Extraction and purification method and application of malus toringoides and malus toringoides tea polyphenol

Abstract

The invention discloses an extraction and purification method and application of malus toringoides leaf and leaf tea polyphenol, and belongs to the field of separation and application of natural plant products. The method comprises the following steps: mixing malus toringoides fruit and leaf dry tea powder with ethanol according to a set material-to-liquid ratio, and carrying out ultrasonic treatment on the mixed solution to obtain a malus toringoides fruit and leaf tea polyphenol crude extract; adding the malus toringoides leaf and leaf polyphenol crude extract into a chromatographic column filled with macroporous resin, and performing elution adsorption with an ethanol solution after adsorption equilibrium is achieved; and recovering the eluent, and freeze-drying to obtain the purified malus toringoides leaf and leaf tea polyphenol extract. The malus toringoides leaf and leaf tea polyphenol extract obtained by the invention has strong free radical scavenging capacity, can effectively inhibit active oxygen of HepG2 cells, and can be used for researching, developing and preparing antioxidant functional foods and medicines.

Description

Technical Field

[0001] This invention belongs to the field of plant natural product separation and application, and more specifically, relates to a method for extracting and purifying Russian tea polyphenols and its application. Background Technology

[0002] Russian tea ( Malus toringoides (Rehd.) Hughes, belonging to the genus Malus in the family Rosaceae, is a perennial shrub or small tree with both ecological value and medicinal potential. In recent years, with the rise of natural product research, Russian tea, due to its leaves and fruits being rich in bioactive components such as polyphenols, amino acids, and trace elements, has gradually become a research hotspot in the fields of food science, pharmacy, and functional food development.

[0003] Plant polyphenols, as an important class of secondary metabolites, are characterized by a benzene ring backbone with multiple hydroxyl groups. These compounds exhibit diversity in nature, with over 8,000 structurally different polyphenols identified to date. Widely distributed in natural dietary foods such as vegetables, berries, fermented teas, grape products, and woody plant seeds, they possess various biological activities and health benefits. Plant polyphenols are diverse and can be mainly classified into phenolic acids (such as chlorogenic acid and gallic acid), flavonoids (including flavonols, flavanones, and isoflavones), and tannins (hydrolyzed tannins and condensed tannins). Flavonoids account for the largest proportion, approximately 60% of the total plant polyphenols.

[0004] Existing research on Russian tea is very limited, mostly focusing on the functional activity of its crude extracts. There is a lack of exploration into the identification of its basic components, the extraction and purification of functional substances, process optimization, and the mechanisms of its functional activity, which hinders its transformation from basic research to industrial application. Currently, there are no in-depth research reports on the extraction, purification process optimization, and bioactivity of Russian tea polyphenols. Summary of the Invention

[0005] The present invention aims to overcome the shortcomings of the prior art and provide a method for extracting and purifying Russian tea polyphenols and their application.

[0006] To achieve the above objectives, the present invention provides a method for extracting and purifying polyphenols from Russian tea, comprising the following steps: 1) Pulverize the dried Russian tea and then sieve it to obtain Russian dried tea powder; mix the Russian dried tea powder with ethanol according to the set material-liquid ratio, sonicate the mixture, filter it after sonication, and obtain crude extract of Russian tea polyphenols. 2) Add the crude extract of Russian tea polyphenols obtained in step 1) to a chromatography column packed with macroporous resin. After reaching adsorption equilibrium, elute with ethanol solution. Recover the eluent and freeze-dry to obtain purified Russian tea polyphenol extract.

[0007] According to a preferred embodiment of the present invention, the ethanol volume percentage concentration in step 1) is 40-80%; the ratio of Russian tea powder to ethanol is 1:20-1:100 g / mL. The ultrasonic temperature is 20-60℃, and the ultrasonic time is 10-30 min. Further, in step 1), the ethanol volume fraction is 40-60%, the material-to-liquid ratio is 1:20-1:60 g / mL, the ultrasonic temperature is 30-50℃, and the ultrasonic time is 10-20 min. More preferably, the ethanol volume fraction is 50%, the material-to-liquid ratio is 1:43.5 g / mL, the ultrasonic temperature is 43℃, and the ultrasonic time is 14 min.

[0008] According to a preferred embodiment of the present invention, in step 2), the macroporous resin is D101 resin. In step 2), the volume fraction of the ethanol solution used for elution and adsorption is 70%.

[0009] The present invention also provides a Russian tea polyphenol extract prepared by the method described above.

[0010] The present invention relates to the application of the Russian tea polyphenol extract in the preparation of products that inhibit ROS levels in HepG2 cells in vitro. It can be further used to prepare antioxidant functional foods or drugs, such as anti-aging products that enhance the antioxidant capacity of tissues, organs, and / or cells.

[0011] Compared with the prior art, the present invention has the following effects and advantages: 1) This invention analyzed the main components and monomeric components in the dry matter of Russian tea and developed a method for extracting and purifying Russian tea polyphenols based on ultrasonic-assisted extraction and macroporous resin purification. In this invention, ultrasonic extraction was applied to the extraction of Russian tea polyphenols, and the optimal extraction process was obtained. This invention further identified D101 as the optimal purification resin. After purification with macroporous resin, the purity of Russian tea polyphenols was significantly improved. Scanning electron microscopy (SEM) confirmed that the purification process did not destroy the polyphenol functional groups and the impurity content was reduced.

[0012] 2) An evaluation using the H2O2-induced HepG2 oxidative stress model revealed that the free radical scavenging ability of purified polyphenols was significantly enhanced. P < 0.05), and can effectively inhibit reactive oxygen species (ROS) induced by H2O2 in HepG2 cells. Attached Figure Description

[0013] Figure 1 The content of the main physicochemical components of Russian tea.

[0014] Figure 2 The results show the effect of single-factor experiments on polyphenol extraction yield.

[0015] Figure 3 The chromatograms of the polyphenol extracts of Russian tea before and after purification are shown. Peak 1 is caffeic acid; peak 2 is p-coumaric acid; peak 3 is rutin; peak 4 is hyperoside; peak 5 isoquercitrin; peak 6 is quercetin; peak 7 is phlorizin; peak 8 is trifolin; peak 9 is quercetin; and peak 10 is phlorizin.

[0016] Figure 4 The images show the microstructure of the Russian tea polyphenol extract before and after purification.

[0017] Figure 5 To investigate the protective effect of purified polyphenols from Russian tea against H2O2-induced HepG2 cell damage.

[0018] Figure 6 To investigate the inhibitory effect of purified polyphenols from Russian tea on H2O2-induced ROS generation in HepG2 cells. Detailed Implementation

[0019] Preferred embodiments of the invention will now be described in more detail. While preferred embodiments of the invention are described below, it should be understood that the invention can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that the invention will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

[0020] Russian tea used in the embodiments of the present invention ( Malus toringoides (Rehd.) Hughes, belonging to the genus Malus of the family Rosaceae, was collected from high-altitude areas at 3200-3600 meters in Ganzi County, Sichuan Province. All reagents used in this invention are commercially available.

[0021] Example 1: Analysis of the main physicochemical components of Russian tea Russian tea sample was ground using a grinder, sieved, and 1.5 g of dried Russian tea powder was accurately weighed and placed in a 250 mL Erlenmeyer flask. An appropriate amount of boiling water was added, and the flask was placed in a 100°C constant temperature water bath for extraction for 45 min. During extraction, the flask was shaken every 15 min to ensure complete extraction. Immediately after extraction, the mixture was filtered under reduced pressure. After the filtrate cooled to room temperature, it was diluted to a final volume of 250 mL with ultrapure water.

[0022] The following detection methods were used to detect and quantitatively analyze the main physicochemical components of Russian tea: a. Determination of total polyphenol content in Russian tea: The total phenol content was determined using the Folin-Ciocalteu method. Gallic acid standard solutions of different concentrations were used as standards. 5.0 mL of 10% Folin-Ciocalteu reagent and 4.0 mL of 7.5% sodium carbonate were added sequentially, and the reaction was carried out in the dark for 60 min. The absorbance was measured at 765 nm, and a standard curve was plotted. 1.0 mL of Russian tea extract was processed using the same method, and the polyphenol content was calculated using the formula: Polyphenol content (mg / g) = C × V × N / M; In the formula: C is the polyphenol concentration (μg / mL), V is the extraction liquid volume (mL), N is the dilution factor, and M is the dry matter mass of the sample (g).

[0023] b. Determination of free amino acid content in Russian-colored tea: The total free amino acid content was determined using the ninhydrin method. Glutamic acid standard solutions of different concentrations were used as standards; after color development with ninhydrin, the absorbance was measured at 570 nm, and a standard curve was plotted. The same procedure was performed on 1.0 mL of Russian tea extract, and the amino acid content was calculated using the formula: Amino acid content (mg / g) = C × V / M; In the formula: C is the amino acid concentration (mg / mL), V is the extraction liquid volume (mL), and M is the dry matter mass of the sample (g).

[0024] c. Determination of soluble protein content in Russian tea The soluble protein content was determined using the Coomassie Brilliant Blue G-250 staining method. Different concentrations of bovine serum albumin standard solutions were used as standards. After reacting with Coomassie Brilliant Blue reagent, the absorbance was measured at 595 nm, and a standard curve was plotted. The same procedure was performed on 0.1 mL of Russian tea extract, and the soluble protein content was calculated using the formula: Soluble protein content (mg / g) = C × V / M; In the formula: C is the protein concentration (mg / mL), V is the extraction liquid volume (mL), and M is the dry matter mass of the sample (g).

[0025] d. Determination of reducing sugar content in Russian-colored tea The reducing sugar content was determined using the 3,5-dinitrosalicylic acid (DNS) colorimetric method. A glucose standard solution was used as a standard, reacted with the DNS colorimetric reagent, heated in a boiling water bath for 5.0 min, cooled, and diluted to 25 mL. The absorbance was measured at 540 nm, and a standard curve was plotted. The same procedure was performed on 0.5 mL of Russian tea extract, and the reducing sugar content was calculated using the formula: Reducing sugar content (mg / g) = C × V / M; In the formula: C is the concentration of reducing sugar (mg / mL), V is the volume of extract (mL), and M is the mass of dry matter in the sample (g).

[0026] Quantitative analysis was performed on the main physicochemical components extracted from the Russian tea in the examples, and the results are as follows: Figure 1As shown, there are significant differences in the content of their main chemical components. Polyphenols, as the core component, have a content as high as 151.98 mg / g, accounting for 51.34% of the total measured components, which is significantly higher than that of free amino acids (18.73 mg / g), soluble proteins (64.42 mg / g), and reducing sugars (60.86 mg / g). P <0.05). The contents of soluble protein and reducing sugar were similar with no significant difference, while the content of free amino acids was only 12.32% of that of polyphenols, indicating that they accounted for a low proportion in Russian tea.

[0027] Example 2: Extraction of crude polyphenols from Russian tea Preliminary experiments showed that using ethanol as the extraction solvent, combined with ultrasonic energy, significantly improved extraction efficiency. Therefore, Example 2 adopted the following procedure: Russian tea sample was ground using a pulverizer, sieved, and 0.2 g of dried Russian tea powder was accurately weighed and placed in an Erlenmeyer flask. Ethanol with a set material-to-liquid ratio was added, and the mixture was ultrasonically sonicated. After ultrasonication, the mixture was filtered to obtain crude extract of Russian tea polyphenols.

[0028] In this embodiment, the optimal process parameters were determined through the following single-factor experiments: Single-factor experiment 1) With the material-to-liquid ratio fixed at 1:60 (g / mL), ultrasonic temperature at 40°C, and ultrasonic time at 15 min, the ethanol concentrations were set at 40%, 50%, 60%, 70%, and 80% to study the effect of ethanol concentration on polyphenol yield.

[0029] Single-factor experiment 2) With the ethanol concentration fixed at 60%, ultrasonic temperature at 40°C, and ultrasonic time at 15 min, the material-liquid ratio was set at 1:20, 1:40, 1:60, 1:80, and 1:100 to study the effect of the material-liquid ratio on the polyphenol yield.

[0030] Single-factor experiment 3) With the ethanol concentration fixed at 60%, the material-to-liquid ratio at 1:60 (g / mL), and the ultrasonic time at 15 min, the ultrasonic temperatures were set at 20°C, 30°C, 40°C, 50°C, and 60°C to study the effect of ultrasonic temperature on polyphenol yield.

[0031] Single-factor experiment 4) With the ethanol concentration fixed at 60%, the material-to-liquid ratio at 1:60 (g / mL) and the ultrasonic temperature at 40°C, the ultrasonic temperatures were set for 10 min, 15 min, 20 min, 25 min and 30 min to study the effect of ultrasonic time on polyphenol yield.

[0032] like Figure 2As shown, ethanol concentration has a significant impact on the extraction yield of polyphenols from Russian tea. When the ethanol concentration increases from 40% to 50%, the polyphenol extraction yield gradually increases, reaching a peak at 50% (164.32 mg / g). After the ethanol concentration exceeds 50%, the extraction yield shows a decreasing trend, possibly because high ethanol concentration reduces solvent polarity, thus inhibiting polyphenol dissolution. The effect of the solid-liquid ratio on the polyphenol extraction yield shows a trend of first increasing and then decreasing. The highest polyphenol yield (157.18 mg / g) is achieved when the solid-liquid ratio is 1:40 (g / mL). A too-small solid-liquid ratio may lead to insufficient solvent, failing to adequately wet the raw material; while a too-large ratio may cause excessive solvent, leading to the dissolution of other impurities and reducing extraction efficiency. The polyphenol extraction yield shows a trend of first increasing and then decreasing with increasing ultrasonic temperature. When the temperature increases from 20°C to 40°C, the extraction yield significantly increases (156.51 mg / g), possibly because the appropriate temperature increase enhances molecular motion and diffusion rates. However, the extraction yield decreased when the temperature exceeded 40°C, presumably due to accelerated polyphenol oxidation or thermal degradation at high temperatures. Ultrasonication within 15 minutes promoted polyphenol dissolution, achieving a yield of 159 mg / g. However, after extending the time to 20 minutes, the extraction yield decreased to 156.84 mg / g, possibly due to polyphenol structural damage or oxidative decomposition caused by prolonged ultrasonication.

[0033] Based on the results of single-factor experiments, a Box-Behnken four-factor, three-level response surface methodology was employed, with ethanol concentration (A), solid-liquid ratio (B), ultrasonic temperature (C), and ultrasonic time (D) as independent variables, and polyphenol extraction yield (Y) as the response value. A quadratic regression model was established by setting up multiple experimental groups. The data were fitted using Design-Expert to obtain the regression equations for ethanol concentration (A), solid-liquid ratio (B), ultrasonic temperature (C), and ultrasonic time (D) versus polyphenol extraction yield (Y): Y=16.88-0.0892A+0.4442B+0.4483C-0.2517D-1.11A 2 -1.52B 2 -0.9448C 2 -0.8623D 2 -0.0325AB+0.0150AC-0.0250AD+0.2625BC-0.0025BD-0.1025CD Significance tests of the model parameters revealed that the linear terms of material-liquid ratio (B), ultrasonic temperature (C), and ultrasonic time (D), as well as the quadratic terms of A2, B2, C2, and D2, significantly affected the polyphenol extraction yield. The ethanol concentration (A) itself did not show a significant effect on the model, but its quadratic term had a significant effect on the model, indicating that the effect of this factor on the extraction yield may have a nonlinear effect.

[0034] Based on response surface methodology and a multiple regression model established using Design-Expert 13.0 software, the optimal conditions for polyphenol extraction from Russian tea were determined to be: 50% ethanol (v / v), a solid-liquid ratio of 1:43.5 g / mL, an ultrasonic temperature of 43°C, and an ultrasonic time of 14 min. Verification experiments showed that the actual polyphenol extraction yield reached 170.57 mg / g under these optimal conditions.

[0035] Example 3: Purification of crude polyphenols from Russian tea and screening with macroporous resin Pre-treated macroporous resins LX-8, D301, HPD600, LX-B14, and D101 were accurately weighed into 250 mL stoppered conical flasks. 100 mL of crude extract of Russian tea polyphenols was quantitatively transferred to establish the adsorption system. The sample was placed in a constant temperature shaking incubator (25±0.5°C, 120 r / min) and shaken continuously for 24 h until adsorption equilibrium was reached. The absorbance of the supernatant after adsorption equilibrium was determined by UV-Vis spectrophotometry. The concentration of remaining polyphenols was calculated according to the standard curve equation, and the adsorption capacity and adsorption rate of each resin were calculated. Subsequent desorption performance testing was performed. After adsorption equilibrium was reached, the resin and residual liquid were separated using a vacuum filtration device. The resin was washed three times with 50 mL of ultrapure water to remove surface adsorbed impurities. 100 mL of 70% ethanol solution was added to the resin for dynamic desorption, and desorption was continued for 24 h in a constant-temperature shaker (25±0.5°C, 120 r / min) until equilibrium was reached. The absorbance was measured at characteristic wavelengths using UV-Vis spectrophotometry, and the concentration of desorbed polyphenols was calculated based on the standard curve. The amount of precipitate and the desorption rate were calculated.

[0036] The static adsorption experimental data are shown in Table 1: Note: Experimental data are expressed as mean ± standard deviation (mean ± SD, n=3). Data in the same column marked with different lowercase letters in the upper right corner indicate statistically significant differences between groups (P < 0.05).

[0037] Static adsorption experimental data showed that D101 and LX-B14 resins exhibited significant adsorption advantages, with equilibrium adsorption capacities of 39.11 mg / g and 39.04 mg / g, respectively, and adsorption rates of 96.29% and 96.09%, respectively, significantly higher than the other three resins. P <0.05). Desorption kinetics studies showed that D101 resin exhibited superior desorption characteristics, with a desorption capacity and desorption rate of 35.29 mg / g and 90.25%, respectively, which were 9.43% and 7.49% higher than those of LX-B14 (32.25 mg / g, 82.65%). P<0.05). D101 resin exhibits significant adsorption performance for phlorizin, an active ingredient in traditional Chinese medicine. Its mechanism of action is mainly attributed to the absence of hydrogen bonds or significant conjugation between the resin matrix and the target molecules, along with weak intermolecular repulsion. This interaction mode reduces competition for adsorption sites, thereby improving the adsorption efficiency of phlorizin.

[0038] Example 4: Purification of Russian tea polyphenols The crude extract of Russian tea polyphenols obtained under the optimal conditions of Example 2 (50% ethanol volume fraction, solid-liquid ratio 1:43.5 g / mL, ultrasonic temperature 43℃, ultrasonic time 14 min) was purified using the following method: 30 g of pretreated D101 resin was packed into a chromatography column (25 mm × 300 mm), and the diluted crude E'se tea extract obtained in Example 2 (9.0 mg / mL, 100 mL) was gradually added using a peristaltic pump at a rate of 1.0 mL / min. After adsorption equilibrium was reached, elution was performed using 70% ethanol solution (v / v) at a flow rate of 1.0 mL / min. The eluent was recovered and freeze-dried to obtain purified E'se tea polyphenols (PES).

[0039] Chromatographic analysis was performed on the purified Russian tea polyphenol extract, such as... Figure 3 As shown, except for caffeic acid, which did not change significantly before and after purification, the other nine characteristic polyphenol monomers of Russian tea showed a significant increase. P <0.05%. Phlorizin still accounts for a very high proportion, increasing from 132.33 mg / g to 597.77 mg / g (4.52 times), followed by isoquercitrin (5.48 times) and quercetin (5.34 times). The contents of other polyphenol monomers such as hyperoside (5.95 times), phlorizin (10.46 times), and trifolin (1.85 times) also increased significantly. P <0.05).

[0040] Scanning electron microscopy was performed on the purified Russian tea polyphenol extract, such as... Figure 4 As shown, the microstructure of Russian tea polyphenols changed after purification. PESP (polyphenol esters) exhibited a more complete structure, predominantly consisting of regular lamellar structures with a smoother surface and fewer impurity particles. In contrast, crude polyphenols had a less uniform and regular structure, containing more impurity particles, a rougher surface, and more irregular crystal shapes. This phenomenon indicates that the surface smoothness and structural integrity of the material were significantly improved after purification.

[0041] Example 5: Inhibitory effect of Russian tea polyphenols on hydrogen peroxide-induced oxidative stress in HepG2 cells a. The protective effect of Russian tea polyphenols against H2O2-induced cellular oxidative damage After HepG2 cells were seeded and cultured for 24 h, the model group and the L / M / H treatment groups were treated with 200 μM H2O2 for 2–6 h to induce oxidative damage. Subsequently, the L / M / H treatment groups were treated with different concentrations of purified polyphenols from Russian tea (L: 10 μg / mL, M: 20 μg / mL, H: 40 μg / mL PESP) for 24 h. The model group and control group were then treated with fresh culture medium. Cell viability was measured after treatment. Figure 5 As shown, in the model group at 2 h, the low concentration (10 μg / mL) of Russian tea polyphenols increased cell viability from 40.19% to 41.18%, while the medium concentration (20 μg / mL) and high concentration (40 μg / mL) treatments of Russian tea polyphenols significantly restored cell viability to 53.53% and 59.65%, respectively. P<0.05 When the H2O2 injury time was extended to 6 h, the high concentration of Russet tea polyphenols could still maintain cell viability at 44.66%, which was 2.08 times higher than that of the H2O2 model group (21.44%). This indicates that the protective effect of Russet tea polyphenols on oxidative stress injury is concentration-dependent, and the high concentration group still maintained significant activity under prolonged injury time.

[0042] b. Effects of Russian tea polyphenols on intracellular ROS levels Intracellular reactive oxygen species (ROS) are mainly generated by mitochondria, endoplasmic reticulum, and nicotinamide adenine dinucleotide phosphate oxidase. When ROS accumulates excessively, it triggers oxidative stress, disrupts the oxidation-antioxidant balance, and leads to widespread damage to cells and tissues.

[0043] like Figure 6 As shown, compared with the control group, under 200 μM H2O2 induction conditions, the intracellular ROS levels in HepG2 cells of the model group were significantly higher than those of the control group at each time point. This indicates that the intracellular ROS level increased significantly after H2O2 treatment, and its endogenous antioxidant system could not effectively remove excess ROS, resulting in a state of oxidative damage. In cells treated with low (L), medium (M), and high (H) concentrations of Russian tea polyphenols, the ROS level was significantly lower than that of the model group. P <0.05), and the inhibitory effect showed a concentration-dependent characteristic. For example, at the 2-hour time point, the ROS levels in the low, medium, and high concentrations of Russian tea polyphenol treatment groups were significantly lower than those in the H2O2-induced model group ( P<0.05). As the oxidative damage time was extended to 4 h and 6 h, the ROS levels in the model group reached 3.65 times and 2.79 times that of the control group, respectively. Under these conditions, low-concentration Russian tea polyphenol treatment could still play a regulatory role, reducing ROS levels to 2.79 times and 2.41 times, respectively, while the high-concentration treatment group further significantly reduced ROS levels to 0.66 times and 0.69 times that of the control group (<0.05). P <0.05). Therefore, the experiment demonstrates that Russian tea polyphenols have a significant intracellular ROS scavenging ability and exhibit a concentration-dependent inhibitory effect.

[0044] The various embodiments of the present invention have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments.

Claims

1. A method for extracting and purifying polyphenols from Russian tea, characterized in that, Includes the following steps: 1) Pulverize the dried Russian tea and then sieve it to obtain Russian dried tea powder; mix the Russian dried tea powder with ethanol according to the set material-liquid ratio, sonicate the mixture, filter it after sonication, and obtain crude extract of Russian tea polyphenols. 2) Add the crude extract of Russian tea polyphenols obtained in step 1) to a chromatography column packed with macroporous resin. After reaching adsorption equilibrium, elute with ethanol solution. Recover the eluent and freeze-dry to obtain purified Russian tea polyphenol extract.

2. The method according to claim 1, characterized in that, In step 1), the volume percentage concentration of ethanol in step 1) is 40-80%; the ratio of Russian dried tea powder to ethanol is 1:20-1:100 g / mL.

3. The method according to claim 1, characterized in that, In step 1), the ultrasonic temperature is 20-60℃ and the ultrasonic time is 10-30 min.

4. The method according to claim 1, characterized in that, In step 1), the ethanol volume fraction is 40-60%, the material-to-liquid ratio is 1:20-1:60 g / mL, the ultrasonic temperature is 30-50℃, and the ultrasonic time is 10-20 min.

5. The method according to claim 1, characterized in that, In step 1), the ethanol volume fraction is 50%, the material-to-liquid ratio is 1:43.5 g / mL, the ultrasonic temperature is 43℃, and the ultrasonic time is 14 min.

6. The method according to claim 1, characterized in that, In step 2), the macroporous resin is D101 resin.

7. The method according to claim 1, characterized in that, In step 2), the volume fraction of the ethanol solution used for elution and adsorption is 70%.

8. The Russian tea polyphenol extract prepared by the method according to any one of claims 1-7.

9. The use of the Russian tea polyphenol extract according to claim 8 in a product for inhibiting ROS levels in HepG2 cells in vitro.

10. The use of the Russian tea polyphenol extract according to claim 8 in the preparation of antioxidant functional foods or medicines.

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