Application of guar gum, fluorescence-enhanced gold nanoclusters and methods for detecting α-glucosidase and screening enzyme inhibitors

Abstract

The present invention provides the application of guar gum, fluorescence-enhanced gold nanoclusters and methods for detecting α-glucosidase and screening enzyme inhibitors, and belongs to the field of nanobiosensor technology. The present invention provides the application of guar gum in improving the fluorescence intensity of gold nanoclusters. In the present invention, guar gum is a natural high molecular polymer extracted from the seeds of guar bean, a leguminous plant, and has the advantages of low price, non-toxicity and good biocompatibility; guar gum is mainly composed of galactose and mannose, and its molecular skeleton is rich in a large number of hydroxyl groups, which can improve the fluorescence emission intensity of gold nanoclusters. On this basis, α-glucosidase activity detection and α-glucosidase inhibitor screening can be realized with high sensitivity and low cost, and it is environmentally friendly and simple to operate, avoiding the modification of fluorescent probes and the use of a large number of organic reagents, and has broad application prospects in the prevention and treatment of diabetes.

Description

Technical Field

[0001] The invention relates to the technical field of nano-biosensing, and in particular to the application of guar gum, fluorescence-enhanced gold nanoclusters and alpha-glucosidase detection and enzyme inhibitor screening methods. Background Art

[0002] Diabetes is a metabolic disease characterized by high blood sugar, which can cause persistent damage to the eyes, kidneys, heart and blood vessels. Diabetes can be divided into type I diabetes, type II diabetes and gestational diabetes. Type II diabetes is related to insufficient insulin secretion or resistance, accounting for more than 90% of current diabetic patients, affecting about 463 million people worldwide. α-Glucosidase (α-Glu) is a carbohydrate hydrolase that promotes the conversion of oligosaccharides and disaccharides in the small intestine into glucose. Excessive α-Glu can lead to hyperglycemia. In recent years, studies have found that α-Glu inhibitors can effectively reduce α-Glu activity, thereby effectively inhibiting the increase in blood sugar levels. At present, α-Glu has become an important enzyme target for the prevention and treatment of type II diabetes, and α-Glu inhibitors such as acarbose have become a few oral drugs for the treatment of type II diabetes. Therefore, α-Glu activity detection and α-Glu inhibitor screening, especially the screening of cheap α-Glu inhibitors with few side effects from natural products, are of great significance for the prevention and treatment of type II diabetes.

[0003] The classic method for α-Glu activity detection and α-Glu inhibitor screening is the p-nitrophenol pyranoglucoside colorimetric method, which has the disadvantages of low sensitivity and large interference. New methods for α-Glu activity determination include high performance liquid chromatography, surface plasmon resonance, electrochemical method, and fluorescence method. Among them, the fluorescence method based on nanoprobes (such as semiconductor quantum dots, carbon quantum dots or copper nanoclusters) has attracted much attention due to its high sensitivity and rapid detection characteristics. However, such methods often have problems such as complex preparation of fluorescent probes, narrow measurement range, poor stability, easy oxidation, and susceptibility to interference. Therefore, the development of more efficient α-Glu fluorescence detection and α-Glu inhibitor screening methods remains a huge challenge.

[0004] Gold nanoclusters are a new type of fluorescent nanomaterial, which is usually less than 3nm in size and consists of several to hundreds of gold atoms (or gold ions) and protective ligands. Gold nanoclusters have the characteristics of simple preparation, good biocompatibility, good stability, and good water solubility. They have broad application prospects in the fields of fluorescence sensing, bioimaging, drug delivery, and disease treatment. However, compared with classical fluorescent dyes and semiconductor quantum dots, the fluorescence quantum yield and fluorescence intensity of gold nanoclusters are generally low, which limits the application range of gold nanoclusters. At present, methods such as metal doping, aggregation-induced fluorescence enhancement, and matrix coating have been used to improve the fluorescence properties of gold nanoclusters. However, these methods often require the use of large amounts of organic solvents, heavy metal ions, etc., which pollute the environment. Summary of the invention

[0005] The purpose of the present invention is to provide an application of guar gum, a fluorescence-enhanced gold nanocluster, and a method for detecting α-Glu and screening an α-Glu inhibitor. The present invention utilizes guar gum to increase the fluorescence emission intensity of the gold nanocluster, and on this basis can achieve high-sensitivity, low-cost detection of α-Glu activity and screening of α-Glu inhibitors, and is environmentally friendly.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The invention provides an application of guar gum in improving the fluorescence intensity of gold nanoclusters.

[0008] Preferably, the protective ligand of the gold nanoclusters is glutathione.

[0009] The invention provides a fluorescence enhanced gold nanocluster. The preparation raw materials include the gold nanocluster, guar gum and water.

[0010] The present invention provides a method for detecting α-glucosidase activity, comprising the following steps:

[0011] Provide fluorescence enhanced gold nanoclusters-Fe 3+ Mix the dispersion, the fluorescence enhanced gold nanocluster-Fe 3+ The fluorescence-enhanced gold nanoclusters in the mixed dispersion are the fluorescence-enhanced gold nanoclusters described in the above technical solution;

[0012] The α-glucosidase solution to be tested, the L-ascorbic acid-2-O-α-D-pyranoglucoside solution and the phosphate buffer are mixed and incubated for the first time to obtain a first incubation solution;

[0013] The first incubation solution is mixed with fluorescence enhanced gold nanocluster-Fe 3+ The mixed dispersions are mixed and incubated for a second time to obtain a second incubation solution;

[0014] The second incubation solution is subjected to fluorescence detection, and the activity of the α-glucosidase to be tested is obtained according to the working curve of the α-glucosidase activity and the fluorescence intensity obtained by the fluorescence detection.

[0015] Preferably, the fluorescence enhanced gold nanocluster-Fe 3+ The concentration of gold nanoclusters in the mixed dispersion is 10-90 μg / mL, the concentration of guar gum is 0.05-1.0 wt.%, and the Fe 3+ The concentration is 0.1 to 100 μmol / L; the fluorescence enhanced gold nanocluster-Fe 3+ The pH value of the mixed dispersion is 4.0 to 9.0.

[0016] Preferably, the temperature of the first incubation is 30-50° C., and the time is 40-60 min; during the first incubation, the pH value of the system is 6.0-8.0, and the concentration of L-ascorbic acid-2-O-α-D-pyranoglucoside is 1-20 mmol / L.

[0017] Preferably, the temperature of the second incubation is 20-30°C, and the time is 10-60 min; the first incubation solution and the fluorescence enhanced gold nanocluster-Fe 3+ The volume ratio of the mixed dispersion is 1:(1-10).

[0018] Preferably, the fluorescence detection is performed at a wavelength of 610 nm.

[0019] The present invention provides a method for screening α-glucosidase inhibitors, comprising the following steps:

[0020] Provide fluorescence enhanced gold nanoclusters-Fe 3+ Mix the dispersion, the fluorescence enhanced gold nanocluster-Fe 3+ The fluorescence-enhanced gold nanoclusters in the mixed dispersion are the fluorescence-enhanced gold nanoclusters described in the above technical solution;

[0021] The α-glucosidase inhibitor solution to be tested, the α-glucosidase solution, the L-ascorbic acid-2-O-α-D-pyranoglucoside solution and the phosphate buffer are mixed for a third incubation to obtain a third incubation solution;

[0022] The third incubation solution is mixed with fluorescence enhanced gold nanocluster-Fe 3+ The mixed dispersions are mixed and incubated for the fourth time to obtain a fourth incubation solution;

[0023] The fourth incubation solution is subjected to fluorescence detection, and a calibration curve of the α-glucosidase inhibitor to be tested is drawn according to the fluorescence intensity obtained by the fluorescence detection. The half inhibition concentration of the α-glucosidase inhibitor to be tested is obtained according to the calibration curve of the α-glucosidase inhibitor to be tested, thereby realizing the screening of α-glucosidase inhibitors.

[0024] Preferably, the α-glucosidase inhibitor to be tested includes acarbose or a plant extract α-glucosidase inhibitor.

[0025] The present invention provides an application of guar gum in improving the fluorescence intensity of gold nanoclusters. In the present invention, guar gum is a natural high molecular polymer extracted from the seeds of guar bean, a leguminous plant, and has the advantages of low price, non-toxicity and good biocompatibility; guar gum is mainly composed of galactose and mannose, and its molecular skeleton is rich in a large number of hydroxyl groups, which can improve the fluorescence emission intensity of gold nanoclusters. On this basis, α-glucosidase activity detection and α-glucosidase inhibitor screening can be realized with high sensitivity and low cost, and it is environmentally friendly and simple to operate, avoiding the modification of fluorescent probes and the use of a large number of organic reagents, and has broad application prospects in the prevention and treatment of diabetes. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The fluorescence spectra of the gold nanocluster dispersion and the guar gum-gold nanocluster mixed dispersion in Example 2 (the guar gum dispersion used has a pH of 8.0 and a concentration of 0.6 wt.%);

[0027] Figure 2 This is a graph showing the effect of different concentrations of guar gum dispersions on the fluorescence emission intensity of gold nanoclusters in Example 2;

[0028] Figure 3 This is a graph showing the effect of guar gum dispersions with different pH values ​​on the fluorescence emission intensity of gold nanoclusters in Example 2;

[0029] Figure 4 The different concentrations of Fe in Example 3 3+ The results of the effect on the fluorescence emission intensity of the guar gum-gold nanocluster mixed dispersion;

[0030] Figure 5 The guar gum-gold nanocluster-Fe 3+ - Fluorescence spectrum of α-Glu mixed dispersion;

[0031] Figure 6 This is the working curve for detecting α-Glu activity in Example 4;

[0032] Figure 7The different interfering substances (cations, anions, glucose, fructose, sucrose and cholesterol) in Example 5 have an influence on the guar gum-gold nanocluster-Fe 3+ - α-Glu mixed dispersion effect results diagram;

[0033] Figure 8 The different interfering substances (amino acids and proteins) in Example 5 have an influence on the guar gum-gold nanocluster-Fe 3+ - α-Glu mixed dispersion effect results diagram;

[0034] Fig. 9 This is the calibration curve for screening α-Glu inhibitor (acarbose) in Example 6;

[0035] Fig.10 This is the calibration curve for screening α-Glu inhibitor (quercetin) in Example 7;

[0036] Fig.11 This is the calibration curve for screening α-Glu inhibitor (rutin) in Example 7;

[0037] Fig.12 This is the calibration curve for screening α-Glu inhibitor (hesperidin) in Example 7. DETAILED DESCRIPTION

[0038] The invention provides an application of guar gum in improving the fluorescence intensity of gold nanoclusters.

[0039] In the present invention, unless otherwise specified, the raw materials involved are commercially available products well known to those skilled in the art or are prepared by methods well known to those skilled in the art.

[0040] In the present invention, the protective ligand of the gold nanoclusters is preferably glutathione. In the present invention, the mass ratio of the gold nanoclusters to guar gum is preferably 1: (2-60), more preferably 1: (5-30), and further preferably 1: 10. The present invention utilizes guar gum to improve the fluorescence intensity of gold nanoclusters. Specifically, adding guar gum to the water dispersion system containing gold nanoclusters can improve the fluorescence intensity of gold nanoclusters. In the present invention, guar gum is a natural high molecular polymer extracted from the seeds of guar beans, a leguminous plant, and has the advantages of low price, non-toxicity and good biocompatibility; guar gum is mainly composed of galactose and mannose, and its molecular skeleton is rich in a large number of hydroxyl groups, which can easily form hydrogen bonds in water, thereby effectively changing the properties of the water dispersion system, specifically, by prolonging the solvent redirection time and inhibiting the intramolecular vibration and rotation of the gold nanocluster protective ligand, the radiation transition rate of the gold nanoclusters is increased and the non-radiation transition rate is reduced, thereby effectively improving the fluorescence emission intensity of the gold nanoclusters.

[0041] The present invention preferably mixes the guar gum dispersion with the gold nanocluster dispersion, so as to increase the fluorescence intensity of the gold nanocluster by the guar gum.

[0042] In the present invention, the concentration of guar gum in the guar gum dispersion is preferably 0.05-1.0wt.%, specifically 0.1wt.%, 0.2wt.%, 0.3wt.%, 0.4wt.%, 0.5wt.%, 0.6wt.%, 0.7wt.%, 0.8wt.%, 0.9wt.% or 1.0wt.%; the solvent of the guar gum dispersion preferably includes water, and in order to facilitate the regulation of pH value, the solvent of the guar gum dispersion is more preferably a Tris buffer solution, the concentration of the Tris buffer solution is preferably 10mmol / L, and the pH value is preferably 4.0-9.0, specifically 4.0, 5.0, 6.0, 7.0, 8.0 or 9.0. In the present invention, preferably at room temperature (25°C), guar gum and Tris buffer solution are mixed and stirred evenly to obtain a guar gum dispersion.

[0043] In the present invention, the concentration of gold nanoclusters in the gold nanocluster dispersion is preferably 0.1-1 mg / mL, more preferably 0.6 mg / mL; the solvent of the gold nanocluster dispersion is preferably water. In the present invention, the preparation method of the gold nanocluster dispersion preferably comprises the following steps: mixing a tetrachloroauric acid aqueous solution, a protective ligand aqueous solution and water, and reacting under stirring conditions to obtain a gold nanocluster dispersion. In the present invention, the tetrachloroauric acid aqueous solution is preferably a newly prepared tetrachloroauric acid aqueous solution, and the concentration of the tetrachloroauric acid aqueous solution is preferably 20 μmol / L; the concentration of the protective ligand aqueous solution is preferably 100 μmol / L; the volume ratio of the tetrachloroauric acid aqueous solution, the protective ligand aqueous solution and water is preferably 0.5:0.15:4.35, the water is preferably ultrapure water, and the tetrachloroauric acid aqueous solution, the protective ligand aqueous solution and water are preferably mixed at room temperature. In the present invention, the reaction temperature is preferably 70°C, the time is preferably 12h; the stirring speed is preferably 500rpm. In the present invention, during the reaction, the color of the reaction system gradually changes from light yellow to bright yellow. In the present invention, when glutathione is used as a protective ligand, it also acts as a reducing agent, and there is no need to use expensive protein protective ligands and external reducing agents.

[0044] After obtaining the guar gum dispersion and the gold nanocluster dispersion, the present invention preferably mixes the guar gum dispersion with the gold nanocluster dispersion to obtain a guar gum-gold nanocluster mixed dispersion. After mixing the guar gum dispersion with the gold nanocluster dispersion, the present invention preferably allows the resulting system to stand to achieve full mixing of the components to obtain a uniform and stable guar gum-gold nanocluster mixed dispersion. In the present invention, the standing is preferably carried out at room temperature, and the standing time is preferably 10 minutes.

[0045] After obtaining the guar gum-gold nanocluster mixed dispersion, the present invention performs fluorescence detection on the guar gum-gold nanocluster mixed dispersion. In the present invention, the fluorescence detection is preferably performed under the condition of a wavelength of 610nm. The present invention can significantly enhance the fluorescence emission intensity of gold nanoclusters by using guar gum; in an embodiment of the present invention, taking the guar gum-gold nanocluster mixed dispersion obtained by mixing 50μL of gold nanocluster dispersion (concentration of 0.6mg / mL) with 500μL of guar gum dispersion as an example, when the concentration of the guar gum dispersion is 0.6wt.% and the pH value is 8.0, the fluorescence emission intensity of the gold nanoclusters is increased by nearly 4 times, and the fluorescence quantum yield is increased from 0.96% to 2%.

[0046] The present invention provides a fluorescence-enhanced gold nanocluster, wherein the raw materials for preparation include gold nanocluster, guar gum and water; in order to facilitate the regulation of pH value, the raw materials for preparation preferably include gold nanocluster, guar gum and Tris buffer solution. In the present invention, the fluorescence-enhanced gold nanocluster is preferably the guar gum-gold nanocluster mixed dispersion described in the above technical solution, which will not be described in detail here.

[0047] The present invention provides a method for detecting α-glucosidase activity, comprising the following steps:

[0048] Provide fluorescence enhanced gold nanoclusters-Fe 3+ Mix the dispersion, the fluorescence enhanced gold nanocluster-Fe 3+ The fluorescence-enhanced gold nanoclusters in the mixed dispersion are the fluorescence-enhanced gold nanoclusters described in the above technical solution;

[0049] The α-glucosidase solution to be tested, the L-ascorbic acid-2-O-α-D-pyranoglucoside solution and the phosphate buffer are mixed and incubated for the first time to obtain a first incubation solution;

[0050] The first incubation solution is mixed with fluorescence enhanced gold nanocluster-Fe 3+ The mixed dispersions are mixed and incubated for a second time to obtain a second incubation solution;

[0051] The second incubation solution is subjected to fluorescence detection, and the activity of the α-glucosidase to be tested is obtained according to the working curve of the α-glucosidase activity and the fluorescence intensity obtained by the fluorescence detection.

[0052] The present invention provides fluorescence enhanced gold nanocluster-Fe 3+ In the present invention, the fluorescence enhanced gold nanocluster-Fe 3+ The concentration of the gold nanoclusters in the mixed dispersion is preferably 10 to 90 μg / mL, more preferably 40 to 60 μg / mL, and further preferably 54 μg / mL; the concentration of the guar gum is preferably 0.05 to 1.0 wt.%, more preferably 0.2 to 0.6 wt.%; the Fe 3+ The concentration of the fluorescence-enhanced gold nanocluster-Fe 3+ The pH value of the mixed dispersion is preferably 4.0 to 9.0, more preferably 6.0 to 8.0. 3+ The solutions were mixed to obtain fluorescence-enhanced gold nanoclusters-Fe 3+ Mixed dispersion. In the present invention, the guar gum dispersion and the gold nanocluster dispersion are preferably the guar gum dispersion and the gold nanocluster dispersion described in the above technical solution, which will not be repeated here. In the present invention, the Fe 3+ Fe in solution 3+ The concentration of Fe is preferably 0.1 to 10 mmol / L, more preferably 0.5 to 1 mmol / L; 3+ The solute of the solution is preferably FeCl3·6H2O, and the solvent is preferably hydrochloric acid. The concentration of the hydrochloric acid is preferably 0.1 to 1 mol / L, more preferably 0.1 to 0.5 mol / L. The present invention preferably uses hydrochloric acid as the solvent in order to inhibit Fe 3+ Hydrolysis in aqueous solution. In the embodiment of the present invention, 50 μL of gold nanocluster dispersion (concentration of 0.6 mg / mL) was mixed with 500 μL of guar gum dispersion (pH=8.0, concentration of 0.6 wt.%), allowed to stand for 10 min at room temperature, and then 10 μL of 1 mmol / L Fe 3+ The solution (solute is FeCl3·6H2O, solvent is 0.1 mol / L hydrochloric acid) was incubated at room temperature for 10 min to obtain fluorescence enhanced gold nanoclusters-Fe 3+ Mixed dispersion. In the present invention, the Fe 3+ The presence of will quench the fluorescence of gold nanoclusters.

[0053] The present invention mixes the α-glucosidase (α-Glu) solution to be tested, L-ascorbic acid-2-O-α-D-pyranoglucoside (AAG) solution and a phosphate buffer solution, performs a first incubation, and obtains a first incubation solution. In the present invention, the pH value of the system during the first incubation is preferably 6.0-8.0, more preferably 7.0; the concentration of AAG is preferably 1-20 mmol / L, more preferably 2-7 mmol / L, and further preferably 3-5 mmol / L. In the present invention, the concentration of the phosphate buffer solution is preferably 0.05-0.2 mol / L, more preferably 0.1 mol / L, and the pH value is preferably 7.0. In an embodiment of the present invention, specifically 100 μL of the α-Glu solution to be tested, 150 μL of AAG solution (concentration of 10 mmol / L) and 200 μL of phosphate buffer solution (concentration of 0.1 mol / L, pH=7.0) are mixed for the first incubation. In the present invention, the temperature of the first incubation is preferably 30-50°C, more preferably 37°C; the time is preferably 40-60 min, more preferably 40-50 min; the first incubation is preferably performed under static conditions. In the present invention, during the first incubation, AAG produces ascorbic acid under the catalysis of α-Glu.

[0054] The first incubation solution and the fluorescence enhanced gold nanocluster-Fe 3+ After mixing the dispersion, the present invention mixes the first incubation solution with the fluorescence enhanced gold nanocluster-Fe 3+ The mixed dispersions were mixed and incubated for the second time to obtain the second incubation solution (i.e., guar gum-gold nanocluster-Fe 3+ -α-Glu mixed dispersion). In the present invention, the first incubation solution and the fluorescence enhanced gold nanocluster-Fe 3+ The volume ratio of the mixed dispersion is preferably 1: (1-10), more preferably 1: (1-3), and further preferably 1: (1.2-1.5). In the present invention, the temperature of the second incubation is preferably 20-30°C, more preferably room temperature; the time is preferably 10-60 min, more preferably 10-20 min. In the present invention, ascorbic acid can effectively restore the Fe 3+ Quenched gold nanocluster fluorescence.

[0055] After obtaining the second incubation solution, the present invention performs fluorescence detection on the second incubation solution, and obtains the activity of the α-glucosidase to be tested according to the working curve of the α-glucosidase activity and the fluorescence intensity obtained by the fluorescence detection. In the present invention, the fluorescence detection is preferably performed at a wavelength of 610 nm. In the present invention, Fe 3+ Dynamic quenching can quench the fluorescence of gold nanoclusters. In the presence of α-Glu and its substrate AAG, α-Glu catalyzes the hydrolysis of AAG to produce ascorbic acid, which can partially restore the fluorescence of gold nanoclusters quenched by Fe.3+ The fluorescence emission of the gold nanoclusters is quenched, so the α-Glu activity can be detected based on the fluorescence method. In the present invention, the working curve of the α-glucosidase activity is specifically the ratio of the fluorescence intensity (I / I0) to the α-glucosidase activity (logC α-Glu ), where I is the fluorescence intensity of the second incubation solution (i.e., the fluorescence intensity of the first incubation solution and the fluorescence enhanced gold nanocluster-Fe 3+ I0 is the fluorescence intensity of the mixed dispersion after the second incubation), and I0 is the fluorescence intensity of the blank dispersion, wherein the blank dispersion is prepared by replacing the α-Glu solution and the AAG solution to be tested with equal volumes of water, and then sequentially undergoing the first incubation and the second incubation according to the above method. The present invention can obtain the fluorescence intensity ratio (I / I0) based on the fluorescence intensity of the second incubation solution, and then can obtain the activity of the α-glucosidase to be tested. The method provided by the present invention is used to detect the activity of α-glucosidase, with a detection range of 2 to 4000U / L and a detection limit of 0.13U / L, having a wider detection range and a lower detection limit, and the method provided by the present invention avoids the modification of the fluorescent probe and the use of a large amount of organic reagents.

[0056] The present invention provides a method for screening α-glucosidase inhibitors, comprising the following steps:

[0057] Provide fluorescence enhanced gold nanoclusters-Fe 3+ Mix the dispersion, the fluorescence enhanced gold nanocluster-Fe 3+ The fluorescence-enhanced gold nanoclusters in the mixed dispersion are the fluorescence-enhanced gold nanoclusters described in the above technical solution;

[0058] The α-glucosidase inhibitor solution to be tested, the α-glucosidase solution, the L-ascorbic acid-2-O-α-D-pyranoglucoside solution and the phosphate buffer are mixed for a third incubation to obtain a third incubation solution;

[0059] The third incubation solution is mixed with fluorescence enhanced gold nanocluster-Fe 3+ The mixed dispersions are mixed and incubated for the fourth time to obtain a fourth incubation solution;

[0060] The fourth incubation solution is subjected to fluorescence detection, and a calibration curve of the α-glucosidase inhibitor is drawn according to the fluorescence intensity obtained by the fluorescence detection. The half inhibition concentration of the α-glucosidase inhibitor to be tested is obtained according to the calibration curve of the α-glucosidase inhibitor, thereby realizing the screening of the α-glucosidase inhibitor.

[0061] The present invention provides fluorescence enhanced gold nanocluster-Fe 3+ Mixed dispersion, in the present invention, the fluorescence enhanced gold nanocluster-Fe 3+The mixed dispersion is preferably the fluorescence enhanced gold nanocluster-Fe 3+ The mixed dispersion is not described in detail here.

[0062] The present invention mixes the α-glucosidase inhibitor solution to be tested, the α-glucosidase solution, the L-ascorbic acid-2-O-α-D-pyranoglucoside solution and the phosphate buffer, performs a third incubation, and obtains a third incubation solution. In the present invention, the pH value of the system during the third incubation is preferably 6.0 to 8.0, and more preferably 7.0; the concentration of α-Glu is preferably 1 to 10 U / mL, and more preferably 2 to 4 U / mL; the concentration of AAG is preferably 1 to 20 mmol / L, and more preferably 2 to 7 mmol / L, and further preferably 3 to 5 mmol / L. In the present invention, the concentration of the phosphate buffer is preferably 0.05 to 0.2 mol / L, and more preferably 0.1 mol / L, and the pH value is preferably 7.0. In the embodiment of the present invention, specifically, 20 μL of the α-Glu inhibitor solution to be tested, 100 μL of α-Glu solution (concentration of 10 U / mL), 150 μL of AAG solution (concentration of 10 mmol / L) and 200 μL of phosphate buffer solution (concentration of 0.1 mol / L, pH=7.0) are mixed for the third incubation. In the present invention, the conditions of the third incubation are preferably the same as those of the first incubation, and will not be repeated here.

[0063] The third incubation solution and the fluorescence enhanced gold nanocluster-Fe 3+ After mixing the dispersion, the third incubation solution is mixed with the fluorescence enhanced gold nanocluster-Fe 3+ The mixed dispersion is mixed and a fourth incubation is performed to obtain a fourth incubation solution. In the present invention, the third incubation solution is mixed with the fluorescence enhanced gold nanocluster-Fe 3+ The volume ratio of the mixed dispersion is preferably 1:(1-10), more preferably 1:(1-3), and further preferably 1:(1-1.2). In the present invention, the conditions of the fourth incubation are preferably the same as those of the second incubation, which will not be described in detail herein.

[0064] After obtaining the fourth incubation solution, the present invention performs fluorescence detection on the fourth incubation solution, draws a calibration curve of the α-glucosidase inhibitor to be measured according to the fluorescence intensity obtained by fluorescence detection, obtains the half inhibition concentration of the α-glucosidase inhibitor to be measured according to the calibration curve of the α-glucosidase inhibitor to be measured, and realizes the screening of α-glucosidase inhibitors. In the present invention, the fluorescence detection is preferably carried out under the condition of a wavelength of 610nm. In the present invention, the calibration curve of the α-glucosidase inhibitor to be measured is preferably a linear curve of the inhibition efficiency (%) of the α-glucosidase inhibitor to be measured and the concentration of the α-glucosidase inhibitor to be measured. According to the present invention, the activity of α-glucosidase can be known according to the fluorescence intensity of the fourth incubation solution, and then the inhibition efficiency of the α-glucosidase inhibitor to be tested can be obtained. Then, preferably, the concentration of the α-glucosidase inhibitor to be tested is used as the horizontal coordinate and the corresponding inhibition efficiency is used as the vertical coordinate to draw a calibration curve of the α-glucosidase inhibitor to be tested. The concentration of the α-glucosidase inhibitor to be tested corresponding to the inhibition of 50% of the α-glucosidase activity is read from the calibration curve of the α-glucosidase inhibitor to be tested, that is, the half inhibition concentration (IC 50 ), on this basis, the screening of α-glucosidase inhibitors can be achieved. In the present invention, the inhibition efficiency of the α-glucosidase inhibitor to be tested is preferably calculated with reference to formula A:

[0065] Inhibition efficiency = [I AuNCs+α-Glu –I AuNCs+α-Glu+待测α-葡萄糖苷酶抑制剂 ] / [I AuNCs+α-Glu –I AuNCs ] Formula A;

[0066] In formula A, I AuNCs+α-Glu Guar gum-gold nanocluster-Fe 3+ -Fluorescence intensity of α-Glu mixed dispersion;

[0067] I AuNCs+α-Glu+待测α-葡萄糖苷酶抑制剂 represents the fluorescence intensity of the fourth incubation solution;

[0068] I AuNCs stands for fluorescence-enhanced gold nanocluster-Fe 3+ Fluorescence intensity of the mixed dispersion.

[0069] The method provided by the present invention is suitable for screening a variety of α-glucosidase inhibitors, wherein the α-glucosidase inhibitor preferably includes acarbose or a plant extract α-glucosidase inhibitor, and the plant extract α-glucosidase inhibitor preferably includes quercetin, rutin, hesperidin, baicalin or theaflavins.

[0070] The technical solutions in the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. 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.

[0071] Example 1 Preparation of gold nanocluster dispersion and guar gum dispersion

[0072] The preparation of the gold nanocluster dispersion comprises the following steps:

[0073] 0.50 mL of a newly prepared 20 μmol / L aqueous solution of tetrachloroauric acid, 0.15 mL of a 100 μmol / L aqueous solution of glutathione (GSH), and 4.35 mL of ultrapure water were mixed at room temperature (25°C), and the resulting mixture was heated to 70°C at a stirring rate of 500 rpm, and the mixture was kept warm for 12 hours. The color of the reaction system gradually changed from light yellow to bright yellow to obtain a gold nanocluster dispersion, in which the gold nanoclusters (GSH-AuNCs) used glutathione as a protective ligand, and the concentration of the gold nanoclusters in the gold nanocluster dispersion was 0.6 mg / mL.

[0074] The preparation of guar gum dispersions with different concentrations and pH values ​​comprises the following steps:

[0075] Weigh 0, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, and 1000 mg of guar gum powder (GG, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.), mix with 100 mL of a 10 mM Tris buffer solution at room temperature, and stir evenly to obtain a guar gum dispersion having a pH of 8.0 and a guar gum concentration of 0, 0.1 wt.%, 0.2 wt.%, 0.3 wt.%, 0.4 wt.%, 0.5 wt.%, 0.6 wt.%, 0.7 wt.%, 0.8 wt.%, 0.9 wt.%, and 1.0 wt.%;

[0076] Weigh 6 parts of guar gum powder with a mass of 600 mg, mix it with 100 mL of Tris buffer solution with a concentration of 50 mM and pH values ​​of 4.0, 5.0, 6.0, 7.0, 8.0, and 9.0 at room temperature, and stir evenly to obtain a guar gum dispersion with a guar gum concentration of 0.6 wt.% and pH values ​​of 4.0, 5.0, 6.0, 7.0, 8.0, and 9.0, respectively.

[0077] The gold nanocluster dispersion and guar gum dispersion used in the following examples are from this example and will not be specifically described later.

[0078] Example 2 Preparation of fluorescence-enhanced gold nanocluster dispersion

[0079] 50 μL of gold nanocluster dispersion was mixed with 500 μL of guar gum dispersion of different concentrations or pH values, respectively, and allowed to stand at room temperature for 10 min. The fluorescence spectrum of the resulting guar gum-gold nanocluster mixed dispersion was measured, and the fluorescence emission intensity at 610 nm was recorded and compared with the fluorescence emission intensity of the gold nanocluster dispersion.

[0080] Figure 1 The fluorescence spectra of the gold nanocluster dispersion and the guar gum-gold nanocluster mixed dispersion (the pH of the guar gum dispersion used is 8.0, and the concentration is 0.6wt.%), where a is the fluorescence spectrum of the gold nanocluster dispersion and b is the fluorescence spectrum of the guar gum-gold nanocluster mixed dispersion. The results show that the addition of guar gum can significantly enhance the fluorescence emission intensity of the gold nanocluster.

[0081] Figure 2 The figure shows the effect of different concentrations of guar gum dispersion on the fluorescence emission intensity of gold nanoclusters, where I0 is the fluorescence intensity of the gold nanocluster dispersion at 610nm, and I is the fluorescence intensity of the guar gum-gold nanocluster mixed dispersion at 610nm. The results show that when the concentration of guar gum dispersion is in the range of 0.1-0.6wt.%, the fluorescence intensity of gold nanoclusters can be enhanced. The best fluorescence enhancement effect is the guar gum dispersion with a concentration of 0.6wt.%. At this time, the fluorescence intensity of gold nanoclusters is increased by nearly 4 times, and the fluorescence quantum yield is increased from 0.96% to 2%. After that, the fluorescence enhancement effect of gold nanoclusters is slightly weakened by further increasing the guar gum concentration.

[0082] Figure 3 The figure shows the effect of guar gum dispersions with different pH values ​​on the fluorescence emission intensity of gold nanoclusters, where I0 is the fluorescence intensity of the gold nanocluster dispersion at 610nm, and I is the fluorescence intensity of the guar gum-gold nanocluster mixed dispersion at 610nm. The results show that when the pH value of the guar gum dispersion is in the range of 4.0 to 9.0, guar gum can increase the fluorescence emission intensity of gold nanoclusters, and the most suitable pH value of the guar gum dispersion is 8.0.

[0083] In summary, the preferred method for preparing the fluorescence-enhanced gold nanocluster dispersion is: mix 50 μL of gold nanocluster dispersion with 500 μL of guar gum dispersion (pH=8.0, concentration of 0.6 wt.%), let stand at room temperature for 10 minutes, and obtain the fluorescence-enhanced gold nanocluster dispersion.

[0084] Example 3 Fluorescence-enhanced gold nanocluster-Fe 3+ Preparation of mixed dispersion

[0085] 10 μL of Fe at different concentrations was added to 550 μL of the fluorescence-enhanced gold nanocluster dispersion prepared in Example 2. 3+ The solution (solute is FeCl3·6H2O, solvent is 0.1M hydrochloric acid) was incubated at room temperature for 10 min to obtain guar gum-gold nanocluster-Fe 3+ Mixed dispersion; the guar gum-gold nanocluster-Fe 3+ Fe in mixed dispersion 3+ The concentrations were 0.36μM, 1.1μM, 1.6μM, 3.6μM, 10μM, 14μM, 18μM, 36μM, 54μM, 72μM, and 90μM, respectively.

[0086] Fe 3+ The fluorescence of gold nanoclusters is quenched by dynamic quenching. Figure 4 For different concentrations of Fe 3+ The fluorescence emission intensity of the guar gum-gold nanocluster mixed dispersion was affected by the fluorescence emission intensity of the guar gum-gold nanocluster mixed dispersion, where I0 is the fluorescence intensity of the guar gum-gold nanocluster mixed dispersion at 610 nm, and I is the fluorescence intensity of the guar gum-gold nanocluster-Fe 3+ The fluorescence intensity of the mixed dispersion at 610 nm. Figure 4 It can be seen that the emission intensity of the guar gum-gold nanocluster mixed dispersion increases with the Fe 3+ concentration gradually decreased and 3+ The plateau phase was reached when the concentration was 18 μM.

[0087] In summary, the preferred fluorescence enhanced gold nanocluster-Fe 3+ The preparation method of the mixed dispersion is as follows: 50 μL of the gold nanocluster dispersion and 500 μL of guar gum dispersion (pH=8.0, concentration of 0.6 wt.%) were mixed, and the mixture was allowed to stand for 10 min at room temperature. 10 μL of 1 mM Fe 3+ The solution (solute is FeCl3·6H2O, solvent is 0.1M hydrochloric acid) was incubated at room temperature for 10 min to obtain fluorescence enhanced gold nanocluster-Fe 3+ The dispersion was mixed.

[0088] Example 4 α-Glu activity detection method

[0089] 100 μL of α-Glu aqueous solution of different concentrations, 150 μL of 10 mM α-Glu aqueous solution and 200 μL of phosphate buffer solution (0.1 M, pH = 7.0) were mixed and allowed to stand at 37°C for 40 min for the first incubation. The first incubation solution was mixed with 560 μL of fluorescence-enhanced gold nanocluster-Fe prepared in Example 3. 3+ Mixed dispersion (ie, guar gum-gold nanoclusters-Fe 3+ The mixed dispersion) was mixed and allowed to stand for 10 min at room temperature for the second incubation. The obtained guar gum-gold nanocluster-Fe 3+ -α-Glu mixed dispersion (i.e., the second incubation solution) was measured, and the fluorescence intensity at 610 nm was recorded; the guar gum-gold nanocluster-Fe 3+ The concentration of α-Glu in the mixed dispersion was 2×10 -4 U / mL, 2×10 -3 U / mL, 0.1U / mL, 0.25U / mL, 1U / mL, 2U / mL, 3U / mL, 4U / mL and 5U / mL.

[0090] At the same time, a blank sample was prepared by mixing 250 μL of water with 200 μL of phosphate buffer solution (0.1 M, pH = 7.0), and standing at 37°C for 40 min. The resulting solution was then mixed with 560 μL of fluorescence-enhanced gold nanocluster-Fe prepared in Example 3. 3+ The mixed dispersions were mixed and allowed to stand for 10 min at room temperature to obtain a blank sample.

[0091] In the presence of α-Glu and its substrate AAG, α-Glu catalyzes the hydrolysis of AAG to produce ascorbic acid, which can partially restore the amount of ascorbic acid lost by Fe 3+ The fluorescence emission of the gold nanoclusters was quenched, so the α-Glu activity could be detected based on the fluorescence method. Figure 5 Guar gum-gold nanoclusters-Fe 3+ - Fluorescence spectrum of α-Glu mixed dispersion, Figure 6 The working curve for detecting α-Glu activity (I0 is the fluorescence intensity of the blank sample at 610 nm, I is the fluorescence intensity of the guar gum-gold nanocluster-Fe 3+ -α-Glu mixed dispersion at 610nm). Figure 6 It can be seen that guar gum-gold nanocluster-Fe 3+-The fluorescence intensity of the α-Glu mixed dispersion increases with the increase of α-Glu activity. The detection range of α-Glu activity is 2 to 4000U / L, and the detection limit is 0.13U / L. The classical method for detecting α-Glu activity is the p-nitrophenol pyranoglucoside colorimetric method, which has a detection limit of 2.5 to 50U / L and a linear range of 1U / L. Compared with the classical method, the method provided by the present invention has a wider detection range and a lower detection limit, and the method provided by the present invention avoids the modification of the fluorescent probe and the use of a large amount of organic reagents.

[0092] Example 5 Selectivity Verification of α-Glu Activity Detection Method

[0093] In order to verify the selectivity of the α-Glu activity detection method, this example tested the effects of various interfering substances that may exist in biological samples on the detection system, including amino acids (Ala, Arg, Gly, His, Lys, Ser and Cys), cations (Na + , K + , Ca 2+ and Fe 2+ ), anions (Cl - 、NO3 - 、SO4 2- and PO4 3- ), proteins (BSA, HSA, ACP and pap), glucose (Glu), fructose (Fru), sucrose (Suc) and cholesterol (Chol).

[0094] 450 μL of the first incubation solution prepared in Example 4 and 560 μL of the fluorescence-enhanced gold nanocluster-Fe prepared in Example 3 were added. 3+ The mixed dispersions were mixed, and then 10 μL of the aqueous dispersions of the various interfering substances mentioned above were added, and the concentration of various interfering substances in the obtained mixed solution was 10 mmol / L; the mixed solution was incubated at room temperature for 10 min, the fluorescence spectrum of the obtained incubation solution was measured, and the fluorescence intensity at 610 nm was recorded.

[0095] At the same time, a blank sample was prepared by mixing 450 μL of the first incubation solution prepared in Example 4 with 560 μL of the fluorescence-enhanced gold nanocluster-Fe prepared in Example 3. 3+ The mixed dispersions were mixed, 10 μL of water was added, and the mixture was allowed to stand at room temperature for 10 min to obtain a blank sample.

[0096] Figure 7 and Figure 8 The blank sample and guar gum-gold nanocluster-Fe under the conditions of different interferences 3+- α-Glu mixed dispersion at 610nm fluorescence intensity. It can be seen from the figure that each interfering substance has no obvious interference effect on the fluorescence detection of α-Glu by the fluorescence enhanced gold nanoclusters described in the present invention, indicating that the method of the present invention has excellent selectivity.

[0097] Example 6 Screening of α-Glu Inhibitors

[0098] Acarbose is a common α-Glu inhibitor and one of the few oral drugs that can be used to treat type II diabetes. This example uses acarbose as an example to study the application of the method of the present invention in the screening of α-Glu inhibitors. The specific steps are as follows:

[0099] 100 μL of α-Glu (10 U / mL) aqueous solution, 150 μL of AAG (10 mM) aqueous solution, 200 μL of phosphate buffer (0.1 M, pH = 7.0) and 20 μL of α-Glu inhibitor (acarbose) of different concentrations were mixed and allowed to stand at 37°C for 40 min for the third incubation; the obtained third incubation solution was mixed with 560 μL of fluorescence enhanced gold nanocluster-Fe prepared in Example 3. 3+ Mixed dispersion (ie, guar gum-gold nanoclusters-Fe 3+ The mixed dispersion) was mixed and allowed to stand for 10 min at room temperature for the fourth incubation. The obtained guar gum-gold nanocluster-Fe 3+ -α-Glu-acarbose mixed dispersion (i.e., the fourth incubation solution) was measured, and the fluorescence intensity at 610 nm was recorded; the guar gum-gold nanocluster-Fe 3+ The concentrations of acarbose in the -α-Glu-acarbose mixed dispersion were 20 μM, 50 μM, 80 μM, 120 μM, 180 μM and 200 μM, respectively.

[0100] According to the fluorescence intensity of the fourth incubation solution under different concentrations of acarbose, the inhibition efficiency of acarbose was calculated according to formula B:

[0101] Inhibition efficiency = [I AuNCs+α-Glu –I AuNCs+α-Glu+阿卡波糖 ] / [I AuNCs+α-Glu –I AuNCs ] Formula B;

[0102] In formula B, I AuNCs+α-Glu Guar gum-gold nanocluster-Fe 3+ +fluorescence intensity of α-Glu mixed dispersion;

[0103] I AuNCs+α-Glu+阿卡波糖 represents the fluorescence intensity of the fourth incubation solution;

[0104] I AuNCsGuar gum-gold nanocluster-Fe 3+ Fluorescence intensity of the mixed dispersion.

[0105] Fig. 9 This is a calibration curve for screening α-Glu inhibitors (acarbose). As shown in the figure, acarbose can inhibit the activity of α-Glu. As the concentration of acarbose increases, the fluorescence intensity of the system gradually decreases.

[0106] Depend on Fig. 9 It can be seen that the IC of acarbose 50 (The concentration of the inhibitor to be tested required to inhibit 50% of α-Glu activity) is 87.3 μM, which is consistent with the existing literature (Li, C.; et al. Anal. Bioanal. Chem. 2021, 413 (9): 2553-2563). This shows that the method provided by the present invention can be effectively used for the screening of acarbose.

[0107] Example 7 Screening of α-Glu Inhibitors from Plant Extracts

[0108] Quercetin, rutin and hesperidin (commercially available products) are plant extract α-Glu inhibitors, and the method provided by the present invention can be used for screening plant extract α-Glu inhibitors. This example first takes quercetin as an example to study the application of the method of the present invention in screening plant extract α-Glu inhibitors, and the specific steps are as follows:

[0109] 100 μL of α-Glu (10 U / mL) aqueous solution, 150 μL of AAG (10 mM) aqueous solution, 200 μL of phosphate buffer (0.1 M, pH = 7.0) and 20 μL of α-Glu inhibitor (quercetin) of different concentrations were mixed and allowed to stand at 37°C for 40 min for the third incubation; the obtained third incubation solution was mixed with 560 μL of fluorescence enhanced gold nanocluster-Fe prepared in Example 3. 3+ Mixed dispersion (ie, guar gum-gold nanoclusters-Fe 3+ The mixed dispersion) was mixed and allowed to stand for 10 min at room temperature for the fourth incubation. The obtained guar gum-gold nanocluster-Fe 3+ -α-Glu-quercetin mixed dispersion (i.e., the fourth incubation solution) was measured for fluorescence spectrum, and the fluorescence intensity at 610 nm was recorded; the guar gum-gold nanocluster-Fe 3+ The quercetin concentrations in the -α-Glu-quercetin mixed dispersion were 6 μM, 8 μM, 10 μM, 15 μM, 20 μM, 30 μM, 40 μM, 50 μM, 60 μM and 70 μM, respectively.

[0110] According to the fluorescence intensity of the fourth incubation solution under different concentrations of quercetin, the inhibition efficiency of quercetin was calculated according to Formula C:

[0111] Inhibition efficiency = [I AuNCs+α-Glu –I AuNCs+α-Glu+槲皮素 ] / [I AuNCs+α-Glu –I AuNCs ] Formula C;

[0112] In formula C, I AuNCs+α-Glu Guar gum-gold nanocluster-Fe 3+ -Fluorescence intensity of α-Glu mixed dispersion;

[0113] I AuNCs+α-Glu+槲皮素 represents the fluorescence intensity of the fourth incubation solution;

[0114] I AuNCs Guar gum-gold nanocluster-Fe 3+ Fluorescence intensity of the mixed dispersion.

[0115] Fig.10 This is a calibration curve for screening α-Glu inhibitors (quercetin). As shown in the figure, quercetin can inhibit the activity of α-Glu. As the concentration of quercetin increases, the fluorescence intensity of the system gradually decreases.

[0116] Depend on Fig.10 It can be seen that the IC of quercetin 50 The concentration of quercetin was 33.4 μM, which is consistent with the existing literature (Liu, D.; et al. 2020. Colloids Surf., B). This shows that the method provided by the present invention can be effectively used for the screening of quercetin.

[0117] According to the above method, the inhibition efficiency of rutin and hesperidin was calculated and the calibration curve was drawn. Figures 11-12 As shown, according to Figures 11-12 It can be seen that the IC values ​​of rutin and hesperidin are 50 They are 56.43 μM and 101.2 μM respectively. These results are consistent with those reported in the literature (Liu, D.; et al. 2020. Colloids Surf., B), further proving the reliability of the method of the present invention.

[0118] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A test α -glucosidase activity method, comprising the following steps: Provide fluorescence enhanced gold nanoclusters-Fe 3+ Mix the dispersion, the fluorescence enhanced gold nanocluster-Fe 3+ The raw materials for preparing fluorescence-enhanced gold nanoclusters in a mixed dispersion include gold nanoclusters, guar gum and water; Will be tested α -glucosidase solution, L-ascorbic acid-2-O- α -D-pyranoglucoside solution is mixed with phosphate buffer and incubated for the first time to obtain a first incubation solution; The first incubation solution is mixed with fluorescence enhanced gold nanocluster-Fe 3+ The mixed dispersions are mixed and incubated for a second time to obtain a second incubation solution; The second incubation solution is subjected to fluorescence detection according to α -The working curve of glucosidase activity and the fluorescence intensity obtained by fluorescence detection are obtained α -glucosidase activity.

2. The method according to claim 1, characterized in that The fluorescence enhanced gold nanocluster-Fe 3+ The concentration of gold nanoclusters in the mixed dispersion was 10-90 μg / mL, the concentration of guar gum was 0.05-1.0 wt.%, and Fe 3+ The concentration of the fluorescence-enhanced gold nanocluster-Fe 3+ The pH value of the mixed dispersion is 4.0~9.

0.

3. The method according to claim 1, characterized in that The temperature of the first incubation is 30-50°C, and the time is 40-60 min; the pH value of the system during the first incubation is 6.0-8.0, L-ascorbic acid-2-O- α The concentration of -D-pyranoglucoside is 1~20 mmol / L.

4. The method according to any one of claims 1 to 3, characterized in that The temperature of the second incubation is 20-30°C and the time is 10-60 min; the first incubation solution and the fluorescence enhanced gold nanocluster-Fe 3+ The volume ratio of the mixed dispersion is 1:(1~10).

5. The method according to claim 1, characterized in that The fluorescence detection is performed at a wavelength of 610 nm.

6. A screening α -glucosidase inhibitor method, comprising the steps of: Provide fluorescence enhanced gold nanoclusters-Fe 3+ Mix the dispersion, the fluorescence enhanced gold nanocluster-Fe 3+ The raw materials for preparing fluorescence-enhanced gold nanoclusters in a mixed dispersion include gold nanoclusters, guar gum and water; Will be tested α -Glucosidase inhibitor solution, α -glucosidase solution, L-ascorbic acid-2-O- α -D-pyranoglucoside solution is mixed with phosphate buffer and incubated for the third time to obtain a third incubation solution; The third incubation solution is mixed with fluorescence enhanced gold nanocluster-Fe 3+ The mixed dispersions are mixed and incubated for the fourth time to obtain a fourth incubation solution; The fourth incubation solution is subjected to fluorescence detection, and the fluorescence intensity obtained by the fluorescence detection is plotted α - Calibration curve of glucosidase inhibitors, according to the α - The calibration curve of the glucosidase inhibitor to be tested is obtained α -The half-inhibitory concentration of glucosidase inhibitors is achieved α -Screening of glucosidase inhibitors.

7. The method according to claim 6, characterized in that The test α - Glucosidase inhibitors including acarbose or plant extracts α -Glucosidase inhibitors.

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