Eutectic solvent and application thereof

Abstract

The invention relates to the technical field of plant effective component extraction, in particular to a deep-eutectic solvent and application thereof, and the deep-eutectic solvent is composed of anhydrous betaine, DL-malic acid and deionized water. The eutecticevaporate solvent is applied to extraction of momordica grosvenori polysaccharide, ultrasonic waves are combined with the eutecticevaporate solvent, in addition, extraction conditions of crude momordica grosvenori polysaccharide are optimized, and a new method is provided for efficient and green extraction of momordica grosvenori polysaccharide. The new method not only expands the application range of DESs in natural product extraction, but also provides important technical reference for industrial development of momordica grosvenori polysaccharide.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant active ingredient extraction, and particularly relates to a deep eutectic solvent and its application.

Background Art

[0002] Deep Eutectic Solvents: First reported by Abbott et al. from the University of Leicester, UK in 2003. It refers to a two-component or three-component eutectic mixture composed of a hydrogen bond acceptor (such as quaternary ammonium salt) and a hydrogen bond donor (such as amide, carboxylic acid, polyol and other compounds) in a certain stoichiometric ratio, and its freezing point is significantly lower than the melting points of the pure substances of each component. This solvent is non-toxic, biodegradable, and the atom utilization rate in the synthesis process reaches 100%, which is a new type of green solvent. The physicochemical properties of deep eutectic solvents are extremely similar to those of ionic liquids, so some people also classify it as a new type of ionic liquid or ionic liquid analog. At present, deep eutectic solvents are mainly applied in two aspects: extraction of bioactive components (such as proteins, nucleic acids, phenols, carrageenan, metal ions, etc.) and extractive distillation (such as separation of aromatics and non-aromatics, separation of alcohol and water, separation of alcohol and ester, etc.).

[0003] Momordica grosvenori, as a natural plant with both medicinal and edible uses, has attracted much attention at home and abroad due to its unique medicinal and edible values. In terms of extraction methods, the research on Momordica grosvenori polysaccharide mainly focuses on hot water extraction method, and some studies are supplemented with strengthening means such as ultrasonic and microwave. Although the traditional hot water extraction technology is simple to operate, it has disadvantages such as wasting time, polluting the environment and high cost. Deep Eutectic Solvents (DESs) have the advantages of low price, simple synthesis, environmental protection and non-toxicity, and are widely used in the field of natural product extraction, but there is no relevant report on extracting polysaccharide from Momordica grosvenori with DESs.

Summary of the Invention

[0004] In view of the above content, it is necessary to provide a deep eutectic solvent and its application. The deep eutectic solvent proposed by the present invention is applied to the extraction of Momordica grosvenori polysaccharide, providing a new method for efficient and green extraction of Momordica grosvenori polysaccharide.

[0005] To achieve the above object, the technical solution adopted by the present invention is:

[0006] A deep eutectic solvent, which is composed of anhydrous betaine, DL-malic acid and deionized water. The hydrogen bond acceptor is anhydrous betaine, and the hydrogen bond donor is DL-malic acid.

[0007] In the present invention, further, the molar ratio of anhydrous betaine to DL-malic acid is 1:2 - 3.

[0008] In the present invention, further, the molar ratio of anhydrous betaine to DL-malic acid is 1:3.

[0009] In the present invention, further, the preparation method of the deep eutectic solvent is as follows: Using anhydrous betaine as the hydrogen bond acceptor and DL-malic acid as the hydrogen bond donor, add them into a beaker. Then add deionized water into the beaker and heat it in a water bath at 75 - 85 °C for 1 - 2 h until the solution becomes clear and transparent, thus obtaining the deep eutectic solvent.

[0010] In the present invention, further, the water content of the deep eutectic solvent is 30%.

[0011] The present invention also provides a method for extracting momordica grosvenori polysaccharide using the above-mentioned deep eutectic solvent, which is characterized in that the method is as follows: Weigh momordica grosvenori powder, mix it with the deep eutectic solvent, then perform ultrasonic extraction on the mixed solution. Subsequently, centrifuge to obtain the supernatant, repeat the operation once for the residue, combine the two filtrates, centrifuge the filtrate at 10000 rpm for 8 - 12 min, collect the supernatant. Then add 1 / 4 volume of Sevag reagent (chloroform: n-butanol = 4:1) to the supernatant, place it on a magnetic stirrer and continuously stir for 25 - 35 min to remove proteins, and repeat this step until no protein precipitate appears in the middle layer. After protein removal is completed, collect the supernatant, perform overnight alcohol precipitation treatment with absolute ethanol at 3 - 5 °C, redissolve the precipitate in deionized water, and use a dialysis membrane to dialyze for 24 h to remove small molecule impurities. Finally, place the dialyzed solution in a freeze dryer for freeze-drying treatment to finally obtain the momordica grosvenori polysaccharide sample.

[0012] In the present invention, further, the liquid-solid ratio of the momordica grosvenori powder to the deep eutectic solvent is 1:20 - 30.

[0013] In the present invention, further, the temperature of the ultrasonic extraction is 50 - 70 °C, the ultrasonic power is 300 - 400 W, and the ultrasonic extraction time is 30 - 40 min.

[0014] In the present invention, further, the temperature of the ultrasonic extraction is 50 °C, the ultrasonic power is 300 W, and the ultrasonic extraction time is 40 min.

[0015] The present invention also provides the application of the momordica grosvenori polysaccharide prepared by the above method, and the momordica grosvenori polysaccharide can be used for preparing hypoglycemic foods and / or drugs.

[0016] The present invention also provides a hypoglycemic food and / or drug, and the hypoglycemic food and / or drug includes the momordica grosvenori polysaccharide prepared according to the above method.

[0017] The present invention has the following beneficial effects:

[0018] 1. The present invention provides a deep eutectic solvent, which is composed of anhydrous betaine, DL-malic acid and deionized water. The applicant first proposes to apply this deep eutectic solvent to extract Momordica grosvenori polysaccharide and finds that the molar ratio of the components and the water content of the deep eutectic solvent have certain effects on the extraction yield of the polysaccharide. Moreover, deep eutectic solvents composed of different hydrogen bond acceptors and hydrogen bond donors also have different effects on the extraction yield of Momordica grosvenori polysaccharide. The extractant of the present application has a high extraction yield, is green, safe, recyclable, has mild reaction conditions and is easy to operate, and has a great cost advantage over conventional extractants.

[0019] 2. The Momordica grosvenori polysaccharide extracted by the deep eutectic solvent and method of the present invention can be applied to the preparation of hypoglycemic foods and / or drugs. Experiments find that the prepared Momordica grosvenori polysaccharide has inhibitory effects on α-glucosidase and α-amylase. Therefore, by inhibiting the activities of α-glucosidase and α-amylase, the digestion process of starch can be effectively delayed, thereby significantly reducing the increase in postprandial blood glucose level and reducing the formation of advanced glycation end products.

Description of the Drawings

[0020] Figure 1 is the extraction yield of polysaccharide under different deep eutectic solvent compositions;

[0021] Figure 2 is the polysaccharide extraction rate under different molar ratios ( Figure 2 (a)) and different water contents ( Figure 2 (b));

[0022] Figure 3 is the influence of different extraction parameters on the yield of Momordica grosvenori polysaccharide; Figure 3 (a) represents the liquid-solid ratio, Figure 3 (b) represents the extraction temperature, Figure 3 (c) represents the ultrasonic power, Figure 3 (d) represents the extraction time.

Detailed Embodiments

[0023] To make the above objects, features and advantages of the present invention more obvious and understandable, the following detailed description of the specific embodiments of the present invention will be given with reference to the accompanying drawings. Many specific details are set forth in the following description in order to fully understand the present invention. However, the present invention can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without departing from the connotation of the present invention. Therefore, the present invention is not limited by the specific embodiments disclosed below.

[0024] Example 1:

[0025] This embodiment provides a deep eutectic solvent, which is mainly composed of anhydrous betaine and DL-malic acid in a molar ratio of 1:2. The preparation method is as follows: Using anhydrous betaine as the hydrogen bond acceptor and DL-malic acid as the hydrogen bond donor, add them into a beaker. Then add deionized water into the beaker and heat it in a water bath at 75 °C for 2 h until the solution becomes clear and transparent, thus obtaining a deep eutectic solvent with a water content of 30%.

[0026] Example 2:

[0027] This embodiment provides a deep eutectic solvent, which is mainly composed of anhydrous betaine and DL-malic acid in a molar ratio of 1:3. The preparation method is as follows: Using anhydrous betaine as the hydrogen bond acceptor and DL-malic acid as the hydrogen bond donor, add them into a beaker. Then add deionized water into the beaker and heat it in a water bath at 80 °C for 2 h until the solution becomes clear and transparent, thus obtaining a deep eutectic solvent with a water content of 30%.

[0028] Example 3:

[0029] This embodiment provides a deep eutectic solvent, which is mainly composed of anhydrous betaine and DL-malic acid in a molar ratio of 1:3. The preparation method is as follows: Using anhydrous betaine as the hydrogen bond acceptor and DL-malic acid as the hydrogen bond donor, add them into a beaker. Then add deionized water into the beaker and heat it in a water bath at 85 °C for 1 h until the solution becomes clear and transparent, thus obtaining a deep eutectic solvent with a water content of 30%.

[0030] Example 4:

[0031] This embodiment provides a preparation method of polysaccharide. The method includes extracting Momordica grosvenori powder with the deep eutectic solvent described in Example 2. The method is as follows: Weigh Momordica grosvenori powder and mix it with the deep eutectic solvent. The liquid-solid ratio of the Momordica grosvenori powder to the deep eutectic solvent is 1:20. Then perform ultrasonic extraction on the mixed solution. The temperature of ultrasonic extraction is 50 °C, the ultrasonic power is 300 W, and the ultrasonic extraction time is 30 min. Subsequently, centrifuge to obtain the supernatant, repeat the operation on the residue once, combine the two filtrates, centrifuge the filtrate at 10000 rpm for 8 min, collect the supernatant. Then add 1 / 4 volume of Sevag reagent (chloroform: n-butanol = 4:1) to the supernatant, place it on a magnetic stirrer and continuously stir for 25 min to remove proteins, and repeat this step until no protein precipitate appears in the middle layer. After protein removal is completed, collect the supernatant, perform overnight alcohol precipitation treatment with absolute ethanol at 3 °C, dissolve the precipitate in deionized water, and use a dialysis membrane to dialyze for 24 h to remove small molecule impurities. Finally, place the dialyzed solution in a freeze dryer for freeze-drying treatment to finally obtain Momordica grosvenori polysaccharide sample.

[0032] Example 5:

[0033] This example provides a method for preparing polysaccharide. The method includes extracting Momordica grosvenori powder with the deep eutectic solvent described in Example 2. The method is as follows: Weigh Momordica grosvenori powder and mix it with the deep eutectic solvent. The liquid-solid ratio of the Momordica grosvenori powder to the deep eutectic solvent is 1:30. Then, perform ultrasonic extraction on the mixed solution. The temperature of ultrasonic extraction is 50 °C, the ultrasonic power is 300 W, and the ultrasonic extraction time is 40 min. Subsequently, centrifuge to obtain the supernatant. Repeat the operation once for the residue. Combine the two filtrates. Weigh Momordica grosvenori powder, mix it with the deep eutectic solvent, then perform ultrasonic extraction on the mixed solution, subsequently centrifuge to obtain the supernatant, repeat the operation once for the residue, combine the two filtrates, centrifuge the filtrate at 10000 rpm for 10 min, collect the supernatant. Subsequently, add 1 / 4 volume of Sevag reagent (chloroform: n-butanol = 4:1) to the supernatant, place it on a magnetic stirrer and continuously stir for 30 min to remove proteins, and repeat this step until no protein precipitate appears in the intermediate layer. After protein removal is completed, collect the supernatant, perform overnight alcohol precipitation treatment with absolute ethanol at 4 °C, dissolve the precipitate in deionized water, and use a dialysis membrane to dialyze for 24 h to remove small molecule impurities. Finally, place the dialyzed solution in a freeze dryer for freeze-drying treatment to finally obtain a Momordica grosvenori polysaccharide sample.

[0034] Example 6:

[0035] This example provides a method for preparing polysaccharide. The method includes extracting Momordica grosvenori powder with the deep eutectic solvent described in Example 2. The method is as follows: Weigh Momordica grosvenori powder and mix it with the deep eutectic solvent. The liquid-solid ratio of the Momordica grosvenori powder to the deep eutectic solvent is 1:30. Then, perform ultrasonic extraction on the mixed solution. The temperature of ultrasonic extraction is 50 °C, the ultrasonic power is 400 W, and the ultrasonic extraction time is 40 min. Subsequently, centrifuge to obtain the supernatant. Repeat the operation once for the residue. Combine the two filtrates. Weigh Momordica grosvenori powder, mix it with the deep eutectic solvent, then perform ultrasonic extraction on the mixed solution, subsequently centrifuge to obtain the supernatant, repeat the operation once for the residue, combine the two filtrates, centrifuge the filtrate at 10000 rpm for 12 min, collect the supernatant. Subsequently, add 1 / 4 volume of Sevag reagent (chloroform: n-butanol = 4:1) to the supernatant, place it on a magnetic stirrer and continuously stir for 35 min to remove proteins, and repeat this step until no protein precipitate appears in the intermediate layer. After protein removal is completed, collect the supernatant, perform overnight alcohol precipitation treatment with absolute ethanol at 5 °C, dissolve the precipitate in deionized water, and use a dialysis membrane to dialyze for 24 h to remove small molecule impurities. Finally, place the dialyzed solution in a freeze dryer for freeze-drying treatment to finally obtain a Momordica grosvenori polysaccharide sample.

[0036] Experimental Example:

[0037] (1) Screening of the type of deep eutectic solvent:

[0038] Taking the yield of Siraitia grosvenorii polysaccharide as the observation value, a suitable deep eutectic solvent was screened out. The experimental fixed parameters were: liquid-solid ratio 20 mL / g, extraction temperature 50 °C, extraction time 40 min, and ultrasonic power 300 W.

[0039] As shown in Table 1, the phenol-sulfuric acid method was used to determine and calculate the yield of Siraitia grosvenorii polysaccharide in different types of DESs to determine the optimal deep eutectic solvent system.

[0040] Table 1 Composition components of deep eutectic solvents

[0041]

[0042] Except for the differences described in Table 1, other extraction methods were the same.

[0043] In addition, the traditional hot water extraction method (water) was set as the control group: 0.5 g of fruit powder was mixed with 17.5 mL of deionized water, and after extraction at 75 °C for 2.5 h, the operation was repeated once. First, the extract was centrifuged at 10000 rpm for 10 min, and the supernatant was collected. Subsequently, 1 / 4 volume of Sevag reagent (chloroform: n-butanol = 4:1) was added to the supernatant, and it was continuously stirred on a magnetic stirrer for 30 min to remove proteins, and this step was repeated until no protein precipitate appeared in the middle layer. After protein removal was completed, the supernatant was collected, and overnight alcohol precipitation treatment was carried out with absolute ethanol at 4 °C. The precipitate was redissolved in deionized water and dialyzed with a dialysis membrane for 24 h to remove small molecule impurities. Finally, the dialyzed solution was placed in a freeze dryer for freeze-drying treatment, and finally a crude Siraitia grosvenorii polysaccharide sample was obtained, and the extraction rate of Siraitia grosvenorii polysaccharide was determined and calculated according to the phenol-sulfuric acid method.

[0044] The yield comparison of extraction agents in each group is as Figure 1 shown. By comparing the extraction yields of eight DESs, it was found that DES-6 composed of betaine and DL-malic acid showed significant polysaccharide extraction ability, and its yield (21.13 ± 0.68%) was significantly higher than that of other solvents and the traditional hot water extraction method (18.48 ± 0.96%). Therefore, DES-6 (anhydrous betaine + DL-malic acid) was selected as the best extraction solvent.

[0045] (2) Screening of the molar ratio of DESs:

[0046] Under the conditions of 30% water content of DESs, solid-liquid ratio of 1:20, extraction temperature of 50 °C, extraction time of 40 min, and ultrasonic power of 300 W, the effects of different molar ratios (HBA:HBD) of DESs, namely 1:1, 1:2, 1:3, 1:4, and 1:5, on the extraction yield of polysaccharides were investigated. Each experiment was repeated three times.

[0047] The results are as Figure 2 (a) shown. In the study of the molar ratio of DESs, with the increase in the proportion of hydrogen bond donor (DL-malic acid), the yield of crude Momordica grosvenori polysaccharides showed a trend of first increasing and then decreasing ( Figure 2 (a)). When the molar ratio of hydrogen bond donor to acceptor was 3:1, the polysaccharide yield reached the maximum value, indicating that the extraction efficiency of DES was the highest under this ratio.

[0048] (3) Screening of the water content of DESs:

[0049] Under the conditions of DESs molar ratio (HBA:HBD) of 1:3, solid-liquid ratio of 1:20, extraction temperature of 50 °C, extraction time of 40 min, and ultrasonic power of 300 W, optimization experiments were carried out by changing the water content of DESs (10%, 20%, 30%, 40%, and 50%) to select the most suitable solvent for extracting Momordica grosvenori polysaccharides.

[0050] The results are as Figure 2 (b) shown. As the water content increased from 10% to 30%, the polysaccharide yield gradually increased and reached the peak at 30%. When the water content continued to increase to 50%, the yield gradually decreased. Therefore, a water content of 30% was determined as the optimal extraction condition.

[0051] Based on the above experimental results, the optimal process conditions for extracting Momordica grosvenori polysaccharides with DESs were finally determined: the molar ratio of betaine to malic acid was 1:3, and the water content was 30%.

[0052] (4) Selection of extraction parameters:

[0053] In the investigation of each factor, when the fixed condition parameters were liquid-solid ratio of 20 mL / g, extraction temperature of 50 °C, extraction time of 40 min, and ultrasonic power of 300 W, the effects of liquid-solid ratio (10, 20, 30, 40, and 50 mL / g), extraction temperature (40, 50, 60, 70, and 80 °C), ultrasonic power (200, 300, 400, 500, and 600 W), and extraction time (20, 30, 40, 50, and 60 min) on the yield of crude Momordica grosvenori polysaccharides were investigated respectively.

[0054] Within a certain range, increasing the liquid-solid ratio can expand the contact area between the raw material and the solvent, thereby promoting the dissolution of the target product. As Figure 3As shown in (a), when the liquid-solid ratio increased from 10:1 to 30:1, the polysaccharide yield increased significantly and reached the maximum at 30:1. However, when it continued to increase, the yield decreased instead. This might be because the solute had reached saturation in the solvent, which not only reduced the mass transfer efficiency but also caused waste of the solvent. Therefore, the liquid-solid ratio of 30:1 was determined as the optimal condition.

[0055] The extraction temperature is one of the key factors affecting the extraction effect of Momordica grosvenori polysaccharide. According to Figure 3 As shown in (b), as the temperature gradually increased from 40 °C to 80 °C, the polysaccharide yield showed a trend of first increasing and then decreasing, and reached the peak at 50 °C. Appropriate increase in temperature can improve the mass transfer efficiency and promote the dissolution of polysaccharides, but too high temperature may lead to the destruction and degradation of the polysaccharide structure. Therefore, 50 °C is considered the ideal temperature for extracting Momordica grosvenori polysaccharide.

[0056] The effect of ultrasonic power on the extraction of Momordica grosvenori polysaccharide is as Figure 3 As shown in (c), when the ultrasonic power increased from 200 W to 600 W, the polysaccharide yield first increased and then decreased, and reached the maximum at 300 W. This phenomenon can be attributed to the cavitation effect generated by appropriate ultrasonic power, which can effectively destroy the cell structure and promote the dissolution of cell contents, thus increasing the yield. However, too high ultrasonic power may lead to the destruction and degradation of the polysaccharide molecular structure. Therefore, 300 W was determined as the optimal power for extracting Momordica grosvenori polysaccharide.

[0057] The extraction time is one of the important parameters affecting the extraction efficiency of Momordica grosvenori polysaccharide, as Figure 3 As shown in (d), as the extraction time extended from 20 min to 40 min, the polysaccharide yield showed a significant upward trend and reached the peak at 40 min. This might be because the continuous penetration of the extraction solution due to the extension of the extraction time promoted the dissolution of polysaccharides. However, when the extraction time continued to increase, the yield decreased instead, which might be due to the degradation of polysaccharides caused by long-term ultrasonic treatment. Therefore, 40 min was determined as the optimal extraction time.

[0058] Based on the research results of the single-factor experiments, four key variables were selected in this study to design a steepest ascent experiment, aiming to further optimize the extraction process of Momordica grosvenori polysaccharide.

[0059] The experimental design and results are shown in Table 2:

[0060] Table 2 Steepest ascent experiment

[0061]

[0062]

[0063] Based on the optimization results of the climbing test, this study clarified the optimal combination of single-factor parameters for the extraction process of Momordica grosvenori polysaccharide: the liquid-solid ratio was set at 1:30, the extraction temperature was controlled at 50 °C, the ultrasonic power was adjusted to 300 W, and the extraction time was fixed at 40 min. Under the control of these parameters, the polysaccharide yield reached the highest value of 26.24 ± 0.43%, so the above parameters can be determined as the optimal extraction parameters of Momordica grosvenori polysaccharide in this application.

[0064] (5) Application of Momordica grosvenori polysaccharide:

[0065] The Momordica grosvenori polysaccharide prepared by the method of this application is applied to the preparation of hypoglycemic foods and / or drugs.

[0066] Further test the hypoglycemic activity of the obtained polysaccharide. The experimental steps are as follows: First, mix 40 μL of Momordica grosvenori polysaccharide solution or positive control drug (acarbose) with different concentrations (0.125, 0.25, 0.5, 1.0, 2.0 mg / mL) with 40 μL of α-glucosidase solution (0.45 U / mL) thoroughly, and incubate at a constant temperature of 37 °C for 10 min. Then add 80 μL of 5 mM p-nitrophenyl-D-α-glucopyranoside (PNPG) solution and continue to react at 37 °C for 20 min. After the reaction, add 100 μL of 0.2 mol / L Na2CO3 solution to the solution and measure the absorbance value at 405 nm, denoted as A1. Use PBS to replace the sample solution and α-glucosidase as the blank control and background control respectively, and their absorbance values are denoted as A0 and A2.

[0067] Calculate the inhibition rate of α-glucosidase according to formula (5.4) to evaluate the inhibitory activity of Momordica grosvenori polysaccharide.

[0068]

[0069] A1 - sample experimental group (containing α-glucosidase, polysaccharide sample or acarbose solution, PNPG solution)

[0070] A2 - sample blank group (containing PBS, polysaccharide solutions of various concentrations, PNPG solution)

[0071] A0 - blank group (containing PBS, α-glucosidase, PNPG solution)

[0072] The specific experimental steps are as follows: First, mix 120 μL of Momordica grosvenori polysaccharide solutions with different concentrations (0.125, 0.25, 0.5, 1.0, 2.0 mg / mL) or the positive control (acarbose) with 60 μL of α-amylase solution (2 U / mL), and react at 37 °C for 15 min. Subsequently, add 120 μL of 1% soluble starch solution to the reaction system and place it at 37 °C for 20 min. After the reaction is terminated, add 300 μL of DNS reagent to the mixture, place it in a boiling water bath for 15 min, cool and dilute, and measure the absorbance value at 540 nm, denoted as A1. Use PBS to replace the sample solution and α-amylase as the blank control and background control respectively, and their absorbance values are denoted as A0 and A2 respectively. Calculate the inhibition rate of α-amylase according to formula (5.4) to evaluate the inhibition effect of Momordica grosvenori polysaccharide.

[0073] Compare the hypoglycemic activities of the polysaccharides obtained by different extraction methods in each group. The grouping is as follows:

[0074] The Momordica grosvenori polysaccharide prepared by the method of Example 5 of this application is the first group;

[0075] Mix 0.5 g of fruit powder with 17.5 mL of deionized water, extract at 75 °C for 2.5 h, and repeat this operation once. First, centrifuge the extract at 10,000 rpm for 10 min, collect the supernatant, then add 1 / 4 volume of Sevag reagent (chloroform: n-butanol = 4:1) to the supernatant, place it on a magnetic stirrer and stir continuously for 30 min to remove proteins, and repeat this step until no protein precipitate appears in the middle layer. After deproteinization is completed, collect the supernatant, perform overnight alcohol precipitation treatment with absolute ethanol at 4 °C, dissolve the precipitate in deionized water, and use a dialysis membrane to dialyze for 24 h to remove small molecule impurities. Finally, place the dialyzed solution in a freeze dryer for freeze-drying treatment to obtain the crude Momordica grosvenori polysaccharide product as the second group;

[0076] Weigh 3.34 g of ammonium sulfate and completely dissolve it in 8.98 mL of deionized water. Subsequently, add 7.015 mL of ethanol to the solution. After thoroughly mixing with a vortex oscillator and allowing it to stand for a period of time, the solution spontaneously separates to form an ATPS composed of 18.7% ammonium sulfate and 31.0% ethanol. Weigh 0.5 g of sample powder and mix it with the ATPS solution at a ratio of 1:32 (g / mL). Place it in an ultrasonic cleaner at 60 °C and extract for 30 min with an ultrasonic power of 300 W. After extraction, perform centrifugation to fully phase-separate the supernatant. Subsequently, repeat the above operation once with Momordica grosvenori residue, and combine the upper and lower phases of the filtrates from the two extractions respectively. Centrifuge the upper and lower phase extracts at 10,000 rpm for 10 min, collect the supernatant, then add 1 / 4 volume of Sevag reagent (chloroform: n-butanol = 4:1) to the supernatant, place it on a magnetic stirrer and continuously stir for 30 min to remove proteins, and repeat this step until no protein precipitate appears in the middle layer. After protein removal, collect the supernatant, perform overnight alcohol precipitation with absolute ethanol at 4 °C, redissolve the precipitate in deionized water, and use a dialysis membrane to dialyze for 24 h to remove small molecule impurities. Finally, place the dialyzed solution in a freeze dryer for freeze-drying treatment to obtain the upper phase polysaccharide as the third group, and the lower phase polysaccharide and water-extracted polysaccharide as the fourth group.

[0077] Compare the inhibition rates of the polysaccharides obtained in the above first to fourth groups against α-glucosidase and α-amylase. The results are shown in Table 3:

[0078] Table 3 Comparison of the hypoglycemic activities of polysaccharides in each group

[0079]

[0080] Research shows that the residues of α-glucosidase and α-amylase can form hydrogen bonds with the carboxyl and hydroxyl groups in the polysaccharide molecule. This interaction hinders the binding of carbohydrates to the enzyme, thereby significantly inhibiting its catalytic activity. In addition, the inhibitory effect of polysaccharides on enzymes is also affected by their molecular weight, glucose content, and higher-order structure (such as triple helix conformation).

[0081] According to the experimental results in Table 3, it is found that the polysaccharides in the first group have the highest inhibitory activities against both α-glucosidase and α-amylase. This may be due to the differences in the chemical structures of the polysaccharides caused by different extraction methods, thereby resulting in differences in biological activities. It shows that the polysaccharides prepared by the method of this application can effectively delay the digestion process of starch, thereby significantly reducing the increase in postprandial blood glucose levels and reducing the generation of advanced glycation end products, and can be applied to the preparation of hypoglycemic foods and / or drugs.

[0082] The above-described embodiments merely represent several implementation manners of the present invention. The description thereof is relatively specific and detailed, but it should not be construed as a limitation to the scope of the present invention.

Claims

1. A deep eutectic solvent, characterized in that: The low eutectic solvent consists of anhydrous betaine, DL-malic acid and deionized water.

2. The deep eutectic solvent according to claim 1, characterized in that The molar ratio of the anhydrous betaine to DL-malic acid is 1:2-3.

3. The deep eutectic solvent according to claim 2, characterized in that The molar ratio of the anhydrous betaine to DL-malic acid is 1:

3.

4. The deep eutectic solvent according to claim 1, characterized in that The preparation method of the low eutectic solvent is as follows: anhydrous betaine is used as a hydrogen bond acceptor and DL-malic acid is used as a hydrogen bond donor, which are added into a beaker, and then deionized water is added into the beaker, and the mixture is heated in a water bath at 75-85° C. for 1-2 hours until the solution becomes clear and transparent, thereby obtaining a low eutectic solvent.

5. The deep eutectic solvent according to claim 4, characterized in that The water content of the deep eutectic solvent is 30%.

6. A method for extracting Momordica grosvenori polysaccharides using the deep eutectic solvent according to any one of claims 1 to 5, characterized in that: The method is as follows: weighing monk fruit powder, mixing it with a low eutectic solvent, then ultrasonically extracting the mixed solution, then centrifuging to obtain a supernatant, repeating the operation once for filtering the residue, combining the two filtrates, centrifuging the filtrate at 10,000 rpm for 8-12 minutes, collecting the supernatant, then adding 1 / 4 volume of Sevag reagent to the supernatant, placing it on a magnetic stirrer and continuously stirring it for 25-35 minutes to remove protein, and repeating this step until no protein precipitation occurs in the middle layer. After deproteinization is completed, the supernatant is collected, and anhydrous ethanol is used for overnight alcohol precipitation at 3-5°C. The precipitate is redissolved in deionized water, and dialyzed for 24 hours using a dialysis membrane to remove small molecular impurities. Finally, the dialyzed solution is placed in a freeze dryer for freeze drying, and finally monk fruit polysaccharide is obtained.

7. The method according to claim 6, characterized in that The liquid-to-solid ratio of the monk fruit powder and the low eutectic solvent is 1:20-30.

8. The method according to claim 6, characterized in that The temperature of the ultrasonic extraction is 50-70°C, the ultrasonic power is 300-400W, and the ultrasonic extraction time is 30-40min.

9. The method according to claim 8, characterized in that The temperature of the ultrasonic extraction is 50° C., the ultrasonic power is 300 W, and the ultrasonic extraction time is 40 min.

10. The Momordica grosvenori polysaccharide prepared according to the method of claim 6, characterized in that: The momordica grosvenori polysaccharide is used for preparing hypoglycemic food and / or medicine.

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