Screening method and application of alpha-amylase inhibitor

Abstract

The invention discloses a screening method and application of an alpha-amylase inhibitor. Belongs to the technical field of glycoside hydrolase inhibitors. According to the invention, the principle that the starch turns blue when meeting iodine is applied to the screening method, and the operation process and the operation steps are simplified to the greatest extent, so that the detection result is more visual, the detection time is greatly shortened, and the labor intensity is reduced. The method can be widely used for screening the alpha-amylase inhibition effect of effective extracts of natural substances and microorganisms, results of colored extracts can be visually seen, and the method is wide in application range, non-toxic and harmless.

Description

Technical Field

[0001] The present invention relates to the technical field of glycoside hydrolase inhibitors, and more specifically to a screening method for alpha-amylase inhibitors and application thereof. Background Art

[0002] Diabetes is a metabolic disorder of the endocrine system. Diabetics cannot secrete or utilize insulin normally, which causes elevated blood sugar levels, which manifests as symptoms such as hyperglycemia, dyslipidemia, visual impairment, and kidney disease. α-Amylase is involved in catalyzing the α-(1,4)-D-glycosidic bonds present in starch, hydrolyzing them into smaller fragments of glucose and other polymers, thereby increasing blood sugar levels in the body, leading to high blood sugar levels, which can further develop into diabetes. By inhibiting the action of α-amylase, it helps reduce problems such as hyperglycemia, obesity, and overweight. Therefore, α-amylase inhibitors are of great significance in the treatment of type 2 diabetes.

[0003] The prior art mostly uses multiple in vivo and in vitro experiments to screen α-amylase inhibitors, but the screening process has high requirements for the purity and dosage of the test compound, and there are defects such as difficulty in screening and high cost. For example, the DNS (3,5-dinitrosalicylic acid) method, according to which α-amylase can catalyze the hydrolysis of starch to generate reducing sugars, further reacts with 3,5-dinitrosalicylic acid to generate a brown-red substance, and the product has a characteristic absorption peak at 540nm, and the activity of α-amylase can be characterized by changes in absorbance. However, the configuration process of DNS is complicated, and 5 substances are needed, among which phenol is corrosive and toxic, and needs to be used with caution. After the preparation is completed, it needs to be placed in the dark for a week before it can be used. In addition, it is necessary to strictly control the reaction conditions, such as temperature, pH, etc., and the sample may contain other reducing substances, which will interfere with the DNS reaction and affect the accuracy of the measurement results.

[0004] For another example, the patent with application number 201510014534.4 and invention name is a method for screening α-amylase inhibitors. In this patent, the sample is applied to a thin layer plate coated with a stationary phase by spotting, and then immersed in a substrate solution and an α-amylase solution in turn. After the reaction, it needs to be placed in a ventilated place or dried with a hot air gun to remove the solvent, and then visualized with a color developer. The process is cumbersome, and there is a problem that the inhibitor spots are not obvious due to the color background, resulting in judgment bias.

[0005] Therefore, how to provide a screening method for α-amylase inhibitors that is safer and more efficient, has a simple process, is easy to operate, has no color background influence, and does not have judgment bias is a technical problem that technical personnel in this field urgently need to solve. Summary of the invention

[0006] In view of this, the present invention provides a method for screening α-amylase inhibitors and its application. The method is simple to operate, can be used for large-scale screening, does not contain toxic or harmful reagents, and can be widely used for screening effective extracts of natural substances and α-amylase inhibitory effects of microorganisms.

[0007] In order to achieve the above object, the present invention adopts the following technical solution:

[0008] A method for screening α-amylase inhibitors comprises the following steps:

[0009] (1) Using agar and starch as raw materials, adding distilled water to prepare a solid primary screening medium for later use;

[0010] (2) Place the solid primary screening culture medium in a plate, wait for it to solidify, punch a hole, add the α-amylase solution and the sample to be tested, mix well, and place at 35-40° C. for 4-12 hours;

[0011] (3) applying a color developing solution to the plate treated in step (2) to perform a color development reaction;

[0012] (4) Observe the color development in the container. If there is no white circle around the hole, it is an α-amylase inhibitor.

[0013] Preferably, the amount of agar added to the solid primary screening culture medium in step (1) is 1.5-2 g / 100 ml water; the amount of starch added is 1-2 g / 100 ml water.

[0014] Preferably, the concentration of the α-amylase solution in step (2) is ≥10 U / ml; the concentration of the sample to be tested is ≥6 mg / ml; and the volume ratio of the α-amylase solution to the sample solution to be tested is 1:1.

[0015] Preferably, the color developing solution in step (3) is an iodine-potassium iodide aqueous solution with an iodine content of 2-20 mg / ml.

[0016] Preferably, the ratio of iodine to potassium iodide in the color developing solution is 1:2-1:5.

[0017] Preferably, the color development time in step (3) is 1-5 min; and the amount of color development solution added is 5-10 ml.

[0018] Preferably, in step (2), control wells of α-amylase solution + distilled water, distilled water, and α-amylase solution + commercially available α-amylase inhibitor may be set to calibrate the experimental results.

[0019] Another object of the present invention is to provide: application of the above screening method in screening α-amylase inhibitors.

[0020] Another object of the present invention is to provide: an α-amylase inhibitor screening kit, comprising a solid primary screening culture medium plate, an α-amylase solution and a color development solution.

[0021] Preferably, the kit may further include a test solution.

[0022] Another object of the present invention is to provide: application of the above kit in screening of α-amylase inhibitors.

[0023] It can be seen from the above technical solution that compared with the prior art, the present invention has the following beneficial effects:

[0024] The invention discloses a screening method for alpha-amylase inhibitors and application thereof, applies the principle that starch turns blue when encountering iodine to the screening method, simplifies the operation process to the greatest extent, makes the detection result more intuitive, greatly shortens the detection time, and reduces the labor intensity; in addition, the invention does not have a color development background, does not involve the problem that the inhibitor spots are not obvious due to the existence of the color development background, and causes judgment deviation, thereby improving the accuracy of judgment; at the same time, the method of the invention can be widely used for screening the alpha-amylase inhibitory effect of effective extracts of natural substances and microorganisms, and the results can be intuitively seen including colored extracts, has a wide range of applications, and is non-toxic and harmless. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0026] Figure 1 The color development results of α-amylase inhibition activity verification in Example 1 are as follows; A represents the color development result after 4 hours of culture; B represents the color development result after 12 hours of culture; wells 1 and 2 in A and B are both 200 μl of distilled water; wells 3 and 4 are both 100 μl of α-amylase solution + 100 μl of distilled water; wells 5 and 6 are both 100 μl of α-amylase + 100 μl of acarbose;

[0027] Figure 2The following is the color development result of Example 2 for verifying the inhibitory activity of α-amylase; wherein wells 1 and 2 in A are both 200 μl of distilled water, wells 3 and 4 are both 100 μl of α-amylase with a concentration of 10 U / ml + 100 μl of distilled water, and wells 5 and 6 are both 100 μl of α-amylase with a concentration of 10 U / ml + 100 μl of acarbose; wells 1 and 2 in B are both 200 μl of distilled water, wells 3 and 4 are both 100 μl of α-amylase with a concentration of 120 U / ml + 100 μl of distilled water, and wells 5 and 6 are both 100 μl of α-amylase with a concentration of 120 U / ml + 100 μl of acarbose;

[0028] Figure 3 The color development result of α-amylase inhibitory activity verification in Example 3 is as follows; wells 1 and 2 are both α-amylase solution + 100 μl distilled water, wells 3 and 4 are both 100 μl α-amylase solution + 100 μl 6 mg / ml acarbose solution, wells 5 and 6 are both 200 μl distilled water, and wells 7 and 8 are both 100 μl α-amylase solution + 100 μl 10 mg / ml acarbose solution;

[0029] Figure 4 The following are the color development results for verifying the inhibitory activity of α-amylase in Example 4; 1 and 2 are 200 μl of distilled water; 3 and 4 are 100 μl of α-amylase + 100 μl of distilled water; 5 and 6 are 100 μl of α-amylase + 100 μl of acarbose; 7 and 8 are 100 μl of α-amylase + 100 μl of Tremella polysaccharide. DETAILED DESCRIPTION

[0030] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] Example 1

[0032] 1. Solution preparation:

[0033] Preparation of solid primary screening culture medium: Dissolve 2g soluble starch and 1.5g agar in 100ml distilled water, heat to boiling, pour into a culture dish, wait for cooling and solidification, and store in a refrigerator at 2-8℃ for later use.

[0034] Preparation of α-amylase solution: According to the enzyme activity, take 66.67 mg of α-amylase powder and mix it with 10 ml of distilled water to prepare it to an activity concentration of 60 U / ml of α-amylase. When using, add distilled water to prepare other concentrations according to the concentration requirements.

[0035] Preparation of color developing solution: Dissolve iodine and potassium iodide in distilled water at a ratio of 1:3 to prepare a color developing solution with an iodine content of 3 mg / ml. Prepare it as needed and store it in a refrigerator at 2-8°C for later use.

[0036] Preparation of test solution: Dissolve 10 mg of acarbose in 1 ml of distilled water to prepare a 10 mg / ml acarbose solution.

[0037] Dissolve 6 mg of acarbose in 1 ml of distilled water to prepare a 6 mg / ml acarbose solution.

[0038] 2. Verification of α-amylase inhibitory activity

[0039] Take 100μl of α-amylase solution + 100μl of distilled water and inject it into the wells punched in advance on the solid primary screening culture medium. Inject 200μl of distilled water and 100μl of α-amylase + 100μl of acarbose solution into the remaining wells respectively. Repeat once and place in a 37℃ incubator for reaction for 4h and 12h. After the reaction is completed, take out the plate, apply the color developing solution on it, and observe it under visible light. The observation results are as follows: Figure 1 shown.

[0040] Result analysis: Figure 1 It can be seen that white circles appeared in the wells of 100μl α-amylase solution + 100μl distilled water after reaction for 4h and 12h, while no white circles appeared in the wells of 200μl distilled water and 100μl α-amylase + 100μl acarbose solution, indicating that acarbose is an α-amylase inhibitor. The method of the present invention can be used to detect and identify α-amylase inhibitors, and the detection process is simple and easy to operate, and the detection results are intuitive and visible to the naked eye. On the other hand, it shows that the culture time used can be 4h-12h.

[0041] Example 2

[0042] 1. Solution configuration

[0043] Preparation of solid primary screening culture medium: Dissolve 1g soluble starch and 2g agar in 100ml distilled water, heat to boiling, pour into a culture dish, wait for cooling and solidification, and store in a refrigerator at 2-8℃ for later use.

[0044] Preparation of α-amylase solution: According to the enzyme activity, take 66.67 mg of α-amylase powder and mix it with 10 ml of distilled water, and prepare it to an activity concentration of 60 U / ml of α-amylase. When using, adjust it to 10 U / mL and 120 U / mL according to the concentration requirements.

[0045] Preparation of color developing solution: Dissolve iodine and potassium iodide in distilled water at a ratio of 1:3 to prepare a color developing solution with an iodine content of 3 mg / ml. Prepare it as needed and store it in a refrigerator at 2-8°C for later use.

[0046] Preparation of test solution: Dissolve 10 mg of acarbose in 1 ml of distilled water to prepare a 10 mg / ml acarbose solution.

[0047] 2. Verification of α-amylase inhibitory activity

[0048] Take 100 μl of the above-mentioned α-amylase solution of different concentrations + 100 μl of distilled water and inject it into the wells extracted from the solid primary screening culture medium. Inject 200 μl of distilled water and 100 μl of α-amylase of different concentrations + 100 μl of acarbose solution into the remaining wells respectively. Repeat once and place in a 35°C incubator for reaction for 12 hours. After the reaction is completed, take out the plate, apply the color developing solution on it, and observe it under visible light. The observation results are as follows: Figure 2 shown.

[0049] Analysis of results: White circles visible to the naked eye appeared in the wells containing 100μl+100μl distilled water of α-amylase solution of different concentrations, while no white circles appeared in the wells containing 200μl distilled water and 100μl α-amylase+100μl acarbose solution, indicating that acarbose is an α-amylase inhibitor. The method of the present invention can be used to detect and identify α-amylase inhibitors, and the detection process is simple and easy to operate, and the detection results are intuitive and visible to the naked eye. On the other hand, it indicates that the concentration of α-amylase contained in the α-amylase solution used only needs to be ≥10U / ml.

[0050] Example 3

[0051] 1. Solution configuration

[0052] The acarbose concentrations used were 6 mg / mL and 10 mg / mL, respectively, and the rest of the solutions were the same as described in Example 1.

[0053] 2. Verification of α-amylase inhibitory activity

[0054] Take 100 μl of the above α-amylase solution + 100 μl of distilled water and inject it into the hole punched by the solid primary screening culture medium extraction, and inject 200 μl of distilled water, 100 μl of α-amylase + 100 μl of acarbose solution of different concentrations into the remaining holes respectively. Repeat once and place in a 40°C incubator to react for 12 hours. After the reaction is completed, take out the plate, apply the color developing solution on it, and observe it under visible light. The observation results are as follows: Figure 3 shown.

[0055] Result analysis: There were no white circles visible to the naked eye in the wells containing different acarbose solutions, indicating that the acarbose used was an α-amylase inhibitor and the acarbose concentration could be 6-10 mg / ml.

[0056] Example 4

[0057] 1. Solution configuration

[0058] Weigh 1 g of Tremella fuciformis, add them into 30 mL of distilled water, treat with ultrasound for 30 minutes, centrifuge, take the supernatant, and freeze-dry to obtain Tremella fuciformis polysaccharide. Dissolve 25 mg of Tremella fuciformis polysaccharide in 1 ml of distilled water to prepare a 25 mg / ml Tremella fuciformis polysaccharide solution. The remaining solutions are α-amylase solution and acarbose solution. The preparation process is the same as that described in Example 1.

[0059] 2. Verification of α-amylase inhibitory activity

[0060] Take 100μl of the above Tremella polysaccharide solution + 100μl of α-amylase solution and inject it into the holes punched in advance in the solid culture medium, and inject 200μl of distilled water, 100μl of α-amylase + 100μl of distilled water, and 100μl of α-amylase + 100μl of acarbose solution into the remaining holes respectively. Repeat once and place in a 37°C incubator for reaction for 12h. After the reaction is completed, take out the plate, apply the color developing solution on it, and observe it under visible light. The observation results are as follows: Figure 4 shown.

[0061] Result analysis: There were no white circles visible to the naked eye in the wells containing the Tremella polysaccharide solution and the acarbose solution, indicating that the Tremella polysaccharide and acarbose used were α-amylase inhibitors. The method of the present invention can be used for the detection and identification of various types of α-amylase inhibitors.

[0062] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0063] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for screening α-amylase inhibitors, characterized in that: The steps include: (1) Using agar and starch as raw materials, adding distilled water to prepare a solid primary screening medium for later use; (2) Place the solid primary screening culture medium in a plate, wait for it to solidify, punch a hole, add the α-amylase solution and the sample to be tested, mix well, and place at 35-40° C. for 4-12 hours; (3) applying a color developing solution to the plate treated in step (2) to perform a color developing reaction; (4) Observe the color development in the container. If there is no white circle around the hole, it is an α-amylase inhibitor.

2. The method for screening α-amylase inhibitors according to claim 1, characterized in that: In the solid primary screening medium of step (1), the amount of agar added in the solid primary screening medium is 1.5-2 g / 100 ml water; the amount of starch added in the solid primary screening medium is 1-2 g / 100 ml water.

3. The method for screening α-amylase inhibitors according to claim 1, characterized in that: Step (2) The concentration of the α-amylase solution is ≥10 U / ml; the concentration of the sample to be tested is ≥6 mg / ml; the volume ratio of the α-amylase solution to the sample solution to be tested is 1:

1.

4. The method for screening α-amylase inhibitors according to claim 1, characterized in that: The color developing solution in step (3) is an iodine-potassium iodide aqueous solution with an iodine content of 2-20 mg / ml.

5. The method for screening α-amylase inhibitors according to claim 4, characterized in that: The ratio of iodine to potassium iodide in the color developing solution is 1:2-1:

5.

6. The method for screening α-amylase inhibitors according to claim 1, characterized in that: The color development time of step (3) is 1-5 minutes; the amount of color development solution added is 5-10 ml.

7. The method for screening α-amylase inhibitors according to claim 1, characterized in that: In step (2), control wells of α-amylase solution + distilled water, distilled water, and α-amylase solution + commercially available α-amylase inhibitor may also be set to calibrate the experimental results.

8. Use of the screening method according to any one of claims 1 to 7 in screening α-amylase inhibitors.

9. An α-amylase inhibitor screening kit, characterized in that: Includes solid primary screening culture medium plate, α-amylase solution and color development solution.

10. Use of the kit according to claim 9 in screening of α-amylase inhibitors.

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