Special anti-oxidation performance determination kit for honey based on Caco-2 cell model and use method of special anti-oxidation performance determination kit

Abstract

The invention relates to a Caco-2 cell model-based antioxidant performance determination kit special for honey and a use method thereof. The kit comprises a reagent A: Caco-2 cell suspension, a reagent B: HBSS buffer solution, a reagent C: DCFH-DA probe solution, a reagent D: free radical generation inducer ABAP, a reagent E: honey sample solution, a reagent F: cell culture medium, and a reagent G: a solvent for diluting a honey sample. The kit disclosed by the invention shows high sensitivity, low variability, excellent repeatability and batch-to-batch consistency in detection of the oxidation resistance of honey, so that the kit becomes a reliable tool for detecting the oxidation resistance activity of honey and other antioxidant samples. The good precision and high repeatability ensure the stability of experimental results, and the method is suitable for large-scale screening and conventional detection application.

Description

Technical Field

[0001] The invention relates to a special honey antioxidant performance determination kit based on a Caco-2 cell model and a use method. Background Art

[0002] Medical research shows that excessive production of free radicals is closely related to the occurrence and development of a variety of diseases, including aging, cancer, cardiovascular disease and other chronic inflammation-related pathological processes. Therefore, exploring the mechanism of action of antioxidants on free radicals has become an important research direction. In recent years, natural antioxidants have attracted much attention due to their high safety and few side effects. Among them, honey has become a research hotspot because of its excellent antioxidant properties due to its active ingredients such as polyphenols and methylglyoxal (MGO). In particular, Manuka honey, with its unique antioxidant activity and ability to scavenge free radicals, has a significant effect in reducing oxidative stress damage and regulating the inflammatory microenvironment, becoming one of the focuses in antioxidant research. However, the existing antioxidant detection methods still have significant deficiencies in accuracy and physiological relevance.

[0003] Existing in vitro antioxidant detection methods are mainly based on chemical reaction systems, such as DPPH, ABTS and FRAP. Although they can quickly evaluate the free radical scavenging ability of antioxidants, they ignore the complex behavior of antioxidants in the body, including metabolism, absorption, bioavailability and other processes. Therefore, the detection results of these methods are often difficult to accurately reflect the true efficacy of antioxidants in the body. In intracellular antioxidant detection, traditional methods such as DCFH-DA method are widely used, but they lack standardization in sample processing, cell exposure time and antioxidant concentration setting, which easily leads to low repeatability and reliability of experimental results. In addition, most of the existing methods are general designs and do not fully consider the characteristics of complex natural products such as honey, such as high sugar content, interference from large molecular proteins and easy loss of active ingredients. This may underestimate the antioxidant capacity of honey during the detection process. In view of the above shortcomings, it is urgent to establish an innovative antioxidant detection method that can not only carry out detection under conditions closer to the human physiological environment, but also optimize the characteristics of complex natural products such as honey to achieve efficient, quantitative and standardized antioxidant capacity evaluation. The new method needs to be optimized in terms of dilution, active ingredient protection, protein interference removal, and concentration setting, and the sensitivity and repeatability of the results should be ensured by unifying the reagent concentration and experimental process. This will not only provide a scientific basis for the functional research of honey, but also lay the foundation for the development and clinical application of natural antioxidants. Summary of the invention

[0004] In view of the problems existing in the prior art, the purpose of the present invention is to provide a honey-specific antioxidant performance determination kit based on a Caco-2 cell model and a method of use. The present invention uses fluorescence detection to measure the antioxidant activity of high antioxidant honey substances, with particular attention paid to the process of converting DCFH-DA into fluorescent compounds in the presence of reactive oxygen species (ROS), which is specifically achieved through the following technical solutions:

[0005] A special honey antioxidant performance assay kit based on a Caco-2 cell model, the kit comprising:

[0006] Reagent A: Caco-2 cell suspension, Reagent B: HBSS buffer for honey dissolution and cell culture, Reagent C: DCFH-DA probe solution for intracellular oxidative stress detection, Reagent D: free radical generation inducer ABAP, Reagent E: honey sample solution, Reagent F: cell culture medium, Reagent F: cell culture medium, Reagent G: solvent for diluting honey samples.

[0007] Furthermore, the concentration of the DCFH-DA probe solution is 12.5 mM, the concentration of ABAP is 60 mM, and the concentration of the honey sample solution is 20 mg / mL.

[0008] Furthermore, the kit also includes a cell plate, which is an opaque 96-well plate with a black bottom and a lid.

[0009] The method for using the above kit is characterized by comprising the following steps:

[0010] 1) Dissolving the honey to be tested in HBSS buffer to prepare honey sample solutions of different concentrations, and subjecting the prepared honey sample solutions to low-temperature heat treatment;

[0011] 2) Reagent B was used to wash the cells, and an antioxidant drug kit was used to screen antioxidant honey, and a blank group, a control group, and an experimental group were set;

[0012] 3) 100 μL of reagent C was added to the blank group and the control group under sterile conditions, and 5 parallels were set up for each group; 100 μL of reagent F (DMEM) was added to the cell plate under sterile conditions;

[0013] 4) The experimental group was added with 50 μL of different honey concentrations and 50 μL of reagent C

[0014] 5) After adding the sample, cover the lid and incubate in a dark incubator for 1 hour;

[0015] 6) Add 100 μL of ABAP to the control group and the experimental group to make a final concentration of 600 μM. Add 100 μL of reagent C to the control group and treat at 37°C for 1 h.

[0016] 7) Measure the fluorescence intensity using a fluorescence microplate reader at an excitation wavelength of 485 nm and an emission wavelength of 538 nm;

[0017] 8) Data processing and calculation of antioxidant activity: Plot a curve of fluorescence intensity versus time for the blank group, control group, and experimental group; analyze the data, use the blank group value to determine whether the cells have been in a stable state during the experiment, and use the control group (treatment containing only ABAP) as a benchmark to calculate the antioxidant activity of each concentration of honey treatment; if the experimental group value < the control group, the honey has antioxidant activity, if the experimental group ≥ the control group, the honey has no antioxidant activity.

[0018] Furthermore, in step 1), the concentration of the honey sample is 5-20 mg / mL, and the low-temperature heat treatment is specifically 60° C. for 30 minutes.

[0019] Furthermore, in step 7), the fluorescence intensity is measured every 5 minutes for 1 hour, and the changes in the fluorescence intensity of each well are recorded.

[0020] The kit of the present invention exhibits high sensitivity, low variability, excellent repeatability and batch-to-batch consistency in the detection of the antioxidant activity of honey, making it a reliable tool for detecting the antioxidant activity of honey and other antioxidant samples. Its good accuracy and high repeatability ensure the stability of the experimental results and are suitable for large-scale screening and routine detection applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The graph shows the change of fluorescence intensity. DETAILED DESCRIPTION

[0022] The present invention is further described below in conjunction with specific embodiments to facilitate a better understanding of the present technical solution.

[0023] Kit composition:

[0024] Reagent A: Caco-2 cell suspension (about 2.5*105 cells / mL);

[0025] Reagent B: HBSS buffer (for honey dissolution and cell culture);

[0026] Reagent C: DCFH-DA probe solution 12.5 mM (suitable for intracellular oxidative stress detection);

[0027] Reagent D: ABAP 60 mM (free radical generation inducer);

[0028] Reagent E: honey sample solution (concentration 20 mg / mL);

[0029] Reagent F: cell culture medium (DMEM with double antibodies and no serum);

[0030] Reagent G: reagent solvent (used to dilute honey sample);

[0031] The kit also contains a cell plate, which is a 96-well plate with an opaque black bottom and a lid. The DCFH-DA reagent needs to be stored at -20°C

[0032] Synchronization of Caco-2 cells: Resuscitate the Caco-2 cell suspension in reagent A and plate at 2.5 × 10 4 The cells were inoculated at a density of 1.54 cells / well in a black flat-bottom 96-well cell culture plate and incubated at 37° C. and 5% CO 2 for 24 hours to obtain cells that can be used for antioxidant experiments.

[0033] How to use the kit:

[0034] 1) Dissolve the honey to be tested in HBSS (Hanks balanced salt solution) to prepare a series of solutions with final concentrations of 5.00, 10.00, 12.50, 15.00, and 20.00 mg / mL of honey samples. The prepared honey solutions are subjected to low-temperature heat treatment (60°C, 30 minutes);

[0035] 2) Reagent B was used to wash the cells, and an antioxidant drug kit was used to screen antioxidant honey, and a blank group, a control group, and an experimental group were set;

[0036] 3) 100 μL of reagent C was added to the blank group and the control group under sterile conditions, and 5 parallels were set up for each group; 100 μL of reagent F (DMEM) was added to the cell plate under sterile conditions;

[0037] 4) The experimental group was added with 50 μL of different tested honey concentrations and 50 μL of reagent C;

[0038] 5) After adding the sample, cover the lid and incubate in a dark incubator for 1 hour;

[0039] 6) Add 100 μL of ABAP to the control group and the experimental group to make a final concentration of 600 μM. Add 100 μL of reagent C to the control group and treat at 37°C for 1 h.

[0040] 7) Measure the fluorescence intensity using a fluorescence microplate reader at an excitation wavelength of 485 nm and an emission wavelength of 538 nm. Measure once every 5 minutes for 1 hour, and record the changes in the fluorescence intensity of each well.

[0041] 8) Data processing and calculation of antioxidant activity: Draw a curve of fluorescence intensity changing with time, and analyze the data of the blank group, control group, and experimental group. Use the blank group value to determine whether the cells have been in a stable state during the experiment, and use the control group (treatment containing only ABAP) as a benchmark to calculate the antioxidant activity of each concentration of honey treatment. If the experimental group value is < the control group, the honey has antioxidant activity, and if it is ≥ the control group, the honey has no antioxidant activity.

[0042] Test kit effect verification: using Manuka honey as an antioxidant

[0043] Experimental steps:

[0044] 1) Cell culture and inoculation: 100 μL Caco-2 cell suspension was plated at 2.5×10 4 The cells / well were seeded in a black flat-bottom 96-well cell culture plate and cultured at 37°C and 5% CO for 24 h;

[0045] 2) Cell washing: After incubation, remove the supernatant from the culture plate and add 100 μL 1× PBS buffer to gently wash the cells once to remove the residual culture medium;

[0046] 3) Honey sample preparation: Honey was dissolved in HBSS buffer to prepare a series of solutions with concentrations of 5.00, 10.00, 12.50, 15.00, and 20.00 mg / mL. The honey solution was then subjected to low-temperature heat treatment (60°C, 30 minutes) to remove protein interference and retain antioxidant active ingredients.

[0047] 4) Fluorescent probe and honey treatment: Prepare 25 μM DCFH-DA fluorescent probe solution, mix well, and add 100 μL of honey solution and DCF-DA probe solution of each concentration to the sample wells in the washed 96-well cell culture plate. Set up control wells to add only DCF-DA probe solution, and add HBSS buffer to blank wells. Set up 5 replicate wells for each sample well, control well, and blank well, and incubate for 1 hour;

[0048] 5) Cell washing and oxidant treatment: After incubation, remove the treatment solution in the wells and wash the cells three times with 200 μL 1×PBS to remove the unabsorbed probe and honey residue. Add 100 μL 600 μM ABAP oxidant solution to the sample wells and control wells respectively, and add an equal amount of HBSS buffer to the blank wells.

[0049] Fluorescence intensity measurement: Use a fluorescence microplate reader to measure the fluorescence intensity at an excitation wavelength of 485 nm and an emission wavelength of 538 nm. Measure once every 5 minutes for 1 hour and record the changes in fluorescence intensity. The experimental results are shown in Figure 1The distribution of the sample wells, control wells and blank wells in the 96-well microplate can be shown in Table 1.

[0050] Table 1: 96-well plate sample distribution

[0051]

[0052] Among them, Blank-blank well; Con-control; S-sample; Si-the i-th concentration of the sample; PBS-cell-free wells around the 96-well plate (to prevent the matrix effect of the edge culture medium volatilization) are used as the benchmark to test whether the cells are stable without stimulation during the entire experiment, based on the fluorescence intensity-time curve between the sample well and the control well.

[0053] CAA unit = 100-(AUC S / AUC C )×100

[0054] AUC S is the area integral of the fluorescence curve generated by the sample group, AUC C is the area integral of the fluorescence curve produced by the control group.

[0055] Stability testing between kits: Accuracy and stability tests were performed on kits of different batches. The results are shown in Table 2.

[0056] Table 2: Comparison of the differences between the quantitative kits and the kits for the three Manuka honey products

[0057]

[0058] From Table 2, we can see that:

[0059] 1) The coefficient of variation (CV) of all samples from 5 mg / mL to 20 mg / mL was less than 10%, and the CV values ​​were low (between 5% and 7%). This indicates that the kit has high precision and low variability, and is suitable for antioxidant performance testing of samples of different concentrations.

[0060] 2) The kit showed excellent repeatability at all concentrations, and the results of three parallel experiments were highly consistent (low CV values), which further verified the reliability and stability of the kit in practical applications.

[0061] 3) Even at a low concentration of 5 mg / mL, the kit was able to provide stable and accurate test results (CV of 8.31%). As the concentration increased, the kit maintained high sensitivity and low variability. For example, at 20 mg / mL, the CV value dropped to 5.74%, demonstrating that the kit had good sensitivity for both low and high concentration samples.

Claims

1. A honey-specific antioxidant performance assay kit based on a Caco-2 cell model, characterized in that: The kit comprises: Reagent A: Caco-2 cell suspension, Reagent B: HBSS buffer for honey dissolution and cell culture, Reagent C: DCFH-DA probe solution for intracellular oxidative stress detection, Reagent D: free radical generation inducer ABAP, Reagent E: honey sample solution, Reagent F: cell culture medium, Reagent G: solvent for diluting honey samples.

2. The honey-specific antioxidant performance assay kit based on the Caco-2 cell model according to claim 1, characterized in that: The concentration of the DCFH-DA probe solution was 12.5 mM, the concentration of ABAP was 60 mM, and the concentration of the honey sample solution was 20 mg / mL.

3. The honey-specific antioxidant performance assay kit based on the Caco-2 cell model according to claim 1, characterized in that: The kit also includes a cell plate, which is an opaque 96-well plate with a black bottom and a lid.

4. The method for using the kit according to any one of claims 1 to 3, characterized in that: The following steps are involved: 1) Dissolving the honey to be tested in HBSS buffer to prepare honey sample solutions of different concentrations, and subjecting the prepared honey sample solutions to low-temperature heat treatment; 2) Reagent B was used to wash the cells, and an antioxidant drug kit was used to screen antioxidant honey, and a blank group, a control group, and an experimental group were set; 3) 100 μL of reagent C was added to the blank group and the control group under sterile conditions, and 5 parallels were set up for each group; 100 μL of reagent F (DMEM) was added to the cell plate under sterile conditions; 4) The experimental group was added with 50 μL of different honey concentrations and 50 μL of reagent C 5) After the sample is added, cover the lid and incubate in a dark incubator for 1 hour; 6) Add 100 μL of ABAP to the control group and the experimental group to make a final concentration of 600 μM. Add 100 μL of reagent C to the control group and treat at 37°C for 1 h. 7) Measure the fluorescence intensity using a fluorescence microplate reader at an excitation wavelength of 485 nm and an emission wavelength of 538 nm; 8) Data processing and calculation of antioxidant activity: Plot a curve of fluorescence intensity versus time for the blank group, control group, and experimental group; analyze the data, use the blank group value to determine whether the cells have been in a stable state during the experiment, and use the control group (treatment containing only ABAP) as a benchmark to calculate the antioxidant activity of each concentration of honey treatment; if the experimental group value < the control group, the honey has antioxidant activity, if the experimental group ≥ the control group, the honey has no antioxidant activity.

5. The method for using the kit according to claim 4, characterized in that: In step 1), the concentration of the honey sample is 5-20 mg / mL, and the low-temperature heat treatment is specifically 60° C. for 30 minutes.

6. The method for using the kit according to claim 4, characterized in that: Step 7) The fluorescence intensity was measured every 5 minutes for 1 hour, and the changes in the fluorescence intensity of each well were recorded.

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