Assessment method and system of alpha-amylase inhibitor based on interaction affinity coefficient
By establishing an interaction affinity coefficient model between the inhibitor and the enzyme, and utilizing micro-thermophoresis technology, the problems of high cost, long time consumption, and instability in the existing inhibitor evaluation methods have been solved, achieving rapid and accurate evaluation of α-amylase inhibitors.
Patent Information
- Application Number
- CN202511885287.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-15
- Publication Date
- 2026-02-27
AI Technical Summary
Existing technologies require the consumption of large amounts of inhibitors when evaluating α-amylase inhibitors, which is costly and time-consuming. Furthermore, substrate differences lead to unstable inhibitory effects, making it difficult to achieve efficient micro-analysis.
By establishing an interaction affinity coefficient model between the inhibitor and the enzyme, micro-thermophoresis (MST) technology is used to analyze the direct binding of the inhibitor and the enzyme, assess its inhibitory ability, avoid the dependence on substrate digestion, and achieve efficient evaluation with only nanogram-level inhibitors.
This method enables highly efficient micro-evaluation of α-amylase inhibitors, avoids the instability of substrate digestion methods, rapidly and accurately determines inhibitory capacity, and reduces analytical costs and time.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the technical field of biological analysis and detection, and relates to a method and system for evaluating alpha-amylase inhibitors based on interaction affinity coefficients, in particular, to a new method and system for efficiently evaluating alpha-amylase inhibitors based on interaction affinity coefficients. BACKGROUND
[0002] Long-term postprandial hyperglycemia is one of the important inducements of glucose metabolism disorder. The intake of staple food is directly related to the fluctuation of postprandial blood glucose level. As the main component of staple food, starch is hydrolyzed into reducing sugars such as maltose, maltotriose and malt oligosaccharides under the action of saliva and pancreatic alpha-amylase, and these intermediates are then further decomposed into glucose by the glucose glycosidase of the small intestinal brush border. Therefore, alpha-amylase is a key enzyme in the process of starch digestion, and inhibiting the activity of this enzyme is considered to be an effective means to regulate postprandial blood glucose level.
[0003] Based on the above mechanism, several drugs targeting carbohydrate hydrolase inhibitors (such as acarbose and voglibose) have been developed and used to alleviate the symptoms of type II diabetes. However, in the long term, the use of these drugs may cause side effects such as diarrhea and abdominal distension. Therefore, developing and exploring starch hydrolysis enzyme inhibitors derived from nature or diet is a promising strategy to control starch digestion.
[0004] In the evaluation and screening of alpha-amylase activity inhibitors, the substrate digestion method is usually used, that is, the enzyme inhibition rate is calculated according to the initial substrate digestion rate under the condition of with or without inhibitors. This method requires the consumption of a large amount of inhibitors, usually milligrams or more, resulting in high analysis cost and long time consumption. More importantly, this method may introduce factors of determination instability, because the difference in substrate enzymolysis mode (i.e. the composition and concentration of alpha-1, 4-glycosidic bonds in the substrate) will cause significant fluctuations in enzyme inhibition effect, for example, the same inhibitor will exhibit different inhibition abilities for alpha-amylase in the digestion system of starch and artificial substrate Gal-G2-CNP. Therefore, it is urgent to develop a high-efficiency micro-analysis method independent of specific substrate digestion for evaluating the inhibition ability of inhibitors on alpha-amylase. SUMMARY
[0005] In order to overcome the defects existing in the prior art, the present application provides a method and system for evaluating alpha-amylase inhibitors based on interaction affinity coefficients. The method is designed according to the principle that the direct binding of inhibitors and enzymes causes enzyme inhibition, and establishes a relationship model between the inhibition ability of inhibitors on enzymes and the interaction affinity coefficient of the two, that is, IC50 -K d between the relationship model, based on this, for the target inhibitor, by analyzing its K d value, according to the established model, judge its inhibition ability. Through this method, based on a few representative inhibitors of enzyme inhibition and binding rule, efficient judgment of other similar inhibitors of inhibition ability. Especially through the MST analysis of the K d value of the inhibitor, only a small amount of inhibitor is consumed, usually nanogram level or less, more importantly, avoid the analysis of the target inhibitor on the substrate digestion inhibition, realize the efficient micro evaluation of enzyme inhibition ability. The following technical solutions are implemented:
[0006] An evaluation method of alpha-amylase inhibitor based on interaction affinity coefficient, comprising the following steps: 1) preparation of gradient concentration inhibitor solution; 2) dissolution and dilution of alpha-amylase; 3) fluorescence labeling of alpha-amylase; 4) incubation of inhibitor and enzyme; 5) micro-thermal swing (MST) analysis of affinity coefficient of direct binding of inhibitor and enzyme; 6) determination of inhibitor inhibition ability; 7) analysis of the relationship between affinity coefficient and inhibition ability; 8) efficient micro evaluation of inhibitor inhibition ability.
[0007] Further, in step 1), the inhibitor stock solution with a concentration of 0.05-0.1 M is prepared using buffer, and HEPES is preferably used to ensure the solubility of the inhibitor, and then the stock solution is diluted with the same buffer to 0.05-100 mM of 12 concentration gradient inhibitors.
[0008] Further, in step 2), the alpha-amylase is dissolved and diluted using buffer, preferably, first dissolved the enzyme using PBS buffer to provide sufficient calcium ion environment for the enzyme to ensure the stability of the enzyme structure, and then dilute the enzyme using HEPES buffer to avoid the generation of flocculation between calcium ions and dye, and the concentration of the diluted enzyme solution is 1-2 μM, preferably 2 μM.
[0009] Further, in step 3), the fluorescence labeling step of alpha-amylase is:
[0010] 3-1) The fluorescence labeling dye is Tris-NTA-RED647 specific to histidine (His), which is prepared into a stock solution of 3-5 mM using HEPES buffer, and before labeling the enzyme, it is diluted into a dye solution with a concentration of 50-100 nM, preferably, the concentration of Tris-NTA-RED647 fluorescence dye is 100 nM.
[0011] 3-2) The α-amylase dilution solution and the dye dilution solution are added to the microcentrifuge tube at a volume ratio of 2:1, incubated at room temperature in the dark for 20-30 min, and the total volume is 24-48 μL.
[0012] 3-3) A small amount of labeled enzyme is diluted 3-4 times with HEPES buffer, and its fluorescence value is detected by MST. If the fluorescence value of the labeled enzyme before dilution can reach 100-200, and the fluorescence value after dilution is proportional to the dilution multiple, it can be judged that the enzyme is successfully labeled.
[0013] Further, in step 4), equal volumes of labeled enzyme and concentration gradient inhibitor are taken and added to the microcentrifuge tube, preferably 5 μL each, and incubated at 4°C for 15 min.
[0014] Further, in step 5), the step of microthermal swing (MST) analysis of the affinity coefficient of the direct binding of the inhibitor to the enzyme is:
[0015] 5-1) A standard MO-L018 capillary is inserted into the enzyme and inhibitor incubation solution, 4 μL of the incubation solution is sucked by capillary siphon, and is balanced to the center area of the capillary.
[0016] 5-2) The capillary is placed in the MST sample disc according to the concentration of the inhibitor, and the concentration of the inhibitor in the capillary gradually increases from top to bottom. After fixation by magnetic strip, the sample disc is placed in the MST analysis chamber.
[0017] 5-3) The interaction affinity is determined by using Nano Temper Monolith NT.115 type MST analyzer, and the instrument parameter settings are as follows: temperature 25°C; infrared laser energy 15-40%, preferably 20%; infrared laser irradiation time of each capillary 25 s; power preferably medium; fluorescence channel Nano-RED; excitation wavelength of the selected dye 650 nm, emission wavelength 670 nm.
[0018] 5-4) After the scanning of all capillaries is completed, the real-time curve of the relative fluorescence ratio and the infrared laser irradiation time (Relative Fluorescence-Times) can be obtained, which is converted into the relationship curve between the binding ratio and the inhibitor concentration (Fraction Bound-Ligand Concentration) by MO. Analysis Software, and the "K d " analysis mode is selected to obtain the interaction affinity coefficient K d value of the inhibitor and α-amylase.
[0019] The real-time curve between relative fluorescence ratio and infrared laser irradiation time is converted into the relationship curve between binding ratio and inhibitor concentration by the calculation of interaction analysis software, which is named MO Affinity Analysis, to obtain the interaction affinity coefficient K d value of the inhibitor and α-amylase.
[0020] Further, in step 6), the inhibition ability determination step of the inhibitor is:
[0021] 6-1) A substrate Gal-G2-CNP solution with a concentration of 5-20 mM is prepared by PBS buffer, preferably with a concentration of 5 mM; an α-amylase solution with a concentration of 10-15 μM is prepared by PBS buffer, preferably with a concentration of 10 μM; an inhibitor stock solution with a concentration of 30-50 mM is prepared by PBS buffer, preferably with a concentration of 30 mM, and is diluted into 5-8 concentration gradients.
[0022] 6-2) Equal volumes of the inhibitor and the α-amylase solution are mixed, preferably 30 μL each, and incubated at 4°C for 10 min, and then 40 μL of the incubation solution is added to the reaction well of a 96-well plate pre-filled with 120 μL of PBS buffer, and each inhibitor concentration is repeated 3 times.
[0023] 6-3) 40 μL of the Gal-G2-CNP solution is added to the reaction well, and the reaction is carried out at 37°C, and the absorbance value at 405 nm is determined at specific time intervals after the reaction, preferably at 0, 3, 6, and 9 min after the reaction, and the initial reaction rate (v) is calculated according to the change rate of the absorbance value with time (δA / t), the inhibition rate (I) is calculated according to the initial reaction rate, and the half maximal inhibitory concentration (IC 50 ) is fitted according to the relationship curve between the inhibition rate and the inhibitor concentration, to characterize the inhibition ability of the inhibitor to the α-amylase.
[0024] Further, in step 7), 6-8 representative inhibitors are selected, preferably small molecule polyphenol compounds are selected as representative inhibitors, and the IC 50 value and K d value of the inhibitor are obtained according to the method in steps 1-6) above, the correlation between the two parameters is established with the K d value of each inhibitor as the variable and the IC 50 value as the dependent variable, to determine the relationship between the inhibition ability of the inhibitor to the enzyme and the interaction affinity coefficient, and to establish an inhibitor evaluation model.
[0025] Further, in step 8), for the evaluation of the inhibitory ability of the target inhibitor, the interaction affinity coefficient K d of the target inhibitor with the alpha-amylase is determined by MST according to the method in steps 1-5), and the IC 50 relationship model established in step 7) is used to judge the inhibitory ability of the inhibitor, thereby achieving efficient and micro evaluation. d
[0026] Compared with the prior art, the present application has at least the following advantages:
[0027] 1) The present application provides a novel method for evaluating alpha-amylase inhibitors, i.e., evaluating the inhibitory ability of the inhibitor by the interaction affinity coefficient of the inhibitor and the enzyme. The method can analyze the correlation between the inhibitory ability of a few representative inhibitors and the interaction affinity coefficient of the two, and establish an evaluation model, thereby achieving the evaluation of the inhibitory ability of the inhibitors of the same category. After the correlation evaluation model is established, the interaction affinity coefficient K d of the target and the enzyme can be quickly determined by MST technology, thereby evaluating and judging the inhibitory ability of the target on the enzyme, without the need to measure the IC 50 value, which embodies the efficiency of the method.
[0028] 2) In the application, the inhibitory ability of the inhibitor on the enzyme is judged according to the measured interaction affinity of the inhibitor and the alpha-amylase, which avoids the fluctuation of the inhibitory activity caused by the difference of the substrate in the substrate digestion method, and embodies the accuracy of the method.
[0029] 3) The present application also establishes a micro heat swing (MST) analysis technology method for the interaction and binding affinity of the inhibitor and the alpha-amylase. The method analyzes the direct binding coefficient of the two by the thermodynamic properties of the complex formed by the binding of the inhibitor and the enzyme. Compared with the conventional fluorescence quenching method (FQ) and isothermal titration calorimetry (ITC) method, MST avoids the indirectness of the binding coefficient obtained by the FQ analysis method, and overcomes the problems of long time consumption (2 h / sample) and large consumption of the inhibitor and the enzyme (inhibitor 1-2 nmol; enzyme 840 nmol) of the ITC analysis method. The MST analysis method consumes 15 min / sample and consumes 0.0002-0.4 nmol of the inhibitor and 0.048 nmol of the enzyme, which embodies the rapidity and micro quantity of the method. BRIEF DESCRIPTION OF DRAWINGS
[0030] Figure 1 The present application is based on the interaction affinity coefficient of the alpha-amylase inhibitor evaluation method;
[0031] Figure 2 This is a real-time curve showing the relationship between fluorescence ratio and infrared laser irradiation time in MST analysis. The curve is based on EGCG, and the curves for other inhibitors are similar.
[0032] Figure 3 This is a curve showing the relationship between the converted binding ratio and the inhibitor concentration in the MST analysis;
[0033] Figure 4 The curves show the relationship between the inhibition rate and concentration of seven representative inhibitors;
[0034] Figure 5 K, as a representative inhibitor d Value and IC 50 The correlation between values and the evaluation model built upon this;
[0035] Figure 6 To illustrate the application of an inhibitor evaluation model using tannic acid and epigallocatechin as examples;
[0036] Figure 7 To verify the inhibitory effect of tannic acid and epigallocatechin. Detailed Implementation
[0037] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.
[0038] A schematic diagram of the evaluation method for α-amylase inhibitors based on interaction affinity coefficients of the present invention is shown below. Figure 1 As shown.
[0039] Example 1: Constructing a highly efficient micro-evaluation model for α-amylase inhibitors using tea polyphenols as an example.
[0040] 1) Preparation of gradient concentration inhibitor solutions: A series of representative tea polyphenols were used as inhibitors, including catechin (C), catechin gallate (CG), gallocatechin (GC), gallocatechin gallate (GCG), epicatechin (EC), epicatechin gallate (ECG), and epigallocatechin gallate (EGCG), totaling seven tea polyphenol monomers. The tea polyphenols were prepared into 0.1 M stock solutions using HEPES buffer and then diluted to 12 gradient concentrations of inhibitors ranging from 0.05 to 100 nM.
[0041] 2) Dissolving and diluting α-amylase: First, use a solution containing Ca... 2+ The α-amylase was dissolved in PBS buffer to a concentration of 4 μM, and then diluted with HEPES buffer to a concentration of 2 μM.
[0042] 3) Fluorescent labeling of α-amylase: The histidine-specific marker Tris-NTA-RED647 was used as the fluorescent labeling dye. A 5 mM stock solution was prepared with HEPES buffer and diluted to a 100 nM dye solution. The α-amylase solution and dye solution were added to a microcentrifuge tube at a volume ratio of 2:1, with a total volume of 24 μL. The tube was incubated at room temperature in the dark for 20 min to allow the dye to fully label the enzyme. A small amount of the labeled enzyme solution was taken and diluted 3 times with HEPES buffer in equal volume proportions. The fluorescence value was detected using a microthermophoresis apparatus to ensure that the fluorescence value reached 100-200 and that the fluorescence value of the diluted solution was proportional to the dilution factor.
[0043] 4) Incubation of inhibitor-enzyme interaction: Take 5 μL of labeled enzyme solution and a series of inhibitors of different concentrations and incubate at 4℃ for 15 min.
[0044] 5) MST analysis of the affinity coefficient between the inhibitor and the enzyme: 4 μL of incubation solution was drawn using a standard MO-L018 capillary tube. Capillaries were placed in the MST sample tray according to inhibitor concentration, with the inhibitor concentration increasing from top to bottom. The capillaries were fixed with magnetic strips, and the sample tray was placed in the micro-thermophoresis analysis chamber. The interaction affinity was determined using a Nano Temper Monolith NT.115 analyzer. Instrument parameters were set as follows: temperature 25℃; infrared laser energy 20%; infrared laser irradiation time for each capillary 25 s; medium power; and the Nano-RED fluorescence channel was selected based on the dye fluorescence characteristics. After scanning each capillary with MST, the real-time curve between the relative fluorescence ratio and the infrared laser irradiation time was generated using MO. Analysis Software. Figure 2 ) is converted into a curve showing the relationship between the binding ratio and the inhibitor concentration. Figure 3 ), through "K d "Analysis mode, select appropriate analytical conditions and concentration fitting points to obtain the interaction affinity coefficient K between the inhibitor and α-amylase." d value.
[0045] 6) Determination of inhibitory activity: 5 mM Gal-G2-CNP substrate solution, 10 μM α-amylase solution, and 30 mM inhibitor solution were prepared using PBS buffer. The inhibitor was then diluted to five gradient concentrations of 1-15 mM. 30 μL of each inhibitor and α-amylase solution were incubated at 4°C for 10 min. 40 μL of the incubation solution was added to the reaction wells of a 96-well plate pre-filled with 120 μL of PBS buffer. Another 40 μL of Gal-G2-CNP solution was added to the reaction wells, and the reaction was carried out at 37°C. The absorbance of the reaction solution was measured using a microplate reader at 0, 3, 6, and 9 min after the start of the reaction. The initial reaction rate was calculated based on the rate of change of absorbance over time, and the inhibition rate was calculated based on the initial reaction rate. The relationship between the inhibition rate and the inhibitor concentration was then analyzed using a curve… Figure 4 ), fitted half-maximal inhibitory concentration (IC50) 50 The value was used to characterize the inhibitory ability of the inhibitor against α-amylase.
[0046] 7) Analysis of the relationship between affinity coefficient and inhibitory ability: For the K of 7 inhibitors d Value and IC 50 Value, in K d The value is a variable, in IC 50 The value is the dependent variable. The correlation between the two parameters is established to determine the relationship between the inhibitor's inhibitory ability on the enzyme and the interaction affinity coefficient between the two, thereby establishing an inhibitor evaluation model. Figure 5 ).
[0047] 8) Inhibitory capacity assessment: In the assessment model of α-amylase inhibitors constructed using tea polyphenols as an example, IC50... 50 Value and K d The values are positively correlated and exhibit good linearity (R0). 2 >0.9)( Figure 5 ), indicating that the smaller K is d The smaller the value, the better the IC. 50 The higher the value, the stronger the inhibitory ability. Given that EGCG is widely reported as an α-amylase inhibitor, and in this example, its inhibitory ability on the enzyme is at a moderate level, according to IC50... 50 Value and K d The correlation of values, if the K of the target inhibitor d If the value is >7, its inhibitory ability on the enzyme is considered weak. If the K value of the target inhibitor is >7, then the inhibitory ability on the enzyme is considered weak. d If the value is <7, it indicates a strong inhibitory ability on the enzyme.
[0048] Example 2: Application of an inhibitor evaluation model using tannic acid and epigallocatechin as examples
[0049] 1) Preparation of gradient concentration inhibitor solutions: Tannins (TA) and epigallocatechin (EGC) were used as target inhibitors. Two polyphenols were prepared into 0.1 M stock solutions using HEPES buffer and then diluted to 2.5 × 10⁻⁶ m³. -5 Twelve gradient concentrations of inhibitors, ranging from -0.05 mM (TA) to 0.05-100 mM (EGC).
[0050] 2) Dissolving and diluting α-amylase: First, use a solution containing Ca... 2+ The α-amylase was dissolved in PBS buffer, and then dissolved in Ca2+-free PBS. 2+ The enzyme was diluted with HEPES buffer to a concentration of 2 μM.
[0051] 3) Fluorescent labeling of α-amylase: Tris-NTA-RED647 was used as the fluorescent labeling dye, and a dye solution with a concentration of 100 nM was prepared and diluted with HEPES buffer. The α-amylase solution and the dye solution were mixed at a volume ratio of 2:1, with a total volume of 24 μL, and incubated at room temperature in the dark for 20 min to allow the dye to fully label the enzyme.
[0052] 4) Incubation of inhibitor-enzyme interaction: Take 5 μL of labeled enzyme solution and a series of inhibitors of different concentrations and incubate at 4℃ for 15 min.
[0053] 5) Micro-thermophoresis analysis of the affinity coefficient between inhibitors and enzymes: 4 μL of incubation solution was drawn using a standard capillary tube. Capillaries were placed in the sample dish according to inhibitor concentration, and the sample dish was placed in the micro-thermophoresis analysis chamber. The interaction affinity was determined using a micro-thermophoresis analyzer with the following instrument parameters: temperature 25℃; infrared laser energy 20%; infrared laser irradiation time per capillary 25 s; medium power; Nano-RED fluorescence channel selected. The results were obtained by using the "K" method. d "Data analysis revealed the interaction affinity coefficients K between the two target inhibitors and α-amylase." d value( Figure 6 Left).
[0054] 6) Evaluation of the inhibitory capacity of tannic acid and epigallocatechin: The Kc of tannic acid was obtained through steps 1-5). d The value is 8.7 × 10 -4 mM, based on the evaluation model established in Example 1, this value is distributed within the range of strong inhibition ability ( Figure 6 (Right), therefore, it is determined that tannic acid is a strong inhibitor, while the K of epigallocatechin... d The value is 15.5 mM, which falls within the range of weak inhibition. Figure 6 (Right), therefore, epigallocatechin is judged to be a weakly active inhibitor.
[0055] Example 3: Verification of the inhibitory effect of tannins and epigallocatechin
[0056] 1) The procedure for determining the inhibitory capacity of tannins and epigallocatechin is as follows:
[0057] 1-1) Prepare 5 mM Gal-G2-CNP substrate solution, 10 μM α-amylase solution, 2 mM tannic acid solution and 30 mM epigallocatechin solution with PBS buffer, and dilute tannic acid and epigallocatechin to 0.0625-1 mM and 1-15 mM respectively, to form 5 gradient concentrations of inhibitors.
[0058] 1-2) Take 30 μL of the inhibitor and α-amylase solution respectively, incubate at 4℃ for 10 min, and then add 40 μL of the incubation solution to the reaction wells of a 96-well plate that has been pre-filled with 120 μL of PBS buffer.
[0059] 1-3) Add 40 μL of Gal-G2-CNP solution to the reaction wells and react at 37℃. Measure the absorbance of the reaction solution at 0, 3, 6, and 9 min after the start of the reaction using a microplate reader. Calculate the initial reaction rate based on the rate of change of absorbance over time, and calculate the inhibition rate based on the initial reaction rate. Analyze the relationship between the inhibition rate and the inhibitor concentration using the curve (…). Figure 7 (Left), fitting the IC50 of tannic acid 50 The value was 0.053 mM, and the IC50 of epigallocatechin was... 50 The value is 40.5 mM ( Figure 7 right).
[0060] 2) Comparative analysis with the results of the evaluation model: The IC50 of tannic acid above 50 The value was much lower than that of the widely reported enzyme inhibitor EGCG (6.23 mM). Figure 7 (Right), demonstrating that tannic acid is a potent inhibitor, consistent with results obtained through the evaluation model; additionally, the above table shows the IC50 of gallocatechin. 50 The value is much higher than EGCG ( Figure 7 (Right) This demonstrates that epigallocatechin is a weakly active inhibitor, which is consistent with the results obtained through the evaluation model.
[0061] Conclusion: Based on the above application results, the method system of the present invention can be effectively used for the accurate evaluation of α-amylase inhibitors and can be applied to the discovery and screening of small molecule inhibitors such as polyphenols. It is a highly efficient method for evaluating trace amounts of enzyme inhibitors.
Claims
1. A method for evaluating α-amylase inhibitors based on interaction affinity coefficients, characterized in that, Includes the following steps: 1) Preparation of inhibitor solutions with gradient concentrations; 2) Dissolution and dilution of α-amylase; 3) Fluorescent labeling of α-amylase; 4) Incubation of inhibitor-enzyme interaction; 5) Micro-thermophoretic analysis of the affinity coefficient between the inhibitor and the enzyme; 6) Determination of the inhibitory capacity; 7) Analysis of the relationship between affinity coefficient and inhibition ability; 8) Highly efficient micro-level assessment of inhibitory capacity.
2. The evaluation method for α-amylase inhibitors based on interaction affinity coefficients according to claim 1, characterized in that, In step 1), when preparing the inhibitor solution, 4-hydroxyethylpiperazine ethanesulfonic acid was selected as the buffer solution, and the concentration of the prepared inhibitor stock solution was 0.05-0.1 M, which was then diluted into 12 concentration gradients.
3. The evaluation method for α-amylase inhibitors based on interaction affinity coefficients according to claim 1, characterized in that, In step 2), α-amylase is dissolved and diluted sequentially with phosphate and 4-hydroxyethylpiperazine ethanesulfonic acid buffer to prepare an enzyme solution with a concentration of 1-2 μM.
4. The evaluation method for α-amylase inhibitors based on interaction affinity coefficients according to claim 1, characterized in that, In step 3), the fluorescent labeling of α-amylase is performed as follows: 3-1) Use the histidine-specific labeling dye Tris-NTA-RED647, prepare a 3-5 mM stock solution with 4-hydroxyethylpiperazine ethanesulfonic acid buffer, and dilute it to 50-100 nM dye solution before fluorescent labeling the enzyme. 3-2) Add the α-amylase dilution and dye dilution to a microcentrifuge tube at a volume ratio of 2:1 and incubate at room temperature in the dark for 20-30 min. 3-3) Take 2-4 μL of labeled enzyme, dilute it and perform fluorescence detection. If the fluorescence value reaches 100-200 and is stable and positively correlated with the dilution factor, then the labeling is considered successful.
5. The method for evaluating α-amylase inhibitors based on interaction affinity coefficients according to claim 1, characterized in that, In step 4), the labeled enzyme and the gradient concentration of inhibitors are added to a microcentrifuge tube at a volume ratio of 1:1 and incubated at 4°C for 15 min.
6. The evaluation method for α-amylase inhibitors based on interaction affinity coefficients according to claim 1, characterized in that, In step 5), the step of analyzing the affinity coefficient between the inhibitor and the enzyme directly using micro-thermophoresis is as follows: 5-1) Use ~4 μL of incubation solution of standard capillary siphon labeled enzyme and inhibitor, and place it in the central region of the capillary; 5-2) Place the capillary tubes into the sample tray of the micro-thermophoresis apparatus in sequence according to the inhibitor concentration, fix them with magnetic strips, and then place them in the analysis chamber of the micro-thermophoresis apparatus. 5-3) The analytical parameters of the micro-thermophoresis apparatus are set as follows: temperature 25℃; infrared laser energy 15-40%; infrared laser irradiation for each capillary for 25 s; power selected as medium; fluorescence channel Nano-RED; dye excitation wavelength 650 nm, emission wavelength 670 nm; 5-4) The real-time curve between the relative fluorescence ratio and the infrared laser irradiation time was converted into a curve showing the relationship between the binding ratio and the inhibitor concentration, thus obtaining the interaction affinity coefficient K between the inhibitor and α-amylase. d value.
7. The method for evaluating α-amylase inhibitors based on interaction affinity coefficients according to claim 1, characterized in that, In step 6), the steps for determining the inhibitory capacity are as follows: 6-1) Prepare a 5-20 mM solution of substrate galactosylmaltose glycoside and a 10-15 μM solution of α-amylase using phosphate buffer; prepare a 30-50 mM stock solution of inhibitor using the same buffer, and then dilute it into 5-8 concentration gradients. 6-2) Mix an equal volume of inhibitor with α-amylase solution and incubate at 4°C for 10 min. Then take 40 μL of the mixture and add it to the reaction wells of a 96-well plate that has been pre-filled with 120 μL of PBS buffer. 6-3) Add 40 μL of galactopyranosylmaltose glycoside solution to the reaction wells and react at 37°C. Measure the absorbance at 405 nm at 0, 3, 6, and 9 min after the reaction. Calculate the initial reaction rate based on the change in absorbance over time, calculate the inhibition rate based on the initial reaction rate, and calculate the inhibitory capacity based on the relationship between the inhibition rate and the inhibitor concentration. Calculate the inhibitory capacity using the half-maximal inhibitory concentration (IC50). 50 Value representation.
8. The method for evaluating α-amylase inhibitors based on interaction affinity coefficients according to claim 1, characterized in that, In step 7), the IC50 of 6-8 representative inhibitors is obtained. 50 Value and K d After determining the values, a numerical statistical trend between the two parameters is established to judge the relationship between the inhibitor's inhibitory ability on the enzyme and the interaction affinity coefficient between the two, thereby establishing an inhibitor evaluation model.
9. An evaluation system for α-amylase inhibitors based on interaction affinity coefficients, characterized in that, include: The method includes: one or more processors; a memory for storing one or more programs; said processors are configured to execute program instructions stored in said memory, which, when executed, perform the evaluation method for α-amylase inhibitors based on interaction affinity coefficients as described in any one of claims 1 to 8.
10. A computer-readable storage medium, characterized in that, The computer-readable storage medium stores a computer program, wherein the computer program, when executed by one or more processors, implements the evaluation method for α-amylase inhibitors based on interaction affinity coefficients as described in any one of claims 1 to 8.