeding area of the gastric mucosa were statistically analyzed using the lmageJ image processing software. The gastric mucosa injury index and the gastric mucosa injury inhibition rate are shown in the table. Compared with the model group, the reduction of the gastric mucosa injury index in the positive control group and the administration group was statistically significant (P<0.01). The gastric mucosa injury inhibition rates of the positive control group and the administration group were 72.55%, 50.69%, 23.20%, 65.85%, and 36.53% respectively. Phyllanthus emblica extract can effectively prevent ethanol-induced gastric mucosa injury and has a certain protective effect on the gastric mucosa. See Table 8 for details.

[0131] Table 8 Effects of Phyllanthus emblica extract on gastric injury index and injury inhibition rate in rats ( n = 6)

[0132]

[0133]

[0134] (Note: Compared with the model group, *P < 0.05, **P < 0.01, ***P < 0.001. Compared with the normal control, # P < 0.05, ## P < 0.01, ### P < 0.001.)

[0135] 2.6 Observation of gastric tissue pathological sections

[0136] Referring to the methods of etc., the liver tissue samples were fixed by soaking in a 4% paraformaldehyde solution by volume fraction. After dehydration, embedding, sectioning and HE staining, the pathological morphology of the rat gastric mucosa was observed under the microscope field of view. Two fields of view were randomly photographed for each section. Based on the LaineL scoring standard with slight improvement, the degree of inflammatory cell infiltration, epithelial cell shedding and edema of the rat gastric mucosa epithelium was evaluated. The scoring standard is shown in Table 9.

[0137] Table 9 Histopathological scoring standard for acute gastric mucosa injury

[0138]

[0139] The mucosal layer of normal control rats was neatly arranged and structurally intact. In the model group, the mucosal layer was damaged, with a large number of apical glandular cells shedding and necrotic in sheets, accompanied by inflammatory infiltration and local bleeding. In the drug administration group, the pathological conditions were alleviated, without obvious glandular shedding, necrosis, and bleeding. The gastric mucosal layer was structurally intact and had good continuity. It was shown that it could effectively relieve alcohol-induced gastric mucosal damage and had a good protective effect. The results are shown in Figure 7 。

[0140] Pathological scores showed that compared with the normal group, the injury scores of various parts of the rats in the model group were significantly increased (P < 0.001), and they were in the pathological grade 4 region. Half of the animals in the positive control group were in the 1 and 2 score regions. The juice polyphenol group and the pomace polysaccharide group dose-dependently reduced the pathological injury. The results suggested that Phyllanthus emblica extracts could promote the healing of the ulcer surface induced by ethanol burning. The results are shown in Table 10.

[0141] Table 10 Histopathological scoring criteria for acute gastric mucosal injury

[0142]

[0143] (Note: Compared with the model group, *P < 0.05, **P < 0.01, ***P < 0.001. Compared with the normal control, # P < 0.05, ## P < 0.01, ### P < 0.001.)

[0144] All data of each group were expressed as ( . One-way ANOVA was performed using SPSS 26.0 software to compare the differences between groups. P < 0.05 was considered statistically significant. Graphs were plotted using GraphPad Prism 9.0 software. After determining that the data were normally distributed, one-way ANOVA (One-Way ANOVA) was used to compare between groups, and Tukey's method was used to analyze the significance of differences. P < 0.05 was used to determine whether there was significance, representing the sample size.

[0145] This study was carried out on the basis of previous studies to study its anti-alcoholism effect and mechanism, further enrich the research content of its anti-alcoholism effect, and provide a certain pharmacological basis for its clinical application expansion and secondary development. ALT and AST are mainly present in hepatocytes. When the liver is damaged, the permeability of the hepatocyte membrane increases, and ALT and AST are released from hepatocytes into the blood, and their contents increase significantly. The two are important indicators for evaluating liver function. In addition, AST is a mitochondrial enzyme. Ethanol will cause oxidative stress and lipid peroxidation reactions during metabolism, and then damage mitochondria, resulting in an increase in serum AST content. When the body ingests alcohol, metabolic systems such as ADH convert ethanol into acetaldehyde, which is then converted into acetic acid by ALDH and enters the tricarboxylic acid cycle for metabolism, and finally produces CO2 and water. Therefore, hangover and liver-protecting products can reduce the damage of ethanol and its metabolites to the liver by accelerating ethanol metabolism.

[0146] The results of this study showed that the polyphenol extract of Phyllanthus emblica fruit juice could reduce the contents of serum ALT and AST, effectively reduce oxidative stress damage. At the same time, it significantly reduced the ethanol content in the blood after drunkenness, enhanced the alcohol metabolism ability, played a certain role in protecting the liver from alcoholic damage, and had a certain protective effect on gastric mucosa damage caused by excessive drinking.

[0147] Example 4 Chemical Characterization of Phyllanthus emblica Extract

[0148] 1. Preparation of Test Solution

[0149] 1.1 Aqueous two-phase upper phase extract of Phyllanthus emblica fruit juice

[0150] Precisely weigh 60 mg of Phyllanthus emblica fruit juice extract, add 10 mL of 80% methanol, ultrasonicate for 30 min (power 250 W, frequency 100 KHz), cool to room temperature and shake well, filter through a 0.22 μm microporous membrane, and take the subsequent filtrate to obtain the solution.

[0151] 1.2 Ultrasonic extraction of Phyllanthus emblica fruit juice stock solution with ethanol (control without aqueous two-phase system)

[0152] Precisely measure 3 mL of fruit juice and 4.5 mL of ethanol, vortex and mix well, place in an ultrasonic cleaner, power 400 W, temperature 40 °C, ultrasonicate for 10 min, cool, centrifuge, take the supernatant, evaporate all solvents by rotary evaporation, add 20 mL of 80% methanol, shake well, filter through a 0.22 μm microporous membrane, and take the subsequent filtrate to obtain the solution.

[0153] 1.3 Ultrasonic extraction of Phyllanthus emblica fresh fruit after liquid nitrogen treatment with ethanol (control of traditional process)

[0154] Precisely weigh 0.103 g of fresh fruit, precisely add 10 mL of 50% ethanol, shake well, weigh, ultrasonically extract for 30 min (power 250 W, frequency 100 KHz), cool, filter, take the filtrate, evaporate all solvents by rotary evaporation, add 10 mL of 80% methanol, shake well, filter through a 0.22 μm microporous membrane, and take the subsequent filtrate to obtain the solution.

[0155] 1.4 Reflux extraction of Phyllanthus emblica fresh fruit after liquid nitrogen treatment with ethanol (control of traditional process)

[0156] Precisely weigh 0.2 g of Phyllanthus emblica fresh fruit, place it in a 100 mL flask, add 20 mL of 50% ethanol, shake well, reflux extract for 2 h, cool, filter, take the filtrate, evaporate all solvents by rotary evaporation, add 20 mL of 80% methanol, shake well, filter through a 0.22 μm microporous membrane, and take the subsequent filtrate to obtain the solution.

[0157] 2. Determination of the Contents of Major Components in Phyllanthus emblica Fruit Juice Extract

[0158] 2.1 Determination of Total Phenol Content

[0159] Take 0.2 mL of the test solution into a 5 mL EP tube, in parallel three times. Add 1 mL of 10% Folin-Ciocalteu reagent and 0.8 mL of 7.5% sodium carbonate solution, and place it in the dark at room temperature for 60 min. Using the corresponding reagent as the blank reference, measure the absorbance at 765 nm, and measure in parallel three times. The experiment is carried out in the dark. Calculate the total polyphenol content (μg / mL) under different conditions according to the standard curve. The results can be referred to Figure 8 。

[0160] 2.2 Determination of Total Sugar Content

[0161] Precisely measure 50 μL of the test solution into a 10 mL EP tube, in parallel 3 portions. Add distilled water to make the solution volume 0.2 mL, vortex and mix well. Add 75 μL of 5% phenol solution, vortex and mix well. Quickly add 600 μL of concentrated sulfuric acid, place it in a water bath at 80 °C and heat for 15 min, then take it out and cool it in an ice water bath for 5 min. Measure the absorbance at a wavelength of 490 nm using a microplate reader. Calculate the total sugar content (mg / mL) under different conditions according to the standard curve. The results can be referred to Figure 9 。

[0162] 2.3 Determination of Vitamin C Content

[0163] Precisely measure 200 μL of the test solution into a 5 mL volumetric flask, and make up the volume to the mark with 0.5 mol / L NaCl solution, mix well thoroughly to obtain the test sample solution. Precisely pipette 1 mL of the test sample solution into a 5 mL volumetric flask, make up the volume with 0.5 mol / L NaCl and mix well, adjust the pH to 4.5 with acetic acid-sodium acetate buffer solution. Using 0.5 mol / L NaCl as the control, measure the absorbance value of the sample at 243 nm; another precisely pipette 1 mL of the test sample solution, add 0.5 mL of copper sulfate solution (100 μg / mL), make up the volume to 5 mL with 0.5 mol / L NaCl, adjust the pH to 4.5 with acetic acid-sodium acetate buffer solution, and measure the absorbance value of the sample at 243 nm. The absorbance of vitamin C is calculated according to the formula as follows:

[0164] A0 = A2 – A1 Equation (3-1)

[0165] In the formula, A0 represents the absorbance of vitamin C in the sample, and A1 and A2 represent the absorbances measured without adding and adding copper sulfate solution to the sample respectively. The results can be referred to Figure 10 。

[0166] 2.4 Results

[0167]

[0168] 3. HPLC Determination of Polyphenolic Chemical Constituents

[0169] 3.1 Chromatographic Conditions:

[0170] Use a YMC-Pack ODS-AC18 (4.6 mm × 250 mm, 5 μm) chromatographic column, with acetonitrile-methanol (4:1) (B) and 0.1% formic acid in water (A) as the mobile phase, a flow rate of 1.0 mL / min, and gradient elution with "0 - 5 min: 0 - 5% B; 5 - 15 min: 5 - 10% B; 15 - 20 min: 10 - 15% B; 20 - 25 min: 15% B; 25 - 30 min: 15 - 20% B; 30 - 35 min: 20% B; 35 - 40 min: 20 - 25% B; 40 - 45 min: 25% B; 45 - 55 min: 25 - 60% B; 55 - 60 min: 60 - 0% B; 60 - 65 min: 0% B"; the column temperature is 30 °C; the injection volume is 10 μL; the detection wavelength is 270 nm.

[0171] 3.2 Standard Curve

[0172]

[0173] 3.3 Results

[0174]

Claims

1. An aqueous two-phase reagent for preparing a polyphenol extract of emblica fruit juice, comprising emblica fruit juice, ethanol and ammonium sulfate, characterized in that: The volume ratio of the emblica fruit juice to the volume of ethanol is 1:1.5-7, and the weight ratio of the ammonium sulfate to the volume of the emblica fruit juice is 0.5-2.5 g / mL.

2. The aqueous two-phase reagent according to claim 1, characterized in that: The volume ratio of the emblica fruit juice to the volume of ethanol is 1:4.8-5.5, and the weight ratio of the ammonium sulfate to the volume of the emblica fruit juice is 1.5-2 g / mL.

3. The aqueous two-phase reagent according to claim 1 or 2, characterized in that: The proportions of the two-phase aqueous reagents are as follows: when the volume of the emblica fruit juice is 3.1 mL, the volume of ethanol is 4.9 mL, and the weight of ammonium sulfate is 1.8 g.

4. A method for obtaining a polyphenol extract of emblica fruit juice using the aqueous two-phase reagent according to any one of claims 1 to 3, characterized in that: The following steps are involved: a) placing the aqueous two-phase reagent in an ultrasonic treatment device and performing sufficient ultrasonic treatment; b) separating the two-phase aqueous reagent after ultrasonic treatment and taking the upper phase, wherein the upper phase comprises total polyphenols, total polysaccharides, vitamin C, gallic acid, corilagin, cheleic acid and ellagic acid.

5. The method according to claim 4, characterized in that: The conditions of the ultrasonic treatment are: power of 300W-500W, and ultrasonic time of 5-10min.

6. The method according to claim 4, characterized in that: The ultrasonic treatment step of the two-phase aqueous reagent also includes: when the volume of the emblica fruit juice is 3.1 mL, the volume of ethanol is 4.9 mL, the weight of ammonium sulfate is 1.8 g, the two-phase aqueous reagent is placed in a 400W ultrasonic treatment device, and ultrasonic treatment is performed for 10 minutes.

7. The method according to claim 4, characterized in that The following steps are also included: The lower phase of the separated two-phase aqueous reagent is recovered and reused.

8. The method according to claim 4, characterized in that The total polyphenol content of the upper phase is measured, 1 mL of 10% Folin phenol reagent and 0.8 mL of 7.5% sodium carbonate solution are added to the upper phase, the corresponding reagents are used as blank references, and the absorbance value is measured at 765 nm; the ratio of each component of the two-phase reagent is adjusted according to the absorbance value, and / or the ultrasonic treatment parameters are adjusted according to the absorbance value.

9. The method according to claim 4, characterized in that The gallic acid content of the upper phase is determined, and the content is calculated according to a standard curve, wherein the regression equation of the standard curve is Y=0.0367X+0.2281, wherein X is the concentration gradient of gallic acid, and Y is the absorbance value; the ratio of each component of the two-phase reagent is adjusted according to the absorbance value, and / or the ultrasonic treatment parameters are adjusted according to the absorbance value.

10. Use of the polyphenol extract of Phyllanthus emblica juice obtained by the method of claim 4 in preparing a medicament for improving acute drunkenness and / or alleviating acute drunkenness-induced liver damage.

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