A buffer and its use in improving stability of resazurin indicator

Abstract

The present application relates to the field of biotechnology, especially to a buffer for improving stability of resazurin, and provides a buffer containing a compound with structure I, and each component is reasonably matched, the buffer is added to a cell culture medium containing resazurin indicator, and the light stability of the culture system is obviously improved, and the color does not change for more than 5 hours under light.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more particularly to a buffer solution and its application in improving the stability of resazu indicator. Background Technology

[0002] Culture medium refers to all substances used for the in vitro culture and preservation of cells for various purposes. Essentially, it artificially simulates the nutritional environment of in vivo growth, enabling cells to grow and reproduce within this environment. It is the material basis for providing cell nutrition and promoting cell growth and proliferation. The main components of cell culture media include: amino acids, water, vitamins, carbohydrates, inorganic ions, and other substances such as nucleic acid degradation products and hormones.

[0003] The combined use of cell culture and chromogenic solutions can be used to monitor the metabolic responses of cells, bacteria, and fungi. For example, after incubation, the presence of microorganisms or their growth and metabolism can be detected by color changes (from blue to red or purple). Commonly used cell culture media include the MEM cell culture medium series, DMEM cell culture medium series, RPMI-1640 cell culture medium series, 199 cell culture medium series, hydrolyzed milk protein cell culture medium, Euler's balanced salt, and F-10 and F-12 cell culture medium series. RPMI-1640 medium supports the growth of various types of cell cultures and has been used as a culture medium for maintaining cell lines and for different cell types.

[0004] Azurlan is a chromogenic solution commonly used in the study of biological materials. Modern biotechnology utilizes cells as carriers; gene therapy, stem cell therapy, and cloning all take place within cells. Cell growth requires a specific nutrient environment, and the nutrient matrix used to maintain cell growth is called culture medium. This refers to all substances used for in vitro culture and preservation of cells for various purposes; essentially, it artificially simulates the nutrient environment of in vivo growth, enabling cells to grow and reproduce within this environment. It is the material basis for providing cell nutrition and promoting cell growth and proliferation. The main components of cell culture medium include: amino acids, water, vitamins, carbohydrates, inorganic ions, and other substances such as nucleic acid degradation products and hormones.

[0005] Resazurin, also known as sodium resazurin, resin azurin, sodium resazurin, and resin phenol azurin, is a blue fluorescent dye used as a redox indicator in the analysis of bacterial activity and the proliferation of bacterial, yeast, or mammalian cells. It is commonly used to measure bacterial and eukaryotic cell viability, as a cell staining agent, and also as an acid-base indicator, with a pH range of 3.8 (orange) to 6.5 (deep purple). In the cytoplasm, resazurin can be reduced from its blue state to a pink, strongly fluorescent form called halogenated resazurin by various reductases, and further reactions result in a colorless and non-fluorescent form of dihydrohalogenated resazurin (see...). Figure 1).

[0006] Rezazon chromogenic solution is added to cell culture media to monitor the metabolic responses of cells, bacteria, and fungi. After incubation, the presence of microorganisms or their growth and metabolism are detected by color changes (from blue to red or purple). Rezazon is redox sensitive, and its stability decreases when mixed with some cell culture media, leading to problems such as the detection system's sensitivity to light, which affects the colorimetric monitoring results of microorganisms. Current solutions involve strict light avoidance during use, but these conditions are demanding, and color changes unrelated to the culture can occur during monitoring due to light exposure, obviously affecting the accuracy of the experimental results. Summary of the Invention

[0007] In view of this, the present invention provides a buffer solution and its application in improving the stability of resazurin. Adding the buffer solution of the present invention to a reaction system using resazurin as an indicator, or using the buffer solution of the present invention to prepare a resazurin colorimetric solution, can significantly improve the light stability of the reaction system.

[0008] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0009] Application of compounds with Formula I structure in improving the stability of azuran

[0010]

[0011] In Formula I, R1 is selected from: -COOH, -COONa, -COOK, -COOCH3, -COOC2H5, -COOC3H7, -COOC3H5, -COOC4H9, -COOC4H7, -COOC5H 11 -COOC5H9, benzyl ester, phenethyl ester, m-methyl phenethyl ester, m-phenol phenethyl ester, 3,4-diphenol phenethyl ester, cinnamyl ester, tartaric acid-2-ester, tartaric acid-2-caffeoyl-3-ester, 1-carboxy-2-(3,4-dihydroxyphenyl)ethyl ester, quinic acid-1-ester, quinic acid-3-ester, quinic acid-4-ester, quinic acid-1-caffeoyl-3-ester, quinic acid-3-caffeoyl-5-ester, quinic acid-3-caffeoyl-4-ester, quinic acid-3,4-dicaffeoyl-5-ester, or cycloarpineenol ester;

[0012] R2 is selected from H, -CN, -COCH3, or phenyl;

[0013] R3 is selected from H, Br, Cl, or -OH;

[0014] R4 is selected from H, Br, Cl, or -OH;

[0015] X is selected from H, -CH3, or -C2H5;

[0016] Y is selected from H, -CH3, or -C2H5.

[0017] In this invention, the resamarium or resamarium indicator is sodium resamarium salt or other forms of salt. In specific embodiments, the resamarium and resamarium indicator used are sodium resamarium salt. Studies have shown that adding the buffer solution of this invention to a culture medium containing resamarium indicator, or using the buffer solution of this invention and resamarium indicator to prepare a resamarium colorimetric solution, can significantly improve the light stability of the reaction system.

[0018] Specifically, the compounds of Formula I are selected from one or more of caffeic acid, methyl caffeate, phenethyl caffeate, ferulic acid, methyl ferulic acid, isopropyl ferulic acid, 3-(2,3,4-trihydroxyphenyl)acrylic acid, 3-bromo-4-hydroxy-5-methoxyphenylacrylic acid, cinnamyl 3,4-dihydroxy-α-cyanocinol, rosmarinic acid, monocaffeoyl tartaric acid, dicaffeoyl tartaric acid, chlorogenic acid, isochlorogenic acid A, isochlorogenic acid B, cryptochlorogenic acid, 1-caffeoylquinic acid, 3,4,5-tricaffeoylquinic acid, or cycloargentinol ferulic acid ester. Studies have shown that buffer solutions prepared from the above compounds can significantly improve the photostability of the resamarium reaction system, increasing the photostability time by up to 30 times.

[0019] Some compounds with structures similar to the above-mentioned compounds, such as betulinol caffeate and 3-methyl-2-butenic acid ester, are less effective in improving the light stability of rezakura, extending the light stability of the rezakura reaction system by approximately three times. The results indicate that only the specific compounds of this invention can significantly improve the light stability of the rezakura reaction system.

[0020] This invention provides a buffer solution for improving the stability of rezamidophos, comprising 0.07M-0.11M of a compound of formula I.

[0021] In some embodiments, the buffer solution comprises components at concentrations of:

[0022] Osmotic pressure regulator 0.2-1 g / L, and / or

[0023] pH buffer 0.05-0.2M, and / or

[0024] Solvent promoter: 0.02wt%-0.06wt%.

[0025] Compared to the compounds mentioned above, betulinol caffeate and 3-methyl-2-butenic acid ester are less effective in improving the light stability of zazablaze, but can extend the light stability of the zazablaze reaction system by about three times.

[0026] In some embodiments, the buffer solution of the present invention comprises water and components of the following concentrations: 0.07M-0.11M of a compound of formula I, 0.2-1 g / L of an osmotic pressure regulator, 0.05-0.2M of a pH buffer, and 0.02wt%-0.06wt% of a solubilizer.

[0027] In some embodiments, the osmotic pressure regulator is bovine serum albumin, citrate, sodium chloride, and glucose; the pH buffer includes at least one pair of disodium hydrogen phosphate-sodium dihydrogen phosphate, disodium hydrogen phosphate-potassium dihydrogen phosphate buffer, potassium dihydrogen phosphate-sodium hydroxide buffer, Tris-hydrochloric acid buffer, and sodium carbonate-sodium bicarbonate buffer; the solubilizer includes at least one of polysorbate 20, polysorbate 60, and polysorbate 80.

[0028] This application may also add growth factors, such as biotin and glutamine, to the buffer solution according to experimental needs to promote bacterial growth. Specifically, the growth factor is selected from at least one of phenylalanine, biotin, and glutamine. Experimental results show that the buffer solution of this application has good photostability after addition. The preferred concentration of the growth factor is 0.5 g / L.

[0029] In some specific embodiments, the buffer solution of the present invention comprises components at the following concentrations:

[0030] Bovine serum albumin 0.5 g / L, disodium hydrogen phosphate-potassium dihydrogen phosphate 0.2 M, phenylalanine 0.5 g / L, phenethyl caffeate (a compound with structure I) 0.08 M, polysorbate 80 0.03 wt%, in water;

[0031] Alternatively, sodium citrate 0.5 g / L, potassium dihydrogen phosphate-sodium hydroxide 0.1 M, ferulic acid (compound of formula I) 0.08 M, polysorbate 60 0.02 wt%, phenylalanine 0.5 g / L, in water as solvent.

[0032] Alternatively, sodium chloride 0.5 g / L, disodium hydrogen phosphate-sodium dihydrogen phosphate 0.1 M, rosmarinic acid (a compound with structure I) 0.08 M, polysorbate 20 0.02 wt%, with water as the solvent.

[0033] Alternatively, sodium chloride 0.5 g / L, Tris-hydrochloric acid buffer 0.2 M, 3-(2,3,4-trihydroxyphenyl)acrylic acid (a compound of formula I) 0.07 M, biotin 0.5 g / L, polysorbate 20 0.02 wt%, in water.

[0034] Alternatively, sodium citrate 0.5 g / L, sodium carbonate-sodium bicarbonate 0.2 M, 3-bromo-4-hydroxy-5-methoxyphenylacrylic acid (a compound with the structure of formula I) 0.07 M, polysorbate 20 0.02 wt%, with water as the solvent.

[0035] Alternatively, the following solutions are prepared: bovine serum albumin 0.5 g / L, disodium hydrogen phosphate-potassium dihydrogen phosphate 0.2 M, cinnamyl 3,4-dihydroxy-α-cyanocinolate (a compound of formula I) 0.09 M, glutamine 0.5 g / L, polysorbate 80 0.03 wt%, with water as the solvent.

[0036] Alternatively, the following solutions are prepared: bovine serum albumin 0.5 g / L, potassium dihydrogen phosphate-sodium hydroxide 0.2 M, phenylalanine 0.5 g / L, monocaffeoyl tartaric acid (a compound with structure I) 0.1 M, polysorbate 80 0.03 wt%, and water as the solvent.

[0037] Alternatively, sodium citrate 0.5 g / L, potassium dihydrogen phosphate-sodium hydroxide 0.1 M, chlorogenic acid (a compound with structure I) 0.08 M, polysorbate 80 0.02 wt%, with water as the solvent.

[0038] Alternatively, sodium citrate 0.5 g / L, disodium hydrogen phosphate-sodium dihydrogen phosphate 0.1 M, 1-caffeoylquinic acid (a compound with structure I) 0.11 M, biotin 0.5 g / L, polysorbate 20 0.02 wt%, in water as solvent.

[0039] Alternatively, the following solutions are prepared: bovine serum albumin 0.5 g / L, disodium hydrogen phosphate-potassium dihydrogen phosphate 0.2 M, cycloartenol ferulic acid ester (a compound with the structure of formula I) 0.11 M, glutamine 0.5 g / L, polysorbate 20 0.03 wt%, with water as the solvent.

[0040] In addition to the above formulations, the buffer solution of the present invention also includes formulations of compounds containing a formula I structure obtained by reducing one or more components based on the above formulations.

[0041] The present invention provides a method for improving the photostability of azadirachtin indicator in a culture medium, comprising: adding the buffer solution described in the present invention to a culture medium containing azadirachtin indicator;

[0042] Alternatively, it may include: dissolving resplenium in the buffer solution described in this invention as a solvent to prepare a resplenium chromogenic solution, and then adding the resplenium chromogenic solution to the culture medium.

[0043] In some embodiments, the amount of buffer solution added is 0.7-2.2 mL of the buffer solution described in this invention per liter of culture medium. In some specific embodiments, 0.7 mL, 1 mL, 1.5 mL, 2 mL or 2.2 mL of the buffer solution described in this invention may be added per liter of culture medium.

[0044] By adding resamaritan and the buffer solution described in this invention using the two methods described above, the final concentration of resamaritan is 1.4-100 mg / L and 0.7-2.2 mL of the buffer solution described in this invention per liter of culture system. In some specific embodiments of this invention, each 500 mL culture system contains 4 mg of resamaritan and 1 mL of the buffer solution described in this invention, i.e., each liter of culture system contains 8 mg of resamaritan and 2 mL of the buffer solution described in this invention.

[0045] In some embodiments, the culture medium is RPMI-1640 medium. In other embodiments, the buffer solution of the present invention remains effective when RPMI-1640 medium is used in combination with other substances or other culture media. RPMI-1640 culture medium has a complex nutrient composition, and after the addition of the indicator resazurin, it easily changes from blue to red under light, affecting the colorimetric monitoring results of microorganisms; therefore, this system must be protected from light. The present invention addresses the light instability of resazurin during use by providing a buffer solution. Adding the buffer solution of the present invention to RPMI-1640 medium containing resazurin indicator significantly improves the light stability of the culture system, maintaining its color for more than 5 hours under light. Attached Figure Description

[0046] Figure 1 The principle of oxidation-reduction color change in azure glaze;

[0047] Figure 2 The color of the reaction system in Example 4 after 0 minutes of light exposure (without light exposure) is shown.

[0048] Figure 3 The color of the reaction system in Example 4 after 10 minutes of light exposure is shown.

[0049] Figure 4 The color of the reaction system in Example 4 after 20 minutes of light exposure is shown.

[0050] Figure 5 The color of the reaction system in Example 4 after 40 minutes of light exposure is shown.

[0051] Figure 6 The color of the reaction system in Example 4 after 5 hours of light exposure is shown.

[0052] Figure 7 The results of comparing the OD values ​​of the experimental groups (Formulas 1-5 of Example 1) and the control group under different light exposure times in Example 4 are shown, corresponding to the data in Tables 1-5;

[0053] Figure 8 The comparison results of OD values ​​of the test groups (Formulas 2-5 of Example 2) and the control group under different light exposure times in Example 6 are shown, corresponding to the data in Table 4-5;

[0054] Figure 9 The comparison results of OD values ​​of the test groups (Formulas 3-4 of Example 3) and the control group under different light exposure times in Example 7 are shown in Table 5-4.

[0055] Figure 10 The comparison results of OD values ​​of the experimental group (Formula 8-5 in Example 8) and the control group under different light exposure times are shown in Table 6-5.

[0056] Figure 11 The comparison results of OD values ​​of the experimental group (Formula 9-4 in Example 9) and the control group under different light exposure times are shown in Table 7-4.

[0057] Figure 12 The comparison results of OD values ​​of the experimental group (Formula 10-5 of Example 10) and the control group under different light exposure times are shown in Table 8-5.

[0058] Figure 13 The comparison results of OD values ​​of the experimental group (Formula 11-5 of Example 11) and the control group under different light exposure times are shown in Table 9-5.

[0059] Figure 14 The comparison results of OD values ​​of the experimental group (Formula 12-4 of Example 12) and the control group under different light exposure times are shown in Table 10-4.

[0060] Figure 15 The comparison results of OD values ​​of the experimental group (Formula 13-5 of Example 13) and the control group under different light exposure times are shown in Table 11-5.

[0061] Figure 16 The comparison results of OD values ​​of the test group (Formula 14-5 of Example 14) and the control group under different light exposure times are shown in Table 12-5.

[0062] Figure 17 Comparison of OD values ​​of experimental group (Comparative Example 1) and control group under different light exposure times;

[0063] Figure 18 Comparison of OD values ​​of experimental group (Comparative Example 2) and control group under different light exposure times. Detailed Implementation

[0064] This invention provides a buffer solution and its application in improving the stability of resamaritan indicator. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired result. It is particularly important to note that this invention uses RPMI-1640 medium as an example. In fact, when similar results occur in other mediums containing the active ingredient RPMI-1640, or when RPMI-1640 medium is directly mixed with other cell culture media, it is easy to conceive of implementing this invention. The color change problem in the resamaritan colorimetric reaction system addressed by the buffer solution provided by this invention is due to a decrease in the stability of resamaritan in the reaction system within a short period of time, rather than a color change caused by culture factors such as cell metabolism. It is also reasonable to infer that the essence of this phenomenon in other types of mediums is the same as that in RPMI-1640 medium. All similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. It should be noted that the method and application of this invention have been described through preferred embodiments. Those skilled in the art can obviously modify or appropriately change and combine the method and application described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention.

[0065] The test materials used in this invention are all common commercial products and can be purchased on the market.

[0066] The present invention will be further illustrated below with reference to the embodiments:

[0067] Example 1: Buffer formulation of the present invention

[0068] Formula 1-1: 0.08M phenethyl caffeate, solvent is water;

[0069] Formulas 1-2: Bovine serum albumin 0.5 g / L, phenethyl caffeate 0.08 M, solvent is water;

[0070] Formulas 1-3: Bovine serum albumin 0.5 g / L, disodium hydrogen phosphate-potassium dihydrogen phosphate 0.2 M, phenethyl caffeate 0.08 M, solvent is water;

[0071] Formulas 1-4: Bovine serum albumin 0.5 g / L, disodium hydrogen phosphate-potassium dihydrogen phosphate 0.2 M, phenylalanine 0.5 g / L, phenethyl caffeate 0.08 M, solvent is water;

[0072] Formulas 1-5: Bovine serum albumin 0.5 g / L, disodium hydrogen phosphate-potassium dihydrogen phosphate 0.2 M, phenylalanine 0.5 g / L, phenethyl caffeate 0.08 M, polysorbate 80 0.03 wt%, solvent is water.

[0073] Example 2: Buffer formulation of the present invention

[0074] Formula 2-1: Ferulic acid 0.08M, solvent is water;

[0075] Formula 2-2: Sodium citrate 0.5 g / L, ferulic acid 0.08 M, solvent is water;

[0076] Formulas 2-3: Sodium citrate 0.5 g / L, potassium dihydrogen phosphate-sodium hydroxide 0.1 M, ferulic acid 0.08 M, solvent is water;

[0077] Formulas 2-4: Sodium citrate 0.5 g / L, potassium dihydrogen phosphate-sodium hydroxide 0.1 M, ferulic acid 0.08 M, phenylalanine 0.5 g / L, solvent is water;

[0078] Formulas 2-5: Sodium citrate 0.5g / L, potassium dihydrogen phosphate-sodium hydroxide 0.1M, ferulic acid 0.08M, polysorbate 60 0.02%, phenylalanine 0.5g / L, solvent is water.

[0079] Example 3: Buffer formulation of the present invention

[0080] Formula 3-1: Rosmarin 0.08M, solvent is water;

[0081] Formula 3-2: Sodium chloride 0.5g / L, rosmarinic acid 0.08M, solvent is water;

[0082] Formula 3-3: Sodium chloride 0.5 g / L, disodium hydrogen phosphate-sodium dihydrogen phosphate 0.1 M, rosmarinic acid 0.08 M, solvent is water;

[0083] Formulas 3-4: Sodium chloride 0.5g / L, disodium hydrogen phosphate-sodium dihydrogen phosphate 0.1M, rosmarinic acid 0.08M, polysorbate 20 0.02%, solvent is water.

[0084] Example 4

[0085] Take 500 ml of RPMI-1640 culture medium (homemade or commercially available), add 2 mL of resazurin chromogenic solution (using water as solvent, resazurin sodium concentration 2 g / L), and then add 0.4 ml of any of the buffer solutions from Example 1; or prepare the resazurin chromogenic solution (resazurin sodium concentration 10 g / L) using 0.4 ml of the buffer solution from Example 1 and 4 mg of resazurin sodium, and then add it to 500 ml of culture medium. Finally, add 4 mg of resazurin and 0.4 mL of any of the buffer solutions from Example 1 to every 500 ml of RPMI-1640 culture medium. Illuminate the entire system with an LED light, and take photos to record the color of the culture medium at 0 minutes, 10 minutes, 20 minutes, 40 minutes, and 5 hours of illumination. The results are shown in [the table below]. Figures 2-6 (Color change diagram of formulas 1-5), the blue OD of the system was detected using a UV spectrophotometer. 600The color retention rate was calculated by dividing the value of the light exposure N (10 / 20 / 30 / 40 / 300) minutes by the value of the light exposure 0 minutes by 100%. The results are shown in Tables 1-1 to 1-5. The control group did not add the buffer solution of the present invention, while the experimental group added the buffer solution of Example 1 of the present invention. Figure 7 The OD values ​​of the control group and experimental group under different light exposure times are shown in Table 1-5.

[0086] Table 1-1 Detection results of the control group and experimental group (buffer solution formulation 1-1)

[0087]

[0088]

[0089] Table 1-2 Detection results of the control group and experimental group (buffer solution formulation 1-2)

[0090]

[0091] Table 1-3 Detection results of the control group and experimental group (buffer solution formulation 1-3)

[0092]

[0093] Table 1-4 Detection results of the control group and experimental group (buffer solution formulations 1-4)

[0094]

[0095] Table 1-5 Detection results of control group and experimental group (buffer solution formulation 1-5)

[0096]

[0097]

[0098] The results showed that the reaction system of *Razorina tenuifolia* with RPMI-1640 culture medium was blue. After 10 minutes of light exposure (laboratory LED light), the experimental group remained unchanged, with a color retention rate of 100.00%, while the control group without buffer turned purplish-red, with a color retention rate of 60.18%. After 20 minutes of light exposure, the experimental group remained unchanged, with a color retention rate of 99.74%, while the control group without buffer turned red, with a color retention rate of 49.74%. After 30 minutes of light exposure, the reaction system in the experimental group remained unchanged, with a color retention rate of 99.61%, while the control group without buffer turned red, with a color retention rate of 29.90%. After 40 minutes, the system with buffer remained unchanged, maintaining a blue color, with a color retention rate of 99.61%, while the system without buffer turned red and became brighter, with a color retention rate of 22.32%. The addition of buffer significantly improved the light stability of the reaction system, maintaining its color unchanged for up to 5 hours, with a color retention rate of 99.21%, while the system without buffer turned red, with a color retention rate decreasing to 21.02%.

[0099] The reaction system with the buffer solution of the present invention has a longer photoreaction time than before. The time it takes for the system to remain unchanged in color after 10 minutes is extended to 5 hours, and the photostability time is increased to 30 times the original time, effectively extending the photostability of the reaction system.

[0100] In addition, experiments have shown that the blue color of the azurite does not change when exposed to light in water, and the blue color remains 100% after 5 hours of light exposure (see Table 2).

[0101] Table 2. Effects of different illumination times on the blue color of azurite aqueous solution.

[0102]

[0103] Example 5: Detection of the effect of the buffer solution of the present invention on bacterial activity.

[0104] The buffer solution used in Example 3 was formulated as follows: sodium chloride 0.5 g / L, disodium hydrogen phosphate-sodium dihydrogen phosphate 0.1 M, rosmarinic acid 0.08 M, and polysorbate 20 0.02%.

[0105] Take 0.5 μL of McBurney's turbidity bacterial suspension of Escherichia coli ATCC25922, and dilute the control group with physiological saline to 1×10⁻⁶. 3 CFU / mL, 1×10 2 CFU / mL, the experimental group was diluted to 1×10⁻⁶ with buffer. 3 CFU / mL, 1×10 2 CFU / mL, incubated at room temperature for 2 hours, plated and counted, the results are as follows:

[0106] Table 3. Detection of the effect of buffer solution on bacterial activity

[0107]

[0108] The above experiments show that there is no significant difference in the number of bacterial colonies between the buffer solution and physiological saline dilution, and the results are consistent, indicating that the buffer solution of the present invention has no effect on bacterial growth and metabolism.

[0109] Example 6

[0110] Take 500 ml of RPMI-1640 culture medium (homemade or commercially available), add 2 mL of resazurin chromogenic solution (using water as solvent, resazurin sodium salt concentration 2 g / L), and then add 0.5 mL of any of the buffer solutions from Example 2; or prepare the resazurin chromogenic solution using 0.5 mL of any of the buffer solutions from Example 2 and 4 mg of resazurin sodium salt, so that each 500 mL of RPMI-1640 culture medium contains 4 mg of resazurin sodium salt and 0.5 mL of the buffer solution from Example 2. Finally, irradiate the entire system with LED light and measure the OD value of the culture medium at 0 minutes, 10 minutes, 20 minutes, 40 minutes, and 5 hours of illumination, calculate the color retention rate, and the results are shown in Tables 4-1 to 4-5. The control group did not add the buffer solution of this invention, while the experimental group added any of the buffer solutions from Example 2 of this invention. Figure 8 The OD values ​​of the control group and experimental group under different light exposure times are shown in Table 4-5.

[0111] Table 4-1 Detection results of the control group and experimental group (buffer solution formulation 2-1)

[0112]

[0113] Table 4-2 Detection results of the control group and experimental group (buffer solution formulation 2-2)

[0114]

[0115] Table 4-3 Detection results of control group and experimental group (buffer solution formulation 2-3)

[0116]

[0117] Table 4-4 Detection results of the control group and experimental group (buffer solution formulation 2-4)

[0118]

[0119] Table 4-5 Detection results of the control group and experimental group (buffer solution formulation 2-5)

[0120]

[0121]

[0122] The results showed that the reaction system of *Razorina tenuifolia* and RPMI-1640 culture medium was blue. After 10 minutes of light exposure (laboratory LED light), the experimental group did not change color, with a color retention rate of 99.88%, while the control group without buffer turned purplish-red, with a color retention rate of 65.21%. After 20 minutes of light exposure, the experimental group did not change color, with a color retention rate of 99.75%, while the control group without buffer turned red, with a color retention rate of 57.98%. After 30 minutes of light exposure, the reaction system in the experimental group did not change color, with a color retention rate of 99.38%, while the control group without buffer turned red, with a color retention rate of 40.65%. After 40 minutes, the system with buffer remained blue, with a color retention rate of 99.50%, while the system without buffer turned red, with a color retention rate of 27.56%. The addition of buffer significantly improved the light stability of the reaction system, maintaining its color for up to 5 hours without turning red, with a color retention rate of 99.63%, while the system without buffer turned red, with a color retention rate decreasing to 23.94%.

[0123] The reaction system with the added buffer solution of the present invention remained red for 10 minutes and 5 hours, respectively, and the photostability time was increased to 30 times the original time, effectively extending the photostability of the reaction system.

[0124] Example 7

[0125] Prepare a 4 g / L resazurin chromogenic solution using any of the buffer formulations in Example 3 as the solvent, and simultaneously prepare a 4 g / L resazurin chromogenic solution using water as a control. Take 1 mL of the chromogenic solution prepared using any of the buffer formulations in Example 3 as the solvent and 1 mL of the resazurin chromogenic solution prepared using water, respectively, and add them to 500 mL of RPMI-1640 culture medium (homemade or commercially available). Place the entire system outdoors in a well-lit area (avoiding direct sunlight), and measure the OD values ​​of the culture medium at 0 minutes, 10 minutes, 20 minutes, 40 minutes, and 5 hours of light exposure, and calculate the color retention rate. The data are shown in Tables 5-1 to 5-4. Figure 9 This is a comparison chart of the detection data in Table 5-4.

[0126] Table 5-1 Detection results of the control group and experimental group (buffer solution formula 3-1)

[0127]

[0128]

[0129] Table 5-2 Detection results of the control group and experimental group (buffer solution formula 3-2)

[0130]

[0131] Table 5-3 Detection results of the control group and experimental group (buffer solution formula 3-3)

[0132]

[0133] Table 5-4 Detection results of the control group and experimental group (buffer solution formulation 3-4)

[0134]

[0135] The results showed that the reaction system of *Razorina tenuifolia* with RPMI-1640 culture medium was blue. The experimental group remained unchanged after 10 minutes of light exposure, with a color retention rate of 100%, while the control group without buffer turned purplish-red, with a color retention rate of 63.69%. After 20 minutes of light exposure, the experimental group remained unchanged, with a color retention rate of 99.88%, while the control group without buffer turned red, with a color retention rate of 37.84%. After 30 minutes of light exposure, the experimental group remained unchanged, with a color retention rate of 99.65%, while the control group without buffer turned red, with a color retention rate of 17.74%. After 40 minutes, the system with buffer remained unchanged, retaining its blue color, with a color retention rate of 99.65%, while the system without buffer turned red, with a color retention rate of 17.39%. The addition of buffer significantly improved the light stability of the reaction system, maintaining its color unchanged for up to 5 hours, with a color retention rate of 99.54%, while the system without buffer turned red, with a color retention rate decreasing to 15.16%.

[0136] The results showed that the reaction system with the buffer solution of Example 3 of the present invention remained red for 10 minutes and 5 hours, respectively, and the photostability time was increased to 30 times the original time, effectively extending the photostability of the reaction system.

[0137] Example 8: Buffer formulation of the present invention

[0138] Formula 8-1: 0.07M 3-(2,3,4-trihydroxyphenyl)acrylic acid (a compound with structure I), in water as solvent.

[0139] Formula 8-2: Sodium chloride 0.5 g / L, 3-(2,3,4-trihydroxyphenyl)acrylic acid (a compound with structure I) 0.07 M, solvent is water.

[0140] Formula 8-3: Sodium chloride 0.5 g / L, Tris-hydrochloric acid buffer 0.2 M, 3-(2,3,4-trihydroxyphenyl)acrylic acid (compound of formula I) 0.07 M, solvent is water.

[0141] Formula 8-4: Sodium chloride 0.5 g / L, Tris-hydrochloric acid buffer 0.2 M, 3-(2,3,4-trihydroxyphenyl)acrylic acid (compound of formula I) 0.07 M, biotin 0.5 g / L, solvent is water.

[0142] Formula 8-5: Sodium chloride 0.5 g / L, Tris-hydrochloric acid buffer 0.2 M, 3-(2,3,4-trihydroxyphenyl)acrylic acid (compound of formula I) 0.07 M, biotin 0.5 g / L, polysorbate 20 0.02 wt%, solvent is water.

[0143] Example 9: Buffer formulation of the present invention

[0144] Formula 9-1: 0.07 M of 3-bromo-4-hydroxy-5-methoxyphenylacrylic acid (a compound with the structure of Formula I), in water as the solvent.

[0145] Formula 9-2: Sodium citrate 0.5 g / L, 3-bromo-4-hydroxy-5-methoxyphenylacrylic acid (a compound with structure I) 0.07 M, solvent is water.

[0146] Formula 9-3: Sodium citrate 0.5 g / L, sodium carbonate-sodium bicarbonate 0.2 M, 3-bromo-4-hydroxy-5-methoxyphenylacrylic acid (a compound with structure I) 0.07 M, solvent is water.

[0147] Formula 9-4: Sodium citrate 0.5 g / L, sodium carbonate-sodium bicarbonate 0.2 M, 3-bromo-4-hydroxy-5-methoxyphenylacrylic acid (compound with structure I) 0.07 M, polysorbate 20 0.02 wt%, solvent is water.

[0148] Example 10: Buffer formulation of the present invention

[0149] Formula 10-1: 0.09 M of cinnamyl 3,4-dihydroxy-α-cyanocinolate (a compound with structure I);

[0150] Formula 10-2: Bovine serum albumin 0.5 g / L, cinnamyl ester of 3,4-dihydroxy-α-cyanocinolate (a compound with structure I) 0.09 M, in water as solvent.

[0151] Formula 10-3: Bovine serum albumin 0.5 g / L, disodium hydrogen phosphate-potassium dihydrogen phosphate 0.2 M, cinnamyl ester of 3,4-dihydroxy-α-cyanocinolate (a compound with structure I) 0.09 M, with water as the solvent.

[0152] Formula 10-4: Bovine serum albumin 0.5 g / L, disodium hydrogen phosphate-potassium dihydrogen phosphate 0.2 M, cinnamyl ester of 3,4-dihydroxy-α-cyanocinolate (a compound with structure I) 0.09 M, glutamine 0.5 g / L, with water as the solvent.

[0153] Formula 10-5: Bovine serum albumin 0.5 g / L, disodium hydrogen phosphate-potassium dihydrogen phosphate 0.2 M, cinnamyl 3,4-dihydroxy-α-cyanocinolate (compound of formula I) 0.09 M, glutamine 0.5 g / L, polysorbate 80 0.03 wt%, solvent is water.

[0154] Example 11 Buffer formulation of the present invention

[0155] Formula 11-1: 0.1 M of monocaffeoyl tartaric acid (a compound with structure I), in water as solvent.

[0156] Formula 11-2: Bovine serum albumin 0.5 g / L, monocaffeoyl tartaric acid (a compound with structure I) 0.1 M, solvent is water.

[0157] Formula 11-3: Bovine serum albumin 0.5 g / L, potassium dihydrogen phosphate-sodium hydroxide 0.2 M, monocaffeoyl tartaric acid (a compound with structure I) 0.1 M, solvent is water.

[0158] Formula 11-4: Bovine serum albumin 0.5 g / L, potassium dihydrogen phosphate-sodium hydroxide 0.2 M, phenylalanine 0.5 g / L, monocaffeoyl tartaric acid (a compound with structure I) 0.1 M, solvent is water.

[0159] Formula 11-5: Bovine serum albumin 0.5 g / L, potassium dihydrogen phosphate-sodium hydroxide 0.2 M, phenylalanine 0.5 g / L, monocaffeoyl tartaric acid (a compound with the structure of formula I) 0.1 M, polysorbate 80 0.03 wt%, and water as the solvent.

[0160] Example 12 Buffer formulation of the present invention

[0161] Formula 12-1: 0.08 M chlorogenic acid (a compound with structure I), in water as solvent;

[0162] Formula 12-2: Sodium citrate 0.5 g / L, chlorogenic acid (a compound with structure I) 0.08 M, solvent is water;

[0163] Formula 12-3: Sodium citrate 0.5 g / L, potassium dihydrogen phosphate-sodium hydroxide 0.1 M, chlorogenic acid (a compound with structure I) 0.08 M, solvent is water;

[0164] Formula 12-4: Sodium citrate 0.5 g / L, potassium dihydrogen phosphate-sodium hydroxide 0.1 M, chlorogenic acid (compound with structure I) 0.08 M, polysorbate 80 0.02 wt%, solvent is water.

[0165] Example 13 Buffer formulation of the present invention

[0166] Formula 13-1: 0.11 M of 1-caffeoylquinic acid (a compound with structure I), in water as the solvent.

[0167] Formula 13-2: Sodium citrate 0.5 g / L, 1-caffeoylquinic acid (a compound with structure I) 0.11 M, solvent is water.

[0168] Formula 13-3: Sodium citrate 0.5 g / L, disodium hydrogen phosphate-sodium dihydrogen phosphate 0.1 M, 1-caffeoylquinic acid (a compound with structure I) 0.11 M, solvent is water.

[0169] Formula 13-4: Sodium citrate 0.5 g / L, disodium hydrogen phosphate 0.1 M, 1-caffeoylquinic acid (a compound with structure I) 0.11 M, biotin 0.5 g / L, solvent is water.

[0170] Formula 13-5: Sodium citrate 0.5 g / L, disodium hydrogen phosphate 0.1 M, 1-caffeoylquinic acid (compound with structure I) 0.11 M, biotin 0.5 g / L, polysorbate 20 0.02 wt%, solvent is water.

[0171] Example 14 Buffer formulation of the present invention

[0172] Formula 14-1: Cyclocarpine ferulate (a compound with structure I) 0.11 M, in water.

[0173] Formula 14-2: Bovine serum albumin 0.5 g / L, cycloargentinol ferulic acid ester (compound with structure I) 0.11 M, solvent is water.

[0174] Formula 14-3: Bovine serum albumin 0.5 g / L, disodium hydrogen phosphate-potassium dihydrogen phosphate 0.2 M, cycloargentenol ferulic acid ester (compound with structure I) 0.11 M, solvent is water.

[0175] Formula 14-4: Bovine serum albumin 0.5 g / L, disodium hydrogen phosphate-potassium dihydrogen phosphate 0.2 M, cycloargentenol ferulic acid ester (compound with structure I) 0.11 M, glutamine 0.5 g / L, solvent is water.

[0176] Formula 14-5: Bovine serum albumin 0.5 g / L, disodium hydrogen phosphate-potassium dihydrogen phosphate 0.2 M, cycloartenol ferulic acid ester (compound with structure I) 0.11 M, glutamine 0.5 g / L, polysorbate 200.03 wt%, solvent is water.

[0177] Comparative Example 1

[0178] Bovine serum albumin 0.5 g / L, disodium hydrogen phosphate-potassium dihydrogen phosphate 0.2 M, phenylalanine 0.5 g / L, 3-methyl-2-buten-caffeic acid ester (compound with formula I) 0.11 M, polysorbate 80 0.03 wt%;

[0179] Ineffective compounds that do not improve stability include: 3,4-dimethoxybenzyl acetone, ferulic acid eicosate, and ferulic acid octadecyl ester.

[0180] Comparative Example 2

[0181] Bovine serum albumin 0.5 g / L, disodium hydrogen phosphate-potassium dihydrogen phosphate 0.2 M, phenylalanine 0.5 g / L, 3,4-dimethoxybenzyl acetone 0.11 M, polysorbate 80 0.03 wt%;

[0182] Test case

[0183] Take 500 mL of RPMI-1640 culture medium (homemade or commercially available), add 2 mL of resazurin chromogenic solution (using water as solvent, resazurin sodium salt concentration 2 g / L), and then add 1 mL of buffer solution (Examples 8-14 or Comparative Examples 1-2); or use the buffer solution to dissolve resazurin sodium salt to prepare resazurin chromogenic solution (resazurin sodium salt concentration 4 g / L), and then add 1 mL of the resazurin chromogenic solution to the culture medium. The final result should be that each 500 mL of RPMI-1640 culture medium contains 4 mg of resazurin and 1 mL of any of the buffer solutions from Examples 8-16. Finally, illuminate the entire system with an LED lamp and detect the blue OD of the system using a UV spectrophotometer. 600 The color retention rate was calculated, and the results are shown in Tables 6-1 to 12-5 and Tables 13 to 14. The control group did not receive buffer solution, while the experimental group received buffer solutions from Examples 8-14 or Comparative Examples 1-2. Results for some formulations in Tables 6-12 are shown below. Figures 10-16 The results in Tables 13 and 14 are shown below. Figures 17-18 .

[0184] Table 6-1 Detection results of the control group and experimental group (formula 8-1) under different light exposure times.

[0185]

[0186] Table 6-2 Detection results of control group and experimental group (formula 8-2) under different light exposure times

[0187]

[0188] Table 6-3 Detection results of control group and experimental group (formula 8-3) under different light exposure times.

[0189]

[0190] Table 6-4 Detection results of control group and experimental group (formula 8-4) under different light exposure times

[0191]

[0192] Table 6-5 Detection results of control group and experimental group (formula 8-5) under different light exposure times.

[0193]

[0194] The results showed that the reaction system of *Razorina tenuifolia* with RPMI-1640 culture medium was blue. After 10 minutes of light exposure (laboratory LED light), the experimental group did not change color, with a color retention rate of 98.21%, while the control group without buffer turned purplish-red, with a color retention rate of 64.63%. After 20 minutes of light exposure, the experimental group did not change color, with a color retention rate of 99.22%, while the control group without buffer turned red, with a color retention rate of 41.36%. After 30 minutes of light exposure, the reaction system in the experimental group did not change color, with a color retention rate of 99.78%, while the control group without buffer turned red, with a color retention rate of 30.96%. After 40 minutes, the system with buffer remained blue, with a color retention rate of 98.54%, while the system without buffer turned red and became brighter, with a color retention rate of 21.36%. The addition of buffer significantly improved the light stability of the reaction system, maintaining its color unchanged for up to 5 hours, with a color retention rate of 98.43%, while the system without buffer turned red, and the color retention rate decreased to 15.25%.

[0195] Table 7-1 Detection of light exposure time in control and experimental groups (Formula 9-1)

[0196]

[0197]

[0198] Table 7-2 Detection of light exposure time in control and experimental groups (Formula 9-2)

[0199]

[0200] Table 7-3 Detection of light exposure time in control and experimental groups (formula 9-3)

[0201]

[0202] Table 7-4 Detection of control and experimental groups under different light exposure times (Formula 9-4)

[0203]

[0204] The results showed that the reaction system of *Razorina tenuifolia* with RPMI-1640 culture medium was blue. After 10 minutes of light exposure (laboratory LED light), the experimental group did not change color, with a color retention rate of 98.96%, while the control group without buffer turned purplish-red, with a color retention rate of 65.26%. After 20 minutes of light exposure, the experimental group did not change color, with a color retention rate of 97.58%, while the control group without buffer turned red, with a color retention rate of 40.23%. After 30 minutes of light exposure, the reaction system in the experimental group did not change color, with a color retention rate of 97.92%, while the control group without buffer turned red, with a color retention rate of 32.91%. After 40 minutes, the system with buffer remained blue, with a color retention rate of 98.15%, while the system without buffer turned red and became brighter, with a color retention rate of 18.86%. The addition of buffer significantly improved the light stability of the reaction system, maintaining its color unchanged for up to 5 hours, with a color retention rate of 98.62%, while the system without buffer turned red, and the color retention rate decreased to 13.94%.

[0205] Table 8-1 Results of light exposure at different times for the control group and experimental group (Formula 10-1)

[0206]

[0207] Table 8-2 Results of light exposure at different times for the control group and experimental group (formula 10-2)

[0208]

[0209] Table 8-3 Results of light exposure at different times for the control group and experimental group (formula 10-3)

[0210]

[0211]

[0212] Table 8-4 Results of light exposure at different times for the control group and experimental group (formula 10-4)

[0213]

[0214] Table 8-5 Results of light exposure at different times for the control group and experimental group (formula 10-5)

[0215]

[0216] The results showed that the reaction system of *Razorina tenuifolia* with RPMI-1640 culture medium was blue. After 10 minutes of light exposure (laboratory LED light), the experimental group did not change color, with a color retention rate of 99.55%, while the control group without buffer turned purplish-red, with a color retention rate of 63.95%. After 20 minutes of light exposure, the experimental group did not change color, with a color retention rate of 99.21%, while the control group without buffer turned red, with a color retention rate of 45.58%. After 30 minutes of light exposure, the reaction system in the experimental group did not change color, with a color retention rate of 99.32%, while the control group without buffer turned red, with a color retention rate of 32.77%. After 40 minutes, the system with buffer remained blue, with a color retention rate of 99.21%, while the system without buffer turned red and became brighter, with a color retention rate of 22.56%. The addition of buffer significantly improved the light stability of the reaction system, maintaining its color unchanged for up to 5 hours, with a color retention rate of 99.66%, while the system without buffer turned red, and the color retention rate decreased to 14.40%.

[0217] Table 9-1 Detection results of control group and experimental group (formula 11-1) under different light exposure times

[0218]

[0219] Table 9-2 Results of light exposure at different times for the control group and experimental group (Formula 11-2)

[0220]

[0221] Table 9-3 Results of light exposure at different times for the control group and experimental group (formula 11-3)

[0222]

[0223] Table 9-4 Results of light exposure at different times for the control group and experimental group (formula 11-4)

[0224]

[0225]

[0226] Table 9-5 Results of light exposure at different times for the control group and experimental group (formula 11-5)

[0227]

[0228] The results showed that the reaction system of *Razorina tenuifolia* with RPMI-1640 culture medium was blue. After 10 minutes of light exposure (laboratory LED light), the experimental group did not change color, with a color retention rate of 98.78%, while the control group without buffer turned purplish-red, with a color retention rate of 65.01%. After 20 minutes of light exposure, the experimental group did not change color, with a color retention rate of 98.78%, while the control group without buffer turned red, with a color retention rate of 41.83%. After 30 minutes of light exposure, the reaction system in the experimental group did not change color, with a color retention rate of 99.33%, while the control group without buffer turned red, with a color retention rate of 32.45%. After 40 minutes, the system with buffer remained blue, with a color retention rate of 99.55%, while the system without buffer turned red and became brighter, with a color retention rate of 18.43%. The addition of buffer significantly improved the light stability of the reaction system, maintaining its color unchanged for up to 5 hours, with a color retention rate of 99.11%, while the system without buffer turned red, and the color retention rate decreased to 13.58%.

[0229] Table 10-1 Results of Detection in Control and Experimental Groups (Formula 12-1) under Different Irradiation Times

[0230]

[0231] Table 10-2 Results of light exposure at different times for the control group and experimental group (Formula 12-2)

[0232]

[0233] Table 10-3 Results of light exposure at different times for the control group and experimental group (formula 12-3)

[0234]

[0235] Table 10-4 Results of light exposure at different times for the control group and experimental group (formula 12-4)

[0236]

[0237] The results showed that the reaction system of *Razorina tenuifolia* with RPMI-1640 culture medium was blue. After 10 minutes of light exposure (laboratory LED light), the experimental group did not change color, with a color retention rate of 98.76%, while the control group without buffer turned purplish-red, with a color retention rate of 64.15%. After 20 minutes of light exposure, the experimental group did not change color, with a color retention rate of 99.10%, while the control group without buffer turned red, with a color retention rate of 45.43%. After 30 minutes of light exposure, the reaction system in the experimental group did not change color, with a color retention rate of 99.66%, while the control group without buffer turned red, with a color retention rate of 31.91%. After 40 minutes, the system with buffer remained blue, with a color retention rate of 99.78%, while the system without buffer turned red and became brighter, with a color retention rate of 21.08%. The addition of buffer significantly improved the light stability of the reaction system, maintaining its color for up to 5 hours without turning red, with a color retention rate of 98.88%, while the system without buffer turned red, and the color retention rate decreased to 13.19%.

[0238] The reaction system with the added buffer solution of Example 12 of the present invention remained red for 10 minutes and 5 hours, respectively, and the photostability time was increased to 30 times the original time, effectively extending the photostability of the reaction system.

[0239] Table 11-1 Detection of light exposure time in control and experimental groups (Formula 13-1)

[0240]

[0241] Table 11-2 Detection of light exposure time in control and experimental groups (Formula 13-2)

[0242]

[0243] Table 11-3 Detection of light exposure time in control and experimental groups (Formula 13-3)

[0244]

[0245]

[0246] Table 11-4 Detection of light exposure time in control and experimental groups (Formula 13-4)

[0247]

[0248] Table 11-5 Detection of light exposure time in control and experimental groups (Formula 13-5)

[0249]

[0250] The results showed that the reaction system of resamaritan and RPMI-1640 culture medium was blue. After 10 minutes of light exposure (laboratory LED light), the experimental group did not change color, with a color retention rate of 99.66%, while the control group without buffer turned purplish-red, with a color retention rate of 60.41%. After 20 minutes of light exposure, the experimental group did not change color, with a color retention rate of 99.20%, while the control group without buffer turned red, with a color retention rate of 42.81%. After 30 minutes of light exposure, the reaction system in the experimental group did not change color, with a color retention rate of 99.43%, while the control group without buffer turned red, with a color retention rate of 31.53%. After 40 minutes, the system with buffer did not change color, remaining blue, with a color retention rate of 99.31%, while the system without buffer turned red and became brighter, with a color retention rate of 19.10%. Adding the buffer solution from Example 13 of this invention significantly improved the light stability of the reaction system, maintaining its color unchanged for up to 5 hours, with a color retention rate of 99.54%, while the system without buffer turned red, with a color retention rate decreasing to 12.43%.

[0251] Table 12-1 Detection of light exposure time in control and experimental groups (Formula 14-1)

[0252]

[0253] Table 12-2 Detection of light exposure time in control and experimental groups (Formula 14-2)

[0254]

[0255] Table 12-3 Detection of light exposure time in control and experimental groups (Formula 14-3)

[0256]

[0257] Table 12-4 Detection of light exposure time in control and experimental groups (Formula 14-4)

[0258]

[0259]

[0260] Table 12-5 Detection of light exposure time in control and experimental groups (Formula 14-5)

[0261]

[0262] The results showed that the reaction system of *Razorina tenuifolia* and RPMI-1640 culture medium was blue. After 10 minutes of light exposure (laboratory LED light), the experimental group did not change color, with a color retention rate of 99.32%, while the control group without buffer turned purplish-red, with a color retention rate of 56.98%. After 20 minutes of light exposure, the experimental group did not change color, with a color retention rate of 99.09%, while the control group without buffer turned red, with a color retention rate of 40.39%. After 30 minutes of light exposure, the reaction system in the experimental group did not change color, with a color retention rate of 99.43%, while the control group without buffer turned red, with a color retention rate of 30.55%. After 40 minutes, the system with buffer remained blue, with a color retention rate of 98.97%, while the system without buffer turned red and became brighter, with a color retention rate of 20.02%. The addition of buffer significantly improved the light stability of the reaction system, maintaining its color unchanged for up to 5 hours, with a color retention rate of 99.54%, while the system without buffer turned red, and the color retention rate decreased to 13.84%.

[0263] From Tables 6-12, Figures 10-16 It is known that by adding a buffer solution containing an effective compound of the present invention (Formula I) to the reaction system, the photoreaction time remained unchanged at 10 minutes and was extended to 5 hours, and the photostability time was increased to 30 times the original time, effectively extending the photostability of the reaction system.

[0264] Table 13 Detection results of control group and experimental group (Comparative Example 1) under different light exposure times

[0265]

[0266]

[0267] The results showed that the reaction system of razor cyanide and RPMI-1640 culture medium was blue. After 10 minutes of light exposure (laboratory LED light), the experimental group did not change color, with a color retention rate of 98.53%, while the control group without buffer turned purplish-red, with a color retention rate of 59.54%. After 20 minutes of light exposure, the experimental group turned purple, with a color retention rate of 86.25%, while the control group without buffer turned red, with a color retention rate of 44.11%. After 30 minutes of light exposure, the reaction system of the experimental group turned purplish-red, with a color retention rate of 71.25%, while the control group without buffer turned red, with a color retention rate of 29.03%. After 40 minutes, the system with buffer turned red, with a color retention rate of 48.82%, while the system without buffer turned red and became brighter red, with a color retention rate of 19.20%. After 5 hours of light exposure, the system turned red, with a color retention rate of 39.68%, while the system without buffer turned red, with a color retention rate decreasing to 13.49%. Adding buffer improved the light stability of the reaction system, but the effect was weak; the color retention rate was less than 90% after 20 minutes of light exposure.

[0268] The reaction system with the addition of buffer solution 1 was subjected to a light reaction time that was extended from 10 minutes without turning red to 30 minutes without turning red. The photostability time was increased, and the photostability of the reaction system was prolonged, but the effect was significantly less than that of the buffer solutions in Examples 1-3 and 8-14.

[0269] Table 14. Detection of light exposure time in the control group and experimental group (Comparative Example 2)

[0270]

[0271] The results showed that the reaction system of razor cyanide and RPMI-1640 culture medium was blue. After 10 minutes of light exposure (laboratory LED light), the experimental group turned purplish-red, with a color retention rate of 63.54%, while the control group without buffer turned purplish-red, with a color retention rate of 59.91%. After 20 minutes of light exposure, the experimental group turned red, with a color retention rate of 37.94%, while the control group without buffer turned red, with a color retention rate of 39.98%. After 30 minutes of light exposure, the reaction system of the experimental group turned red, with a color retention rate of 29.49%, while the control group without buffer turned red, with a color retention rate of 26.04%. After 40 minutes, the system with buffer turned red and became brighter red, with a color retention rate of 21.14%, while the system without buffer turned red and became brighter red, with a color retention rate of 18.89%. After 5 hours of light exposure, the experimental group turned red, with a color retention rate of 15.31%, while the system without buffer turned red, with a color retention rate decreasing to 12.10%. The addition of ineffective buffer did not significantly improve the light stability of the reaction system.

[0272] The reaction system with buffer solution containing the ineffective compound 3,4-dimethoxybenzyl acetone showed no significant difference in photostability time compared to the system without buffer solution.

[0273] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of compounds with the structure of Formula I in improving the stability of razorazon. In Formula I, R1 is selected from: -COOH, -COONa, -COOK, -COOCH3, -COOC2H5, -COOC3H7, -COOC3H5, -COOC4H9, -COOC4H7, -COOC5H 11 -COOC5H9, benzyl ester, phenethyl ester, m-methyl phenethyl ester, m-phenol phenethyl ester, 3,4-diphenol phenethyl ester, cinnamyl ester, tartaric acid-2-ester, tartaric acid-2-caffeoyl-3-ester, 1-carboxy-2-(3,4-dihydroxyphenyl)ethyl ester, quinic acid-1-ester, quinic acid-3-ester, quinic acid-4-ester, quinic acid-1-caffeoyl-3-ester, quinic acid-3-caffeoyl-5-ester, quinic acid-3-caffeoyl-4-ester, quinic acid-3,4-dicaffeoyl-5-ester, or cycloarpineenol ester; R2 is selected from H, -CN, -COCH3, or phenyl; R3 is selected from H, Br, Cl, or -OH; R4 is selected from H, Br, Cl, or -OH; X is selected from H, -CH3, or -C2H5; Y is selected from H, -CH3, or -C2H5.

2. The application according to claim 1, characterized in that, The compound of Formula I is selected from one or more of caffeic acid, methyl caffeate, phenethyl caffeate, ferulic acid, methyl ferulic acid, isopropyl ferulic acid, 3-(2,3,4-trihydroxyphenyl)acrylic acid, 3-bromo-4-hydroxy-5-methoxyphenylacrylic acid, cinnamyl 3,4-dihydroxy-α-cyanocinol, rosmarinic acid, monocaffeoyl tartaric acid, dicaffeoyl tartaric acid, chlorogenic acid, isochlorogenic acid A, isochlorogenic acid B, cryptochlorogenic acid, 1-caffeoylquinic acid, 3,4,5-tricaffeoylquinic acid, or cycloargentinol ferulic acid ester.

3. A buffer solution for improving the stability of rezuril, characterized in that, Compounds with a structure of formula I, ranging from 0.07 M to 0.11 M; In Formula I, R1 is selected from: -COOH, -COONa, -COOK, -COOCH3, -COOC2H5, -COOC3H7, -COOC3H5, -COOC4H9, -COOC4H7, -COOC5H 11 -COOC5H9, benzyl ester, phenethyl ester, m-methyl phenethyl ester, m-phenol phenethyl ester, 3,4-diphenol phenethyl ester, cinnamyl ester, tartaric acid-2-ester, tartaric acid-2-caffeoyl-3-ester, 1-carboxy-2-(3,4-dihydroxyphenyl)ethyl ester, quinic acid-1-ester, quinic acid-3-ester, quinic acid-4-ester, quinic acid-1-caffeoyl-3-ester, quinic acid-3-caffeoyl-5-ester, quinic acid-3-caffeoyl-4-ester, quinic acid-3,4-dicaffeoyl-5-ester, or cycloarpineenol ester; R2 is selected from H, -CN, -COCH3, or phenyl; R3 is selected from H, Br, Cl, or -OH; R4 is selected from H, Br, Cl, or -OH; X is selected from H, -CH3, or -C2H5; Y is selected from H, -CH3, or -C2H5.

4. The buffer solution according to claim 3, characterized in that, The compound of Formula I is selected from one or more of caffeic acid, methyl caffeate, phenethyl caffeate, ferulic acid, methyl ferulic acid, isopropyl ferulic acid, 3-(2,3,4-trihydroxyphenyl)acrylic acid, 3-bromo-4-hydroxy-5-methoxyphenylacrylic acid, cinnamyl 3,4-dihydroxy-α-cyanocinol, rosmarinic acid, monocaffeoyl tartaric acid, dicaffeoyl tartaric acid, chlorogenic acid, isochlorogenic acid A, isochlorogenic acid B, cryptochlorogenic acid, 1-caffeoylquinic acid, 3,4,5-tricaffeoylquinic acid, or cycloargentinol ferulic acid ester.

5. The buffer solution according to claim 3 or 4, characterized in that, This includes osmotic pressure regulators and / or pH regulators and / or solubilizers.

6. The buffer solution according to claim 5, characterized in that, The components include the following concentrations: Osmotic pressure regulator 0.2-1 g / L and / or pH buffer 0.05-0.2M and / or Solvent promoter: 0.02wt%-0.06wt%.

7. The buffer solution according to any one of claims 3 to 5, characterized in that, The osmotic pressure regulator includes at least one of bovine serum albumin, citrate, sodium chloride, and glucose; the pH buffer includes at least one pair of disodium hydrogen phosphate-sodium dihydrogen phosphate, disodium hydrogen phosphate-potassium dihydrogen phosphate buffer, potassium dihydrogen phosphate-sodium hydroxide buffer, Tris-hydrochloric acid buffer, and sodium carbonate-sodium bicarbonate buffer; the solubilizer includes at least one of polysorbate 20, polysorbate 60, and polysorbate 80.

8. The buffer solution according to claim 7, characterized in that, It also includes growth factors, which are selected from at least one of phenylalanine, biotin, and glutamine.

9. A method for improving the stability of azurlan indicator in a culture medium, characterized in that, include: Add the buffer solution according to any one of claims 3 to 8 to the cell culture medium containing rezamidoide indicator; Alternatively, it may include: mixing the buffer solution according to any one of claims 3 to 8 with the resazu indicator to prepare a resazu colorimetric solution, and then adding the resazu colorimetric solution to the cell culture medium.

10. The method according to claim 9, characterized in that, The buffer solution is added at a rate of 0.7-2.2 mL per liter of cell culture medium, according to any one of claims 3 to 8.

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