Method for rapidly evaluating actual oxidation resistance of polyphenol complex system and application

Abstract

The invention provides a method for rapidly evaluating the actual oxidation resistance of a polyphenol complex system and application. The method comprises the following three main processes: 1, simulating gastrointestinal digestion; 2, measuring oxygen free radical absorption capacity (ORAC) of related digestion products; and 3, drawing a standard curve by taking Trolox as a standard substance, and calculating the actual ORAC value of each group of samples. According to the method, an in-vitro simulation digestion system is combined with an ORAC method, and related experimental processes and conditions are close to physiological environments, so that the method has the advantages of simplicity, convenience and rapidness in operation, accuracy in measurement, high sensitivity and the like. Animal experiments prove that the antioxidant capacity rule obtained by the method has certain correlation with the antioxidant capacity change rule in the animal in-vivo digestion process.

Description

Technical Field

[0001] The present invention relates to the field of antioxidant efficacy evaluation methods, and particularly to a rapid evaluation method for the actual antioxidant capacity of an important class of antioxidant components (polyphenols). Background Art

[0002] With the increasing attention of modern people to health, antioxidants, as a key substance that can protect cells from oxidative damage, have gradually received wide attention. Polyphenols are a class of plant secondary metabolites that widely exist in fruits and vegetables and have multiple biological activities such as antioxidant, anti-inflammatory, and anti-cancer effects, playing an extremely important role in preventing chronic diseases and promoting human health.

[0003] Although polyphenolic compounds have significant antioxidant capacity, due to their complex molecular structure, easy oxidation, poor solubility, and low bioavailability in the body, their effects in practical applications are greatly limited. In addition, the transformation process of polyphenols in the digestive tract and the changes in their biological activities after binding to other components (such as proteins) are also key factors affecting their biological activities and antioxidant effects. Most polyphenols have the disadvantages of poor water solubility, poor stability, low bioavailability, bitter taste, etc., which greatly limit the application of polyphenols in the food industry.

[0004] At present, a large number of studies have proven that the solubility and stability of polyphenols can be improved through nano-encapsulation technology and microencapsulation technology, thereby effectively protecting phenolic substances from the influence of the external environment. Antioxidant capacity is the basic active function of polyphenols. In the free state, it is relatively easy to detect the antioxidant capacity of polyphenols. However, after encapsulation, wall materials will cover the polyphenol activity to a certain extent. During the digestion process of encapsulated polyphenols in the human body, as the wall materials are digested and hydrolyzed, the antioxidant activity of the polyphenols in the content can be more completely revealed. Therefore, there is a problem here: directly measuring the antioxidant capacity of polyphenol-protein complexes or microcapsules may not be completely consistent with their antioxidant effects in the human body.

[0005] At present, antioxidant detection methods tend to be diversified and can be roughly divided into three categories: chemical methods, cell methods, and in vivo detection methods. Chemical methods include 2,2-diphenyl-1-picrylhydrazyl radical scavenging method (abbreviated as DPPH radical scavenging ability), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) cation radical scavenging method (abbreviated as ABTS radical scavenging ability), ferric ion reducing ability, etc. Although they are fast and simple, the principles are quite different and the reaction systems are not unified, so the relevant results lack representativeness. Cell methods have a high degree of correlation with the antioxidant reactions in actual organisms, but do not take into account factors such as the digestion and absorption of polyphenols in the stomach or intestines, so they have certain limitations (Journal of Future Foods, 2024, 4(3): 193-204). Although in vivo antioxidant detection methods can take into account factors such as the digestion, metabolism, and absorption of polyphenols, the experimental process is more complex and the experimental cycle is longer, so they are not suitable for the preliminary screening and research and development of foods or drugs either.

[0006] By consulting the antioxidant evaluation methods disclosed in domestic and foreign papers and patents, it can be seen that although chemical methods based on DPPH radical scavenging ability, ABTS radical scavenging ability, etc. are the most widely used, there is a lack of a simple, fast, and antioxidant evaluation method that conforms to the actual antioxidant ability of the complex polyphenol system. Moreover, whether the chemical evaluation results used in in vitro analysis can be directly applied to biological systems has not been solved so far. Patent CN105483203A discloses a method using Escherichia coli as a model and measuring the bacterial survival rate by turbidimetry to reflect antioxidant activity, but this method also does not consider the influence of the digestion process on the antioxidant ability of the target substance, and for the microcapsule system encapsulating polyphenols, bacteria cannot directly absorb. Moreover, for colored substances, it is also difficult to remove the influence of the substance itself on the absorbance, so there are certain limitations.

[0007] In summary, the existing antioxidant test methods lack a unified test standard, and it is difficult to compare the data between different methods, nor can they truly reflect the health effects of polyphenols protected by encapsulation. Therefore, it is necessary to develop and explore a simple, accurate, and fast method according to the specificity of polyphenolic compounds. The present invention proposes a rapid antioxidant ability determination method for polyphenol-protein composite systems, aiming to simulate the digestion process, combine the ORAC method for antioxidant evaluation, and evaluate the correlation between this evaluation method and the antioxidant evaluation of the digestion process in mice, so as to efficiently and accurately evaluate the antioxidant effect of such systems during digestion, and provide important data support and theoretical basis for the development of related foods, health products, and drugs. Summary of the Invention

[0008] In view of the deficiencies of existing polyphenol antioxidant detection methods, the present invention provides a method for rapidly evaluating the actual antioxidant capacity of a polyphenol complex system. This method innovatively combines an in vitro simulated digestion system with the ORAC method, and the relevant experimental processes and conditions are close to the physiological environment, thus having the advantages of simple and rapid operation, accurate measurement, high sensitivity, etc. This method is applicable to the detection of the antioxidant capacity of polyphenol complex systems, and the obtained data can be compared and studied with a database. Compared with existing antioxidant methods such as the 2,2-diphenyl-1-picrylhydrazyl radical scavenging method (abbreviated as DPPH radical scavenging capacity) and the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) cation radical scavenging method (abbreviated as ABTS radical scavenging capacity), the present invention is more in line with the in vivo environment and actual digestion conditions, and can be compared with the ORAC values of different substances. Verified by animal experiments, the antioxidant capacity law obtained by this method has a certain correlation with the change law of antioxidant capacity during the digestion process in animals.

[0009] To achieve the object of the present invention, an embodiment of the present invention provides a test method for rapidly measuring the actual antioxidant capacity of a polyphenol-protein complex system.

[0010] This method is as follows: Prepare a solution or suspension of the polyphenol-containing complex system to be tested, measure the oxygen radical absorbance capacity after simulated gastrointestinal digestion, and detect the degree of inhibition of the fluorescence quenching reaction induced by oxygen radicals by polyphenols under certain conditions, so as to quantify the free radical scavenging ability of the antioxidant. Using sodium fluorescein as a fluorescent label, AAPH as an oxygen radical generator and fluorescence quenching agent, use an enzyme-linked immunosorbent assay (ELISA) reader to detect the fluorescence intensity value at regular intervals, calculate the area of the delayed part of the fluorescence quenching curve (NetAUC) of the sample group, draw a standard curve with Trolox as a standard product to calculate the actual ORAC value of each group of samples, and use the ORAC value to reflect the antioxidant activity of the sample.

[0011] Specifically:

[0012] Step 1: Using deionized water as a solvent, dissolve or disperse the polyphenol-containing substance into a solution or suspension, subject the solution or suspension to simulated gastric digestion by constant temperature oscillation at 36.5 - 37.5 °C for 60 - 120 min, and simulate small intestine digestion for 120 - 180 min, with deionized water as a blank control;

[0013] Step 2: Measure the oxygen radical absorbance capacity (ORAC) of the solution before digestion, at 60 min of digestion, at 120 min of digestion, and at 180 min of digestion. The specific steps include:

[0014] d. Using Trolox as a standard product, dilute the taken solution with PBS buffer;

[0015] e. Using sodium fluorescein as the fluorescent label and AAPH as the fluorescent quencher, incubate at 36.5 - 37.5 °C for 10 - 30 min;

[0016] f. Use a microplate reader to detect the fluorescence intensity values at regular intervals, with an excitation wavelength of 485 nm and an emission wavelength of 538 nm, and continuously detect until the fluorescence intensity drops below 5% of the initial value;

[0017] Step 3: Draw a standard curve using Trolox as the standard product to calculate the actual ORAC values of each group of samples. According to the area under the fluorescence value curve (AUC) of the sample group and the blank control group, calculate the fluorescence protection area of the sample group, that is, the net area NetAUC of the delayed part of the fluorescence quenching curve; calculate the NetAUC value according to the following formula;

[0018] AUC = 0.5 × [2 × (f0 + f i + …… + f n -1 + f n ) - f0 - f n × Δt

[0019] NetAUC = AUC sample - AUC AAPH+

[0020]

[0021] In the formula: The ORAC value represents the oxygen radical absorbance capacity; AUC represents the area under the fluorescence value curve; AUC sample is the area under the fluorescence curve measured and calculated for the sample group; AUC AAPH+ is the area under the fluorescence curve measured and calculated for the blank control group; f i represents the relative fluorescence intensity value in the i-th round; Δt represents the measurement time interval; NetAUC represents the net value of the area under the fluorescence curve; c Trolox represents the Trolox concentration, and c sample represents the sample concentration.

[0022] Preferably, the polyphenol-containing complex system is a substance containing polyphenols such as plant extracts, synthetic polyphenols, or food ingredients containing polyphenolic compounds, and the concentration of polyphenols in the substance is 0.01 - 10 mg / mL.

[0023] Preferably, the solvent used for the solution or suspension is deionized water.

[0024] Preferably, in step 1, the conditions for simulating gastric digestion are: the pH value of the simulated gastric juice is 1.5 - 3.0, and pepsin is added to simulate gastric digestion.

[0025] Preferably, in step 1, the simulated small intestine digestion conditions are as follows: the pH value of the simulated intestinal fluid is 6.8 - 7.4, and pancreatic enzymes and bile salts are added to simulate digestion in the small intestine.

[0026] Preferably, in step 1, the total duration of the simulated gastric and intestinal digestion is 3 - 4 h.

[0027] Preferably, in step 2, the reference substance is dissolved in PBS with a pH of 7.4 before detection. The concentration of sodium fluorescein is 10 - 50 μmol / L, and the concentration of AAPH is 20 - 60 mg / mL.

[0028] Preferably, in step 2, the time interval for detecting the fluorescence intensity can be 2 - 5 min, the detection temperature is 37 °C, and the total detection duration is preferably 1 - 3 h.

[0029] Preferably, the calculation result of the Trolox equivalent is the Trolox equivalent per gram of polyphenol or per milliliter of sample (μmol TE / g or μmol TE / mL).

[0030] The present invention can be applied to the evaluation of the antioxidant capacity of polyphenol - protein complex systems or microcapsules encapsulating polyphenols. The protein in the complex system can be casein, whey protein, soy protein, pea protein, etc., and the microcapsule wall material can be maltodextrin, resistant starch, trehalose, etc.

[0031] The present invention can be used to evaluate the antioxidant properties of foods, health products, and drugs containing polyphenols or polyphenol - protein complexes.

[0032] The present invention provides a test method for rapidly measuring the actual antioxidant capacity of polyphenol - protein complex systems. The antioxidant properties of complex systems are tested using a unified test standard. The larger the ORAC value, the better the free radical scavenging effect and the stronger the antioxidant capacity of polyphenols.

[0033] The change in the ORAC value of polyphenols reflects the change in the actual antioxidant capacity of polyphenols during the digestion process.

[0034] In summary, the present invention proposes a rapid antioxidant capacity determination method for polyphenol - protein complex systems, aiming to evaluate antioxidant properties by simulating the digestion process and combining with the ORAC method, and evaluate the correlation between this evaluation method and the antioxidant evaluation of the digestion process in mice. It can efficiently and accurately evaluate the antioxidant effect of such systems during the digestion process, providing important data support and theoretical basis for the development of related foods, health products, and drugs.

[0035] Compared with the prior art, the beneficial effects of the present technology mainly lie in:

[0036] (1) By simulating the digestion process and combining with the ORAC method for antioxidant evaluation, this method can accurately evaluate the antioxidant capacity of complex systems such as polyphenol-protein composite systems and polyphenol microcapsules after the digestion process in a relatively short time.

[0037] (2) This method can truly reflect the change of antioxidant capacity of polyphenol-protein composite systems during the in vivo digestion process. By comparing and verifying with the antioxidant evaluation of the in vivo digestion process of mice, this method provides a more relevant and higher-precision evaluation, and can more reliably reflect the antioxidant effect.

[0038] (3) By simulating the digestion environment, this method avoids complex animal experiment operations, making the antioxidant test process more simple and fast, and has high operability and application value.

[0039] (4) This technology can provide scientific data support for the research and development of foods, health products, and drugs in related fields, help developers better understand and evaluate their antioxidant efficacy, and promote product optimization. Description of the Drawings

[0040] Figure 1 It is the fluorescence decay curve and standard curve of Trolox series concentrations in Example 1.

[0041] Figure 2 It is the content change of free polyphenols during in vitro simulated digestion process in Example 1 and the determination results of antioxidant methods in Example 1, Comparative Example 1, Comparative Example 2, and Comparative Example 3.

[0042] Figure 3 It is the content change of polyphenol-protein composite nanoparticles during in vitro simulated digestion process in Example 1 and the determination results of antioxidant methods in Example 1, Comparative Example 1, Comparative Example 2, and Comparative Example 3.

[0043] Figure 4 It is the content change of polyphenol microcapsules during in vitro simulated digestion process in Example 1 and the determination results of antioxidant methods in Example 1, Comparative Example 1, Comparative Example 2, and Comparative Example 3.

[0044] Figure 5 It is the antioxidant capacity (T-AOC) of the sample in the digestive juice at different times after gavage in mice in Example 1.

[0045] Figure 6 It is the polyphenol content in the in vivo digestive juice of the sample at different times after gavage in mice in Example 1. Detailed Embodiments

[0046] In the description of the present invention, it should be noted that unless otherwise clearly specified and defined, the term "comprising" or its variants such as "including" or "having" etc. will be understood to include the stated elements or components, without excluding other elements or other components. In some embodiments, raw materials, components, methods, means, etc. well-known to those skilled in the art are not described in detail, and the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0047] Example 1

[0048] A method for evaluating the antioxidant capacity of a polyphenol complex system:

[0049] The sample preparation method is as follows:

[0050] (1) Free complexed polyphenol group: Weigh 10 mg of polyphenols, add deionized water and mix well to prepare a uniform solution or suspension of 1 mg / ml. The polyphenols used are a complex combination of quercetin and caffeic acid in a molar ratio of 1:1. The polyphenols with a quercetin to caffeic acid molar ratio of 1:1 are denoted as F.

[0051] (2) Polyphenol-protein composite particle group: Prepare polyphenol-protein composite nanoparticles. Dissolve the complex mixture of quercetin and caffeic acid in a molar ratio of 1:1 in ethanol to form a solution of 2 mg / ml, add it to the soy protein isolate solution, with a protein to polyphenol mass ratio of 5:1, and dialyze at 4°C for 24 h to remove ethanol to prepare a composite particle dispersion of sodium caseinate and the complexed polyphenols, denoted as SCF.

[0052] (3) Polyphenol microcapsule group: Add a mass-compounded mixture of trehalose and maltodextrin as a spray-drying wall material to the SCF obtained in step (2). The mass of the added wall material is 2 times the mass of the solids in the SCF solution (the SCF solid concentration is 20 mg / ml). After mixing for 2 h, spray-dry to prepare microcapsules containing polyphenols, denoted as SCF-MT2.

[0053] Simulated gastrointestinal digestion treatment:

[0054] (1) Prepare simulated gastric juice: Accurately weigh 0.2574 g of KCl, 0.0612 g of KH2PO4, 1.0500 g of NaHCO3, 1.3806 g of NaCl, 0.0122 g of MgCl2(H2O)6, and 0.0240 g of (NH4)2CO3 in a beaker, add 400 mL of deionized water and stir well, and adjust the pH to 2.0.

[0055] (2) Preparation of simulated intestinal fluid: Accurately weigh 0.2536 g of KCl, 0.0544 g of KH2PO4, 3.5700 g of NaHCO3, 1.1232 g of NaCl, and 0.0336 g of MgCl2(H2O)6 into a beaker, add 400 mL of deionized water, stir evenly, and adjust the pH to 7.0.

[0056] (3) Gastric digestion: Place the samples of each group in a 37°C water bath, add simulated gastric fluid (pH 2.0, containing 2000 U / mL of pepsin), and oscillate for 60 min to simulate the gastric digestion process. Samples are taken at 0 min and 60 min of simulated gastric digestion.

[0057] (4) Intestinal digestion: After gastric digestion is completed, adjust the pH of the samples to 7.0, add simulated intestinal fluid (containing pancreatin, 100 U / mL) and a 2% bile salt suspension by mass concentration, and continue to oscillate for 120 min to simulate the small intestine digestion process. Samples are taken at 60 min and 120 min of simulated intestinal digestion.

[0058] (5) After terminating digestion, immediately cool the samples to 4°C for subsequent analysis.

[0059] Measurement of oxygen radical absorbance capacity (ORAC):

[0060] (1) Preparation work:

[0061] Prepare PBS buffer (pH 7.4, 75 mmol / L) as the reactant solvent.

[0062] Prepare fluorescein sodium salt solution (concentration: 10 μmol / L) as the fluorescent label.

[0063] Prepare AAPH solution (concentration: 48 mg / ml) as the oxygen radical generator.

[0064] Prepare Trolox standard solution (concentration range: 5 - 100 μmol / L) for drawing the standard curve.

[0065] (2) Measurement steps:

[0066] Take an appropriate amount of sample digest and dilute it to an appropriate concentration with PBS buffer.

[0067] In a 96-well microplate, add 140 μL of fluorescein sodium salt solution and sample solution in a volume ratio of 6:1 to each well. In the control group AAPH+, replace the sample solution with deionized water. After pre-incubating at 37°C for 20 min, add 40 μL of AAPH solution to each well except the blank control group AAPH-. The AAPH- group adds PBS buffer, and immediately start recording the change in fluorescence intensity.

[0068] The fluorescence intensity was measured using an enzyme-labeled instrument with an excitation wavelength of 485 nm and an emission wavelength of 538 nm. The measurement was performed every 4 min for 120 min.

[0069] (3) Data processing:

[0070] The relative fluorescence intensity curve was drawn, and the fluorescence protection area of the sample group, that is, the area of the delayed part of the fluorescence quenching curve (NetAUC) was calculated based on the area under the fluorescence value curve (AUC) of the sample group and the blank control group. The ORAC value of the sample was calculated using the Trolox standard curve, and the result was expressed in Trolox equivalent (μmol TE / g).

[0071] AUC = 0.5 × [2 × (f0 + f i +……+f n -1+f n )-f0-f n ]×Δt

[0072] NetAUC=AUC sample -AUC AAPH+

[0073]

[0074] Where: ORAC value represents oxygen free radical absorption capacity; AUC represents the area under the fluorescence curve; fi represents the relative fluorescence intensity value of the i-th round; Δt represents the measurement time interval; NetAUC represents the net value of the area under the fluorescence curve; c Trolox represents Trolox concentration, c sample Represents the sample concentration.

[0075] The fluorescence decay curves of the Trolox series concentrations in Example 1 and the standard curve are as follows: Figure 1 As shown. The NetAUC value can be obtained according to the above formula and substituted into the standard curve to obtain the Trolox equivalent, which can be converted into the ORAC value according to the formula. The fluorescence intensity values measured at different digestion times for F, SCF, and SCF-MT2 were processed to obtain AUC and NetAUC, and the ORAC value was further calculated from the concentration, as shown in Table 1.

[0076] Table 1 ORAC values of samples in each group

[0077]

[0078] Comparative Example 1

[0079] The sample preparation method and simulated digestion steps were according to Example 1.

[0080] The DPPH free radical scavenging ability was determined as follows:

[0081] Prepare a 48 mg / L DPPH solution with absolute ethanol. Add 2.00 mL of the DPPH solution and 1.00 mL of the sample to be tested into test tube 1, shake well, react in the dark for 30 min, and measure the absorbance at 517 nm using a visible spectrophotometer. This is the absorbance A of the sample to be tested. s Use vitamin C as the standard.

[0082] Add 2.00 mL of the DPPH solution and 1.00 mL of absolute ethanol to test tube 2, shake well, react in the dark for 30 min, and measure the absorbance at 517 nm. This is the absorbance A0 of the initial solution.

[0083] Add 1.0 mL of the sample to be tested and 2.00 mL of absolute ethanol to the test tube with the sample to be tested, shake well, react in the dark for 30 min, and measure the absorbance value at 517 nm. This is the absorbance A of the reference solution. r .

[0084] The DPPH radical scavenging rate of the sample (DPPH / %; %) is calculated according to the following formula.

[0085]

[0086] In this comparative example, the chemical method DPPH method is used, with vitamin C as the standard, to make a standard curve of standard product concentration - net absorbance, and the results are expressed in terms of vitamin C equivalent (μmol V C / g). Through the measurement results of the sample absorbance value, substitute it into the formula to obtain the DPPH radical scavenging rate, and substitute it into the standard curve to convert it into vitamin C equivalent. Calculate the DPPH values of F, SCF, and SCF-MT2 at different digestion times, as shown in Table 2.

[0087] Table 2 DPPH values of each group of samples

[0088]

[0089] Comparative Example 2

[0090] The sample preparation method and the simulated digestion steps are according to Example 1.

[0091] The operation for measuring the ABTS radical scavenging ability is as follows:

[0092] Mix 100 μL of ABTS solution and oxidant solution to prepare the ABTS stock solution, and store it in the dark at room temperature for 12 - 16 hours before use. Before use, dilute the ABTS working stock solution with PBS to obtain the ABTS working solution. It is required that after subtracting the corresponding PBS blank control from the absorbance of the ABTS working solution, A734 is 0.7 ± 0.05. Using vitamin C as the standard product, add the ABTS working solution and the standard solution in a ratio of 20:1, and add 10 μL of various samples to the sample detection wells, then mix gently. Incubate at room temperature for 2 - 6 minutes and then measure A734. The absorbance of the test solution is denoted as A1, and the absorbance of the reference solution is denoted as A0. The ABTS radical scavenging rate of the sample (ABTS / %; %) is calculated according to the following formula.

[0093]

[0094] This comparative example uses the chemical method ABTS method, with vitamin C as the standard product, to make a standard curve of standard product concentration - net absorbance, and the results are expressed in vitamin C equivalents (μmol V C / g). Through the measurement results of the sample absorbance values, substitute them into the formula to obtain the ABTS radical scavenging rate, and substitute them into the standard curve to convert to vitamin C equivalents. Calculate the ABTS values of F, SCF, and SCF-MT2 at different digestion times, as shown in Table 3.

[0095] Table 3 ABTS values of each group of samples

[0096]

[0097]

[0098] Comparative Example 3

[0099] The sample preparation method and the simulated digestion steps are the same as in Example 1.

[0100] The operation for measuring the ferric ion reducing power is as follows:

[0101] Take 2.5 mL of the sample, with vitamin C as the standard product, and add 2.5 mL of 0.2 mol / L PBS solution with a pH of 6.6 and 1% potassium ferricyanide solution respectively, mix well, incubate in a water bath at 50 °C for 20 min, then take it out and quickly cool it in ice water; then add 2.5 mL of 10% trichloroacetic acid solution, mix well, centrifuge at 3000 r / min for 10 min, take 2.5 mL of the supernatant into a test tube, add 2.5 mL of distilled water and 0.5 mL of 0.1% ferric chloride solution, vortex and mix well, let it stand for 10 min, and measure the absorbance value A1 at 700 nm; use 2.5 mL of distilled water instead of the sample solution as the sample control group, measure the absorbance value A2, and the reducing power is expressed by the following formula:

[0102] A = A0 - A1

[0103] This comparative example uses the reducing power method in chemical methods. Using vitamin C as the standard product, a standard curve of standard product concentration - net absorbance is made, and the results are expressed in terms of vitamin C equivalent (μmol VC / g). By measuring the absorbance value of the sample and substituting it into the formula to obtain the net absorbance value, and then substituting it into the standard curve to convert it into vitamin C equivalent. Calculate the ABTS values of F, SCF, and SCF-MT2 at different digestion times, as shown in Table 4.

[0104] Table 4 Reducing power values of each group of samples

[0105]

[0106] Simulated digestion verification of correlation:

[0107] The Pearson correlation coefficient is often used to measure the strength and direction of the linear relationship between two variables. The value of the Pearson coefficient ranges from -1 to +1. The closer the value is to +1, the stronger the positive correlation between the two; close to -1 indicates a negative correlation, and close to 0 indicates no correlation. A Pearson coefficient of 0 - 0.3 represents a weak correlation between variables, a Pearson coefficient of 0.3 - 0.7 represents a moderate correlation between variables, and a Pearson coefficient of 0.7 - 1.0 represents a strong correlation between variables.

[0108] Taking the free compound polyphenols of Sample 1 as the research object, the content changes of free polyphenols during in vitro simulated digestion in Example 1 and the determination results of the antioxidant methods in Example 1, Comparative Example 1, Comparative Example 2, and Comparative Example 3 are obtained, as Figure 2 shown ( Figure 2 the content changes of free polyphenols during in vitro simulated digestion, the determination results of antioxidant methods. See Figure (a) for Example 1, Figure (b) for Comparative Example 1, Figure (c) for Comparative Example 2, and Figure (d) for Comparative Example 3). It can be found that there is a correlation in the results obtained by the methods of each example. The Pearson correlation coefficient is used to evaluate the correlation between the polyphenol content and Figure 2 the results of the four antioxidant methods in. The results are shown in Table 5. It can be seen that during digestion, as the polyphenol content decreases, the antioxidant property of a unit amount of polyphenol increases, indicating that during digestion, the antioxidant property of polyphenols is enhanced.

[0109] Table 5 Pearson correlation coefficients between polyphenol retention rate (F, %) of the free polyphenol group and the determination results of four antioxidant methods

[0110]

[0111] Note: ** indicates significant correlation at the 0.01 level, and * indicates significant correlation at the 0.05 level.

[0112] A correlation coefficient greater than 0.7 indicates a strong correlation between the methods and contents of Example 1 and Comparative Example 2, and the correlation is significant. This high correlation proves the accuracy and reliability of the method. However, the consistency between the methods of Comparative Example 2 and Example 3 and the polyphenol content is worse than that of Example 1 and Comparative Example 2.

[0113] The DPPH method shows a strong positive correlation with the polyphenol content. This may be because during the simulated digestion of free polyphenols, the composition of the digestive juice is relatively simple, and DPPH free radicals can react freely with polyphenols, thus showing good correlation. The ORAC method is applicable to alcohol-soluble and water-soluble systems, and the reaction uses a fluorescent indicator, which is less interfered by other impurities, so it shows good correlation.

[0114] Furthermore, taking the polyphenol-protein composite nanoparticles of Sample 2 as the research object, the content changes during the in vitro simulated digestion process and the determination results of the antioxidant methods of Example 1, Comparative Example 1, Comparative Example 2, and Comparative Example 3 are measured, as Figure 3 shown ( Figure 3 are the content changes and the determination results of the antioxidant methods during the in vitro simulated digestion of polyphenol-protein composite nanoparticles. See Figure (a) for Example 1, Figure (b) for Comparative Example 1, Figure (c) for Comparative Example 2, and Figure (d) for Comparative Example 3).

[0115] As digestion progresses, the polyphenol concentration gradually decreases, and the antioxidant shows different patterns. The Pearson correlation coefficient is used to evaluate the correlation between its content during digestion and the results of the four antioxidant methods. The results are shown in Table 6.

[0116] Table 6 Pearson correlation coefficients between the polyphenol retention rate (F, %) of the polyphenol-protein composite nanoparticle group and the determination results of the four antioxidant methods

[0117]

[0118]

[0119] Note: ** indicates significant correlation at the 0.01 level, and * indicates significant correlation at the 0.05 level.

[0120] A correlation coefficient greater than 0.9 indicates a strong correlation between the method of Example 1 and the content, and the correlation is significant. This high correlation proves the accuracy and reliability of the method. The methods of Comparative Example 1, Example 2, and Example 3 have a worse consistency with the polyphenol content compared to Example 1. And the ORAC results are all somewhat correlated with the results of other comparative examples. This result proves to a certain extent that the experimental method of this experiment is applicable to measuring the antioxidant level of the polyphenol-protein complex system.

[0121] Furthermore, taking the polyphenol nanomicrocapsules of Sample 3 as the research object, the content changes during in vitro simulated digestion and the measurement results of the antioxidant methods of Example 1, Comparative Example 1, Comparative Example 2, and Comparative Example 3 are as follows Figure 4 shown (the content changes during in vitro simulated digestion of polyphenol microcapsules, the measurement results of antioxidant methods, see Figure (a) for Example 1, Figure (b) for Comparative Example 1, Figure (c) for Comparative Example 2, and Figure (d) for Comparative Example 3). It can be seen from the figure that the content change rate of Sample 3 is slower than that of Sample 2, indicating that Sample 3 has better stability. The Pearson correlation coefficient is used to evaluate the correlation between its content during digestion and the results of the four antioxidant methods, and the results are shown in Table 7. It can be seen that during digestion, as the polyphenol content decreases, the antioxidant property of polyphenols increases.

[0122] Table 7 Pearson correlation coefficients between the polyphenol retention rate (F, %) of the polyphenol microcapsule group and the measurement results of four antioxidant methods

[0123]

[0124] Note: ** indicates significant correlation at the 0.01 level, and * indicates significant correlation at the 0.05 level.

[0125] A correlation coefficient greater than 0.9 indicates a strong correlation between the method of Example 1 and the content. This high correlation proves the accuracy and reliability of the method. The methods of Comparative Example 1, Example 2, and Example 3 have a worse consistency with the polyphenol content compared to Example 1.

[0126] Furthermore, by comparing Tables 1, 2, and 3, it is found that the DPPH and ABTS methods are not stable when targeting different polyphenol systems, and their performance under different polyphenol systems may be affected by the type, structure, color, or solvent environment of polyphenols, resulting in fluctuations in the results under different systems. And the ABTS method has a weak or moderate correlation with the content and other methods, indicating that the ABTS method is not suitable for evaluating the antioxidant activity of complex polyphenol systems.

[0127] In contrast, the correlation of the ORAC method in different polyphenol systems always remains negative. This phenomenon shows that the ORAC method has a relatively consistent trend for the antioxidant evaluation of polyphenol systems. Therefore, the application of Example 1 in different polyphenol systems may be more reliable, can provide more consistent and repeatable results, and help provide a more scientific basis for the evaluation of the antioxidant effects of polyphenol compounds.

[0128] Animal experiments verify the relevance:

[0129] Furthermore, the reliability of the results of Example 1 was verified by in vivo digestion in animal experiments. Mice were used as experimental subjects, and the free polyphenols and polyphenol microcapsules prepared in Example 1 were used as oral gavage drugs to conduct in vivo digestion studies. Through this in vivo digestion experiment, the metabolic engineering of polyphenols in a real biological environment can be monitored, and their stability and antioxidant effects during the digestion process can be studied, which is helpful to verify the reliability of the antioxidant evaluation method.

[0130] (1) Experimental design:

[0131] Healthy 8-week-old male KM mice, weighing 40±4 g, were selected and randomly and evenly divided into 2 groups (n=21), namely, a free polyphenol group and a polyphenol microcapsule group. Four mice from the same litter were added as normal mice blank controls. The grouping and intervention methods are shown in Table 8.

[0132] Table 8 Animal experiment groups and intervention methods

[0133]

[0134] The free polyphenols in the table are F prepared in Example 1, and the polyphenol microcapsules are SCF-MT2 prepared in Example 1.

[0135] The temperature of the animal breeding room was maintained at 24-26°C and the humidity was 40%-60%. The animals were adaptively fed for 1 week after arrival at the animal room. The animals were free to eat and drink. They were fasted for 12 hours before drug gavage, but not water. The animals were gavaged intensively on the eighth day.

[0136] (2) Index detection:

[0137] The mice were lightly anesthetized with ether at 30min, 1h, 2h, 3h, 4h, 8h, and 24h after gavage, and then killed by dislocation. The complete gastrointestinal tissue from the esophagus to the anus of the mouse with a digestion time ranging from 30min to 8h was frozen with liquid nitrogen and then freeze-dried. After grinding, 10mL of normal saline was added, and the tissue was extracted by shaking for 1h. The total antioxidant capacity (T-AOC) detection kit was used to determine the total antioxidant capacity of the tissue grinding fluid. The T-AOC in the digestive fluid of the sample in Example 1 was measured at different times after gavage of mice. The results are shown as follows: Figure 5 shown.

[0138] Take a part of the ground gastrointestinal tissue fluid, add 9 times the volume of methanol, sonicate for 10 min, oscillate and extract polyphenols for 1 h, centrifuge at 8000 g for 10 min, filter through a membrane, and then use HPLC to detect the polyphenol concentration in the digestive fluid. The mobile phase is 100% methanol and 0.1% formic acid solution.

[0139] The polyphenol content in the in vivo digestive fluid of the sample in Example 1 at different times after gavage of mice is as Figure 6 shown.

[0140] (3) Correlation analysis:

[0141] Compare the correlation between the in vitro ORAC value and the in vivo antioxidant enzyme activity and oxidative stress markers, and use Pearson correlation analysis in statistics to verify the applicability of the method in the evaluation of the antioxidant capacity of the polyphenol complex system.

[0142] It can be seen from Figure 5 that during the digestion process of free polyphenols, the antioxidant capacity of the digestive fluid gradually decreases at the initial stage of digestion, the change is not significant within the first 1 h, and it decreases rapidly after 1 h and returns to the normal level, which may be related to the degradation of polyphenols in the digestive fluid.

[0143] It can be found from Figure 6 that as digestion progresses, the content of polyphenols continuously decreases, and the decreasing rate of free polyphenols is faster than that of polyphenol microcapsules. When digestion proceeds for 4 h, the content of free polyphenols approaches 0, because polyphenols are completely digested and excreted, while polyphenol microcapsules can prolong the retention time. When digestion proceeds for 4 h, 20% of the content remains. The correlation between the polyphenol content and antioxidant capacity during the digestion process in mice after gavage of free polyphenols is shown in Table 9.

[0144] As the digestion process progresses, polyphenolic compounds are absorbed by the mouse intestine and enter the systemic circulation, and some are metabolized and utilized or excreted through urine and other pathways, resulting in a gradual decrease in the concentration of polyphenols in the body. There is a strong positive correlation with a correlation coefficient of up to 0.913 between the changes in the polyphenol content of in vivo digestion and in vitro simulated digestion, indicating that the in vitro simulated digestion model is applicable.

[0145] Table 9 Pearson correlation coefficients of the content and antioxidant capacity during in vitro digestion of free polyphenols and in vivo digestion in mice

[0146]

[0147] Note: ** indicates significant correlation at the 0.01 level, and * indicates significant correlation at the 0.05 level.

[0148] Furthermore, the antioxidant evaluation method in Example 1 showed a significant negative correlation (-0.729) with the antioxidant evaluation of in vivo digestion. The correlation coefficient |R| > 0.7, indicating that the measurement method in Example 1 was consistent with the antioxidant changes obtained from in vivo digestion in animals.

[0149] This negative correlation result may be because in the process of simulating in vitro digestion, the utilization and excretion processes of polyphenols are not involved. Therefore, the polyphenols and their metabolites remaining in the digestive fluid can still exhibit the ORAC oxygen radical absorption capacity. In animals, however, the absorption, utilization, and excretion processes of polyphenols in the body will result in the loss of this part of polyphenols, thus showing a decrease in antioxidant capacity in the digestive fluid. Therefore, from the perspective of the correlation results, the method in Example 1 can still evaluate the antioxidant activity of polyphenols to a certain extent.

[0150] After gavage with polyphenol microcapsules, the correlation between the polyphenol content and antioxidant activity during the in vivo digestion process in mice is shown in Table 10. From Figure 5 、 Figure 6 it can be found that the microcapsules can improve the antioxidant capacity of the digestive fluid, with a better effect than free polyphenols, and have a good protective effect on polyphenols, manifested as a slower decline in content.

[0151] Table 10 Pearson correlation coefficients of the content and antioxidant activity during in vitro digestion of polyphenol microcapsules and in vivo digestion in mice

[0152]

[0153] Note: ** indicates significant correlation at the 0.01 level, and * indicates significant correlation at the 0.05 level.

[0154] In addition, after gavage with polyphenol microcapsules, there was a strong positive correlation of up to 0.987 between the changes in polyphenol content during in vivo digestion and in vitro simulated digestion, indicating that the in vitro simulated digestion model can be used to simulate the digestion of polyphenol-containing substances in animals.

[0155] Furthermore, the antioxidant evaluation method in Example 1 showed a significant positive correlation (0.958) with the antioxidant evaluation of in vivo digestion. This positive correlation indicates that as in vivo digestion progresses, the ORAC oxygen radical absorption activity measured by the method in Example 1 is consistent with the trend of antioxidant activity in the in vivo digestive fluid, and this trend has been quantified to a certain extent. This phenomenon may be closely related to the absorption, utilization, and excretion processes of polyphenols in the body. Therefore, the method in Example 1 can still evaluate the antioxidant activity of polyphenols to a certain extent.

[0156] Through the above method, it is found that the antioxidant capacity of the polyphenol complex system after simulated digestion can be accurately measured. The ORAC value has a strong correlation with the antioxidant activity in animal experiments (correlation coefficient |R| > 0.7), and there is a strong positive correlation between the simulated digestion of polyphenols and the change in the content of polyphenols digested in vivo in animal experiments (correlation coefficient R > 0.7).

[0157] In summary, the evaluation method of the present invention is applicable to the rapid determination of the antioxidant capacity of the polyphenol complex system.

[0158] Example 2

[0159] A method for rapidly evaluating the actual antioxidant capacity of a polyphenol complex system specifically includes the following steps:

[0160] The samples are as follows:

[0161] (1) Grape polyphenol group: Weigh 160 mg of grape polyphenols, add 10 mL of deionized water and mix well to prepare a uniform suspension.

[0162] (2) Compound polyphenol group: Weigh 160 mg of compound polyphenols composed of grape polyphenols, tea polyphenols and cranberry proanthocyanidins. The mass ratio of grape polyphenols, tea polyphenols and cranberry proanthocyanidins in the compound polyphenols is 1:2:1. Add 10 mL of deionized water and mix well to prepare a uniform suspension.

[0163] (3) Compound polyphenol milk group: Weigh 320 mg of compound polyphenols composed of grape polyphenols, tea polyphenols and cranberry proanthocyanidins. The mass ratio of grape polyphenols, tea polyphenols and cranberry proanthocyanidins in the compound polyphenols is 1:2:1. Dissolve it in 10 mL of hot water to obtain a polyphenol solution. Weigh 0.5 g of skim milk powder and 1 g of sodium caseinate, first dissolve them in 10 mL of hot water and then add them to the polyphenol solution. Mix them under stirring at 90 °C for 1 h, and then cool and stir for 30 min to obtain heat-induced protein-polyphenol nanocomposite milk.

[0164] (4) Fruit extract polyphenol revitalizing drink: A drink with a polyphenol content of 1.6%, provided by Yunhui Health Technology Co., Ltd.

[0165] Antioxidant capacity detection:

[0166] Take 5 mL of the sample diluted to a concentration of 2 mg / ml, and perform simulated gastrointestinal digestion treatment and ORAC detection on the samples in Example 2 according to the method of Example 1.

[0167] The main purpose of this embodiment is to verify the applicability of the method of the present invention in detecting mixed polyphenols and polyphenol-containing beverages on the market. The test results of this method are shown in Table 11 and Table 12. There is an obvious correlation between the oxygen radical absorbance capacity value of a substance and its antioxidant activity. The level of the oxygen radical absorbance capacity value can indirectly reflect the strength of the antioxidant capacity.

[0168] Table 11 ORAC values of common antioxidants during in vitro simulated digestion

[0169]

[0170] Table 12 ORAC values of protein-polyphenol complexes during in vitro simulated digestion

[0171]

[0172] As can be seen from Table 11 and Table 12, among the above various products, the ORAC value of compound polyphenol milk changes greatly before and after digestion, indicating that protein-embedded polyphenols cover up the antioxidant capacity of polyphenols to a certain extent, and this part of the antioxidant capacity is reflected after digestion. This shows that the method of Example 1 in this experiment can be used to detect the antioxidant capacity of commercially available samples containing polyphenols and has applicability to various complex polyphenol systems.

[0173] Among various polyphenol samples, with the progress of digestion, the antioxidant capacity of polyphenols increases, indicating that polyphenols gradually exhibit stronger antioxidant capacity in the simulated digestive fluid. This may be because under the action of digestive enzymes, polyphenols combine with peptides formed by protein hydrolysis to generate some products with high antioxidant activity, thus enhancing their antioxidant effect. In addition, polyphenols may show better solubility in the bile salt micelles in the digestive fluid, improving their contact opportunities with free radicals in the digestive fluid and further enhancing their antioxidant capacity.

[0174] The method of the present invention reveals the mechanism of the enhanced antioxidant capacity of polyphenols in the digestive fluid through simulating the digestion process, provides an efficient and reliable evaluation method, helps to deeply understand the changes in the antioxidant effect of polyphenols during the in vivo digestion process, and provides a scientific basis for the development of related foods, health products and drugs.

[0175] Any person skilled in the relevant art may use the technical content disclosed above to make changes or modifications into equivalent embodiments with equivalent changes and apply them to other fields. However, as long as they do not depart from the content of the technical solution of the present invention, any simple modification, equivalent change and modification made to the above embodiments based on the technical essence of the present invention still fall within the protection scope of the technical solution of the present invention.

Claims

1. A method for rapidly evaluating the actual antioxidant capacity of a polyphenol complex system, characterized in that: It includes the following steps: Step 1: Using deionized water as a solvent, dissolve or disperse the polyphenol-containing substance into a solution or suspension. Subject the solution or suspension to simulated gastric digestion by constant shaking at 36.5 - 37.5 °C for 60 - 120 min, and simulated small intestine digestion for 120 - 180 min, with deionized water as a blank control; Step 2: Take the solutions before digestion, after 60 min of digestion, after 120 min of digestion, and after 180 min of digestion to measure the oxygen radical absorbance capacity (ORAC). The specific steps include: a. Using Trolox as a standard, dilute the taken solution with PBS buffer; b. Using sodium fluorescein as a fluorescent label and AAPH as a fluorescent quencher, incubate at 36.5 - 37.5 °C for 10 - 30 min; c. Use a microplate reader to detect the fluorescence intensity value at regular intervals. The excitation wavelength is 485 nm and the emission wavelength is 538 nm. Continuously detect until the fluorescence intensity drops below 5% of the initial value; Step 3: Draw a standard curve with Trolox as a standard to calculate the actual ORAC value of each group of samples. According to the area under the fluorescence value curve (AUC) of the sample group and the blank control group (deionized water), calculate the fluorescence protection area of the sample group, that is, the area of the delayed part of the fluorescence quenching curve NetAUC; Calculate the NetAUC value according to the following formula; AUC = 0.5×[2×(f0 + f i + …… + f n - 1 + f n ) - f0 - f n ×Δt NetAUC = AUC sample - AUC AAPH Wherein: the ORAC value represents the oxygen radical absorbance capacity; the AUC represents the area under the fluorescence value curve; AUC sample is the area under the fluorescence curve obtained by measurement and calculation of the sample group; AUC AAPH+ is the area under the fluorescence curve obtained by measurement and calculation of the blank control group; f i represents the relative fluorescence intensity value of the i-th round; Δt represents the measurement time interval; NetAUC represents the net value of the area under the fluorescence curve; c Trolox represents the Trolox concentration, c sample represents the sample concentration.

2. The method for rapidly evaluating the actual antioxidant capacity of a polyphenol complex system according to claim 1, wherein The polyphenol-containing substance is a plant extract, a synthetic polyphenol, or a food ingredient containing polyphenolic compounds. The concentration of polyphenols in the polyphenol-containing substance is 0.01 - 10 mg / mL.

3. The method for rapidly evaluating the actual antioxidant capacity of a polyphenol complex system according to claim 1, characterized in that, In Step 1, the conditions for simulated gastric digestion are: the pH value of the simulated gastric juice is 1.5 - 3.0, and pepsin is added to simulate gastric digestion.

4. The method for rapidly evaluating the actual antioxidant capacity of a polyphenol complex system according to claim 1, characterized in that, In Step 1, the conditions for simulated small intestine digestion are: the pH value of the simulated intestinal juice is 6.8 - 7.4, and pancreatic enzymes and bile salts are added to simulate small intestine digestion.

5. The method for rapidly evaluating the actual antioxidant capacity of a polyphenol complex system according to claim 1, characterized in that, In Step 2, the concentration of sodium fluorescein is 10 - 50 μmol / L, and the concentration of AAPH is 20 - 60 mg / mL.

6. The method for rapidly evaluating the actual antioxidant capacity of a polyphenol complex system according to claim 1, wherein In Step 2, the interval time for the microplate reader to detect the fluorescence intensity is 2 - 5 min, the detection temperature is 37 °C, and the total detection time is preferably 1 - 3 h.

7. The method for rapidly evaluating the actual antioxidant capacity of a polyphenol complex system according to claim 1, characterized in that, The calculation result of the Trolox equivalent is μmol TE / g, FW or μmol TE / mL, FW of the Trolox equivalent per gram of polyphenols or per milliliter of fresh weight sample.

8. The antioxidant capacity evaluation method according to any one of claims 1 - 7 is applied to the evaluation of the antioxidant capacity of a polyphenol - protein complex system or a microcapsule encapsulating polyphenols.

9. The application according to claim 8, wherein The protein in the composite system is casein, whey protein, soy protein, or pea protein; the microcapsule wall material is maltodextrin, resistant starch, or trehalose.

10. The antioxidant capacity evaluation method according to any one of claims 1 - 7 is applied to the evaluation of the antioxidant properties of foods, health products, or drugs containing polyphenols or polyphenol - protein complexes.

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