CN110208355B - Method for measuring interaction efficiency of quinone substances and carboxymethyl lysine in solution - Google Patents

Abstract

The invention discloses a method for measuring the interaction efficiency of quinone substances and carboxymethyl lysine (CML) in solution. The method measures the cyclic voltammetry curves of the catechols solution before and after the addition of CML, records the peak current and voltage characteristic values of the catechols solution before and after the addition of CML, and then calculates the reduction rate of the reduction peak current by calculating , the interaction efficiency between quinones in solution and CML can be obtained. In the method of the invention, after the phenolic substances are oxidized to form quinones, they can quickly interact with the CML in the system to reduce the side reactions of the quinones; the amount of substances attached to the electrode surface is small, and the detection accuracy is high; In the prior art, the adhesion of polyphenols and their oxidation products to the working electrode, the related side reactions of quinones and other factors interfere with the interaction between quinones and CML; at the same time, the assay method of the invention is simple, the analysis time is short, and the reproducibility is good. and convenience, low cost of use, and wide application.

Description

A method for determining the interaction efficiency of quinones and carboxymethyl lysine in solution

technical field

The invention relates to the technical field of food safety detection and analytical chemistry, and more particularly, to a method for determining the interaction efficiency of quinones and carboxymethyl lysine (CML) in solution.

Background technique

CML (carboxymethyl lysine) is a typical advanced glycation end product (AGEs) that is most widely studied at present, and it exists widely in various foods. CML can accumulate in tissues after being digested and absorbed, and existing studies have proved its potential harm to human health. Therefore, research on the means to reduce the content of CML can effectively reduce its potential harm to human health.

While catechols play an antioxidant role, they will be oxidized to form corresponding o-phthaloquinones. As strong electrophiles, o-phthaloquinones are prone to Michael addition reaction with amino groups on CML. So as to achieve the purpose of eliminating the system CML. At present, the main method for determining the interaction efficiency between o-phthaloquinones and CML is to use the controlled potential electrolysis Coulomb method to oxidize the catechols in the solution to obtain the corresponding o-phthaloquinones, and then use Interaction kinetics between o-phthaloquinones and CML were determined by stopped-flow spectroscopy. The preparation of o-phthaloquinones by controlled-potential electrolytic coulomb method requires that the cyclic voltammetric behavior of the selected catechols on the working electrode is reversible and does not have obvious adsorption. At present, only the corresponding quinones (4-methyl phthaloquinone) have been successfully prepared from 4-methyl catechol. effect.

Most of the polyphenols containing catechol structure, such as catechins, rutin, quercetin, etc., are easy to be adsorbed on the surface of the working electrode, and the quinones formed by oxidation are prone to polymerization and heavy weight. Therefore, there is no possibility to prepare the corresponding quinones by the controlled potential electrolysis Coulomb method. Therefore, it is impossible to measure the interaction of the quinones obtained by the oxidation of the above-mentioned polyphenols in the solution with CML by the conventional means.

Therefore, in view of the shortcomings of the above detection methods, it is necessary to provide a detection method with a wider range of use.

SUMMARY OF THE INVENTION

The object of the present invention is to, in view of the prior art, in the process of detecting the interaction efficiency between quinones and CML by using controlled potential electrolysis coulomb-stopped-flow spectroscopy, there are fewer types of applicable catechols, and its application Due to the limited scope, a method for determining the interaction efficiency of quinones in solution with CML is provided. The method of the invention utilizes the electrochemical activity of catechols, measures the reduction peak current of catechols before and after adding CML by cyclic voltammetry, calculates the reduction rate of the reduction peak current, and obtains the quinones in the solution The interaction efficiency between the substance and CML; the method of the present invention is suitable for most catechol substances, and the determination method is simple, convenient, accurate and reliable in detection results, and has stability and repeatability. The detection and evaluation of the interaction efficiency with CML has broad application prospects.

Above-mentioned purpose of the present invention is achieved through the following scheme:

A method for measuring carboxymethyl lysine (CML) content in a solution, comprising the steps:

S1. Dissolving catechols in buffer to obtain solution A; dissolving CML in buffer to obtain solution B; dissolving catechols and CML in buffer to obtain solution C;

S2. Use glassy carbon electrode as working electrode, platinum wire as counter electrode, silver/saturated silver chloride as reference electrode, each electrode is connected with electrochemical workstation, build a three-electrode system, use cyclic voltammetry to measure solution A, Solution B and solution C are detected, the cyclic voltammetry curve of each solution is obtained, and the peak current and voltage characteristic values of each solution are recorded respectively;

S3. According to the peak current and voltage characteristic values measured in step S2, calculate according to the following formula, and then the interaction efficiency of quinones in the solution and CML can be measured:

Interaction efficiency (reduction peak current reduction rate, %)=(I _pC1(phenol) -I _{pC1(phenol+CML)} )/I _pC1(phenol) ;

The I _{pC1 (phenol)} is the reduction peak-to-peak current of the solution A; the I _{pC1 (phenol+CML)} is the reduction peak-to-peak current of the solution C.

Since the structure of catechols is electrochemically active, in cyclic voltammetry scanning, when the voltage is scanned in a positive direction, catechols are oxidized on the electrode surface to form corresponding quinones, and in the subsequent negative During the forward scanning process, the quinones are reduced to the original phenols on the electrode surface, forming a pair of redox peaks; when the CML in the solution system reacts with the quinones, resulting in the consumption of the quinones, the negative scanning During the process, the reduction peak current of solution C was significantly lower than that of solution A. By measuring the reduction rate of reduction peak current in the cyclic voltammetry curves of phenols before and after adding CML, the interaction efficiency of quinones and CML can be calculated.

When the original controlled potential electrolytic Coulomb method is used for detection, by applying a certain voltage, the phenol in the system is completely oxidized to form quinone after a period of time (about 40 minutes), and then the quinone reacts with CML to determine its interaction efficiency; The oxidation of phenolic substances to quinones takes a long time, which requires the generated quinones to be very stable. Otherwise, when the quinones undergo a series of side reactions or adhere to the electrode to hinder the subsequent preparation process, they will It affects the accuracy of the test results, and when the quinones adhere to the electrode for a long time, it will cause irreversible damage to the mesh glass carbon electrode. However, the whole scanning process of the cyclic voltammetry adopted in the present invention does not exceed 3 minutes. Once the phenolic substances are oxidized on the electrodes to form quinone substances, the quinone substances can react with the CML coexisting in the system, and the possibility of side reactions is reduced. It will not adhere to the surface of the electrode for a long time, which reduces the damage to the electrode.

Preferably, the catechol substances are catechol, 4-methyl catechol, protocatechuic acid, ethyl protocatechuate, caffeic acid, chlorogenic acid, rosmarinic acid, One or more of rutin, (+)-catechin, (-)-epicatechin, luteolin or quercetin. The detection method of the invention can be applied to a variety of catechol substances and has a wide application range.

Preferably, during the cyclic voltammetry test, the scanning potential range of the electrochemical workstation is -0.8-1.2V; the scanning rate is 0.01-0.10V/s; the sensitivity is 10 ^-5 A/V; the initial scanning direction is forward ; The number of scanning segments is 2; the temperature is 25±1℃.

Preferably, before the solution A, the solution B and the solution C are detected, the oxygen in the solution needs to be removed first.

More preferably, before the solution A, solution B and solution C are detected, nitrogen gas is respectively introduced into each solution to remove oxygen.

More preferably, the introduction time of the nitrogen gas is 5-10 min.

Preferably, the diameter of the glassy carbon working electrode is 3 mm; before each scan, the glassy carbon working electrode needs to be polished on the chamois to which the alumina suspension with a particle size of 0.05 μm is dropped, and then washed with distilled water. Before the test, grinding the glassy carbon electrode can effectively remove the attachments on the electrode surface, ensure that the electrode surface is clean before the test, and improve the detection accuracy.

Preferably, the buffer is formulated with phosphate.

Preferably, the pH of the buffer solution is 5.0-8.0, and the concentration of phosphate is 0.2M.

Preferably, when the solute in solution A is luteolin or quercetin, the solvent is a mixture of 0.2M phosphate buffer and ethanol, and the volume ratio is 4:1.

Preferably, in the solutions A and C, the concentration of catechols is 0.5mM.

Preferably, in the solutions B and C, the concentration of CML is 0.5-7.5 mM.

Compared with the prior art, the present invention has the following beneficial effects:

(1) The present invention utilizes cyclic voltammetry, so that after phenolic substances are oxidized to form quinone substances, they can quickly interact with CML in the system to reduce side reactions of quinone substances; Grinding can effectively remove the attachments on the electrode surface, improve the detection accuracy, and reduce the interference of polyphenols and their oxidation products on the working electrode, the side reactions related to quinones in the prior art, and other factors on the interaction between quinones and CML.

(2) the method of the present invention is simple, the analysis time is short, the repeatability and convenience are good, the cost of use is low, and the method has a good application prospect in the field of interaction research between quinones and CML, and makes up for the existing technical means Only the weak point of the interaction of 4-methylophthaloquinone with CML could be determined.

Description of drawings

Figure 1 is a cyclic voltammogram of the interaction of catechol with CML (pH 7.0, scan rate 0.01 V/s).

Figure 2 is a cyclic voltammogram of the interaction of 4-methylcatechol with CML (pH 7.0, scan rate 0.01 V/s).

Figure 3 is a cyclic voltammogram of the interaction of protocatechuic acid with CML (pH 7.0, scan rate 0.01 V/s).

Figure 4 is a cyclic voltammogram of the interaction of protocatechuic acid with CML (pH 7.0, scan rate 0.05 V/s).

Figure 5 is a cyclic voltammogram of the interaction of ethyl protocatechuate with CML (pH 7.0, scan rate 0.05 V/s).

Figure 6 is a cyclic voltammogram of the interaction of caffeic acid with CML (pH 7.0, scan rate 0.01 V/s).

Figure 7 is a cyclic voltammogram of the interaction of chlorogenic acid with CML (pH 7.0, scan rate 0.01 V/s).

Figure 8 is a cyclic voltammogram of the interaction of rosmarinic acid with CML (pH 7.0, scan rate 0.01 V/s).

Figure 9 is a cyclic voltammogram of the interaction of rutin with CML (pH 7.0, scan rate 0.05 V/s).

Figure 10 is a cyclic voltammogram of (+)-catechin interaction with CML (pH 7.0, scan rate 0.05 V/s).

Figure 11 is a cyclic voltammogram of the interaction of (-)-epicatechin with CML (pH 7.0, scan rate 0.05 V/s).

Figure 12 is a cyclic voltammogram of the interaction of luteolin with CML (pH 7.0, scan rate 0.05 V/s).

Figure 13 is a cyclic voltammogram of the interaction of quercetin with CML (pH 7.0, scan rate 0.05 V/s).

Detailed ways

The present invention will be further elaborated below with reference to specific embodiments, which are only used to explain the present invention, but not to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents, etc. used are commercially available reagents and materials unless otherwise specified.

Example 1

A method for determining the interaction efficiency of quinones in solution and CML is tested according to the following steps:

S1. Dissolving catechols in buffer to obtain solution A; dissolving CML in buffer to obtain solution B; dissolving catechols and CML in buffer to obtain solution C;

S2. Using glassy carbon electrode (diameter 3mm) as working electrode, platinum wire as counter electrode, silver/saturated silver chloride as reference electrode, each electrode is connected to electrochemical workstation to construct a three-electrode system, using cyclic voltammetry Detect solution A, solution B and solution C respectively, obtain the cyclic voltammetry curve of each solution, and record the peak current and voltage characteristic values of each solution respectively; ~10min high-purity nitrogen to remove oxygen; glassy carbon working electrode needs to be polished on the chamois to which the alumina suspension with a particle size of 0.05 μm is added dropwise before each scan, and then washed with distilled water; the scanning potential range of the electrochemical workstation -0.8~1.2V; scan rate: 0.01~0.10V/s; sensitivity: 10 ^-5 A/V; initial scan direction is forward; number of scan segments is 2; temperature is 25±1℃;

S3. According to the peak current and voltage characteristic values measured in step S2, calculate according to the following formula, and the interaction efficiency between quinones and CML can be measured as:

Interaction efficiency=(reduction peak current reduction rate, %)=(I _pC1(phenol) -I _{pC1(phenol+CML)} )/I _pC1(phenol) ;

The I _{pC1 (phenol)} is the reduction peak-to-peak current of the solution A; the I _{pC1 (phenol+CML)} is the reduction peak-to-peak current of the solution C.

1. Investigate the effect of buffer pH, scan rate and CML concentration on the detection results

According to the process of the above step S1, use NaH ₂ PO ₄ and Na ₂ HPO ₄ to prepare 0.2M phosphate buffer solution with pH values of 5.0, 7.0 and 8.0 respectively, and then dissolve a certain amount of catechol or 4-methyl ortho- Hydroquinone, configured to obtain solution A, wherein the concentration of catechol or 4-methyl catechol is 0.5mM;

Dissolve a certain amount of CML in 0.2M phosphate buffer solution to prepare solution B, wherein the concentration of CML is 0.5-7.5mM;

Dissolve a certain amount of catechol (or 4-methyl catechol) and CML in the buffer, stir and mix with magnetic force to prepare solution C, ensure that catechol (or 4-methyl catechol (or 4-methyl catechol) in solution C Hydroquinone) at a concentration of 0.5 mM, and CML at a concentration of 0.5 to 7.5 mM.

According to the detection process in the above-mentioned step S2, different scanning rates (0.01, 0.05 and 0.10V/s) were changed for detection, and the measured results were shown in Table 1 and Table 2, wherein Table 1 is the o-phthaloquinone under different conditions. The detection results of the interaction efficiency with CML (5.0mM), Table 2 is the detection results of the interaction efficiency between 4-methyl phthaloquinone and CML (5.0mM) under different conditions, and Table 3 is 4-methyl phthaloquinone The detection results of the interaction efficiency of quinone with different concentrations of CML (0.5-7.5mM) at pH 7.0 at a scan rate of 0.01V/s. The greater the reduction rate of reduction current, the greater the degree of reaction, that is, the higher the efficiency of the reaction between quinones and CML.

Table 1 Interaction efficiencies of o-phthaloquinone and CML under different conditions

Table 2 The interaction efficiency of 4-methylophthaloquinone with CML under different conditions

It can be seen from Table 1 and Table 2 that the method of the present invention has better stability under the conditions of pH 7.0 and 8.0, the RSD value is between 0.00 and 9.22, and the stability is better; at pH 5.0, due to quinone substances and The low interaction efficiency between CML resulted in poor method stability and high RSD value. Therefore, this method is more suitable for the detection of the interaction efficiency between quinones and CML in pH 7.0 and 8.0 solution systems.

The invention judges the interaction between CML and quinones by measuring the reduction rate of the reduction peak current in the cyclic voltammetry curve of phenolic substances before and after adding CML, and the greater the reduction rate, the higher the interaction efficiency. The order of the interaction efficiency of o-phthaloquinone or 4-methylo-phthaloquinone with CML under the conditions of different pH and the same scan rate is: pH 8.0>pH 7.0>pH 5.0; Preparation of 4-methyl phthaloquinone, and then using stopped-flow spectroscopy to determine the interaction efficiency between 4-methyl phthaloquinone and CML under different pH conditions, the results are consistent (Food Chemistry, 2018, 244:25-28), That is, pH 8.0>pH 7.0>pH 5.0, so the method for determining the interaction efficiency of quinones in solution and CML involved in the present invention is more accurate.

Table 3 Interaction efficiencies of 4-methylo-phthaloquinone with different concentrations of CML at pH 7.0 and scan rate 0.01 V/s

It can be seen from Table 3 that at pH 7.0 and scan rate 0.01V/s, the method used to determine the interaction efficiency of 4-methylo-phthaloquinone with different concentrations of CML has good stability, and the RSD value ranges from 0.35 to 5.62. With the increase of CML content in the system, the interaction efficiency of 4-methyl phthaloquinone with CML increased significantly, which was in line with the theoretical expectation.

2. To explore the effect of different substituents on the benzene ring on the interaction efficiency of quinones and CML

The test process refers to the test process in Example 1, the difference is that solution A is respectively catechol, 4-methylcatechol or protocatechuic acid dissolved in phosphate buffer (0.2M, pH7. 0) Prepared, the concentration of polyphenols is 0.5mM; solution C is respectively catechol, 4-methylcatechol or protocatechuic acid and CML dissolved in phosphate buffer (0.2M, pH7 .0), wherein the concentration of polyphenols is 0.5 mM, and the concentration of CML is 5.0 mM. During the test, the operating parameters of the electrochemical workstation are: scan potential range, -0.8-1.2V; scan rate, 0.01V/s; sensitivity, 10 ^-5 A/V; initial scan direction, forward; number of scan segments, 2 . The cyclic voltammetry curve of each solution was obtained, and the peak current and voltage characteristic values of each solution were recorded respectively, and calculated according to the formula in Example 1. The calculation results are shown in Table 4.

Table 4. Interaction efficiencies of different quinones and CML

It can be seen from Table 4 that under the same conditions, CML has the highest interaction efficiency with quinone protocatechuic acid formed by oxidation of protocatechuic acid, and the interaction efficiency with quinone formed by oxidation of catechol is in the middle. The 4-methyl phthaloquinone formed by the oxidation of 4-methyl catechol had the lowest interaction efficiency. The methyl group on the benzene ring has an electron-donating effect, which increases the electron cloud density of quinones, thus reducing the activity of its electrophilic reaction; the carboxyl group on the benzene ring has an electron-withdrawing effect, which reduces the electrons of quinones. cloud density, thus enhancing the activity of its electrophilic reaction. In conclusion, the test results of the present invention are in line with theoretical expectations, and have high stability (RSD value range is 0.00-9.22).

3. The degree of reaction between protocatechuic acid and protocatechuic acid carboxyethyl esterification to corresponding quinones and CML

The test process refers to the test process in Example 1, the difference is that solution A is prepared by dissolving protocatechuic acid or ethyl protocatechuate in phosphate buffer (0.2M, pH7.0), respectively, and the polyphenol The concentrations of these substances are all 0.5mM; solution C is prepared by dissolving protocatechuic acid or ethyl protocatechuate and CML in phosphate buffer (0.2M, pH7.0) respectively, and the concentration of polyphenols is 0.5 mM, the concentration of CML was 5.0 mM. During the test, the operating parameters of the electrochemical workstation are: scan potential range, -0.8-1.2V; scan rate, 0.05V/s; sensitivity, 10 ^-5 A/V; initial scan direction, forward; number of scan segments, 2 . The cyclic voltammetry curve of each solution was obtained, and the peak current and voltage characteristic values of each solution were recorded respectively, and calculated according to the formula in Example 1. The calculation results are shown in Table 5.

Table 5 Interaction efficiencies of different catechol quinones and CML

As can be seen from Table 5, under the same conditions, the interaction efficiency ratio between CML and protocatechuic acid quinone formed by oxidation of protocatechuic acid and the interaction efficiency of protocatechuic acid ethyl quinone formed by oxidation of protocatechuic acid ethyl ester The working efficiency is high, and the ethyl esterification of the carboxyl group weakens the electron-pulling effect of the carboxyl group, so its electrophilic reaction ability declines. The test result of the present invention meets the theoretical expectation and has high stability (the RSD value range is 0.39-6.22) .

4. The degree of reaction between quinones formed by oxidation of different phenolic acids and CML

The test process refers to the test process in Example 1, the difference is that the solution A is prepared by dissolving caffeic acid, chlorogenic acid or rosmarinic acid in phosphate buffer (0.2M, pH7.0), respectively, and the polyphenol The concentrations of these substances are all 0.5mM; solution C is prepared by dissolving caffeic acid, chlorogenic acid or rosmarinic acid and CML in phosphate buffer (0.2M, pH 7.0) respectively, wherein the concentration of polyphenols is 0.5 mM, the concentration of CML was 5.0 mM. During the test, the operating parameters of the electrochemical workstation are: scan potential range, -0.8-1.2V; scan rate, 0.01V/s; sensitivity, 10 ^-5 A/V; initial scan direction, forward; number of scan segments, 2 . The cyclic voltammetry curve of each solution was obtained, and the peak current and voltage characteristic values of each solution were recorded respectively, and calculated according to the formula in Example 1. The calculation results are shown in Table 6.

Table 6 Reaction degree of quinones formed by oxidation of different phenolic acids and CML

It can be seen from Table 6 that under the same conditions, the interaction efficiency of CML with caffeic acid quinone formed by oxidation of caffeic acid is higher than that of quinones oxidized by chlorogenic acid or rosmarinic acid, chlorogenic acid quinone and rosemary The steric hindrance effect brought by the large substituent on the acid quinone benzene ring weakens its interaction efficiency with CML. The test results of the present invention are in line with theoretical expectations and have high stability (RSD value range is 2.80-7.27).

5. The degree of reaction between quinones formed by oxidation of flavonoids and CML

The test process refers to the test process in Example 1, the difference is that solution A is obtained by dissolving rutin, (+)-catechin or (-)-epicatechin in phosphate buffer (0.2M, pH7.0), or take luteolin or quercetin and dissolve it in a mixture of 0.2M phosphate buffer and ethanol (volume ratio: 4:1, pH 7.0), and the concentration of polyphenols is 0.5mM ; Solution C is respectively prepared by rutin, (+)-catechin or (-)-epicatechin and CML dissolved in phosphate buffer (0.2M, pH7.0), or luteolin or quercetin Cortexin and CML were prepared by dissolving in a mixture of 0.2M phosphate buffer and ethanol (volume ratio: 4:1, pH 7.0), wherein the concentration of polyphenols was 0.5 mM, and the concentration of CML was 5.0 mM. During the test, the operating parameters of the electrochemical workstation are: scan potential range, -0.8-1.2V; scan rate, 0.05V/s; sensitivity, 10 ^-5 A/V; initial scan direction, forward; number of scan segments, 2 . The cyclic voltammetry curve of each solution was obtained, and the peak current and voltage characteristic values of each solution were recorded respectively, and calculated according to the formula in Example 1. The calculation results are shown in Table 7.

Table 7. Interaction efficiency of quinones formed by oxidation of flavonoids and CML

As can be seen from Table 7, under the same conditions, the interaction efficiency between CML and the luteolin quinone formed by the oxidation of luteolin is the highest among the selected flavonoids, which is related to the lower electron cloud density of its benzene ring, and the test results of the present invention are consistent with The theoretical expectation is that the RSD value ranges from 5.48 to 42.73, and some flavonoids have poor stability in the test.

To sum up, the present invention is suitable for detecting the reaction efficiency of quinones formed by oxidation of CML and a variety of different phenolic substances, which is in line with theoretical expectations, indicating that the detection results are accurate, and the RSD value of the detection results is small, indicating that the detection method and the detection results. Has stability.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit the protection scope of the present invention. For those of ordinary skill in the art, on the basis of the above descriptions and ideas, the Variations or changes in other different forms are not required and cannot be exhaustive of all implementations here. Any modifications, equivalent replacements and improvements made within the spirit and principle of the present invention shall be included within the protection scope of the claims of the present invention.

Claims (4)

1. a method for measuring the interaction efficiency of quinones in solution and carboxymethyl lysine, is characterized in that, comprises the steps: S1. Dissolving catechols in buffer to obtain solution A; dissolving CML in buffer to obtain solution B; dissolving catechols and CML in buffer to obtain solution C; S2. Use glassy carbon electrode as working electrode, platinum wire as counter electrode, silver/saturated silver chloride as reference electrode, each electrode is connected with electrochemical workstation, build a three-electrode system, use cyclic voltammetry to measure solution A, Solution B and solution C are detected, the cyclic voltammetry curve of each solution is obtained, and the peak current and voltage characteristic values of each solution are recorded respectively; S3. According to the peak current and voltage characteristic values measured in step S2, calculate according to the following formula, and then the interaction efficiency of quinones in the solution and CML can be measured: Interaction efficiency/%=(I _pC1(phenol) -I _{pC1(phenol+CML)} )/I _pC1(phenol) ; Wherein the I _{pC1 (phenol)} is the reduction peak-to-peak current of the phenolic catechol structural unit in the solution A; the I _{pC1 (phenol+CML)} is the phenolic phthalate in the solution C. The reduction peak-to-peak current of the phenolic structural unit; The catechol substances are catechol, protocatechuic acid, ethyl protocatechuate, caffeic acid, chlorogenic acid, rosmarinic acid, rutin, (+)-catechin, ( -) - one or more of epicatechin, luteolin or quercetin; During the cyclic voltammetry test, the scanning potential range of the electrochemical workstation was -0.8~1.2V; the scanning rate was 0.01~0.10V/s; the sensitivity was 10 ^-5 A/V; the initial scanning direction was positive; the number of scanning segments is 2; the temperature is 25±1℃; The buffer solution is prepared with phosphate; the pH of the buffer solution is 7.0-8.0, and the concentration of phosphate is 0.2M; In the solutions A and C, the concentration of catechols is 0.5 mM; in the solutions B and C, the concentration of CML is 0.5-7.5 mM; The diameter of the glassy carbon working electrode is 3 mm; the glassy carbon working electrode needs to be polished on the chamois to which the alumina suspension with a particle size of 0.05 μm is added dropwise before each scan, and then washed with distilled water; When the solute in solution A is luteolin or quercetin, the solvent is a mixture of 0.2M phosphate buffer and ethanol, and the volume ratio is 4:1. 2. The method for measuring the interaction efficiency of quinones in solution and CML according to claim 1, characterized in that, before the solution A, solution B and solution C are detected, oxygen in the solution needs to be removed. 3. the method for the interaction efficiency of quinones and CML in the measurement solution according to claim 2, is characterized in that, before described buffer solution A, buffer solution B and mixed solution C detect, pass through in each solution respectively. Nitrogen was introduced to remove oxygen. 4 . The method for measuring the interaction efficiency of quinones in solution and CML according to claim 3 , wherein the introduction time of the nitrogen gas is 5-10 min. 5 .

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