Application of sorghum 3-deoxyanthocyanin extract in preparation of alpha-amylase and alpha-glucosidase inhibitor

Abstract

The invention belongs to the technical field of agricultural and sideline product processing and utilization, and discloses application of sorghum 3-deoxyanthocyanin extract in preparation of alpha-amylase and alpha-glucosidase inhibitors. The 3-deoxyanthocyanin extracted from sorghum can effectively inhibit alpha-amylase and alpha-glucosidase and can be used for preparing an alpha-amylase and alpha-glucosidase inhibitor, and the inhibitor can replace traditional hypoglycemic drugs, is natural, healthy, free of toxic and side effects and low in cost and has good social benefits. In the process of preparing the sorghum 3-deoxy anthocyanin extract, ball milling is adopted to prepare DES, and an ultrasonic-assisted anti-solvent method is cooperated, so that the method is environment-friendly, the average particle size of 3-deoxy anthocyanin can be remarkably reduced, and the inhibition capability on alpha-amylase and alpha-glucosidase is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of processing and utilization of agricultural and sideline products, and particularly relates to the application of sorghum 3-deoxyanthocyanin extract in the preparation of α-amylase and α-glucosidase inhibitors. Background Art

[0002] Diabetes is a chronic metabolic disorder and is considered an important global public health problem. Excessive intake and rapid digestion of carbohydrates can lead to elevated postprandial blood glucose levels, thereby increasing the risk of the disease. Therefore, dietary strategies are crucial for the prevention and management of diabetes.

[0003] For example, phenolic substances in various fruits and vegetables have been proven beneficial for reducing blood glucose. In recent years, the impact of grains on health has attracted extensive attention. α-Amylase and α-glucosidase are key enzymes in carbohydrate metabolism and can cause elevated blood glucose levels. In recent years, studies at home and abroad have proposed using natural polyphenols as an alternative method to inhibit glycoside hydrolases, thereby delaying starch digestion and glucose absorption. Glycosidase inhibitors from natural grains have low costs and no side effects and have great development potential, which is in sharp contrast to the high costs and frequent gastrointestinal side effects associated with traditional hypoglycemic drugs (such as acarbose, voglibose, and miglitol).

[0004] Sorghum is an important economic crop in China and the fifth largest food crop in the world. The color-forming substances 3-deoxyanthocyanins (3-DAS) in bran and grains are typical bioactive substances that distinguish sorghum from other grains and are mainly composed of luteolinidin, apigeninidin, and their methoxy derivatives. The content of 3-DAS in sorghum bran can reach 4.7 - 16 mg / g and has good antioxidant, anti-inflammatory, and anti-tumor activities. 3-DAS is an analogue of anthocyanin in structure but has irreplaceable superiority over anthocyanin in chemical and biochemical properties. On the one hand, due to the lack of a hydroxyl group at the highly active C-3 position, 3-DAS is more stable than anthocyanins in most plants and is not easily affected by physical and chemical factors such as temperature, light, and pH value; on the other hand, 3-DAS in sorghum mainly exists in the form of free glycosyl ligands, so the bioavailability is relatively high, and it has broad application prospects in the fields of food and biomedicine.

[0005] However, the current basic research on sorghum-derived 3-DAS remains limited. Patent CN118496188A discloses a method for extracting proanthocyanidins from sorghum using an aqueous threonine solution, which has antioxidant and anti-aging effects. Patent CN112385840A discloses a method for preparing low-GL cereal-derived nutritional special dietary foods and their processing from grain cereals rich in 3-DAS. There is currently no detailed literature on the inhibitory effect of sorghum 3-DAS on glycoside hydrolases (α-amylase and α-glucosidase). In addition, more plant active substance extracts come from the extraction with organic reagents. Therefore, the dietary utilization value of 3-DAS is greatly underestimated, and the efforts in deep processing have encountered a "bottleneck" state. The present invention can provide valuable insights for optimizing the utilization of sorghum and its by-products, and at the same time establish a solid foundation and empirical evidence for integrating sorghum-derived 3-DAS into the development of low glycemic index foods. It can also be widely applied in the fields of ordinary foods, health foods, foods for special medical purposes, and pharmaceuticals, providing new solutions for foods for people with obesity, diabetes, etc. Summary of the Invention

[0006] To solve the problems in the background art, the present invention provides the application of extracting active ingredients from sorghum in α-amylase and α-glucosidase inhibitors.

[0007] To achieve the above object, the technical solution adopted by the present invention is: Application of sorghum 3-deoxyanthocyanin extract in the preparation of α-amylase and α-glucosidase inhibitors.

[0008] Preferably, the average particle size of 3-deoxyanthocyanins in the sorghum 3-deoxyanthocyanin extract is 100-300 nm.

[0009] Preferably, the preparation method of the sorghum 3-deoxyanthocyanin extract is as follows: Dry and crush sorghum seeds and their processing by-products into sorghum powder; Mix a hydrogen bond donor and a hydrogen bond acceptor and perform ball milling to obtain a deep eutectic solvent (DES); Mix the deep eutectic solvent, deionized water and sorghum powder, perform ultrasonic extraction, then centrifuge and retain the supernatant to obtain a sorghum 3-deoxyanthocyanin extract solution; Treat the sorghum 3-deoxyanthocyanin extract solution by ultrasonic-assisted antisolvent method to obtain a sorghum 3-deoxyanthocyanin suspension, and obtain a sorghum 3-deoxyanthocyanin extract after freeze-drying.

[0010] Preferably, the hydrogen bond acceptor includes any one of choline chloride, acetylcholine and betaine, and the hydrogen bond donor includes any one of oxalic acid, glutaric acid, lactic acid, tartaric acid, citric acid, malonic acid, malic acid, acetylsalicylic acid and L-ascorbic acid.

[0011] Preferably, the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is (2:1) to (1:2).

[0012] Preferably, the mass ratio of the balls to the material in the ball milling is (1 - 30):1, the rotation speed of the ball milling is 300 - 900 rpm, and the ball milling time is 40 - 120 min.

[0013] Preferably, in the ultrasonic - assisted antisolvent method, after mixing the sorghum 3 - deoxyanthocyanin extract with the antisolvent according to a volume ratio of 1:(1 - 25), it is dispersed under ultrasonic for 5 - 30 min.

[0014] Preferably, the ultrasonic power in the ultrasonic - assisted antisolvent method is 200 - 500 w.

[0015] Preferably, the antisolvent is deionized water.

[0016] Preferably, the ultrasonic power of the ultrasonic extraction is 200 - 500 w, the extraction temperature is 30 - 45 °C, and the material - liquid ratio of the sorghum powder, the deep eutectic solvent, and deionized water is (5 - 30):1 g / mL.

[0017] Compared with the prior art, the present invention has the following effects: The 3 - deoxyanthocyanin extracted from sorghum provided by the present invention can effectively inhibit α - amylase and α - glucosidase, and can be used to prepare inhibitors of α - amylase and α - glucosidase. This inhibitor can replace traditional hypoglycemic drugs, is natural, healthy, non - toxic and has low cost, and has good social benefits.

[0018] In the process of preparing the sorghum 3 - deoxyanthocyanin extract, the present invention uses ball milling to prepare DES and combines it with the ultrasonic - assisted antisolvent method. Not only is the method environmentally friendly, but it can also significantly reduce the average particle size of 3 - deoxyanthocyanin and improve the inhibitory ability against α - amylase and α - glucosidase. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 In (a), it is the inhibitory effect diagram of the sorghum 3 - DAS extract obtained with choline chloride - malic acid as DES in Example 1 and Comparative Example 2, and acarbose at different concentrations on α - amylase; in (b), it is the inhibitory effect diagram of the sorghum 3 - DAS extract obtained with choline chloride - malic acid as DES in Example 1 and Comparative Example 2, and acarbose at different concentrations on α - glucosidase. Figure 2Figure (a) shows the particle size distribution of 3-DAS in the sorghum 3-DAS extract obtained with choline chloride-malonic acid as the DES in Comparative Example 1; (b) shows the particle size distribution of 3-DAS in the sorghum 3-DAS extract obtained with choline chloride-malonic acid as the DES in Example 1. Figure 3 Figure (a) shows the inhibition effect diagrams of the sorghum 3-DAS extract obtained with choline chloride-malonic acid as the DES in Example 1 and Comparative Example 1, and acarbose on α-amylase at different concentrations; (b) shows the inhibition effect diagrams of the sorghum 3-DAS extract obtained with choline chloride-malonic acid as the DES in Example 1 and Comparative Example 1, and acarbose on α-glucosidase at different concentrations. Detailed implementation manners

[0020] To make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with specific implementation manners. It should be understood that these descriptions are merely exemplary and are not intended to limit the scope of the present invention. In addition, in the following description, the descriptions of well-known structures and technologies are omitted to avoid unnecessarily confusing the concepts of the present invention. For those not specified in the examples, they are carried out under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments not specified by the manufacturer are all conventional products that can be obtained through commercial purchase.

[0021] The implementation manner of the present invention provides an application of a sorghum 3-deoxyanthocyanin extract in the preparation of an α-amylase and an α-glucosidase inhibitor.

[0022] The average particle size of 3-deoxyanthocyanins in the sorghum 3-deoxyanthocyanin extract is 100-300 nm. The nano-scale particles have a high surface area, good solubility, and high biological activity, can significantly improve the inhibition ability against α-amylase and α-glucosidase, and are also more conducive to human absorption.

[0023] The preparation method of the sorghum 3-deoxyanthocyanin extract in the implementation manner of the present invention is as follows: Dry and crush sorghum seeds and their processing by-products into sorghum powder; mix a hydrogen bond donor and a hydrogen bond acceptor and then carry out ball milling to obtain a deep eutectic solvent; mix the deep eutectic solvent, deionized water and sorghum powder, after ultrasonic extraction, centrifuge and retain the supernatant to obtain a sorghum 3-deoxyanthocyanin extract; treat the sorghum 3-deoxyanthocyanin extract by ultrasonic-assisted antisolvent method to obtain a sorghum 3-deoxyanthocyanin suspension, and obtain a sorghum 3-deoxyanthocyanin extract after freeze-drying.

[0024] The key to this preparation method lies in the use of ball milling to prepare DES and ultrasonic-assisted antisolvent dispersion: preparing DES by ball milling can increase the extraction content of sorghum 3-DAS compared with preparing DES by heating, reduce the IC 50 value of the inhibition of α-amylase and α-glucosidase, and improve the enzyme inhibition ability. Treating the sorghum 3-deoxyanthocyanin extract by the ultrasonic-assisted antisolvent method can reduce the average particle size of 3-DAS to nanoscale particles, significantly improve the inhibition ability against α-amylase and α-glucosidase. The average particle size of 3-DAS in the sorghum 3-DAS extract provided by the present invention is much smaller than that of 3-DAS precipitated directly without ultrasonic dispersion, and the enzyme inhibition activity is also significantly improved, and both are superior to the enzyme inhibition effects of commercially available inhibitors.

[0025] Regarding the specific parameters of ball milling, those skilled in the art can select appropriate parameters according to the desired effects. In some preferred embodiments, the mass ratio of balls to materials for ball milling is (1~30):1, the ball milling speed is 300~900 rpm, and the ball milling time is 40~120 min.

[0026] In the process of preparing DES, the hydrogen bond acceptor and hydrogen bond donor are common components in the art. For example, the hydrogen bond acceptor includes any one of choline chloride, acetylcholine, and betaine, and the hydrogen bond donor includes any one of oxalic acid, glutaric acid, lactic acid, tartaric acid, citric acid, malonic acid, malic acid, acetylsalicylic acid, and L-ascorbic acid.

[0027] There is no special limitation on the dosages of the hydrogen bond acceptor and hydrogen bond donor, and those skilled in the art can select them according to the desired effects. In some preferred embodiments, the molar ratio of the hydrogen bond acceptor to the hydrogen bond donor is (3:1)~(1:3).

[0028] In some preferred embodiments, the ultrasonic power for ultrasonic extraction is 200~500 w, the extraction temperature is 30~45 °C, and the material-liquid ratio of sorghum powder, deep eutectic solvent, and deionized water is (5~30):1 g / mL. The extraction time is generally 30~90 min. It should be noted that the material-liquid ratio of sorghum powder, deep eutectic solvent, and deionized water refers to the ratio of the mass of sorghum powder to the total volume of the deep eutectic solvent and deionized water, and the volume of deionized water accounts for 5%~25% of the total volume. At this deionized water content, the viscosity of DES is lower, which is more conducive to subsequent extraction.

[0029] There is no special limitation on the specific parameters of centrifugation, and those skilled in the art can select appropriate centrifugation parameters according to the centrifugation object.

[0030] In some preferred embodiments, in order to further improve the extraction efficiency, the sorghum powder can be the powder after ball milling.

[0031] In some preferred embodiments, the sorghum processing by-products include glumes, bran, etc.

[0032] Specifically, for the ultrasonic-assisted anti-solvent method, after mixing the sorghum 3-deoxyanthocyanin extract with the anti-solvent at a volume ratio of 1: (1-25), it is dispersed under ultrasonic waves for 5-30 min, and the ultrasonic power is 200-500 w.

[0033] For the mixing method of the sorghum 3-deoxyanthocyanin extract and the anti-solvent, it is more preferably to add the sorghum 3-deoxyanthocyanin extract dropwise to the anti-solvent, and ultrasonic stirring can be carried out simultaneously during the addition process.

[0034] In some preferred embodiments, the anti-solvent is deionized water.

[0035] To make the technical solution of the present invention clearer, the following provides a plurality of specific examples to illustrate the application and effect of the sorghum 3-deoxyanthocyanin extract in the preparation of α-amylase and α-glucosidase inhibitors.

[0036] The following detection methods are used: (1) Determination of the content of sorghum 3-DAS extract The pH differential method is used to determine the content of 3-DAS in the sorghum 3-DAS extract. The methanol solution of the sorghum 3-DAS extract is diluted to different concentrations with buffers of pH 1.0 and 4.5 respectively to determine the dilution factor (within the linear range of the spectrophotometer of 0.2-1.2), and the absorbance values are measured at the maximum absorption wavelengths of 483 nm and 700 nm of sorghum 3-DAS. Finally, in combination with the Fuleki formula (1), the content of 3-DAs is calculated based on the apigeninidin monomer.

[0037] (1); In the formula: A = (A 483 nm -A 700 nm ) pH1.0 -(A 483 nm -A 700 nm ) pH4.5 ; A 483 nm and A 700 nm are the absorbance values of the dilution solution at 483 nm and 700 nm, (A 483nm -A 700 nm ) pH1.0 is the difference in absorbance of the dilution solution at different concentrations when pH is 1; (A 483 nm -A 700 nm ) pH4.5is the difference in absorbance of the dilution solution when the pH is 4.5; A is the difference in absorbance under different pH conditions. MW is the relative molecular mass, and the MW of Apigeninidin is 255.24 g / mol; DF is the dilution factor, and its value is the volume after dilution / the volume before dilution; ε is the molar extinction coefficient: 30400 L·mol –1   × cm -1 ; L is the optical path / cm; V is the total volume of the extraction solution / mL; m is the sample mass / g.

[0038] (2)Determination of the inhibitory activities of sorghum 3-DAS extract against α-amylase and α-glucosidase Mix 250 μL of 8 U / mL α-amylase phosphate solution (0.1 mol / L, pH = 6.8) with 250 μL of sorghum 3-DAS extract sample solution with a series of gradient concentrations (1, 5, 10, 25, 50, 100, and 200 μg / mL). At the same time, use the acarbose solution with the same concentration gradient as the positive control, and incubate at 37 °C for 30 min; then add the starch solution with a mass concentration of 10 mg / mL, and react at 37 °C for 3 min; finally, add 500 μL of DNS color reagent, mix well, place in boiling water and react for 5 min, then immediately place in an ice-water bath to cool to room temperature, dilute to an appropriate concentration, and use a multi-functional microplate reader to read the absorbance at 540 nm.

[0039] Mix 50 μL of 0.5 U / mL α-glucosidase solution (0.1 mol / L, pH = 6.8) preheated to 37 °C with 50 μL of sorghum 3-DAS extract methanol solution with a series of gradient concentrations (0.1, 0.5, 1, 2, 5, 7.5, and 10 μg / mL) in a 96-well plate. At the same time, use the acarbose solution with the same concentration gradient as the positive control, pre-incubate at 37 °C for 15 min, add 100 μL of PNPG substrate solution with a mass concentration of 3.0 mg / mL, mix well, and incubate at 37 °C for 15 min. Finally, add 50 μL of Na with a concentration of 0.67 mol / L 2 CO 3 to terminate the reaction, and use a multi-functional microplate reader to measure the absorbance of the sample at 405 nm. Calculate the inhibition rate according to the following formula, and use Origin software to obtain the corresponding IC 50 value.

[0040] ; Where: Aa is the absorbance value of the sample group, Ab is the absorbance value of the sample blank group (an equal volume of methanol solution replaces the sample solution), Ac is the absorbance value of the control group (an equal volume of phosphate buffer replaces the enzyme solution), and Ad is the absorbance value of the control blank group (an equal volume of methanol solution and an equal volume of phosphate buffer replace the sample and the enzyme solution).

[0041] Example 1 Preparation of Enzyme Inhibitor from Sorghum 3-DAS Extract Dry the sorghum grains and their processing by-products, control their water content below 7%, crush them and pass through an 80-mesh sieve to obtain sorghum powder.

[0042] Mix choline chloride and organic carboxylic acid (specific components are shown in Table 1) in a molar ratio of 1:2 and then carry out ball milling to obtain DES. The mass ratio of the ball to the material is 1:1, the ball milling speed is 900 rpm, and the ball milling time is 40 min; mix DES, deionized water and sorghum powder in a solid-liquid ratio of 5:1 g / mL (deionized water is 25% of the total volume), oscillate and extract for 30 min under the conditions of an ultrasonic power of 200 w and a temperature of 35 °C, then centrifuge at 8000 rpm for 15 min, and discard the bottom solids to obtain the sorghum 3-DAS extract.

[0043] Under the condition of an ultrasonic power of 200 w, dropwise add the sorghum 3-DAS extract to deionized water. The volume ratio of the sorghum 3-DAS extract to deionized water is 1:5. After complete dropping, continue ultrasonic dispersion for 5 min to form a sorghum 3-DAS suspension. After freeze-drying the sorghum 3-DAS suspension, obtain the nano-sized particle enzyme inhibitor of the sorghum 3-DAS extract.

[0044] Determine the content of 3-DAS, the average particle size in the sorghum 3-DAS extract extracted in this example, and the IC 50 value of its inhibitory activity against α-amylase and α-glucosidase. The results are shown in Table 1.

[0045] Table 1: Effects of Different Extraction Solvents on the Content, Size of Sorghum 3-DAS and the IC 50 Value of the Inhibitory Activity of Sorghum 3-DAS against α-Amylase and α-Glucosidase .

[0046] As can be seen from Table 1, among all the extraction reagents, choline chloride-malic acid DES shows the best extraction effect. The size of the prepared sorghum 3-DAS nanoparticle enzyme preparation is the smallest, and the IC 50 value for α-amylase is in the range of 18.98 - 39.90 μg / mL, and the IC 50 value for α-glucosidase is in the range of 0.85 - 1.32 μg / mL, and the IC 50All the values were lower than those of the positive control acarbose, indicating that the sorghum 3-DAS nanoparticle enzyme preparation had excellent glycoside hydrolase inhibitory ability and had the potential to be applied in the fields of obesity and diabetes.

[0047] The inhibitory effects of the sorghum 3-DAS extract and acarbose obtained when the above DES was choline chloride-malic acid on α-amylase and α-glucosidase at different concentrations are respectively as Figure 1 shown by "ball milling to prepare DES" and "acarbose" in (a) and 1 (b), Figure 3 shown by "after ultrasonic treatment" and "acarbose" in (a) and 3 (b); the particle size distribution of 3-DAS in the sorghum 3-DAS extract obtained when the above DES was choline chloride-malic acid is as Figure 2 shown in (b).

[0048] Example 2 Preparation of sorghum 3-DAS extract enzyme inhibitor Dry the sorghum grains and their processing by-products, control their water content below 7%, crush them and pass through an 80-mesh sieve to obtain sorghum powder.

[0049] Mix choline chloride and malic acid in a molar ratio of 2:1 and then carry out ball milling to obtain DES, with a ball-to-material mass ratio of 30:1, a ball milling speed of 300 rpm, and a ball milling time of 120 min; mix DES, deionized water and sorghum powder in a solid-liquid ratio of 30:1 g / mL (deionized water is 5% of the total volume), oscillate and extract for 90 min under the conditions of an ultrasonic power of 500 w and a temperature of 45 °C, and then centrifuge at 8000 rpm for 15 min to discard the bottom solids to obtain the sorghum 3-DAS extract.

[0050] Under the condition of an ultrasonic power of 500 w, dropwise add the sorghum 3-DAS extract to deionized water, and set the volume ratio of the sorghum 3-DAS extract to deionized water as a gradient of (1:1) to (1:25) (specifically shown in Table 2). After complete dropping, continue ultrasonic dispersion for 30 min to form a sorghum 3-DAS suspension. After freeze-drying the sorghum 3-DAS suspension, a sorghum 3-DAS extract nano-sized particle enzyme inhibitor is obtained.

[0051] Determine the content, average particle size of 3DAS in the sorghum 3-DAS extract extracted in this example and the IC 50 value of its inhibitory activity against α-amylase and α-glucosidase. The results are shown in Table 2.

[0052] Table 2: Effects of different volume ratios of sorghum 3-DAS extract to deionized water on the content, size of sorghum 3-DAS and the IC 50 value of sorghum 3-DAS inhibitory activity against α-amylase and α-glucosidase .

[0053] As can be seen from Table 2, when preparing the sorghum 3-DAS nanoparticle enzyme preparation by the anti-solvent precipitation method, the volume ratio of the sorghum 3-DAS extract to deionized water is an important factor. Experiments show that when the volume ratio of the sorghum 3-DAS extract to deionized water is set to 1:15, the size of the prepared sorghum 3-DAS nanoparticles is the smallest, and the inhibitory ability against the two glycoside hydrolases is the strongest, and the IC 50 value is less than acarbose.

[0054] Example 3 Preparation of Sorghum 3-DAS Extract Enzyme Inhibitor Dry the sorghum grains and their processing by-products, control the moisture content below 7%, crush them and pass through an 80-mesh sieve to obtain sorghum powder.

[0055] Mix choline chloride and malic acid in a molar ratio of 1:1 and then carry out ball milling to obtain DES, with a ball-to-material mass ratio of 20:1, a ball milling speed of 500 rpm, and a ball milling time of 50 min; mix DES, deionized water and sorghum powder according to a solid-to-liquid ratio of 15:1 g / mL (deionized water is 15% of the total volume), oscillate and extract for 60 min under the conditions of an ultrasonic power of 300 w and a temperature of 40 °C, then centrifuge at 8000 rpm for 15 min, discard the bottom solids, and obtain the sorghum 3-DAS extract.

[0056] Under the conditions of different ultrasonic powers (200 - 500 w, see Table 3 for details), slowly add the sorghum 3-DAS extract dropwise to deionized water, and set the volume ratio of the sorghum 3-DAS extract to deionized water to 1:15. After complete addition, continue ultrasonic dispersion for 10 min to form a sorghum 3-DAS suspension. After freeze-drying the 3-DAS suspension, a sorghum 3-DAS extract nano-sized particle enzyme inhibitor is obtained.

[0057] Determine the content of 3-DAS, the average particle size, and the IC 50 value of its inhibitory activity against α-amylase and α-glucosidase in the sorghum 3-DAS extract extracted in this example, and the results are shown in Table 3.

[0058] Table 3: Effects of Different Ultrasonic Powers on the Content, Size of Sorghum 3-DAS and the IC 50 Value of Its Inhibitory Activity against Sorghum 3-DAS Inhibiting α-Amylase and α-Glucosidase .

[0059] As can be seen from Table 3, under the optimized conditions, the effects of different ultrasonic powers (200 - 500 w) on the content and size of sorghum 3 - DAS, and the IC 50 values of the inhibitory activities of sorghum 3 - DAS against α - amylase and α - glucosidase were studied. It was found that at 400 w, the size of sorghum 3 - DAS nanoparticles was the smallest, reaching 0.098 μm, and the IC 50 values against the two glycoside hydrolases also decreased. The decrease in particle size enhanced the inhibitory ability of sorghum 3 - DAS against α - amylase and α - glucosidase. In this example, the application of sorghum 3 - DAS enzyme preparation was optimized.

[0060] Comparative Example 1 Different from the method described in Example 1, after obtaining the sorghum 3 - DAS extract, the steps of drop - wise adding the sorghum 3 - DAS extract to deionized water and ultrasonic treatment were cancelled. Instead, deionized water was directly added to the sorghum 3 - DAS extract, and after precipitation for 24 h, it was freeze - dried to obtain the sorghum 3 - DAS extract enzyme preparation. Other experimental steps were the same as those in Example 1.

[0061] The content of 3 - DAS, the average particle size, and the IC 50 values of the inhibitory activities of the sorghum 3 - DAS extract obtained in this comparative example against α - amylase and α - glucosidase were measured, and the results are shown in Table 4.

[0062] Table 4: Effects of Canceling Ultrasonic Treatment on the Content and Size of Sorghum 3 - DAS and the IC 50 Values of the Inhibitory Activities of Sorghum 3 - DAS against α - Amylase and α - Glucosidase .

[0063] As can be seen from Comparative Example 1, the sorghum 3 - DAS extracted by DES without ultrasonic stirring and dispersion had a larger average particle size and larger IC 50 values, that is, the inhibitory ability against α - amylase and α - glucosidase decreased significantly. After the particle size increased, it was not conducive to digestion and dissolution, and the enzyme - inhibiting effect could not be exerted. This shows that the ultrasonic - assisted anti - solvent precipitation method is a key step in the preparation of sorghum 3 - DAS nanoparticles enzyme preparation. The IC 50 values of all sorghum 3 - DAS extracts against α - amylase and α - glucosidase were less than those of their positive control acarbose, indicating that the sorghum 3 - DAS extract is a good inhibitor of α - amylase and α - glucosidase.

[0064] The inhibitory effects of the sorghum 3 - DAS extract obtained using choline chloride - malic acid as DES on α - amylase and α - glucosidase at different concentrations are as shown in Figure 3as shown by "before ultrasound"; the particle size distribution of 3-DAS in the sorghum 3-DAS extract obtained using choline chloride-malonic acid as the DES is as shown in Figure 2 (a).

[0065] Comparative Example 2 Different from the method described in Example 1, the preparation of DES by ball milling was cancelled, and the DES was prepared by heating in an oil bath at 60 °C. Other experimental steps were the same as those in Example 1.

[0066] The content, average particle size of 3-DAS in the sorghum 3-DAS extract extracted in this comparative example, and the IC 50 value of its inhibitory activity against α-amylase and α-glucosidase were measured, and the results are shown in Table 5.

[0067] Table 5: Influence of cancelling ball milling in the preparation of DES on the content, size of sorghum 3-DAS, and the IC 50 value of the inhibitory activity of sorghum 3-DAS against α-amylase and α-glucosidase .

[0068] It can be seen from Comparative Example 2 that after cancelling ball milling in the DES preparation process, while the extraction content of sorghum 3-DAS decreased, the average particle size of sorghum 3-DAS nanoparticles increased, and the IC 50 value also increased, that is, the inhibitory ability against α-amylase and α-glucosidase decreased significantly. This shows that the 3-DAS prepared by ball milling to prepare DES is a key step in the preparation of α-amylase and α-glucosidase inhibitors. And the IC 50 values of all sorghum 3-DAS extracts against α-amylase and α-glucosidase were less than those of their positive control acarbose, indicating that the sorghum 3-DAS extract is a good α-amylase and α-glucosidase inhibitor and has the potential to be applied in the fields of weight loss and hypoglycemic food and drugs.

[0069] The inhibitory effects of the sorghum 3-DAS extract obtained using choline chloride-malonic acid as the DES on α-amylase and α-glucosidase at different concentrations are as shown in Figure 1 "heating to prepare DES".

[0070] Comparative Example 3 Different from the method described in Example 1, the preparation of DES by ball milling was cancelled, and the DES was prepared by heating in an oil bath at 60 °C. The step of dropping the sorghum 3-DAS extract into deionized water and ultrasonic treatment was cancelled, and deionized water was directly added to the sorghum 3-DAS extract. After precipitation for 24 h, the sorghum 3-DAS extract enzyme preparation was freeze-dried. Other experimental steps were the same as those in Example 1.

[0071] The content of 3-DAS, average particle size in the sorghum 3-DAS extract obtained in this comparative example, and the IC 50 value of its inhibitory activities against α-amylase and α-glucosidase were measured, and the results are shown in Table 6.

[0072] Table 6: Effects of canceling ball milling for DES preparation and ultrasonic dispersion on the content and size of sorghum 3-DAS and the IC 50 value of the inhibitory activities of sorghum 3-DAS against α-amylase and α-glucosidase .

[0073] As can be seen from Table 6, after canceling the ball milling in the DES preparation process and the ultrasonic treatment of the sorghum 3-DAS extract with deionized water, the extraction content of the sorghum 3-DAS extracted decreased significantly, the average particle size of the sorghum 3-DAS particles increased significantly, and the IC 50 value increased, that is, the inhibitory ability against α-amylase and α-glucosidase decreased significantly. This indicates that the synergistic effect of ball milling in the DES preparation process and the ultrasonic dispersion of the sorghum 3-DAS extract with deionized water is a key step in the preparation of sorghum 3-DAS α-amylase and α-glucosidase inhibitors.

[0074] From Figures 1 to 3 it can be seen that ball milling for DES preparation as an extraction reagent for 3-DAS can effectively enhance the biological inhibitory ability of 3-DAS against enzymes. Therefore, the ball milling step is a key step to enhance the enzyme inhibition effect; the particle size of the sorghum 3-DAS nanoparticles decreases after ultrasonic assistance (sorghum 3-DAS is added to the antisolvent); the inhibitory effect of the sorghum 3-DAS nanoparticle extract prepared by ultrasonic antisolvent precipitation on α-amylase and α-glucosidase is significantly improved, indicating that the ultrasonic antisolvent precipitation method is a key step in the preparation of 3-DAS enzyme preparations.

[0075] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of sorghum 3-deoxyanthocyanidin extract in the preparation of α-amylase and α-glucosidase inhibitors.

2. The use according to claim 1, characterized in that The average particle size of 3-deoxyanthocyanidin in sorghum 3-deoxyanthocyanidin extract is 100~300 nm.

3. The use according to claim 1, characterized in that The preparation method of sorghum 3-deoxyanthocyanidin extract is as follows: drying and crushing sorghum seeds and their processing byproducts into sorghum powder; The hydrogen bond donor and the hydrogen bond acceptor are mixed and ball milled to obtain a deep eutectic solvent; The deep eutectic solvent, deionized water and sorghum powder are mixed, and after ultrasonic extraction, the mixture is centrifuged and the supernatant is retained to obtain a sorghum 3-deoxyanthocyanidin extract; The sorghum 3-deoxyanthocyanidin extract was treated with an ultrasound-assisted anti-solvent method to obtain a sorghum 3-deoxyanthocyanidin suspension, which was then freeze-dried to obtain a sorghum 3-deoxyanthocyanidin extract.

4. The use according to claim 3, characterized in that The hydrogen bond acceptor includes any one of choline chloride, acetylcholine and betaine, and the hydrogen bond donor includes any one of oxalic acid, glutaric acid, lactic acid, tartaric acid, citric acid, malonic acid, malic acid, acetylsalicylic acid and L-ascorbic acid.

5. The use according to claim 3 or 4, characterized in that: The molar ratio of hydrogen bond acceptor to hydrogen bond donor is (2:1)~(1:2).

6. The use according to claim 3, characterized in that The ball-to-material mass ratio of the ball mill is (1-30):1, the ball mill speed is 300-900 rpm, and the ball mill time is 40-120 min.

7. The use according to claim 3, characterized in that The ultrasonic-assisted anti-solvent method comprises mixing the sorghum 3-deoxyanthocyanidin extract with the anti-solvent in a volume ratio of 1: (1-25), and then dispersing the mixture under ultrasound for 5-30 minutes.

8. The use according to claim 7, characterized in that The ultrasonic power in the ultrasound-assisted antisolvent method is 200~500 w.

9. The use according to claim 3 or 7, characterized in that: The antisolvent was deionized water.

10. The use according to claim 3, characterized in that The ultrasonic power of the ultrasonic extraction is 200-500 W, the extraction temperature is 30-45 ° C, and the material-liquid ratio of sorghum powder to low eutectic solvent and deionized water is (5-30):1 g / mL.

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