Method for detecting epigallocatechin gallate content

Abstract

The application belongs to the technical field of detection, and particularly relates to a detection method of epigallocatechin gallate content. The application provides a detection method of epigallocatechin gallate (EGCG) content based on salicylic acid-copper nanoscale enzyme. The salicylic acid-copper nanoscale enzyme has excellent oxidase simulation activity on one hand, and can efficiently catalyze TMB oxidation, so as to realize high-sensitivity detection of EGCG content. On the other hand, the nanoscale enzyme relies on inorganic coordination hybrid structure, has strong environmental tolerance, excellent storage stability, and can be recycled and reused multiple times. Based on the above characteristics, the detection method provided by the application is simple to operate, does not need complex instruments and equipment, has high sensitivity, is particularly suitable for on-site rapid detection and large-scale popularization and application, and has broad practical application prospects in complex matrices such as tea, tea beverages, health products and biological samples.

Description

Technical Field

[0001] This application belongs to the field of detection technology, specifically relating to a method for detecting the content of epigallocatechin gallate. Background Technology

[0002] EGCG (epigallocatechin gallate) is one of the most important active ingredients in tea, possessing various biological activities such as antioxidant, anti-inflammatory, and anti-cancer properties, and is widely used in health products, pharmaceuticals, and functional foods. With the increasing market demand for EGCG-related products, accurate and rapid detection of its content is particularly important. Currently, the main methods for EGCG content detection include high-performance liquid chromatography (HPLC), spectrophotometry, and the Folin-Ciocalteu method, which play a crucial role in laboratory analysis.

[0003] However, while existing HPLC methods offer high accuracy, they are hampered by expensive equipment, complex operation, and long detection cycles, making them unsuitable for rapid detection needs. Spectrophotometry, though relatively simple to operate, suffers from low sensitivity, poor specificity, and susceptibility to interfering substances. The Folin-Ciocalteu method requires additional, expensive standards. Furthermore, the natural oxidases used in traditional detection methods are prone to denaturation and inactivation, require stringent storage conditions, exhibit significant batch-to-batch variability, and carry the risk of endotoxin contamination, limiting their application and widespread adoption in practical detection.

[0004] Therefore, it is necessary to develop a new EGCG detection method that combines high sensitivity and ease of operation to meet the demand for rapid and accurate detection of EGCG content in practical applications. Summary of the Invention

[0005] Based on this, one embodiment of this application provides a method for detecting the content of epigallocatechin gallate.

[0006] This application provides a method for detecting the content of epigallocatechin gallate, comprising:

[0007] A mixture was prepared by mixing the salicylic acid-copper nanozyme solution with the sample to be tested.

[0008] A colorimetric substrate for horseradish peroxidase was added to the mixture, and a colorimetric reaction was carried out to prepare a reaction solution.

[0009] The absorbance of the reaction solution at wavelengths of 645nm-650nm was measured, and the EGCG content was calculated based on the linear relationship between the EGCG concentration and the system absorbance. The salicylic acid-copper nanozyme was prepared from polyvinylpyrrolidone, copper salt, and salicylic acid.

[0010] The molar ratio of the copper salt to the salicylic acid is (21-25):(16-20).

[0011] In some embodiments, the preparation method of the salicylic acid-copper nanozyme includes: mixing an aqueous solution of polyvinylpyrrolidone with an aqueous solution of copper salt, adjusting the pH value to 10-12, and stirring; then adding an aqueous solution of salicylic acid and continuing to stir to prepare a salicylic acid-copper nanozyme solution.

[0012] In some embodiments, the copper salt comprises copper chloride.

[0013] In some embodiments, the polyvinylpyrrolidone includes polyvinylpyrrolidone K30.

[0014] In some embodiments, the stirring process includes a first stirring process and a second stirring process.

[0015] In some embodiments, the parameters of the first stirring treatment include: a rotation speed of 400 r / min to 600 r / min and a stirring time of 1 h to 3 h.

[0016] In some embodiments, the parameters of the second stirring process include: a rotation speed of 150 r / min - 250 r / min and a stirring time of 20 h - 30 h.

[0017] In some embodiments, the concentration of polyvinylpyrrolidone in the aqueous solution is 4 w / v%-5 w / v.

[0018] In some embodiments, the concentration of copper salt in the aqueous copper salt solution is 0.2 w / v%-0.3 w / v.

[0019] In some embodiments, the concentration of salicylic acid in the aqueous salicylic acid solution is 0.8 w / v%-1.0 w / v.

[0020] In some embodiments, the chromogenic substrate of the horseradish peroxidase includes 3,3',5,5'-tetramethylbenzidine.

[0021] In some embodiments, the concentration of 3,3',5,5'-tetramethylbenzidine is 200 μmol / L to 500 μmol / L.

[0022] In some embodiments, the conditions for the colorimetric reaction include: pH 4.0-5.0, temperature 20°C-30°C, and time 3-10 min.

[0023] In some embodiments, the copper salt comprises copper chloride.

[0024] In some embodiments, the polyvinylpyrrolidone includes polyvinylpyrrolidone K30.

[0025] In some embodiments, the molecular weight of the vinylpyrrolidone K30 ranges from 40,000 to 60,000.

[0026] In some embodiments, the sample to be tested includes one or more of oral beverages, health products, cosmetics, and pharmaceutical preparations.

[0027] This application provides a method for detecting epigallocatechin gallate (EGCG) content based on a salicylate-copper nanozyme. This salicylate-copper nanozyme exhibits excellent oxidase-mimicking activity, efficiently catalyzing the oxidation of TMB, thus achieving highly sensitive EGCG content detection. Furthermore, relying on an inorganic coordination hybrid structure, this nanozyme possesses strong environmental tolerance, excellent storage stability, and can be repeatedly recycled. Based on these characteristics, the detection method provided in this application is simple to operate, requires no complex instruments, and has high sensitivity, making it particularly suitable for rapid on-site detection and large-scale application. It shows broad practical application prospects in complex matrices such as tea, tea beverages, health products, and biological samples. Attached Figure Description

[0028] To more clearly illustrate the technical solutions in the embodiments of this application and to more completely understand this application and its beneficial effects, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0029] Figure 1 This application provides an embodiment of the oxidase simulation activity verification of salicylic acid-copper nanozyme.

[0030] Figure 2 Double reciprocal curves for verifying the enzyme-simulated catalytic activity of salicylic acid-copper nanozyme provided in an embodiment of this application;

[0031] Figure 3 A linear response curve of EGCG provided in one embodiment of this application;

[0032] Figure 4 The colorimetric detection method based on salicylic acid-copper nanozyme provided in one embodiment of this application is used to detect EGCG in different beverage systems in actual samples. Detailed Implementation

[0033] The present application will be further described in detail below with reference to the embodiments and examples. It should be understood that these embodiments and examples are for illustrative purposes only and are not intended to limit the scope of the present application. The purpose of providing these embodiments and examples is to enable a more thorough and comprehensive understanding of the disclosure of the present application. It should also be understood that the present application can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various modifications or alterations without departing from the spirit of the present application, and the equivalent forms obtained also fall within the protection scope of the present application. Furthermore, numerous specific details are set forth in the following description to provide a fuller understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.

[0034] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0035] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0036] The terms "and / or," "or / and," and "and / or" as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical AND," and also undoubtedly includes technical solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0037] In this application, the terms "multiple", "various", "multiple times", "multi-dimensional", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0038] The terms “combinations of,” “any combination of,” and “any combination of” used in this article include all suitable combinations of any two or more of the listed items.

[0039] In this document, the term "suitable" as used in phrases such as "suitable combination," "suitable method," and "any suitable method" refers to the ability to implement the technical solution of this application, solve the technical problem of this application, and achieve the expected technical effect of this application.

[0040] In this application, terms such as "further," "even further," and "particularly" are used to describe purposes and indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0041] In this application, "optionally," "optionally," and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without." If there are multiple "optional" entries in a technical solution, unless otherwise specified, and there are no contradictions or mutual constraints, each "optional" entry shall be independent.

[0042] In this application, the technical features described in an open-ended manner include both closed technical solutions composed of the listed features and open technical solutions composed of the listed features.

[0043] In this application, numerical intervals (i.e., numerical ranges) are involved. Unless otherwise specified, the selected numerical distributions within the aforementioned numerical intervals are considered continuous and include the two endpoints (i.e., the minimum and maximum values) of the numerical range, as well as every value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints. In this document, this is equivalent to directly listing every integer. For example, if t is an integer selected from 1 to 10, it means that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Furthermore, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0044] Unless otherwise specified, the temperature parameters in this application are permitted to be either constant-temperature treatment or variations within a certain temperature range. It should be understood that the constant-temperature treatment allows temperature fluctuations within the precision range of the instrument control, such as ±5℃, ±4℃, ±3℃, ±2℃, or ±1℃.

[0045] In this application, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass-volume percentage.

[0046] All references to documents mentioned in this application are incorporated herein by reference as if each document were individually incorporated herein by reference. Unless they conflict with the inventive purpose and / or technical solution of this application, all cited documents are incorporated herein by reference in their entirety and for all purposes. When citing documents in this application, the definitions of relevant technical features, terms, nouns, phrases, etc., are also incorporated herein by reference. When citing documents in this application, examples and preferred embodiments of the cited technical features may also be incorporated herein by reference, but only to the extent that they enable the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, this application shall prevail or modifications shall be made adaptably to the description in this application.

[0047] To address the core technical problems of traditional EGCG detection methods, such as low sensitivity, poor stability, slow response speed, high cost, and complex operation, this application provides a method for detecting the content of epigallocatechin gallate.

[0048] The first aspect of this application provides a method for detecting the content of epigallocatechin gallate, comprising:

[0049] A salicylic acid-copper nanozyme solution was mixed with the sample to be tested to prepare a mixture; a chromogenic substrate for horseradish peroxidase was added to the mixture to carry out a colorimetric reaction to prepare a reaction solution; the absorbance of the reaction solution at wavelengths of 645nm-650nm was measured, and the EGCG content was calculated based on the linear relationship between the EGCG concentration and the system absorbance; the salicylic acid-copper nanozyme was prepared from polyvinylpyrrolidone, copper salt and salicylic acid.

[0050] The molar ratio of the copper salt to the salicylic acid is (21-25):(16-20). For example, the molar ratio is (21, 22, 23, 24, 25):(16, 17, 18, 19, 20).

[0051] This method utilizes the excellent catalytic performance of salicylic acid-copper nanozyme and the strong reducing ability of EGCG to achieve rapid and accurate detection of EGCG content. It features high detection sensitivity, strong stability, fast response speed, low cost, and simple operation, and its overall performance is significantly better than traditional HPLC, spectrophotometry, and Folin-Ciocalteu method.

[0052] Salicylic acid-copper nanozymes exhibit peroxidase and laccase-like activities, efficiently catalyzing the oxidation of chromogenic substrates to produce colored products. Due to the strong reducing power of EGCG, this oxidation process can be effectively inhibited, and the degree of inhibition is positively correlated with the EGCG content. The EGCG content can be accurately estimated by measuring changes in absorbance at characteristic wavelengths. Based on its inorganic coordination hybrid structure, the salicylic acid-copper nanozyme possesses extremely strong environmental tolerance, able to withstand harsh conditions such as high and low temperatures, a wide range of acid and alkali conditions, high salt content, and organic solvents. It exhibits excellent storage stability and can be repeatedly recycled, avoiding the drawbacks of natural enzymes such as easy denaturation and inactivation, and demanding storage conditions.

[0053] In some embodiments, the chromogenic substrate includes 3,3',5,5'-tetramethylbenzidine; optionally, the concentration of the 3,3',5,5'-tetramethylbenzidine is 200 μmol / L-500 μmol / L. TMB is a commonly used chromogenic substrate that can be oxidized to the blue product oxTMB under the catalysis of salicylic acid-copper nanozyme. This product has a characteristic absorption peak at a characteristic wavelength, and the change in absorbance has a good linear relationship with the EGCG content, with a linear correlation coefficient R. 2 The concentration can reach 0.9949, exhibiting high detection sensitivity and accuracy. In some embodiments, the TMB concentration can be selected from any value among 200 μmol / L, 300 μmol / L, 400 μmol / L, and 500 μmol / L.

[0054] In some embodiments, the characteristic wavelength is 645nm-650nm. 645nm-650nm is the characteristic absorption peak wavelength range of oxTMB. Measuring absorbance within this wavelength range can effectively avoid interference from other substances, improving the specificity and accuracy of detection. Experimental results show that within the EGCG concentration range of 0-1 mg / mL, the absorbance value exhibits a good linear relationship with the concentration, with a regression equation of y = -0.0745x + 1.5139, providing a reliable basis for the quantitative detection of EGCG. In some embodiments, the characteristic wavelength can be selected from any value among 645nm, 648nm, and 650nm.

[0055] In some embodiments, the colorimetric reaction conditions include: pH 4.0-5.0, temperature 20℃-30℃, and time 3 min-10 min. Suitable pH, temperature, and reaction time are key conditions for ensuring detection sensitivity and accuracy. The salicylic acid-copper nanozyme exhibits the highest catalytic activity within the pH range of 4.0-5.0; the temperature range of 20℃-30℃ facilitates rapid reaction; and the reaction time of 3 min-10 min ensures both sufficient reaction time and meets the requirements for rapid detection.

[0056] In some embodiments, the copper salt comprises copper chloride dihydrate. Copper chloride dihydrate is a commonly used, inexpensive, and readily available copper source that stably releases copper ions in aqueous solution, which coordinate with salicylic acid to form nanozymes with oxidase activity. Experiments show that the salicylic acid-copper nanozyme prepared using copper chloride dihydrate has a Km value of 2.58 × 10⁻⁶ for TMB. -2 mmol·L -1 This indicates that it has excellent affinity for the substrate TMB and extremely high catalytic activity.

[0057] In some embodiments, the polyvinylpyrrolidone comprises polyvinylpyrrolidone K30. The molecular weight of the polyvinylpyrrolidone K30 is in the range of 40,000-60,000. For example, the molecular weight range is 40,000, 45,000, 50,000, 55,000, and 60,000.

[0058] Polyvinylpyrrolidone K30 is a commonly used water-soluble polymer with good stability and dispersibility. It can effectively control the particle size distribution of nanozymes, prevent nanoparticle aggregation, and improve the stability and catalytic activity of nanozymes.

[0059] In some embodiments, the sample to be tested is one or more of oral beverages, health supplements, cosmetics, and pharmaceutical preparations. This method has a wide range of applications and can be used to detect EGCG content in various real-world samples, demonstrating good practicality and application prospects.

[0060] This application also provides a method for preparing a salicylic acid-copper nanozyme solution, comprising: mixing an aqueous solution of polyvinylpyrrolidone with an aqueous solution of a copper salt, adjusting the pH to 10-12, and stirring; then adding an aqueous solution of salicylic acid and continuing stirring to prepare the salicylic acid-copper nanozyme; optionally, the copper salt comprises copper chloride dihydrate; optionally, the polyvinylpyrrolidone comprises polyvinylpyrrolidone K30. This method is simple, uses inexpensive and readily available raw materials, and has mild preparation conditions, making it suitable for large-scale production. The prepared salicylic acid-copper nanozyme exhibits excellent oxidase mimicry activity and stability.

[0061] In some embodiments, the stirring process includes a first stirring process and a second stirring process. The parameters for the first stirring process include: a rotation speed of 400 r / min-600 r / min and a stirring time of 1 h-3 h; and / or the parameters for the second stirring process include: a rotation speed of 150 r / min-250 r / min and a stirring time of 20 h-30 h. These are optimal stirring parameters that can prepare salicylic acid-copper nanozymes with uniform particle size, good dispersibility, and high catalytic activity. In some embodiments, the first stirring speed can be selected from any value among 400 r / min, 500 r / min, and 600 r / min, and the stirring time can be selected from any value among 1 h, 2 h, and 3 h; the second stirring speed can be selected from any value among 150 r / min, 200 r / min, and 250 r / min, and the stirring time can be selected from any value among 20 h, 24 h, and 30 h.

[0062] In some embodiments, the concentration of polyvinylpyrrolidone K30 in the aqueous solution is 4w / v%-5w / v.

[0063] In some embodiments, the concentration of copper chloride dihydrate in the aqueous solution is 0.2 w / v%-0.3 w / v; and / or the concentration of salicylic acid in the aqueous solution is 0.8 w / v%-1.0 w / v.

[0064] In some embodiments, the concentration of polyvinylpyrrolidone K30 can be selected from any value among 4 w / v%, 4.5 w / v%, and 5 w / v%; the concentration of copper chloride dihydrate can be selected from any value among 0.2 w / v%, 0.25 w / v%, and 0.3 w / v%; and the concentration of salicylic acid can be selected from any value among 0.8 w / v%, 0.9 w / v%, and 1.0 w / v%.

[0065] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this application, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0066] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0067] It should be understood that in the various embodiments of this application, the order of the above-mentioned processes does not imply the order of execution. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0068] Example 1

[0069] This embodiment provides a method for detecting the content of epigallocatechin gallate and a method for preparing salicylic acid-copper nanozyme solution.

[0070] The raw materials for preparing the salicylic acid-copper nanozyme solution include: polyvinylpyrrolidone K30 (purchased from Sinopharm Group, content 1.2g, concentration 5w / v%), copper chloride dihydrate (CuCl2·2H2O, purchased from Sinopharm Group, content 0.4g, concentration 0.21w / v%), salicylic acid (purchased from Sinopharm Group, content 0.25g, concentration 1.0w / v%), and deionized water (balance).

[0071] The preparation method of salicylic acid-copper nanozyme solution includes: dissolving 1.2g of polyvinylpyrrolidone K30 in 24mL of deionized water to prepare an aqueous solution of polyvinylpyrrolidone K30; dissolving 0.4g of copper chloride dihydrate in 192mL of deionized water to prepare an aqueous solution of copper chloride dihydrate; mixing the above two aqueous solutions, adjusting the pH value to 11, and stirring at 500r / min for 2h; adding 25mL of aqueous solution containing 0.25g of salicylic acid to the above mixed solution, and stirring at 200r / min for 24h to prepare salicylic acid-copper nanozyme; centrifuging the resulting green solution at 10000r / min, discarding the supernatant, and drying the precipitate at 60℃ to obtain a powder product.

[0072] The method for detecting EGCG content includes: mixing 10 μL of salicylic acid-copper nanozyme solution diluted 60 times with 50 μL of the sample to be tested to prepare a mixture; adding 200 μL of 3,3',5,5'-tetramethylbenzidine solution with a concentration of 416 μmol / L to the mixture and then performing a colorimetric reaction; the colorimetric reaction conditions are pH 4.5, temperature 25℃, and time 5 min; the EGCG content is estimated by measuring the change in absorbance at a wavelength of 648 nm.

[0073] Example 2

[0074] This embodiment provides a method for detecting the content of epigallocatechin gallate and a method for preparing salicylic acid-copper nanozyme solution.

[0075] The raw materials for preparing the salicylic acid-copper nanozyme solution include: polyvinylpyrrolidone K30 (purchased from Sinopharm Group, content 1.5g, concentration 6.25w / v%), copper chloride dihydrate (CuCl2·2H2O, purchased from Sinopharm Group, content 0.4g, concentration 0.21w / v%), salicylic acid (purchased from Sinopharm Group, content 0.25g, concentration 1.0w / v%), and deionized water (balance).

[0076] The preparation method of salicylic acid-copper nanozyme solution includes: dissolving 1.5g of polyvinylpyrrolidone K30 in 24mL of deionized water to prepare an aqueous solution of polyvinylpyrrolidone K30; dissolving 0.4g of copper chloride dihydrate in 192mL of deionized water to prepare an aqueous solution of copper chloride dihydrate; mixing the above two aqueous solutions, adjusting the pH value to 11, and stirring at 500r / min for 2h; adding 25mL of aqueous solution containing 0.25g of salicylic acid to the above mixed solution, and stirring at 200r / min for 24h to prepare salicylic acid-copper nanozyme; centrifuging the resulting green solution at 10000r / min, discarding the supernatant, and drying the precipitate at 60℃ to obtain a powder product.

[0077] The method for detecting EGCG content is the same as in Example 1.

[0078] Example 3

[0079] This embodiment provides a method for detecting the content of epigallocatechin gallate and a method for preparing salicylic acid-copper nanozyme solution.

[0080] The raw materials for preparing the salicylic acid-copper nanozyme solution include: polyvinylpyrrolidone K30 (purchased from Sinopharm Group, content 1.2g, concentration 5w / v%), copper chloride dihydrate (CuCl2·2H2O, purchased from Sinopharm Group, content 0.6g, concentration 0.31w / v%), salicylic acid (purchased from Sinopharm Group, content 0.25g, concentration 1.0w / v%), and deionized water (balance).

[0081] The preparation method of salicylic acid-copper nanozyme solution includes: dissolving 1.2g of polyvinylpyrrolidone K30 in 24mL of deionized water to prepare an aqueous solution of polyvinylpyrrolidone K30; dissolving 0.6g of copper chloride dihydrate in 192mL of deionized water to prepare an aqueous solution of copper chloride dihydrate; mixing the above two aqueous solutions, adjusting the pH value to 11, and stirring at 500r / min for 2h; adding 25mL of aqueous solution containing 0.25g of salicylic acid to the above mixed solution, and stirring at 200r / min for 24h to prepare salicylic acid-copper nanozyme; centrifuging the resulting green solution at 10000r / min, discarding the supernatant, and drying the precipitate at 60℃ to obtain a powder product.

[0082] The method for detecting EGCG content is the same as in Example 1.

[0083] Example 4

[0084] This embodiment provides a method for detecting the content of epigallocatechin gallate and a method for preparing salicylic acid-copper nanozyme solution.

[0085] The raw materials for preparing the salicylic acid-copper nanozyme solution include: polyvinylpyrrolidone K30 (purchased from Sinopharm Group, content 1.2g, concentration 5w / v%), copper chloride dihydrate (CuCl2·2H2O, purchased from Sinopharm Group, content 0.4g, concentration 0.21w / v%), salicylic acid (purchased from Sinopharm Group, content 0.3g, concentration 1.2w / v%), and deionized water (balance).

[0086] The preparation method of salicylic acid-copper nanozyme solution includes: dissolving 1.2g of polyvinylpyrrolidone K30 in 24mL of deionized water to prepare an aqueous solution of polyvinylpyrrolidone K30; dissolving 0.4g of copper chloride dihydrate in 192mL of deionized water to prepare an aqueous solution of copper chloride dihydrate; mixing the above two aqueous solutions, adjusting the pH value to 11, and stirring at 500r / min for 2h; adding 25mL of aqueous solution containing 0.3g of salicylic acid to the above mixed solution, and stirring at 200r / min for 24h to prepare salicylic acid-copper nanozyme; centrifuging the resulting green solution at 10000r / min, discarding the supernatant, and drying the precipitate at 60℃ to obtain a powder product.

[0087] The method for detecting EGCG content is the same as in Example 1.

[0088] Comparative Example 1: This comparative example did not use salicylic acid-copper nanozyme.

[0089] The difference between this comparative example and Example 1 is that salicylic acid-copper nanozyme was not used in the detection system; other components and processes were the same as in Example 1. Specifically, 10 μL of deionized water was mixed with 50 μL of the sample to be tested to prepare a mixture; 200 μL of a 416 μmol / L solution of 3,3',5,5'-tetramethylbenzidine was added to the mixture, followed by a colorimetric reaction; the colorimetric reaction conditions were pH 4.5, temperature 25°C, and time 5 min; the EGCG content was calculated by measuring the change in absorbance at a wavelength of 648 nm.

[0090] Comparative Example 2: This comparative example uses natural horseradish peroxidase instead of salicylic acid-copper nanoenzyme.

[0091] The difference between this comparative example and Example 1 is that natural horseradish peroxidase is used instead of salicylate-copper nanozyme; other components and processes are the same as in Example 1. Specifically, 10 μL of horseradish peroxidase solution diluted 60 times (0.1 mg / mL) was mixed with 50 μL of the sample to be tested to prepare a mixture; 200 μL of 3,3',5,5'-tetramethylbenzidine solution with a concentration of 416 μmol / L was added to the mixture for a colorimetric reaction; the colorimetric reaction conditions were pH 4.5, temperature 25°C, and time 5 min; the EGCG content was calculated by measuring the change in absorbance at a wavelength of 648 nm.

[0092] Result verification:

[0093] I. Verification of Salicylic Acid-Copper Nanoenzyme Oxidase Activity

[0094] To verify whether the salicylic acid-copper nanozymes prepared in each example and comparative example have oxidase-mimicking activity, eight experimental systems were designed according to the table below. The samples were reacted at 25℃ for 5 min. After the reaction, the ultraviolet absorption spectrum of the system was measured at 648 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0095] Table 1: Details of different sample systems

[0096]

[0097] To investigate whether salicylic acid-copper nanozymes possess oxidase-mimicking activity, this experiment set up six parallel experimental systems, and measured the absorbance of each system at a wavelength of 648 nm. The experimental results are as follows: Figure 1 As shown, neither the TMB+H2O system nor the salicylic acid-copper nanozyme+H2O system exhibited any color development, and their absorbance values ​​were close to 0. The absence of color development in the TMB+H2O system indicates that individual TMB molecules do not undergo auto-oxidation to generate oxidized TMB (oxTMB) under the experimental conditions. The lack of color development in the salicylic acid-copper nanozyme+H2O system suggests that the IrO2 nanozyme itself does not possess inherent color development capabilities, thus eliminating the interference of the nanozyme's own color on the experimental results. Samples 1-4 demonstrate the effectiveness of Examples 1-4. Upon addition of the salicylic acid-copper nanozyme solution to the TMB system, the reaction system immediately turned blue, and a distinct characteristic absorption peak appeared at 648 nm. This absorption peak is characteristic of oxTMB, indicating that TMB was successfully oxidized to oxTMB. Samples 7-8 show that Comparative Examples 1-2 had no catalytic effect due to the failure to successfully synthesize the salicylic acid-copper nanozyme. Furthermore, if natural horseradish peroxidase was directly added according to this process, enzyme inactivation occurred, with absorption peaks similar to Sample 5. These experimental results confirm that the salicylic acid-copper nanozyme possesses significant oxidase-mimicking activity.

[0098] II. Enzyme-simulated catalytic activity verification of salicylic acid-copper nanozymes

[0099] To further investigate the enzyme-mimicking catalytic activity of salicylic acid-copper nanozymes, steady-state kinetics were studied using 3,3',5,5'-tetramethylbenzidine (TMB) as a substrate. The specific experimental procedures are as follows:

[0100] First, 200 μL of TMB solution at different concentrations was added to each well of the 96-well plate, with concentration gradients of 416 μmol / L, 208 μmol / L, 104 μmol / L, 52 μmol / L, 26 μmol / L, 13 μmol / L, 6.5 μmol / L, and 3.25 μmol / L. Then, 10 μL of salicylic acid-copper nanozyme solution diluted 60 times was added to each well, and the reaction system was kept at 25 ℃ and pH=4.5 for 5 min.

[0101] A blank control group was set up, which did not add TMB working solution, but only added an equal volume of buffer solution and salicylate-copper nanozyme solution; three parallel experiments were set up for each TMB concentration gradient to reduce experimental error. After the reaction, the absorbance of each reaction system was measured at 648 nm using a microplate reader (A). 648 ).

[0102] According to the Lambert-Beer law, as shown in equation (1), the concentration of oxidized TMB (oxTMB) generated in the system within 5 min of the reaction is calculated by measuring the absorbance value, and then used for fitting and analysis of subsequent steady-state kinetic parameters.

[0103] A= bc (1)

[0104] In equation (1): A is the absorbance of the system;

[0105] Let be the molar absorptivity of the absorbing substance; the molar absorptivity of oxTMB is 3.9 × 10⁻⁶. 4 L / mol / cm;

[0106] b represents the thickness of the absorbent layer, which is 0.3 cm from the concave meniscus to the top of the 96-well plate.

[0107] c represents the concentration of oxTMB produced, in μmol / L;

[0108] Divide the calculated c by the reaction time to obtain the amount of oxTMB produced per unit time (V, mmol / L / min). According to the formula (see equation (2)), with and Plot a double reciprocal curve and determine K based on the slope of the curve. m .

[0109] = · + (2)

[0110] In equation (2): V0 is the initial velocity, mmol / L / min;

[0111] Vmax is the maximum reaction rate, in mmol / L / min;

[0112] [S] represents the substrate concentration, in mmol / L;

[0113] K m is the Michaelis constant, mmol / L.

[0114] The results are as follows Figure 2 As shown, the Km value characterizes the enzyme's affinity for the substrate; a smaller value indicates higher affinity. Kinetic fitting results show that the Km value of the salicylate-copper nanozyme for TMB is 2.58 × 10⁻⁶. -2 mmol L -1 It is evident that the salicylic acid-copper nanozyme possesses both excellent affinity for the substrate TMB and extremely high catalytic activity, and its oxidase mimicry performance is at an advanced level among similar materials.

[0115] III. Sensitivity Verification of Salicylic Acid-Copper Nanozyme for EGCG Detection

[0116] To evaluate the detection sensitivity of salicylic acid-copper nanozyme for EGCG, its linear detection range was determined in this experiment. The experimental procedure was as follows: 10 μL of salicylic acid-copper nanozyme solution diluted 60 times was added sequentially to a 96-well plate, followed by 50 μL of EGCG standard solutions of different mass concentrations (concentration gradient of 0 mg / mL). -1 0.13 mg·mL -1 0.37 mg·mL -1 0.44 mg·mL -1 0.48 mg·mL -1 0.59 mg·mL -1 0.81 mg·mL -1A reaction system was constructed using 200 μL of a 416 μol / L TMB solution. The reaction system was incubated at pH 4.5 and 25°C for 5 min. After the reaction, the absorbance was measured at 648 nm using a microplate reader. A blank control group without TMB solution was also included to eliminate interference from other components. Data processing was performed by plotting the mass concentration of hydroxytyrosol on the x-axis and the corresponding absorbance on the y-axis to create a standard curve, which was then calculated.

[0117] Experimental results are as follows Figure 3 As shown, by Figure 3 It can be seen that as the EGCG concentration gradually increases, the absorbance of the detection system shows a significant decreasing trend, and within the EGCG concentration range of 0-1 mg / mL, a good linear correlation exists between the absorbance and the concentration. This linear relationship can be described by the regression equation y = -0.0745x + 1.5139, where the linear correlation coefficient R0 is... 2 =0.9949.

[0118] The closer the linear correlation coefficient is to 1, the better the linear fit. In this experiment, R0 2 =0.9949, indicating an extremely high goodness of fit, further confirming the reliable linear relationship between EGCG concentration and system absorbance within the aforementioned concentration range. In summary, the colorimetric assay based on salicylic acid-copper nanozymes can be used for the quantitative detection of EGCG, and this method possesses a certain degree of accuracy, providing experimental evidence for the rapid and convenient detection of EGCG.

[0119] IV. Detection of EGCG in Real Samples Using a Colorimetric Detection Method Based on Salicylic Acid-Copper Nanozymes

[0120] To verify the feasibility and reliability of the developed colorimetric detection method based on salicylic acid-copper nanozyme in detecting EGCG in actual samples, a spiking experiment was conducted. Three common oral beverages (collagen drink, hyaluronic acid drink, and coconut juice drink) were selected as experimental samples. The specific procedures were as follows: First, the three beverages were diluted 100 times with ultrapure water. After initial filtration through filter paper, they were further filtered through a 0.45 μm filter membrane to remove impurities, obtaining clear diluted beverage solutions for later use.

[0121] Accurately weigh a certain amount of EGCG sample and add it to the three beverage diluents prepared above to obtain spiked sample solutions with final EGCG concentrations of 0.79 mg / mL, 0.54 mg / mL, and 0.14 mg / mL, respectively. Then, according to the established detection system, add 10 μL of a 60-fold diluted salicylate-copper nanozyme solution, 50 μL of EGCG spiked recovery solution, and 200 μL of 416 μol / L TMB chromogenic solution to each well of a 96-well plate. The 96-well plate is then incubated at 25 ℃ for 5 min. After the reaction, the absorbance of each reaction system is measured at 648 nm using a microplate reader. Three parallel experiments are set up for each concentration to ensure the reliability of the experimental results.

[0122] Depend on Figure 4 The test data shows that the developed colorimetric detection method based on salicylic acid-copper nanozyme can be used to detect EGCG content in different systems in practical applications. The detected value in collagen drinks was closest to the standard addition amount. However, hyaluronic acid drinks and coconut juice drinks may contain components that affect the activity of hydroxytyrosol, slightly impacting the detection accuracy.

[0123] The embodiments described above are merely illustrative of several implementation methods of this application, intended to facilitate a detailed understanding of the technical solutions of this application, but should not be construed as limiting the scope of protection of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the scope of protection of this application. Furthermore, it should be understood that after reading the above teachings of this application, those skilled in the art can make various alterations or modifications to this application, and the equivalent forms obtained also fall within the scope of protection of this application. It should also be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided in this application through logical analysis, reasoning, or limited experimentation are all within the scope of protection of the appended claims. Therefore, the scope of protection of this patent application should be determined by the content of the appended claims, and the specification can be used to interpret the content of the claims.

Claims

1. A method for detecting the content of epigallocatechin gallate, characterized in that, include: A mixture was prepared by mixing the salicylic acid-copper nanozyme solution with the sample to be tested. A colorimetric substrate for horseradish peroxidase was added to the mixture, and a colorimetric reaction was carried out to prepare a reaction solution. The absorbance of the reaction solution at wavelengths of 645nm-650nm was measured, and the EGCG content was calculated based on the linear relationship between EGCG concentration and system absorbance; and... The salicylic acid-copper nanozyme is prepared from polyvinylpyrrolidone, copper salt and salicylic acid; The molar ratio of the copper salt to the salicylic acid is (21-25):(16-20).

2. The method for detecting the content of epigallocatechin gallate ester according to claim 1, characterized in that, The preparation method of the salicylic acid-copper nanozyme includes: Mix the aqueous solution of polyvinylpyrrolidone with the aqueous solution of copper salt, adjust the pH value to 10-12, and stir. Add salicylic acid aqueous solution and continue stirring to prepare salicylic acid-copper nanoenzyme solution; Optionally, the copper salt includes copper chloride; Optionally, the polyvinylpyrrolidone includes polyvinylpyrrolidone K30.

3. The method for detecting the content of epigallocatechin gallate according to claim 2, characterized in that, The mixing process includes a first mixing process and a second mixing process; The parameters for the first stirring treatment include: a rotation speed of 400 r / min - 600 r / min, and a stirring time of 1 h - 3 h; and / or The parameters for the second stirring treatment include: a rotation speed of 150 r / min - 250 r / min and a stirring time of 20 h - 30 h.

4. The method for detecting the content of epigallocatechin gallate according to claim 2, characterized in that, The concentration of polyvinylpyrrolidone in the aqueous solution is 4 w / v%-5 w / v%; and / or The concentration of copper salt in the copper salt aqueous solution is 0.2 w / v%-0.3 w / v%; and / or The concentration of salicylic acid in the aqueous solution is 0.8 w / v%-1.0 w / v.

5. The method for detecting the content of epigallocatechin gallate ester according to any one of claims 1 to 4, characterized in that, The chromogenic substrate of the horseradish peroxidase includes 3,3',5,5'-tetramethylbenzidine.

6. The method for detecting the content of epigallocatechin gallate ester according to any one of claims 1 to 4, characterized in that, The concentration of the 3,3',5,5'-tetramethylbenzidine is 200 μmol / L-500 μmol / L.

7. The method for detecting the content of epigallocatechin gallate ester according to any one of claims 1 to 4, characterized in that, The conditions for the colorimetric reaction include: pH 4.0-5.0, temperature 20℃-30℃, and time 3min-10min.

8. The method for detecting the content of epigallocatechin gallate ester according to any one of claims 1 to 4, characterized in that, The copper salt includes copper chloride.

9. The method for detecting the content of epigallocatechin gallate ester according to any one of claims 1 to 4, characterized in that, The polyvinylpyrrolidone includes polyvinylpyrrolidone K30; Optionally, the molecular weight of the vinylpyrrolidone K30 is in the range of 40,000-60,000.

10. The method for detecting the content of epigallocatechin gallate ester according to any one of claims 1 to 4, characterized in that, The samples to be tested include one or more of the following: oral beverages, health products, cosmetics, and pharmaceutical preparations.

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