Preparation method of alpha-glucosidase inhibitor, alpha-glucosidase inhibitor and application

Abstract

The invention discloses a preparation method of an alpha-glucosidase inhibitor. The preparation method comprises the following steps: preparing crude enzyme liquid by taking crown pears as raw materials; and mixing the crude enzyme solution with a tea polyphenol solution to carry out enzymatic reaction, carrying out enzyme deactivation after the reaction is finished, and obtaining a reaction solution after enzyme deactivation or concentrating and drying the reaction solution to obtain powder, namely the alpha-glucosidase inhibitor. The alpha-glucosidase inhibitor prepared by the preparation method disclosed by the invention is natural in components, has a good inhibition effect on alpha-glucosidase, and has a relatively good application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of alpha-glucosidase inhibitors, and in particular to a preparation method of an alpha-glucosidase inhibitor, an alpha-glucosidase inhibitor and an application thereof. Background Art

[0002] Diabetes is a metabolic disease in which glucose concentration accumulates in the blood due to impaired insulin secretion or the body's inability to effectively utilize insulin produced by the pancreas. α-glucosidase inhibitors can delay the release of glucose from dietary complex carbohydrates and delay glucose absorption, resulting in lower postprandial blood glucose levels and inhibiting postprandial hyperglycemia.

[0003] At present, the α-glucosidase inhibitors used clinically mainly include acarbose, voglibose and miglitol. These drugs are mainly derived from microbial metabolites or chemical synthesis. However, some studies have also explored the extraction of α-glucosidase inhibitors from natural plants and animals. For example, the Chinese patent with publication number CN118662558A mentions a composition for inhibiting α-glucosidase activity, which contains cinnamon and hawthorn, cinnamon is cinnamon leaf extract, hawthorn is hawthorn fruit extract, and the optimal weight ratio of cinnamon and hawthorn is 1:1. The composition has a certain effect of inhibiting α-glucosidase. The patent with publication number CN104922173A discloses the use of balsam pear extract to inhibit α-glucosidase, and its inhibitory effect is better than the reference substance acarbose.

[0004] In addition, according to existing research, tea products have certain α-glucosidase inhibitory activity. For example, the Chinese patent with publication number CN104906264B mentions an α-glucosidase activity inhibitor, which is a black tea pigment, including theaflavins, thearubigins and theabrownins; wherein the black tea pigment is a natural product obtained by extracting Yixing black tea with an organic solvent. Green tea polyphenols have been proven by other existing studies to have good antioxidant activity, anti-inflammatory, hypoglycemic, hypolipidemic and lipid metabolism regulating effects, and are widely used in the treatment of metabolic diseases, including hypertension, hyperlipidemia, and diabetes. However, green tea polyphenols have low α-glucosidase inhibitory activity. my country is the birthplace of green tea. According to statistics, the annual output is as high as 1.934 million, which is far higher than black tea and other categories. How to further make full use of green tea raw materials and improve the economic value of green tea additional products is the focus of research and development. The present invention aims to propose a preparation method of α-glucosidase inhibitor and α-glucosidase inhibitor to develop a new α-glucosidase inhibitor with good inhibitory effect. Summary of the invention

[0005] The technical problem solved by the present invention is to provide a preparation method of α-glucosidase inhibitor and α-glucosidase inhibitor to solve the problems in the above-mentioned background technology.

[0006] The technical problem solved by the present invention is achieved by adopting the following technical solutions:

[0007] A method for preparing an alpha-glucosidase inhibitor comprises the following steps: using crown pear as a raw material to prepare a crude enzyme solution; mixing the crude enzyme solution with a tea polyphenol solution to carry out an enzymatic reaction; inactivating the enzyme after the reaction is completed; and the reaction solution obtained after the enzyme inactivation or the powder obtained by concentrating and drying the reaction solution is the alpha-glucosidase inhibitor.

[0008] Furthermore, green tea polyphenols with a polyphenol content of ≥98% are dissolved in a buffer solution to obtain a tea polyphenol solution, wherein the mass concentration of the tea polyphenols in the tea polyphenol solution is 0.2-0.4 mg / mL.

[0009] Furthermore, the enzyme activity of the crude enzyme solution is 0.5-1.5 U / mL, and the volume ratio of the crude enzyme solution to the tea polyphenol solution is 1:45-55.

[0010] Furthermore, the enzymatic reaction temperature is 10-50°C, the reaction time is 20-100 min, and the reaction pH is 3-7.

[0011] Preferably, the mass concentration of tea polyphenols in the tea polyphenol solution is 0.25-0.35 mg / mL, the enzyme activity of the crude enzyme solution is 0.75-1.2 U / mL, and the volume ratio of the crude enzyme solution to the tea polyphenol solution is 1:50; the enzymatic reaction temperature is 28-32°C, the reaction time is 55-65 min, and the reaction pH is 3.8-4.2.

[0012] Furthermore, the preparation method of the crude enzyme solution comprises: peeling a crown pear and cutting it into pieces to obtain pear pieces, pre-freezing the pear pieces and then adding a color preservative and a buffer solution, crushing them, filtering them and performing a first centrifugation treatment, and obtaining a supernatant; adding ammonium sulfate powder to the supernatant to prepare a solution with a saturation of 70% ammonium sulfate, refrigerating the solution in a refrigerator overnight and then performing a second centrifugation treatment, obtaining a precipitate, and dissolving the precipitate with a buffer solution to obtain a crude enzyme solution.

[0013] Furthermore, during the first centrifugal treatment, the temperature is 3-5°C, the rotation speed is 7500-8500 r / min, and the centrifugal time is 8-12 min; during the second centrifugal treatment, the temperature is 3-5°C, the rotation speed is 7500-8500 r / min, and the centrifugal time is 18-22 min.

[0014] Further, the following steps are included:

[0015] Preparation of crude enzyme solution: peel the crown pear and cut it into pieces to obtain pear blocks, pre-freeze the pear blocks, add color preservative and buffer, crush them, filter them and perform the first centrifugation to obtain the supernatant; add ammonium sulfate powder to the supernatant to prepare a solution with 70% ammonium sulfate saturation, refrigerate the solution in a refrigerator overnight, perform the second centrifugation, obtain the precipitate, and dissolve the precipitate with buffer to obtain the crude enzyme solution;

[0016] Enzymatic reaction: dissolve green tea polyphenol powder in a buffer solution to obtain a polyphenol solution, mix the polyphenol solution with a crude enzyme solution to carry out an enzymatic reaction, and after the reaction is completed, heat the reaction solution in a boiling water bath for 5 minutes to inactivate the enzyme to terminate the reaction, and the reaction solution obtained after inactivating the enzyme or the powder obtained by concentrating and drying the reaction solution is the α-glucosidase inhibitor.

[0017] Furthermore, the buffer is a citric acid-phosphate buffer, and the pH value of the citric acid-phosphate buffer is 4-5.

[0018] An alpha-glucosidase inhibitor is prepared by the method as described above, wherein the key components of the alpha-glucosidase inhibitor include TFDG, TF-3-G, TF-3'-G, TSB, TSA, TSD, TF1, and theaflatolide; the inhibition rate of the alpha-glucosidase inhibitor on alpha-glucosidase is greater than 85%.

[0019] Beneficial effects:

[0020] The method for preparing an alpha-glucosidase inhibitor of the present invention uses crown pear as a raw material to prepare a crude enzyme solution, which is then catalyzed by tea polyphenols to perform an enzymatic reaction to obtain a pure natural alpha-glucosidase inhibitor. The inhibition rate of the alpha-glucosidase inhibitor on alpha-glucosidase is greater than 85%.

[0021] In the preparation process of the α-glucosidase inhibitor preparation method of the present invention, the preparation of the crude enzyme solution and the control of the reaction strips in the enzymatic reaction process are extremely critical. Only with the overall coordination of the raw materials and the process can a product with a good α-glucosidase inhibition rate be obtained. In addition, the preparation nodes of this aspect are easy to control, the preparation process is not complicated, the raw material cost is low, and it is suitable for batch processing and production.

[0022] The alpha-glucosidase inhibitor of the present invention has pure natural raw materials, has good inhibitory effect on alpha-glucosidase, and is suitable for popularization and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the statistical result of α-glucosidase inhibition of products by different tea polyphenols concentrations in single factor experiment. Figure 2 This is the statistical result of α-glucosidase inhibition of the product by different enzymatic reaction times in single factor experiment.

[0024] Figure 3 This is the statistical result of α-glucosidase inhibition of the product by different enzymatic reaction temperatures in single factor experiment.

[0025] Figure 4 This is the statistical result of α-glucosidase inhibition of the product by different enzymatic reaction pH in single factor experiment. Figure 5 The secondary metabolite class composition of α-glucosidase inhibitors and tea polyphenols raw materials in differential analysis.

[0026] Figure 6 is the Pearson correlation coefficient of α-glucosidase inhibitors and tea polyphenols raw materials in differential analysis.

[0027] Figure 7 This is the analysis of the differential secondary metabolites of α-glucosidase inhibitors and tea polyphenols in the differential analysis. (A. Volcano diagram of differential secondary metabolites; B. Bar graph of the differential multiples of 8 types of secondary metabolites; C. Bar graph of the differential multiples of tannin secondary metabolites; D. Bar graph of the differential multiples of terpenoid metabolites; E. Bar graph of the differential multiples of catechins, theaflavins and their derivatives).

[0028] Figure 8 The docking results of the key component of α-glucosidase inhibitor and substrate PNPG with α-glucosidase (A is TFDG; B is TF-3-G; C is TF-3'-G; D is TSB; E is TSA; F is TSD; G is TF1; H is theahuanggalloin). DETAILED DESCRIPTION

[0029] In order to make the technical means, creative features, objectives and effects achieved by the present invention easy to understand, the present invention is further described below in conjunction with specific embodiments.

[0030] The preparation method of the α-glucosidase inhibitor of the present invention comprises the following steps:

[0031] Preparation of crude enzyme solution: Peel the crown pear and cut it into pieces to obtain pear pieces, weigh 100 g of pear pieces, pre-freeze the pear pieces at -20°C for 2 h, add appropriate amounts of PVPP and VC as color preservatives after pre-freezing, and add 100 mL of pre-cooled citric acid-phosphate buffer, the pH value of which is 5.6; then mash the pieces, filter them with four layers of gauze, and perform low-temperature high-speed centrifugation on the filtrate at 4°C and 8000 r / min, and take the supernatant after centrifugation for 10 min.

[0032] Slowly and evenly add ammonium sulfate powder to the supernatant to prepare a solution with 70% ammonium sulfate saturation. The resulting solution is placed in a 4°C refrigerator overnight and then subjected to low-temperature high-speed centrifugation at 4°C and 8000 r / min for 20 min. The precipitate is collected and dissolved with an appropriate amount of citric acid-phosphate buffer with a pH value of 5.6 to obtain a crude enzyme solution with an enzyme activity of 0.94 U / mL.

[0033] Enzymatic reaction: taking green tea polyphenol powder with a polyphenol content of ≥ 98%, dissolving the green tea polyphenol powder with 50 ml of citric acid-phosphate buffer, using ultrasound to assist dissolution during the dissolution process, to obtain a polyphenol solution;

[0034] 1 ml of the crude enzyme solution was added to the tea polyphenol solution to perform an enzymatic reaction. After the enzymatic reaction was completed, the reaction solution was placed in a boiling water bath and heated for 5 minutes to inactivate the enzyme, thereby terminating the enzymatic reaction. The obtained reaction solution was concentrated and freeze-dried to obtain an α-glucosidase inhibitor product.

[0035] (I) Single factor experiment

[0036] Select tea polyphenol concentration, pH value, reaction time and reaction temperature for single factor test. Fix the reaction temperature at 30℃, pH 4.0 and reaction time at 60min to explore the inhibitory effect of tea polyphenol concentration (0.2, 0.25, 0.3, 0.35, 0.4mg / mL) on α-glucosidase activity in tea polyphenol solution. Fix the tea polyphenol concentration at 0.30mg / mL, reaction time at 60min and pH 4.0 to explore the inhibitory effect of reaction temperature (10, 20, 30, 40, 50℃) on α-glucosidase activity. Fix the tea polyphenol concentration at 0.30mg / mL, reaction time at 60min and reaction temperature at 30℃ to explore the inhibitory effect of pH value (3, 4, 5, 6, 7) on α-glucosidase activity. The concentration of tea polyphenols was fixed at 0.30 mg / mL, the reaction temperature was 30°C, and the pH was 4.0, and the inhibitory effect of reaction time (20, 40, 60, 80, 100 min) on α-glucosidase activity was investigated.

[0037] The method for detecting the inhibition rate of the product on α-glucosidase activity is as follows: add 90uL phosphate buffer (pH = 6.9) to a 96-well plate, add 20uL OTP (31.25ug / mL) and 20uL α-glucosidase solution (2.0U / mL) in sequence, preheat at 37°C in the dark for 15min, then add 30uL 37°C preheated pNPG solution (2.5mmol / mL) as a substrate, mix well and continue to react at 37°C for 15min, finally add 80uL saturated Na2CO3 to terminate the reaction, and use an enzyme reader to measure its absorbance at a wavelength of 405nm. Use phosphate buffer as a blank control, and repeat each experiment 3 times. Calculate the inhibitory activity of the sample on α-glucosidase according to the following formula.

[0038]

[0039] Where: A1 is the absorbance of the blank group, and A2 is the absorbance of the experimental group.

[0040] The results are as follows Figure 1 to Figure 4 shown.

[0041] The degree of enzymatic oxidation of the substrate TP by polyphenol oxidase varies under different conditions, and different degrees of oxidation represent different product compositions. Figure 1 It can be seen that when the concentration of tea polyphenols in the reaction substrate is 0.3 mg / mL, the enzymatic oxidation degree of the crude enzyme solution on the tea polyphenol solution is the most thorough; the product has the best inhibitory effect on α-glucosidase, with an inhibition rate of 88.43%. As the concentration of tea polyphenols in the reaction substrate increases, the activity of the crude enzyme solution is inhibited, the oxidation degree of tea polyphenols is reduced, and the inhibitory effect of the product on α-glucosidase decreases, and the inhibition rate decreases to 78.10%.

[0042] Depend on Figure 2 and Figure 3 It can be seen that when the reaction time is 60 min and the reaction temperature is 30 °C, the product has the best inhibitory effect on α-glucosidase, with inhibition rates of 87.18% and 86.92%, respectively. Subsequently, the increase in reaction time and reaction temperature leads to product decomposition, and the inhibition rates decrease to 77.98% and 80.27%, respectively.

[0043] pH affects the inhibitory effect of α-glucosidase by affecting the stability of the substrate tea polyphenols and the product. Figure 4 As shown, when the pH is 4, the product has the best inhibitory effect on α-glucosidase, with an inhibition rate of 81.82%. As the pH value increases, tea polyphenols and the product become unstable, thereby affecting the inhibitory effect of the product on α-glucosidase. At a pH of 6, the inhibition rate gradually decreases to 75.47%.

[0044] (II) Response surface optimization experiment

[0045] Based on the results of the single factor test, a Box-Behnken response surface test with 4 factors and 3 levels was conducted on tea polyphenols concentration, reaction temperature, pH, and reaction time using Design Expert 8.0.6 software to determine the optimal experimental conditions for the PPO enzymatic synthesis of OTP with α-glucosidase inhibition. The response surface test factors and levels are shown in Table 1.

[0046] Table 1 Box-Behnken test design factors and levels

[0047]

[0048] The method for detecting the inhibition rate of the product on α-glucosidase activity is the same as above. The results are shown in Table 2.

[0049] Table 2 Response surface experimental design and results

[0050]

[0051]

[0052] As shown in Table 2, in the present invention, different tea polyphenol concentrations and enzymatic conditions have a key influence on the final product and the inhibition rate of α-glucosidase.

[0053] (III) Differential analysis of α-glucosidase inhibitors and tea polyphenols raw materials.

[0054] The α-glucosidase inhibitor and tea polyphenol raw materials obtained under the conditions of tea polyphenol concentration of 0.3 mg / mL, enzymatic reaction time of 57 min, enzymatic reaction temperature of 30.5°C, and enzymatic reaction pH of 4.13 were differentially analyzed.

[0055] Depend on Figure 5 It can be seen that 1372 secondary metabolites were identified from α-glucosidase inhibitors and tea polyphenols samples, including flavonoids (575), followed by phenolic acids (274), others (159), alkaloids (118), lignans and coumarins (102), tannins (49), terpenoids (47), quinones (28), etc.

[0056] To further explore the differences in secondary metabolites between α-glucosidase inhibitors and tea polyphenols samples, multivariate statistical analysis was performed, and the Pearson coefficient r was used as an evaluation index for the correlation between biological replicates and TPs, e.g. Figure 6 As shown, the correlation between α-glucosidase inhibitors and tea polyphenols samples was <0.86, indicating that there were significant differences in secondary metabolites before and after the oxidation of tea polyphenols by the Crown Pear crude enzyme solution.

[0057] Further screening of differential secondary metabolites revealed 161 significantly different metabolites in the secondary metabolites of α-glucosidase inhibitors and tea polyphenols, accounting for 11.73% of all identified secondary metabolites. Figure 7 As shown, among the secondary metabolites, 75 are downward-accumulating and 86 are upward-accumulating ( Figure 7 A); the significant order of up-regulated secondary metabolites was flavonoids (31) > phenolic acids (19) > tannins (15) > lignans and coumarins (7) > terpenoids (4) > alkaloids (4) > others (4) > quinones (2), and the significant order of down-regulated secondary metabolites was flavonoids (39) > phenolic acids (15) > others (5) > alkaloids (6) > lignans and coumarins (5) > quinones (2) > terpenoids (2) > tannins (1) ( Figure 7 B). The effect of pear PPO-catalyzed TP on tannins and terpenoids was more obvious. Except for tannins (proanthocyanidin A6) and terpenoids (scutebarbolide C, glucosyl 6-hydroxy-2,6-dimethyl-2E,7-octadienoic acid) which accumulated downward, the rest of tannins and terpenoids accumulated upward ( Figure 7 C. Figure 7 D). In summary, it is shown that in the present invention, after the green tea polyphenols are subjected to enzymatic reaction by the Crown Pear crude enzyme solution under specific conditions, a complex composite product is obtained, and the composite product has a good inhibitory effect on α-glucosidase.

[0058] (IV) Molecular docking experiment

[0059] Molecular docking simulation is used to further test the nature of the interaction between the key components of the α-glucosidase inhibitor obtained by the present invention and α-glucosidase. As shown in Table 3, the binding energy of TFDG, TF-3-G, TSF, TF-3'-G, TSB, TSA, TSD, TF1, thea yellow galloline, and thea yellow acid with α-glucosidase is less than -7.0 kcal·mol-1, indicating that they have a strong affinity for α-glucosidase, can form a stable complex with α-glucosidase, and are potential active ingredients.

[0060] Table 3 Docking results of key components of α-glucosidase inhibitor and substrate PNPG with α-glucosidase

[0061]

[0062] Furthermore, the interaction force analysis between ligand and protein using Pymol and LigPlus software showed that TFDG, TF-3-G, TF-3'-G, TSB, TSA, TSD, TF1 and thea yellow gallol are the key components of OTP inhibiting α-glucosidase, and the inhibitory mechanism of OTP differential metabolites on α-glucosidase can be explored through molecular docking simulation; as shown in Table 3, the binding energies of TFDG, TF-3-G, TF-3'-G, TSB, TSA, TSD, TF1 and thea yellow gallol with α-glucosidase are all less than -8.0 kcal·mol-1, while the binding energy of acarbose is -7.6 kcal / mol, indicating that it has a strong affinity for α-glucosidase and may be more stable than acarbose in binding to α-glucosidase, and is a potential active ingredient.

[0063] Figure 8 It shows that 8 differential metabolites are inserted into the active site of α-glucosidase and interact with various amino acid residues. As shown in Table 3, hydrogen bonds and hydrophobic interactions exist in the docking model, and the lengths of hydrogen bonds formed between the differential metabolites and the amino acid residues near the active site are all within 1.5~2.0 mm. The formation of short hydrogen bonds greatly promotes the formation of complexes between differential metabolites and α-glucosidase, hindering the binding of α-glucosidase and substrate; the cavity composed of amino acid residues tightly wraps the differential metabolite small molecules, produces hydrophobic interactions with the differential metabolites, reduces the involvement of water, and is conducive to the formation of stable complexes. From the perspective of the active site, the differential metabolites bind to the amino acid residues in the active site of α-glucosidase to inhibit its catalytic activity. The catalytic domain of α-glucosidase mainly exists in the 358-720 amino acid residues, and the binding amino acid residues in the active site of α-glucosidase contain arginine (Arg), tryptophan (Trp) and cysteine ​​(Cys)

[41] . OTP differential metabolites bind to α-glucosidase residues in the range of Asp203-Asp777. TFDG binds to four potential active sites of α-glucosidase (Arg 647, Arg 712, Arg 653, Arg 730), TF-3'-G binds to one potential active site of α-glucosidase (Arg 730), TSB binds to two potential active sites of α-glucosidase (Arg 647, Arg 653), TSA and TSD bind to one potential active site of α-glucosidase (Arg283), TF1 binds to one potential active site of α-glucosidase (Arg 730), and theahuanggaloline binds to one potential active site of α-glucosidase (Arg 202). The above results indicate that these differential metabolites occupy the potential active sites of α-glucosidase through hydrogen bonds and hydrophobic interactions, thereby hindering the formation of a complex between α-glucosidase and substrate.

[0064] These results indicate that the inhibitors of the present invention, including the above 8 OTP key substances and their combinations, form complexes with α-glucosidase through hydrogen bonds and hydrophobic interactions, thereby changing the spatial structure of the enzyme and further reducing the enzymatic activity of α-glucosidase.

[0065] Example 1

[0066] In this embodiment, the preparation method of the α-glucosidase inhibitor comprises the following steps:

[0067] Preparation of crude enzyme solution: Peel the crown pear and cut it into pieces to obtain pear pieces, weigh 100 g of pear pieces, pre-freeze the pear pieces at -20°C for 2 h, add appropriate amounts of PVPP and VC as color preservatives after pre-freezing, and add 100 mL of pre-cooled citric acid-phosphate buffer, the pH value of which is 5.6; then mash the pieces, filter them with four layers of gauze, and perform low-temperature high-speed centrifugation on the filtrate at 4°C and 8000 r / min, and take the supernatant after centrifugation for 10 min.

[0068] Slowly and evenly add to the supernatant to make a solution with 70% ammonium sulfate saturation, and the resulting solution is placed in a 4°C refrigerator overnight and then subjected to low-temperature high-speed centrifugation at 4°C, 8000 r / min for 20 min, and the precipitate is collected and dissolved with an appropriate amount of pH 5.6 citric acid-phosphate buffer to obtain a crude enzyme solution with an enzyme activity of 0.94 U / mL;

[0069] Enzymatic reaction: take green tea polyphenol powder with polyphenol content ≥ 98%, dissolve the green tea polyphenol powder in 50 ml of citric acid-phosphate buffer, and use ultrasound to assist dissolution during the dissolution process to obtain a polyphenol solution;

[0070] 1 ml of the crude enzyme solution was added to the tea polyphenol solution to perform an enzymatic reaction. After the enzymatic reaction was completed, the reaction solution was placed in a boiling water bath and heated for 5 minutes to inactivate the enzyme, thereby terminating the enzymatic reaction. The obtained reaction solution was concentrated and freeze-dried to obtain an α-glucosidase inhibitor product.

[0071] In this example, the concentration of tea polyphenols is 0.3 mg / mL, the enzymatic reaction time is 57 min, the enzymatic reaction temperature is 30.5° C., and the enzymatic reaction pH value is 4.1. After testing, the inhibition rate of the product on α-glucosidase is 87.25%.

[0072] Comparative Example 1

[0073] In this comparative example, fragrant pear was used as raw material, and the other operation steps were the same. The inhibition rate of the product on α-glucosidase was 59.58%.

[0074] Comparative Example 2

[0075] In this control example, during the preparation of the crude enzyme solution, the crown pears were directly peeled and pulped, filtered and concentrated to obtain a crude enzyme solution with the same enzyme activity, and the next step of the test was carried out, and the rest of the operations were the same. After testing, the inhibition rate of the product on α-glucosidase was 75.34%.

[0076] As can be seen from the above, in the present invention, in addition to the tea polyphenol concentration, the temperature, pH and reaction time of the enzymatic reaction, the selection of raw material types and the preparation process of the crude enzyme solution have a more critical impact on the final product composition and product efficacy.

[0077] The above shows and describes the basic principles, main features and advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A method for preparing an α-glucosidase inhibitor, characterized in that: The crude enzyme solution is prepared by taking Crown Pear as raw material; the crude enzyme solution is mixed with tea polyphenol solution to carry out enzymatic reaction, and the enzyme is inactivated after the reaction is completed. The reaction solution obtained after the enzyme inactivation or the powder obtained by concentrating and drying the reaction solution is the alpha-glucosidase inhibitor.

2. The method for preparing an α-glucosidase inhibitor according to claim 1, characterized in that: The green tea polyphenols with a polyphenol content of ≥98% are dissolved in a buffer solution to obtain a tea polyphenol solution, wherein the mass concentration of the tea polyphenols in the tea polyphenol solution is 0.2-0.4 mg / mL.

3. The method for preparing an α-glucosidase inhibitor according to claim 1, characterized in that: The enzyme activity of the crude enzyme solution is 0.5-1.5 U / mL, and the volume ratio of the crude enzyme solution to the tea polyphenol solution is 1:45-55.

4. The method for preparing an α-glucosidase inhibitor according to claim 3, characterized in that: The temperature of the enzymatic reaction is 10-50°C, the reaction time is 20-100 minutes, and the reaction pH is 3-7.

5. The method for preparing an α-glucosidase inhibitor according to claim 1, characterized in that: The preparation method of the crude enzyme solution comprises: peeling a crown pear and cutting it into pieces to obtain pear pieces, pre-freezing the pear pieces, adding a color preservative and a buffer solution, crushing them, filtering them, and performing a first centrifugation to obtain a supernatant; adding ammonium sulfate powder to the supernatant to prepare a solution with a saturation of 70% ammonium sulfate, refrigerating the solution in a refrigerator overnight, performing a second centrifugation, obtaining a precipitate, and dissolving the precipitate with a buffer solution to obtain a crude enzyme solution.

6. The method for preparing an α-glucosidase inhibitor according to claim 5, characterized in that: During the first centrifugal treatment, the temperature is 3-5°C, the rotation speed is 7500-8500 r / min, and the centrifugal time is 8-12 min; during the second centrifugal treatment, the temperature is 3-5°C, the rotation speed is 7500-8500 r / min, and the centrifugal time is 18-22 min.

7. The method for preparing an α-glucosidase inhibitor according to claim 1, characterized in that: The steps include: Preparation of crude enzyme solution: peel the crown pear and cut it into pieces to obtain pear blocks, pre-freeze the pear blocks, add color preservative and buffer, crush them, filter them and perform the first centrifugation to obtain the supernatant; add ammonium sulfate powder to the supernatant to prepare a solution with 70% ammonium sulfate saturation, refrigerate the solution in a refrigerator overnight, perform the second centrifugation, obtain the precipitate, and dissolve the precipitate with buffer to obtain the crude enzyme solution; Enzymatic reaction: dissolve green tea polyphenol powder in a buffer solution to obtain a polyphenol solution, mix the polyphenol solution with a crude enzyme solution to carry out an enzymatic reaction, and after the reaction is completed, heat the reaction solution in a boiling water bath for 5 minutes to inactivate the enzyme to terminate the reaction, and the reaction solution obtained after inactivating the enzyme or the powder obtained by concentrating and drying the reaction solution is the α-glucosidase inhibitor.

8. The method for preparing an α-glucosidase inhibitor according to claim 5, characterized in that: The buffer is a citric acid-phosphate buffer, and the pH value of the citric acid-phosphate buffer is 5-6.

9. An α-glucosidase inhibitor, characterized in that The invention is prepared by the method according to any one of claims 1 to 8, wherein the key components of the α-glucosidase inhibitor include TFDG, TF-3-G, TF-3'-G, TSB, TSA, TSD, TF1 and theagrogallin; and the inhibition rate of the α-glucosidase inhibitor on α-glucosidase is greater than 85%.

10. Use of the α-glucosidase inhibitor according to claim 9 in the preparation of a drug for treating diabetes.

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