Method and kit for detecting inhibitory activity of non-enzymatic glycosylation end product

Abstract

The invention provides a method for detecting the inhibitory activity of non-enzymatic glycosylation end products (AGEs). The method comprises the following steps: preparing a mixed solution containing bovine serum albumin and dihydroxyacetone as a control system; adding a to-be-detected substance into the mixed solution containing bovine serum albumin and dihydroxyacetone to serve as a to-be-detected system; carrying out incubation reaction on the control system and the to-be-detected system at 25-55 DEG C; respectively detecting fluorescence values of the control system and the to-be-detected system; and according to the change of the fluorescence value before and after the to-be-detected substance is added, evaluating whether the to-be-detected substance has an inhibiting effect on the generation of the non-enzymatic glycosylation end product or not. The invention also provides a kit for detecting the inhibitory activity of the non-enzymatic glycosylation end product. According to the detection method and the kit, the incubation reaction time of AGEs is shortened from 7 days to 24 hours, the detection time is greatly shortened, the detection efficiency is improved, and a new choice is provided for high-throughput screening of AGEs inhibitors.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology detection, and specifically relates to a method and a kit for high-throughput detection of non-enzymatic glycosylation end products (AGEs) inhibitory activity. Background Art

[0002] Non-enzymatic glycosylation is a series of complex non-enzymatic reactions, in which reducing sugars react with amino groups in the body to generate unstable Schiff bases, which then rearrange to form relatively stable Amadori rearrangement products, called early glycosylation products; the Amadori rearrangement products react with the amino groups of proteins, and the sugar part undergoes a dehydration reaction to form unsaturated aldehyde and ketone compounds; the aldehyde and ketone compounds then react with the free amino groups of proteins, and the proteins are denatured due to intermolecular or intramolecular cross-linking and cleavage, ultimately generating irreversible non-enzymatic glycosylation end products AGEs (Advanced Glycation End Products).

[0003] AGEs are a class of compounds formed in organisms through non-enzymatic reactions. These reactions usually occur between proteins, lipids or nucleic acids and reducing sugars (such as glucose, fructose, etc.). Current studies have shown that the formation of AGEs is a slow process. AGEs accelerate the aging of the human body and lead to the occurrence and development of many chronic degenerative diseases. For example, skin aging (the accumulation of AGEs can cause sagging, aging and dull skin), diabetes (the formation of AGEs in diabetic patients is accelerated, and is related to diabetes-related complications such as retinopathy, nephropathy and neuropathy), cardiovascular disease (AGEs can promote the development of atherosclerosis and increase the risk of cardiovascular disease), neurodegenerative diseases (in neurodegenerative diseases such as Alzheimer's disease, the accumulation of AGEs is related to nerve damage), etc. At present, the study of AGEs is an active field. Scientists are exploring the role of AGEs in diseases and how to reduce the impact of AGEs through intervention, so the study of AGE detection methods is of great significance.

[0004] AGEs have the characteristic of spontaneous fluorescence. Under the irradiation of near-ultraviolet light, they can emit fluorescence in the visible light band. Therefore, by measuring the fluorescence spectrum of AGEs and observing the changes in fluorescence values, the amount of AGEs generated can be judged. This fluorescence spectroscopy method generally uses the method of in vitro protein and reducing sugar reaction to evaluate the impact on non-enzymatic glycation end products AGEs, which is faster and more convenient than the slow glycation reaction detection in the body.

[0005] CN102478503A discloses a method for detecting the mechanism of action of ursolic acid in inhibiting AGEs. The method uses 20mM aminoguanidine as a positive control, adopts a 5g / L BSA and 50mM d-glucose model, cultures in a 37°C incubator for 40 days, and then measures the AGEs fluorescence value to determine the inhibitory effect of ursolic acid on AGEs.

[0006] CN116549527A discloses a composition for inhibiting skin glycation. The patent uses aminoguanidine as a positive control, and adopts 10 mg / mL BSA and 0.5 M glucose to react at 37°C for 21 days. The degree of inhibition of the composition on advanced glycation products (AGEs) is determined by measuring the fluorescence intensity.

[0007] CN112220893A discloses the effect of a bamboo sap extract on the formation of AGEs. With 0.1 mg / ml aminoguanidine as the positive control, two detection models were adopted: 30 mg / mL BSA and 1.5 M fructose, and 30 mg / mL BSA and 60 mM MGO. After 3 ml of the reaction solution was incubated in a 37°C incubator for 6 days, the degree of inhibition of the extract on advanced glycation products (AGEs) was determined by measuring the fluorescence intensity.

[0008] CN114672527A discloses a fermentation extraction method and application of ganoderma lucidum polysaccharide, and the anti-glycation property of ganoderma lucidum polysaccharide extract is tested by the BSA and fructose model of test example 2. After incubation at 37°C for 21 days, the fluorescence intensity was measured on the 7th, 14th and 21st days, and it was found that the strongest inhibitory effect was found on the 7th day.

[0009] Jariyapongskul A et al. reported that a-Mangostin, a mangosteen extract, has clear anti-glycation activity (see Non-Patent Document 1).

[0010] As mentioned above, the models currently used for in vitro non-enzymatic glycosylation detection usually include models of protein and reducing sugars such as glucose or fructose, or models of protein and methylglyoxal (MGO). Among them, the model of protein and glucose or fructose detects the effect of the sample on the entire process of non-enzymatic glycosylation, from the initial stage to the late stage. This type of model has a slow reaction in the initial stage and a long experimental cycle, which is not suitable for rapid screening of samples. The model of protein and MGO is a model established for the late stage of non-enzymatic glycosylation. The advantage is that the reaction time is much shorter than that of the reducing sugar type. The disadvantage is also obvious, that is, it is impossible to detect samples that act on the upstream of the non-enzymatic glycosylation process, which may cause omissions in sample screening or low product detection activity.

[0011] Non-patent literature 1: Jariyapongskul A, Areebambud C, Suksamrarn S, MekseepralardC (2015) Alpha-mangostin attenuation of hyperglycemia-induced ocularhypoperfusion and blood retinal barrier leakage in the early stage of type2diabetes rats. Biomed Res Int 2015:785826) Summary of the invention

[0012] Technical Problems to be Solved by the Invention

[0013] In view of the fact that the current in vitro non-enzymatic glycosylation detection model has a long experimental cycle, low detection efficiency, and incomplete detection, it cannot meet the needs of high-throughput screening of samples. The present invention provides a new in vitro non-enzymatic glycosylation detection model, detection method, and detection kit, which can more efficiently and comprehensively screen effective AGEs inhibitors.

[0014] Technical solutions to technical problems

[0015] The present invention includes the following technical solutions:

[0016] The present invention provides a method for detecting non-enzymatic glycosylation end product inhibitory activity, comprising the following steps:

[0017] A mixed solution containing bovine serum albumin and dihydroxyacetone was prepared as a control system;

[0018] Adding the object to be tested into the mixed solution containing bovine serum albumin and dihydroxyacetone as the test system;

[0019] The control system and the test system are incubated at 25°C to 55°C;

[0020] Respectively detect the fluorescence values ​​of the control system and the system to be tested;

[0021] Based on the change in fluorescence value before and after adding the test substance, it is evaluated whether the test substance has an inhibitory effect on the formation of non-enzymatic glycosylation end products.

[0022] In the detection method of the present invention, the solvent of the mixed solution containing bovine serum albumin and dihydroxyacetone is a phosphate buffered saline solution.

[0023] In the detection method of the present invention, the temperature range of the incubation reaction is 37°C±5°C.

[0024] The detection method of the present invention further comprises calculating the inhibition rate of the test substance on non-enzymatic glycosylation end products by the following formula:

[0025] Inhibition rate (%) = [1-(RFU 待测物 -RFU 空白对照 ) / (RFU 阴性对照 -RFU 空白对照 )]×100

[0026] Among them, RFU 空白对照 RFU is the fluorescence value detected by BSA and dihydroxyacetone at 0 hours of reaction. 阴性对照 RFU is the fluorescence value after bovine serum albumin and dihydroxyacetone react for a period of time. 待测物 It is the fluorescence value after adding the analyte to bovine serum albumin and dihydroxyacetone and reacting for a period of time.

[0027] In the detection method of the present invention, the reaction period is more than 24 hours.

[0028] In the detection method of the present invention, in the mixed solution containing bovine serum albumin and dihydroxyacetone, the concentration of bovine serum albumin is not less than 5 mg / ml, and the concentration of dihydroxyacetone is not less than 10 mg / ml; more preferably, the concentration of bovine serum albumin is 10-100 mg / ml, and the concentration of dihydroxyacetone is 20-200 mg / ml.

[0029] In the detection method of the present invention, the incubation reaction conditions include: 96-well plate, 100-200ul per well.

[0030] In the detection method of the present invention, the incubation reaction is carried out under light-proof conditions.

[0031] The present invention also provides a kit for detecting the inhibitory activity of non-enzymatic glycosylation end products, comprising a bovine serum albumin solution with a concentration of 10 mg / ml or more and a dihydroxyacetone solution with a concentration of 20 mg / ml or more and instructions for use, and the two solutions are mixed when used. The concentration of the bovine serum albumin solution is more preferably 20-100 mg / ml, and the concentration of the dihydroxyacetone solution is more preferably 40-200 mg / ml, and the two solutions are mixed at a volume ratio of 1:2-2:1 when used.

[0032] Beneficial technical effects

[0033] Compared with the traditional BSA-reducing sugar model, the BSA-DHA model and detection method provided by the present invention shorten the incubation reaction time of AGEs from 7-21 days to 24 hours-3 days, greatly shortening the detection time and improving the detection efficiency. Compared with the commonly used BSA-MGO model, the BSA-DHA model and detection method provided by the present invention are more comprehensive and complete for the detection of diversified samples, providing a new option for high-throughput screening of effective anti-sugar components. The method of the present invention can directly perform incubation reaction on a 96-well plate, and directly perform detection after the reaction is completed, eliminating the need to take out a certain volume from each group of reaction solution and put it in the well plate for detection, simplifying the operation, and being more conducive to high-throughput detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 : AGEs fluorescence value after 3 days of reaction in BSA-DHA system.

[0035] Figure 2 : Fluorescence values ​​of BSA-DHA system reaction on days 1-3.

[0036] Figure 3 : The inhibitory effect of aminoguanidine on the formation of non-enzymatic glycation end products (AGEs) was detected using BSA-DHA model and BSA-MGO model. DETAILED DESCRIPTION

[0037] The present invention is described in detail below with reference to the examples and drawings. The examples provide detailed implementation methods and processes to fully disclose and demonstrate that the invention can be implemented. The examples do not represent limitations on the invention, and the protection scope of the present invention is not limited to the following examples.

[0038] Experimental methods without specific conditions in the examples of the specification are usually carried out according to conventional conditions in the art or conditions recommended by the manufacturer. Instruments, reagents and drugs without sources in the examples of the specification can be commercially available.

[0039] In the present invention, unless otherwise specified, "above", "below", "within" means including the number itself. When "comprising", "including", "containing" are used to describe an embodiment herein, other similar embodiments described as "consisting of" and / or "consisting essentially of" are also provided. When "consisting essentially of" is used to describe an embodiment herein, other similar embodiments described as "consisting of" are also provided.

[0040] The term "and / or" as used herein in phrases such as "A and / or B" is intended to include both A and B; A or B; A (alone); and B (alone). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to include each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0041] The "±" used in phrases such as "A±B" in this article means all values ​​within the range from AB to A+B. For example, 37℃±5℃ means all values ​​between 32-42℃ and values ​​within the range of 32-42℃ after rounding.

[0042] In this article, "non-enzymatic glycosylation" and "glycation" are different names for the same concept with the same meaning. Therefore, "non-enzymatic glycosylation reaction" and "glycation reaction" are sometimes used interchangeably, and "non-enzymatic glycosylation end products" and "glycation products" are sometimes used interchangeably.

[0043] Experimental principle of the present invention

[0044] The inventors found that bovine serum albumin (BSA) and dihydroxyacetone (DHA) can interact to produce non-enzymatic glycation end products (AGEs). AGEs have the characteristic of fluorescence luminescence. The test compound is co-incubated with BSA and DHA, and the change in the fluorescence value in the system before and after the addition of the test compound is detected to evaluate whether the test compound has an inhibitory effect on the formation of AGEs, thereby screening out active substances with an inhibitory effect on the formation of AGEs.

[0045] The method for detecting the inhibitory activity of non-enzymatic glycosylation end products of the present invention achieves the purpose of high-throughput screening of AGEs inhibitors through the above experimental principle. Specifically, the method for detecting the inhibitory activity of non-enzymatic glycosylation end products of the present invention comprises the following steps:

[0046] A mixed solution containing bovine serum albumin and dihydroxyacetone was prepared as a control system;

[0047] Adding the object to be tested into the mixed solution containing bovine serum albumin and dihydroxyacetone as the test system;

[0048] The control system and the test system are incubated at 25°C to 55°C;

[0049] Respectively detect the fluorescence values ​​of the control system and the system to be tested;

[0050] Based on the change in fluorescence value before and after the addition of the analyte, that is, the change in fluorescence value of the control system before the addition of the analyte and the analyte after the addition of the analyte, it is evaluated whether the analyte has an inhibitory effect on the formation of non-enzymatic glycation end products, thereby screening out active substances with an inhibitory effect on AGEs formation.

[0051] The mixed solution containing bovine serum albumin and dihydroxyacetone can be prepared by separately preparing a PBS solution of bovine serum albumin and a PBS solution of dihydroxyacetone, and then mixing them in a certain volume ratio (e.g., 1:2, 1:1 or 2:1), or the bovine serum albumin and dihydroxyacetone can be mixed and then directly prepared with a PBS solution.

[0052] Concentrations of BSA and DHA in the BSA-DHA model

[0053] One embodiment of the present invention shows that the higher the concentration of BSA and DHA, the more complete the saccharification reaction. For example, after equal volumes of 20 mg / ml or more BSA and 50-100 mg / ml DHA were mixed, the saccharification reaction reached saturation on the third day. After equal volumes of 40 mg / ml BSA and 50-100 mg / ml DHA were mixed, the saccharification reaction reached saturation on the first day.

[0054] In the mixed reaction system containing BSA and DHA of the present invention, the concentration of BSA is not less than 5 mg / ml, and the concentration of DHA is not less than 10 mg / ml; more preferably, the concentration of BSA is not less than 10 mg / ml, and the concentration of DHA is not less than 20 mg / ml.

[0055] In the present invention, the upper limit of the concentration of BSA and DHA is not particularly limited. From the perspective of economy and material saving, for example, it can be 100 mg / ml or less.

[0056] The present invention also provides a kit for detecting the inhibitory activity of non-enzymatic glycosylation end products, comprising a bovine serum albumin solution with a concentration of 20-100 mg / ml and a dihydroxyacetone solution with a concentration of 40-200 mg / ml and instructions for use. The two solutions are mixed when used, preferably in a volume ratio of 1:2-2:1, and more preferably in a ratio of 1:1.

[0057] Example

[0058] Example 1: Simulating non-enzymatic glycosylation reactions in vivo

[0059] Reagent preparation: Take bovine serum albumin powder (BSA) and dissolve it with PBS. After complete dissolution, filter it with a 0.22um filter membrane to sterilize it to obtain a sterile 40mg / mL BSA solution, which is diluted with PBS to BSA solutions of different concentrations, namely 20mg / mL, 10mg / mL, 5mg / mL, and 1mg / mL; take dihydroxyacetone powder (DHA) and dissolve it with PBS. After complete dissolution, filter it with a 0.22um filter membrane to sterilize it to obtain a sterile 100mg / mL solution, which is diluted with PBS to solutions of different concentrations, namely 50mg / mL, 10mg / mL, 1mg / mL, and 0.1mg / mL.

[0060] Operation: Add different concentrations of BSA solution and DHA solution in a volume ratio of 1:1, shake and mix, then add to a 96-well plate, 100ul per well, seal the plate with a sealing film, and incubate in a 37°C incubator away from light. Take out the plate for fluorescence detection on days 0, 1, 2, and 3. The fluorescence spectrum of the microplate reader is set to an excitation wavelength of 340nm and an emission wavelength of 440nm. The fluorescence intensity detection results are as follows: Figure 1 and Figure 2 shown. Figure 1 It is the relative fluorescence value (Relative Fluorescence Units; RFU) of different concentrations of BSA solutions and different concentrations of DHA solutions on the third day of incubation reaction. Figure 2 It is the fluorescence value of 40 mg / ml BSA and different concentrations of DHA system after incubation for 1-3 days.

[0061] from Figure 1 It can be seen that the BSA-DHA model of the present invention has reached the plateau phase of the reaction on the third day, indicating that the saccharification reaction of 20-40 mg / ml BSA and 50-100 mg / ml DHA has reached a saturated state on the third day. Figure 2 There was no significant change in the fluorescence values ​​of 40 mg / ml BSA and 50-100 mg / ml DHA on days 1-3, indicating that the reaction at this concentration had reached saturation on day 1. Dihydroxyacetone, as a reducing polyhydroxy ketose, reacts more rapidly than the anti-sugar model of reducing sugars such as glucose or fructose combined with BSA used in traditional models.

[0062] Example 2: Detection of the inhibitory effect of aminoguanidine on AGEs

[0063] As a glycation blocker, aminoguanidine is often used as a positive reference for anti-glycemic models. In this example, aminoguanidine was used as a test substance to examine the inhibitory effect of aminoguanidine on AGEs using the traditional BSA-MGO model and the BSA-DHA model of the present invention.

[0064] Reagent preparation: Dissolve bovine serum albumin powder (BSA) with PBS, filter and sterilize with a 0.22um filter membrane after complete dissolution to obtain a sterile 40mg / mL BSA solution; Dissolve dihydroxyacetone powder (DHA) with PBS, filter and sterilize with a 0.22um filter membrane after complete dissolution to obtain a sterile 50mg / mL DHA solution. Dilute methylglyoxal (MGO) solution with PBS to obtain a 10mg / ml MGO solution. Dissolve aminoguanidine hydrochloride (AG) powder with PBS to a 20mg / ml solution.

[0065] Procedure: Add 50mg / mL DHA and 40mg / mL BSA in a ratio of 1:1 (V / V), shake and mix, then add to a 96-well plate, 100ul per well, add the test substance aminoguanidine hydrochloride (AG), the final concentration is 200ug / ml. After shaking and mixing, seal the plate with a sealing film and incubate in a 37℃ constant temperature box away from light. Perform fluorescence detection at 0h and 24h.

[0066] In the same way, 10 mg / mL MGO and 40 mg / mL BSA were added to a 96-well plate at a ratio of 1:1 (V / V), 100 ul per well. The test substance aminoguanidine hydrochloride (AG) was added to a final concentration of 200 ug / ml, and the plate was sealed with a sealing film after oscillation and then placed in a constant temperature box incubated in the dark for 1-2 days.

[0067] After incubation, fluorescence detection was performed, and the fluorescence spectrum of the microplate reader was set to an excitation wavelength of 340 nm and an emission wavelength of 440 nm. Six parallels were set for each concentration gradient of each sample, and the average fluorescence value of each group was calculated using statistics to analyze the inhibition rate of AGEs generation in each group. The results are shown in Figure 3 And as shown in Table 1.

[0068] The calculation formula of AGEs inhibition rate is as follows:

[0069] AGEs inhibition rate (%) = [1-(RFU 待测物 -RFU 空白对照 ) / (RFU 阴性对照 -RFU 空白对照 )]×100

[0070] Among them, RFU 空白对照 is the fluorescence value of BSA and DHA / MGO detected at 0h, RFU 阴性对照 RFU is the fluorescence value detected after the complete reaction of BSA and DHA / MGO. 待测物 It is the fluorescence value detected after the analyte is added to BSA and DH / MGOA and the reaction is complete. In this embodiment, the analyte is aminoguanidine.

[0071] Table 1: Inhibition rate of aminoguanidine on AGEs in two models

[0072] Inhibition rate % BSA-DHA model (Mean±RSD) BSA-MGO model (Mean±RSD) 200ug / ml AG 74.53±0.53 78.06±0.24

[0073] from Figure 3 As can be seen from Table 1, aminoguanidine has a significant inhibitory effect on the formation of AGEs in the BSA and DHA models. According to the calculation formula of the AGEs inhibition rate of the present invention, the inhibition rate of 200ug / ml aminoguanidine is 74.53%. As a verification, in the BSA-MGO model, the inhibition rate of 200ug / ml aminoguanidine was 78.06%. The aminoguanidine inhibition rates measured by the two models are basically the same, indicating that the BSA-DHA model method of the present invention is reliable and accurate, and can be used as a detection model for non-enzymatic glycation end products AGEs. Moreover, compared with the reaction conditions of the BSA-glucose model in the CN116549527A patent, which is reacted at 37°C for 21 days, and the BSA-fructose model and BSA-MGO model in the CN112220893A patent, which are incubated at 37°C for 6 days, the BSA-DHA model of the present invention can effectively detect the inhibitory activity of aminoguanidine by incubating at 37°C for 24h, which greatly shortens the reaction time and improves the detection efficiency.

[0074] Example 3: Effects of testing samples on AGEs in different models

[0075] Reagent preparation: Take bovine serum albumin powder (BSA) and dissolve it with PBS. After complete dissolution, filter it with a 0.22um filter membrane to sterilize it and obtain a sterile 40mg / mL BSA solution. Take dihydroxyacetone powder (DHA) and dissolve it with PBS. After complete dissolution, filter it with a 0.22um filter membrane to sterilize it and obtain a sterile 50mg / mL DHA solution. Take methylglyoxal (MGO) solution and dilute it with PBS to obtain a 10mg / ml MGO solution. Take aminoguanidine hydrochloride powder and dissolve it with PBS to a 20mg / ml solution. Take L-ascorbic acid powder and dissolve it with PBS to obtain a 50mg / ml solution. Take mangosteen extract α-mangostin powder and dissolve it with DMSO to obtain a 10mg / ml solution.

[0076] Procedure: Add 50mg / mL DHA and 40mg / mL BSA in a ratio of 1:1 (V / V), shake and mix, then add to a 96-well plate, 100ul per well. Add AG solution to the positive control group, and add L-ascorbic acid solution and a-Mangostin solution to the test group, respectively. After shaking and mixing, seal the plate with a sealing film and then incubate in a 37℃ incubator away from light for 24 hours.

[0077] In the same way, 10 mg / mL MGO and 40 mg / mL BSA were added at a ratio of 1:1 (V / V), and then added to a 96-well plate, 100 ul per well. AG solution was added to the positive control group, and L-ascorbic acid solution and a-Mangostin solution of mangosteen extract were added to the test group, respectively. After shaking and mixing, the plate was sealed with a sealing film and then placed in a 37°C incubator in the dark for 1-2 days.

[0078] After incubation was complete, fluorescence detection was performed, and the fluorescence spectrum of the microplate reader was set to an excitation wavelength of 340 nm and an emission wavelength of 440 nm. Six parallels were set for each sample, and the average fluorescence value of each group was calculated using statistics to analyze the AGEs generation inhibition rate of each group. The calculation results are shown in Table 2.

[0079] The calculation formula of AGEs inhibition rate is as follows:

[0080] AGEs inhibition rate (%) = [1-(RFU 待测物 -RFU 空白对照 ) / (RFU 阴性对照 -RFU 空白对照 )]×100

[0081] Among them, RFU 待测物 、RFU 空白对照 and RFU 阴性对照 Same as the definition in Example 2.

[0082] Table 2: Inhibition rate of samples on AGEs generation under different models (n=6)

[0083]

[0084] As can be seen from Table 2, the inhibition rates of the positive control AG in the two models were basically the same, and there was no significant difference from the results in Example 2, which once again verified the repeatability of the method.

[0085] According to the report of the above-mentioned non-patent document 1, α-mangostin, a mangosteen extract, has clear anti-glycation activity. The results of this example show that α-mangostin does have a significant inhibitory effect on the formation of AGEs, and the anti-glycation activity of α-mangostin in the BSA-DHA model at the same concentration is about 10% higher than that in the BSA-MGO model.

[0086] L-ascorbic acid is a typical antioxidant, and some reports claim that it also has a certain effect in anti-glycation, but there is currently no clear measured data to prove its effectiveness in in vitro anti-glycation detection. This example detects the effect of L-ascorbic acid on AGEs in two models. The results show that in the BSA-MGO model, L-ascorbic acid exhibits very low anti-glycation activity, but in the BSA-DHA model, it exhibits strong anti-glycation activity. The inhibition rate of 500 μg / ml L-ascorbic acid on AGEs reaches more than 60%, which clarifies the effective effect of L-ascorbic acid in anti-glycation.

[0087] This example fully demonstrates that the BSA-DHA model can more comprehensively detect the inhibitory effect of samples on non-enzymatic glycosylation end products AGEs compared to the BSA-MGO model, and is suitable for a variety of sample detection and screening.

[0088] It should be noted that the present invention is not limited to the above implementation modes and examples. The above implementation modes and examples are only examples, and implementation modes that have substantially the same structure as the technical idea and exert the same effect within the scope of the technical solution of the present application are all included in the technical scope of the present application. In addition, within the scope of the subject matter of the present application, various modifications and substitutions of the implementation modes / examples that can be thought of by those skilled in the art, and other methods formed by combining some of the constituent elements in the implementation modes are also included in the scope of the present application, and the scope of protection of the present invention is defined by the attached claims.

Claims

1. A method for detecting the inhibitory activity of non-enzymatic glycosylation end products, comprising the following steps: A mixed solution containing bovine serum albumin and dihydroxyacetone was prepared as a control system; Adding the object to be tested into the mixed solution containing bovine serum albumin and dihydroxyacetone as the system to be tested; The control system and the test system are incubated at 25°C to 55°C; Respectively detect the fluorescence values ​​of the control system and the system to be tested; Based on the change in fluorescence value before and after adding the test substance, it is evaluated whether the test substance has an inhibitory effect on the formation of non-enzymatic glycosylation end products.

2. The detection method according to claim 1, wherein the solvent of the mixed solution containing bovine serum albumin and dihydroxyacetone is a phosphate buffer solution.

3. The detection method according to claim 1 or 2, wherein the temperature range of the incubation reaction is 37°C ± 5°C.

4. The detection method according to claim 1 or 2, further comprising calculating the inhibition rate of the analyte on non-enzymatic glycosylation end products by the following formula: Inhibition rate (%) = [1-(RFU 待测物 -RFU 空白对照 ) / (RFU 阴性对照 -RFU 空白对照 )]×100 in, RFU 空白对照 RFU is the fluorescence value detected by BSA and dihydroxyacetone at 0 hours of reaction. 阴性对照 RFU is the fluorescence value after bovine serum albumin and dihydroxyacetone react for a period of time. 待测物 It is the fluorescence value after adding the analyte to bovine serum albumin and dihydroxyacetone and reacting for a period of time.

5. The detection method according to claim 4, wherein the reaction period is more than 24 hours.

6. The detection method according to claim 1 or 2, wherein in the mixed solution containing bovine serum albumin and dihydroxyacetone, the concentration of the bovine serum albumin is above 5 mg / ml, and the concentration of the dihydroxyacetone is above 10 mg / ml.

7. The detection method according to claim 1 or 2, wherein in the mixed solution containing bovine serum albumin and dihydroxyacetone, the concentration of the bovine serum albumin is 10-100 mg / ml, and the concentration of the dihydroxyacetone is 20-200 mg / ml.

8. The detection method according to claim 1 or 2, wherein the incubation reaction conditions further comprise: 96-well plate, 100-200ul per well, and / or Carry out under light-proof conditions.

9. A kit for detecting the inhibitory activity of non-enzymatic glycosylation end products, comprising a bovine serum albumin solution with a concentration of 10 mg / ml or more and a dihydroxyacetone solution with a concentration of 20 mg / ml or more and instructions for use, the two solutions are mixed when used.

10. The kit according to claim 9, wherein the concentration of the bovine serum albumin solution is 20-100 mg / ml, the concentration of the dihydroxyacetone solution is 40-200 mg / ml, and they are mixed in a volume ratio of 1:2-2:1 during use.

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