Method for evaluating antioxidant effect of exogenous antioxidant by taking saccharomyces cerevisiae as model

Abstract

The invention provides a method for evaluating the antioxidant effect of an exogenous antioxidant by taking saccharomyces cerevisiae as a model, and belongs to the technical field of antioxidant evaluation. The invention provides application of yeast cells in preparation of a cell model for evaluating the antioxidant effect of an exogenous antioxidant, and the yeast cells are used as the cell model for evaluating the in-vivo antioxidant activity of the exogenous antioxidant. The method for evaluating the antioxidant effect of the exogenous antioxidant comprises the following steps: taking yeast cells as a model, and determining the influence of the exogenous antioxidant on the in-vivo activity of the yeast cells.

Description

Technical Field

[0001] The invention belongs to the technical field of antioxidant evaluation, and in particular relates to a method for evaluating the antioxidant effect of exogenous antioxidants by taking saccharomyces cerevisiae as a model. Background Art

[0002] Under normal circumstances, the production of oxidants and antioxidants in the human body should be in a dynamic balance. However, under the stimulation of the external environment, the production of oxidants in the body exceeds the clearance capacity of the antioxidant system, and the balance is destroyed, resulting in the generation of a large number of reactive oxygen species and free radicals, which will attack cell proteins, lipids, and nucleic acids, causing damage to cell structure and function. This reaction is called oxidative stress, and oxidative stress is one of the pathogenesis of many diseases in the body. Excessive accumulation of ROS is difficult to eliminate by the body's own antioxidant system, so seeking a safe and effective exogenous antioxidant to reduce ROS levels is an effective way to alleviate oxidative stress.

[0003] At present, the evaluation methods of antioxidant interactions at home and abroad include the summation method, direct comparison method and response surface method. However, the summation method and direct comparison method are mainly applicable to the situation where the antioxidant activity of a single component is similar, and are not applicable to the situation where the dose and antioxidant activity are not linearly correlated. The response surface method can make up for the shortcomings of the summation method and the direct comparison method, but in order to obtain a more accurate response surface equation, multiple groups of experiments are often required, which is not suitable for the analysis of expensive standards or small amounts of compounds. Therefore, a simpler, faster and more comprehensive evaluation method for antioxidant interactions is urgently needed. Summary of the invention

[0004] The present invention provides a method for evaluating the antioxidant effect of an exogenous antioxidant using saccharomyces cerevisiae as a model, which can quickly and easily verify the in vivo antioxidant effect of the exogenous antioxidant.

[0005] The present invention provides the use of yeast cells in preparing a cell model for evaluating the antioxidant effect of exogenous antioxidants.

[0006] In a specific embodiment of the present invention, the yeast cell includes Saccharomyces cerevisiae and a homologous gene-deficient strain of the Saccharomyces cerevisiae.

[0007] The invention provides a method for evaluating the antioxidant effect of an exogenous antioxidant, comprising the following steps: using yeast cells as a model to determine the influence of the exogenous antioxidant on the in vivo activity of the yeast cells.

[0008] In a specific embodiment of the present invention, the method further comprises determining the composition and in vitro activity of the exogenous antioxidant.

[0009] In a specific embodiment of the present invention, determining the composition of the exogenous antioxidant includes determining the total protein content and the total flavonoid content of the exogenous antioxidant.

[0010] In a specific embodiment of the present invention, determining the in vitro activity of the exogenous antioxidant comprises determining at least one of the following: DPPH free radical scavenging ability, hydroxyl free radical scavenging ability and ABTS free radical scavenging ability.

[0011] In a specific embodiment of the present invention, determining the in vivo activity effect of the yeast cells comprises determining at least one of the following: toxicity analysis of the yeast cells, tolerance analysis in an oxidative environment, determination of cell membrane lipid peroxidation levels in an oxidative environment, and determination of intracellular peroxidation levels.

[0012] In a specific embodiment of the present invention, the tolerance analysis under an oxidative environment includes setting up a control group and an experimental group. In the experimental group, the yeast cells are incubated in a medium added with the exogenous antioxidant for a period of time, and then the oxidant is added for culture. The bacterial solution is diluted and inoculated into the medium, and the number of colonies and cell survival are counted.

[0013] The control group was operated in the same manner as the experimental group except that the exogenous antioxidant was not added.

[0014] In a specific embodiment of the present invention, the method for measuring the level of cell membrane lipid peroxidation includes the TCA method.

[0015] In a specific embodiment of the present invention, the method for measuring the intracellular peroxidation level includes the fluorescent probe DCFH-DA method.

[0016] Beneficial effects: The present invention provides an application of yeast cells in preparing a cell model for evaluating the antioxidant effect of exogenous antioxidants. The present invention uses yeast cells as a cell model to evaluate the in vivo antioxidant activity of exogenous antioxidants.

[0017] The present invention establishes a method for evaluating the antioxidant effect of an exogenous antioxidant, comprising the following steps: using yeast cells as a model, determining the in vivo activity effect of the exogenous antioxidant on the yeast cells. The yeast cells selected by the present invention have the advantages of a short growth cycle and easy expansion and cultivation. The present invention uses Saccharomyces cerevisiae as a model to explore whether earth dragon protein plant solid beverage (EPPD) has the ability to increase tolerance to oxidative stress and to analyze the tolerance mechanism to oxidative stress. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Standard curves of BSA (a) and rutin (b);

[0019] Figure 2Remove DPPH and OH for EPPD - and ABTS free radical capacity assay results;

[0020] Figure 3 The figure is the result of cytotoxicity of EPPD to Saccharomyces cerevisiae;

[0021] Figure 4 This is the result diagram of the effect of EPPD on the survival rate of Saccharomyces cerevisiae;

[0022] Figure 5 This is the result diagram of the effect of EPPD on the lipid peroxidation level of the cell membrane of Saccharomyces cerevisiae;

[0023] Figure 6 This is a graph showing the effect of EPPD on the intracellular peroxidation level of Saccharomyces cerevisiae. DETAILED DESCRIPTION

[0024] The present invention provides the use of yeast cells in preparing a cell model for evaluating the antioxidant effect of exogenous antioxidants.

[0025] In one embodiment of the present invention, the yeast cell includes Saccharomyces cerevisiae and homologous gene-deficient strains of the Saccharomyces cerevisiae. In one embodiment of the present invention, the selected wild-type strain of Saccharomyces cerevisiae BY4741 (MATa, his3, leu2, met15 and ura3) was purchased from Euroscarf, Germany, and its homologous gene-deficient strains Sod1Δ, Gsh1Δ, Ctt1Δ, Gtt1Δ and Gtt2Δ were donated by Professor Pereira of Federal do Rio de Janeiro University, Brazil, and these strains have been disclosed in literature materials (Gao Y, Fang L, Wang X, Lan R, Wang M, Du G, et al. Antioxidant Activity Evaluation of Dietary Flavonoid Hyperoside Using Saccharomyces Cerevisiae as a Model. Molecules. 2019; 24 (4): 788.; Wang M, Meng D, Zhang P, Wang X, Du G, Brennan C, et al. Antioxidant Protection of Nobiletin, 5-Demethylnobiletin, Tangeretin, and 5-Demethyltangeretin from Citrus Peel in Saccharomyces cerevisiae. Journal of Agricultural and Food Chemistry. 2018; 66(12): 3155-60.; Zhao W, Wang L, Yang F, Zhang N, Fan J, Qin S, et al. Antioxidant activity assessment of Yingjisha sweet almond oil. International Journal of Food Science & Technology. 2022; 57(3): 1773-81.). The stock solution of the saccharomyces cerevisiae and the homologous gene-deficient strain of the saccharomyces cerevisiae of the present invention is stored in a solid 2% YPD medium, cultured at 28° C. and 160 rpm, and can be measured by OD 600 The growth of the strain is measured by the absorbance at . The YPD medium of the present invention comprises the following items: 1% (m / v) yeast powder, 2% (m / v) glucose and 2% (m / v) tryptone.

[0026] The invention provides a method for evaluating the antioxidant effect of an exogenous antioxidant, comprising the following steps: using yeast cells as a model to determine the influence of the exogenous antioxidant on the in vivo activity of the yeast cells.

[0027] In a specific embodiment of the present invention, it also includes determining the composition and in vitro activity of the exogenous antioxidant, wherein determining the composition of the exogenous antioxidant includes determining the total protein content and total flavonoid content of the exogenous antioxidant. For the total protein content of the present invention, the BCA kit is used in the embodiment to determine the total protein content in the earth dragon protein plant solid beverage; for the total flavonoid content, the aluminum nitrate colorimetric method is used in the embodiment, with rutin as the standard, rutin concentration as the horizontal coordinate, and the absorbance at 510nm as the vertical coordinate to draw a rutin standard curve and determine the total flavonoid content.

[0028] In a specific embodiment of the present invention, determining the in vitro activity of the exogenous antioxidant comprises determining at least one of the following: DPPH free radical scavenging ability, hydroxyl free radical scavenging ability and ABTS free radical scavenging ability.

[0029] In a specific embodiment of the present invention, determining the in vivo activity effect of the yeast cells comprises determining at least one of the following: toxicity analysis of the yeast cells, tolerance analysis in an oxidative environment, determination of cell membrane lipid peroxidation levels in an oxidative environment, and determination of intracellular peroxidation levels.

[0030] When performing toxicity analysis in the embodiment of the present invention, the brewer's yeast cultured in the YPD culture medium was diluted, and a control group and an experimental group were set up. Different concentrations of exogenous antioxidants were added to the experimental group, and then inoculated into the YPD solid culture medium after incubation. After culturing at 28° C., the number of colonies and cell survival were counted, and the control group was not treated in any way.

[0031] When performing tolerance analysis in the embodiment of the present invention, a control group and an experimental group are also set up. In the experimental group, the yeast cells are incubated in a culture medium added with the exogenous antioxidant for a period of time, and then the oxide culture is added. The bacterial solution is diluted and inoculated into the culture medium, and the number of colonies and cell survival are counted. In the control group, except that the exogenous antioxidant is not added, the rest of the operations are the same as those of the experimental group.

[0032] In one embodiment of the present invention, the method for determining the level of cell membrane lipid peroxidation includes the TCA method. In one embodiment of the present invention, the method for determining the level of intracellular peroxidation includes the fluorescent probe DCFH-DA method.

[0033] In order to further illustrate the present invention, the method for evaluating the antioxidant effect of exogenous antioxidants using Saccharomyces cerevisiae as a model provided by the present invention is described in detail below in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0034] Unless otherwise specified, the materials, reagents and methods used in the examples of the present invention are conventional materials, reagents and methods in the art.

[0035] 1. Drugs and reagents: Earth dragon protein plant solid beverage (EPPD) was provided by Earth Dragon Power Bioengineering Co., Ltd.; 30% H2O2 and CdSO4 were purchased from Tianjin Damao Chemical Reagent Factory; yeast powder, tryptone, and agar powder were purchased from Tianjin Solomon Biotechnology Co., Ltd.; glucose was purchased from Tianjin Huihang Chemical Technology Co., Ltd.;

[0036] 2. Saccharomyces cerevisiae strains, culture media, and growth conditions: The wild-type Saccharomyces cerevisiae strain BY4741 (MATa, his3, leu2, met15, and ura3) was purchased from Euroscarf, Germany, and its homologous gene-deficient strains Sod1Δ, Gsh1Δ, Ctt1Δ, Gtt1Δ, and Gtt2Δ were donated by Professor Pereira of the Federal do Rio de Janeiro University, Brazil.

[0037] The stock solution of the yeast strain was stored in solid 2% YPD medium (1% yeast powder, 2% glucose, 2% peptone and 2% agar), and Saccharomyces cerevisiae was cultured at 28°C, 160rpm, in 2% YPD (1% yeast powder, 2% glucose, 2% tryptone) medium, and its growth was measured by absorbance at 600nm.

[0038] 3. Experimental data were processed using GraphPadPrism 5.0. The results were expressed as Mean±SD, and One-Way ANOVA was used for inter-group difference analysis. The inter-group difference analysis was annotated with letter notation, and different letters indicated significant differences between the treatment groups.

[0039] Example 1

[0040] 1. Component Analysis

[0041] 1.1 Determination of total protein content

[0042] The total protein content in earthworm protein plant solid beverage was determined using the BCA kit.

[0043] 1.2 Determination of total flavonoid content

[0044] Aluminum nitrate colorimetric method was used, with rutin as standard, rutin concentration as the abscissa, and absorbance at 510 nm as the ordinate to draw a rutin standard curve and determine the total flavonoids content.

[0045] The results are as follows Figure 1 As shown, the BSA standard curve equation is y = 0.0009565x + 0.1251, R 2 =0.9956; the standard curve equation of rutin is y=0.4012x+0.002905, R 2 =0.9980. The above two curve equations show that the total protein content in EPPD is 221.33 mg / g, and the total flavonoid content is 4.069 mg / g. This proves that protein and flavonoid components account for a large proportion in EPPD; animal protein and flavonoid substances have good antioxidant effects.

[0046] 2. In vitro activity analysis

[0047] 2.1 DPPH free radical scavenging ability test

[0048] Mix 2 ml of 0.1 mM DPPH solution and 2 ml of EPPD solution of different concentrations (0-8 mg / ml) thoroughly, store at 37°C in the dark for 0.5 h, measure the absorbance value A1 at 517 nm, use water as blank control and VC as positive control group. The clearance calculation formula is as follows:

[0049]

[0050] Where A1 is the absorbance of DPPH solution and EPPD solution; A2 is the absorbance of EPPD solution and ethanol; A0 is the absorbance of water and DPPH solution.

[0051] 2.2 Hydroxyl radical scavenging ability test

[0052] To 1ml of EPPD solution of different concentrations, add 1ml of 5mM ferrous sulfate solution, 1ml of 5mM hydrogen peroxide solution, and 1ml of 5mM salicylic acid solution in sequence, mix thoroughly, react at room temperature for 1h, and measure the absorbance A1 at 517nm. Use water as blank control and VC as positive control group. The clearance calculation formula is as follows:

[0053]

[0054] Where A1 is the absorbance value of the sample solution; A2 is the absorbance value of the hydrogen peroxide solution replaced by water; A0 is the absorbance value of the sample solution replaced by water.

[0055] 2.3 ABTS free radical scavenging ability test

[0056] Add 3 ml of 7.4 mM ABTS diluent to 1 ml of EPPD solution of different concentrations, mix thoroughly, and place in dark place at room temperature for 10 min. Measure the absorbance value A1 of the mixture at 734 nm. Use water as blank control and VC as positive control. The clearance calculation formula is as follows:

[0057]

[0058] Where A1 is the absorbance value of the sample solution; A2 is the absorbance value when PBS buffer replaces ABTS diluent; A0 is the absorbance value when water replaces the sample solution.

[0059] EPPD to DPPH, OH - , ABTS free radicals have strong scavenging ability. Figure 2 As shown in Figure 2, with the increase of EPPD concentration, the removal of DPPH and OH - , the ability of ABTS free radicals is also gradually increasing. When the EPPD concentration is 8mg / ml, the DPPH free radical scavenging rate reaches 97.28%, OH - The free radical scavenging rate is about 80%, and VC at a lower concentration can reach about 90% for the three free radicals. Under the same scavenging ability, EPPD is much greater than VC concentration, which shows that EPPD has a much higher scavenging capacity for DPPH, OH - Although the free radical scavenging ability of ABTS is not as good as that of VC, it also has good in vitro antioxidant ability.

[0060] 3. In vivo activity analysis

[0061] 3.1 Toxicity test

[0062] Pick a single colony of activated Saccharomyces cerevisiae and culture it overnight (12-16 hours) in a test tube containing fresh YPD culture medium. Dilute the culture medium appropriately with new culture medium and add different concentrations of EPPD solution to it. Incubate at 28℃ and 200rpm for 2 hours and then inoculate on solid YPD culture medium. Observe at 28℃ for 72 hours, count the number of colonies, and analyze the survival of cells. The survival rate of the blank group without treatment is 100%, and the survival rate of yeast cells in the treated group is the ratio of the number of colonies in the treated group to that in the blank group.

[0063] The results of EPPD toxicity test on yeast cells are as follows Figure 3 As shown, EPPD concentrations in the range of 0.1 to 5 mg / ml have no toxic effect on yeast cells and can even promote the growth of yeast cells. Therefore, the sample solution in the range of 0.1 to 5 mg / ml can be used for subsequent experiments. Therefore, in the subsequent experiments, three concentrations of 0.1, 1, and 5 mg / ml (low, medium, and high) were selected to pre-protect yeast cells.

[0064] 3.2 Tolerance test

[0065] The tolerance test method was modified from the toxicity test. After incubating the brewer's yeast in a medium with (or without) EPPD for 2 hours, 2mM H2O2 and 3mM CdSO4 were added and cultured for 1 hour. The bacterial solution was diluted and inoculated into a solid culture medium. The culture was observed at 28°C for 72 hours, the number of colonies was counted, and the survival of the cells was analyzed. The survival rate of the blank group without treatment was 100%, and the survival rate of the yeast cells in each treatment group was calculated.

[0066] H2O2 and CdSO4 were used as oxidative stressors to study the effect of EPPD on the survival of yeast cells. Figure 4 As shown in a and c, the survival rate of wild-type yeast cells (WT) was significantly reduced after being stimulated by oxidants, while the survival rate of yeast cells pre-protected by EPPD was improved compared with the oxidative model group, which indicates that EPPD can improve the oxidative stress damage of yeast cells caused by H2O2 and CdSO4.

[0067] The same experiment was performed on yeast strains with antioxidant-related gene deletions (sod1Δ, gsh1Δ, gtt1Δ, gtt2Δ, ctt1Δ). Figure 4 As shown in Figure b, gsh1Δ and ctt1Δ strains showed increased tolerance, with a 10% to 20% increase in survival rate; however, no such effect was observed in sod1Δ, gtt1Δ, and gtt2Δ strains, indicating that the protective effect of EPPD on yeast cells may be achieved through the sod1, gtt1, and gtt2 genes. Figure 4 As shown in Figure d, all tested strains showed increased survival rate after EPPD pretreatment, but the gtt2Δ strain was not sensitive to CdSO4. It can also be considered that the gtt2 gene may be involved in the protective effect of EPPD against CdSO4 stress.

[0068] 3.3 Determination of cell membrane lipid peroxidation level

[0069] The TCA method was used to measure the level of cell membrane lipid peroxidation. Modifications were made based on the tolerance experiment. After the yeast cells were exposed to 2mM H2O2 and 3mM CdSO4 for 1h, the bacterial solution was centrifuged, the precipitate was washed with pure water, the cell precipitate was weighed, 10% TCA was added in a fixed ratio and crushed in a cell crusher, the supernatant was centrifuged, 0.1mM EDTA and 1% TBA were added, mixed evenly, and then placed in a metal bath for 15min, and then quickly placed in an ice bath. When cooled to room temperature, the absorbance value at 532nm was measured.

[0070] The lipid peroxidation level was the ratio of the absorbance of the blank control group, oxidation model group, and EPPD pre-protection group.

[0071] After the body is stimulated by exogenous oxidants, the redox homeostasis in the body is unbalanced, and a large amount of ROS is generated to attack cells, causing damage to the organism. The cell membrane is the first target of ROS attacking cells. ROS reacts with cell membrane lipids to generate malondialdehyde (MDA), causing cell leakage and death. Therefore, MDA is often used as a marker of membrane lipid peroxidation to detect the level of cell membrane lipid peroxidation.

[0072] Effects of EPPD on the level of lipid peroxidation in Saccharomyces cerevisiae cells Figure 5 As shown in the results, under the stress conditions of H2O2 and CdSO4, the cell membrane lipid peroxidation level of the WT strain increased to varying degrees, while EPPD pretreatment could inhibit yeast cell membrane lipid peroxidation, which was consistent with the observed survival results. This indicates that the lipid peroxidation reaction induced by H2O2 and CdSO4 occurs on the cell membrane, and EPPD can improve the integrity of the cell membrane.

[0073] like Figure 5 As shown in a, the gsh1Δ and ctt1Δ strains behaved in the same way as the WT strain, while the sod1Δ, gtt1Δ, and gtt2Δ strains did not show any inhibition of yeast cell membrane lipid peroxidation. Figure 5 As shown in middle b, under CdSO4 stress, the behaviors of sod1Δ, gtt2Δ and ctt1Δ strains were similar to the WT strain, while gsh1Δ and gtt1Δ strains also tended to inhibit cell membrane lipid peroxidation, but without obvious effects.

[0074] 3.4 Determination of intracellular peroxidation levels

[0075] Fluorescent probe DCFH-DA (purchased from Beijing Solebow Technology Co., Ltd.) was used to detect intracellular peroxidation levels. After treatment in 3.2, 5 mM DCFH-DA fluorescent probe was added to the bacterial solution and cultured at 28°C, 200 r / min shaking incubator until the probe was completely absorbed, centrifuged to obtain the precipitate, washed with cold PBS, and then added with 0.5 ml of pure water. The cells were broken in a cell disruptor, the supernatant was centrifuged, and the supernatant was diluted with pure water. The fluorescence value was measured at an excitation light of 488 nm and an emission light of 525 nm.

[0076] The intracellular peroxidation level was the ratio of the fluorescence values ​​of the blank control group, oxidation model group, and EPPD pre-protection group.

[0077] The DCFH-DA fluorescent probe was used to detect the intracellular peroxidation level of yeast cells. The DCFH-DA probe has good lipid solubility and membrane permeability. It is oxidized to DCF when encountering ROS and emits strong fluorescence within a specific wavelength range. People often use fluorescence values ​​to measure the intracellular ROS level. ROS is mainly generated in mitochondria, and excessive accumulation causes functional damage to cells or even death. Natural compounds play an important role in clearing intercellular ROS, and EPPD may have a clearing effect on clearing intercellular ROS.

[0078] Effects of EPPD on intracellular peroxidation levels in Saccharomyces cerevisiae Figure 6 As shown in the data, under the stress of H2O2 and CdSO4, the intracellular peroxidation level of the WT strain increased significantly, and after EPPD pre-protection, the intracellular peroxidation level of the yeast cells was effectively reduced. The data showed that EPPD does have the ability to remove intercellular ROS. Figure 6 As shown in (a), under H2O2 stress, the responses of gsh1Δ, gtt1Δ and ctt1Δ strains were the same as those of the WT strain, while the responses of sod1Δ and gtt2Δ strains were different, which may indicate that these two genes are related to the clearance of intracellular ROS. Figure 6 As shown in (b), the addition of EPPD in all strains can inhibit the increase in cellular peroxidation levels, so under CdSO4 stress, the increase in EPPD tolerance to yeast cells may not be achieved by reducing the level of ROS between cells.

[0079] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Application of yeast cells in preparing cell models for evaluating the antioxidant effects of exogenous antioxidants.

2. The use according to claim 1, characterized in that: The yeast cells include Saccharomyces cerevisiae and homologous gene-deficient strains of the Saccharomyces cerevisiae.

3. A method for evaluating the antioxidant effect of an exogenous antioxidant, characterized in that: The following steps are involved: Yeast cells were used as a model to determine the effects of exogenous antioxidants on the in vivo activity of the yeast cells.

4. The method according to claim 3, characterized in that: Also included is determining the composition and in vitro activity of the exogenous antioxidant.

5. The method according to claim 4, characterized in that: Determining the components of the exogenous antioxidant includes determining the total protein content and the total flavonoid content of the exogenous antioxidant.

6. The method according to claim 4, characterized in that: Determining the in vitro activity of the exogenous antioxidant includes determining at least one of the following: DPPH free radical scavenging ability, hydroxyl free radical scavenging ability and ABTS free radical scavenging ability.

7. The method according to claim 3, characterized in that: Determining the in vivo activity effect of the yeast cells comprises determining at least one of the following: toxicity analysis of the yeast cells, tolerance analysis under an oxidative environment, determination of cell membrane lipid peroxidation levels under an oxidative environment, and determination of intracellular peroxidation levels.

8. The method according to claim 7, characterized in that: The tolerance analysis under the oxidative environment includes setting up a control group and an experimental group. In the experimental group, the yeast cells are incubated in a culture medium with the exogenous antioxidant for a period of time, and then the oxidant is added for culture. The bacterial solution is diluted and inoculated into the culture medium, and the number of colonies and cell survival are counted. The control group was operated in the same manner as the experimental group except that the exogenous antioxidant was not added.

9. The method according to claim 7, characterized in that: The method for measuring the cell membrane lipid peroxidation level includes the TCA method.

10. The method according to claim 7, characterized in that: The method for measuring the intracellular peroxidation level includes the fluorescent probe DCFH-DA method.

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