Preparation method of mulberry leaf extract for controlling blood sugar and weight and clinical application thereof

Abstract

The application discloses a preparation method of mulberry leaf extract for controlling blood sugar and weight and clinical application thereof, and belongs to the technical field of mulberry leaf extract. The preparation method comprises the following steps: water extraction, ultrasonic secondary extraction, step-by-step separation of an auxiliary precipitant, and finally microfiltration, ultrafiltration, concentration and spray drying to obtain the mulberry leaf extract. The auxiliary precipitant is a polyethylene glycol grafted polyethylene imine compound. In the step-by-step separation, the auxiliary precipitant can promote the precipitation of mulberry leaf protein through steric hindrance and hydrophobic effect at a low concentration, and can adhere to and wrap impurity molecules such as mulberry leaf polysaccharides and tannins in the mulberry leaf extract solution at a high concentration. Finally, the impurities are removed through centrifugal filtration to obtain an extract solution rich in mulberry leaf alkaloids. Through the step-by-step separation, the mulberry leaf protein in the extract solution can be better reserved, the extraction efficiency is improved, and the mulberry leaf extract rich in both the mulberry leaf protein and the mulberry leaf alkaloids is obtained.

Description

Technical Field

[0001] This invention belongs to the technical field of mulberry leaf extract, specifically relating to a method for preparing mulberry leaf extract for controlling blood sugar and weight, and its clinical application. Background Technology

[0002] With changes in social structure, faster pace of life, altered dietary habits and food composition, and an aging population, the spectrum of human diseases has undergone significant transformations. The incidence of chronic diseases related to diet and nutrition, such as diabetes, cardiovascular disease, obesity, and hyperlipidemia, is constantly rising, becoming a major global public health issue. As one of the chronic diseases most closely related to diet and nutrition, the scientific dietary control of diabetes has received widespread attention.

[0003] Mulberry leaf extract refers to substances extracted from pure natural mulberry leaves. Rich in various active ingredients, it can help regulate the body's metabolic processes and maintain normal blood sugar levels. However, traditional mulberry leaf extraction processes often focus on maximizing the extraction efficiency of single components, neglecting the simultaneous acquisition of other important active ingredients and overlooking the synergistic effects between the overall components of the mulberry leaf.

[0004] Furthermore, current mulberry leaf extraction processes often employ processing aids such as acids, resulting in complex processes and significant environmental pollution. Therefore, obtaining a mulberry leaf extract that retains multiple active ingredients from mulberry leaves and using it as a raw material to develop formulations with blood sugar and weight control effects is of great significance. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing mulberry leaf extract for controlling blood sugar and weight, and its clinical application, so as to solve the problem of simultaneously improving the extraction efficiency of alkaloids and mulberry leaf proteins in mulberry leaf extract.

[0006] The objective of this invention can be achieved through the following technical solutions:

[0007] In a first aspect, the present invention provides a method for preparing mulberry leaf extract for controlling blood sugar and weight, comprising the following process steps:

[0008] S1. Mulberry leaves are extracted with water to obtain mulberry leaf aqueous extract and mulberry leaf residue;

[0009] S2. Disperse the mulberry leaf residue in water, sonicate, raise the temperature to 35-45℃, heat for 30-50 minutes, and centrifuge to obtain crude mulberry leaf protein extract;

[0010] S3. Mix the aqueous extract of mulberry leaves and the crude protein extract of mulberry leaves to obtain the crude extract of mulberry leaves;

[0011] S4. Add a precipitant to the crude mulberry leaf extract and separate the solids.

[0012] S5. Continue to add a precipitant to the crude mulberry leaf extract, adjust the pH of the solution to 5.5-6.5, centrifuge to obtain the mulberry leaf extract;

[0013] S6. Dissolve the solids in mulberry leaf extract, and obtain mulberry leaf extract by microfiltration, ultrafiltration, concentration and spray drying;

[0014] The precipitant is a polyethylene glycol-grafted polyethyleneimine complex.

[0015] Preferably, in step S2, the ratio of mulberry leaf residue to water is 1g:(25-30)mL.

[0016] Preferably, microfiltration is performed using a tubular separation device with a rotation speed of 10,000 to 12,000 r / min.

[0017] Preferably, ultrafiltration is performed using a ceramic membrane separation device with 32-45 pores, a separation accuracy of 90-105 nm, a pore size of 3.3-3.7 mm, and a membrane tube length of 0.85-1.25 m.

[0018] By adopting the above technical solution, water extraction of mulberry leaves can effectively obtain a variety of bioactive components from mulberry leaves. This not only preserves the natural properties of mulberry leaves but also effectively avoids the use of organic solvents. A large number of nutrients in mulberry leaves, such as flavonoids, polyphenols, and alkaloids, are dissolved in the aqueous solution. Among them, alkaloids can inhibit α-glucosidase to prevent further digestion and absorption of carbohydrates, thereby reducing postprandial blood glucose peaks, improving lipid metabolism, and enhancing the body's immunity.

[0019] Mulberry leaf proteins can assist alkaloids, their interaction helping alkaloids exert better effects. They can also help improve insulin signaling pathways, enhance cellular response to insulin, and increase the body's efficiency in utilizing glucose, thus achieving blood sugar and weight control. However, due to the relatively large molecular size and complex structure of mulberry leaf proteins, they do not easily penetrate cell walls and cell membranes. Water extraction alone limits their release from mulberry leaf tissue. Therefore, after water extraction, the mulberry leaf residue still contains a large amount of undissolved mulberry leaf protein components.

[0020] Ultrasonic treatment of mulberry leaf residue can significantly improve the dissolution rate of proteins by disrupting cell walls, thus obtaining a crude extract of mulberry leaf protein.

[0021] The crude mulberry leaf extract still contains a large number of impurities. First, a precipitant, namely polyethylene glycol-grafted polyethyleneimine complex, is used for secondary separation. The first separation yields protein solids, while the second separation uses the precipitant to adsorb impurities such as mulberry polysaccharides and tannins in the crude mulberry leaf extract, mainly retaining the active ingredients, including alkaloids. Next, the solids are dissolved in the separated extract. Finally, a two-stage filtration process is used to further reduce impurities in the extract, yielding a mulberry leaf extract rich in mulberry leaf protein and mulberry leaf alkaloids.

[0022] Preferably, in step S4, the mass fraction of the precipitant in the crude mulberry leaf extract is controlled to be 15-20%; in step S5, the mass fraction of the precipitant in the crude mulberry leaf extract is controlled to be 35-40%.

[0023] By adopting the above technical solution, in the process of separation aided by the precipitant, the precipitant is first controlled to be dispersed at a low concentration in the crude mulberry leaf extract. At this time, the precipitant molecules will form a network structure in the solution, which will have a spatial repulsion effect with the mulberry leaf protein molecules, causing the mulberry leaf protein molecules to aggregate and precipitate. Moreover, the polyethylene glycol-grafted polyethyleneimine complex has hydrophilicity, which can reduce the hydration layer on the surface of the protein molecules and promote the precipitation process. The low concentration dispersion can also ensure that the mulberry leaf protein will not be over-aggregated or deformed due to the effect of the grafted polyethyleneimine segments, thereby maintaining the biological activity of the mulberry leaf protein. The solid obtained by separation is obtained by precipitation of mulberry leaf protein.

[0024] Subsequently, a precipitant was added to increase its mass fraction in the crude mulberry leaf extract, resulting in a high concentration dispersion of the precipitant. At this point, the high molecular weight polyethylene glycol-grafted polyethyleneimine complex could form larger polymers in the crude mulberry leaf extract. These polymers adsorbed and encapsulated impurity molecules such as mulberry polysaccharides and tannins in the mulberry leaf extract. Finally, the impurities were removed by centrifugation and filtration, yielding an extract solution rich in mulberry leaf alkaloids. Through step-by-step separation, the mulberry leaf protein in the extract could be better preserved, improving extraction efficiency and resulting in a mulberry leaf extract rich in both mulberry leaf protein and mulberry leaf alkaloids.

[0025] Preferably, the raw materials for the polyethylene glycol-grafted polyethyleneimine composite include polyethylene glycol and polyethyleneimine in a mass ratio of 1:(0.2-0.3).

[0026] Preferably, the molecular weight of polyethylene glycol is 4000-6000 g / mol.

[0027] Preferably, the polyethylene glycol-grafted polyethyleneimine composite is prepared according to the following method:

[0028] Polyethylene glycol was added to water and stirred to dissolve it. The temperature was raised to 80-85°C, and an epoxy silane coupling agent was added. After stirring for 2-3 hours, a pre-reaction solution was obtained. Polyethylene imine was dissolved in water and mixed with the pre-reaction solution. The mixture was stirred for 15-20 hours, and finally dried and ground to obtain a polyethylene glycol-grafted polyethylene imine composite.

[0029] Preferably, the amount of epoxy silane coupling agent added is 5-10% of the mass of polyethylene glycol.

[0030] Preferably, the epoxy silane coupling agent includes one or more combinations of γ-glycidoxypropyltrimethoxysilane and γ-glycidoxypropyltriethoxysilane.

[0031] High-concentration dispersion of precipitating agents in crude mulberry leaf extract can help adsorb and encapsulate impurity molecules, but at the same time, it can affect the small-molecule mulberry leaf alkaloids, causing them to bind with polyethylene glycol, thus affecting the extraction efficiency and content of mulberry leaf alkaloids.

[0032] By employing the above technical solution, polyethyleneimine segments are grafted onto the polyethylene glycol molecular chain through the action of an epoxy silane coupling agent. Since the main components of mulberry leaf alkaloids include polyhydroxy alkaloids such as 1-deoxynojirimycin, which exhibit positive charge characteristics under acidic conditions, while impurity molecules such as mulberry leaf polysaccharides and tannins maintain negative charge characteristics under acidic conditions, grafting polyethyleneimine onto the polyethylene glycol molecular chain allows the numerous primary, secondary, and tertiary amine groups contained in polyethyleneimine to be protonated in aqueous solution, forming positively charged ammonium ions. This also results in a certain branched structure, effectively increasing the number of cationic sites on the polyethylene glycol molecular chain, increasing the contact area with impurity molecules, and strengthening the electrostatic attraction between impurities and negatively charged macromolecular impurities. This, in turn, improves the selective adsorption of the precipitant in the mulberry leaf extract.

[0033] The positively charged functional groups in polyethyleneimine can not only increase the number of cation exchange sites, but also increase the binding strength between the precipitant and impurity molecules through non-covalent interactions such as hydrogen bonds. This can effectively separate impurity molecules from the crude mulberry leaf extract and improve the efficacy of mulberry leaf extract in controlling blood sugar and weight.

[0034] Furthermore, because polyethyleneimine has a certain branched structure, it can effectively adsorb large molecular impurities while using steric hindrance to restrict the approach and binding of small molecular alkaloids, thereby increasing the effective content of mulberry leaf alkaloids in mulberry leaf extract.

[0035] Secondly, the present invention provides a clinical application of mulberry leaf extract for controlling blood sugar and weight, wherein the mulberry leaf extract is prepared according to the above preparation method; the clinical application includes any one of controlling postprandial blood glucose levels, improving lipid metabolism, supporting intestinal health, and weight loss.

[0036] The beneficial effects of this invention are:

[0037] 1. In the preparation method of the mulberry leaf extract for controlling blood sugar and weight of the present invention, a precipitant is used to separate the crude mulberry leaf extract. The precipitant is a polyethylene glycol-grafted polyethyleneimine complex. Polyethyleneimine provides a large number of cation exchange sites for polyethylene glycol, which can form a strong electrostatic attraction between the mulberry leaf polysaccharide and tannin and other impurity molecules, thereby improving the selective adsorption capacity of the precipitant for impurity molecules. Moreover, its branched structure can also use the steric hindrance effect to restrict the approach and binding of small molecule alkaloids, thereby increasing the content of mulberry leaf alkaloids in the obtained mulberry leaf extract.

[0038] 2. In the preparation method of the mulberry leaf extract for controlling blood sugar and weight of the present invention, the concentration of the precipitant in the crude mulberry leaf extract is controlled by adding it twice, thereby separating the effective components in the crude mulberry leaf extract in steps. Specifically, under low concentration conditions, the precipitant can promote the precipitation of mulberry leaf protein through steric hindrance and hydrophobic interaction to form a solid, avoiding the influence of subsequent processing; under high concentration conditions, the high molecular weight precipitant can attach to and encapsulate impurity molecules such as mulberry leaf polysaccharides and tannins in the mulberry leaf extract. Finally, after centrifugation and filtration to remove impurities, an extract solution rich in mulberry leaf alkaloids is obtained. Through step-by-step separation, the mulberry leaf protein in the extract can be better preserved, the extraction efficiency can be improved, and a mulberry leaf extract rich in both mulberry leaf protein and mulberry leaf alkaloids can be obtained. Detailed Implementation

[0039] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0040] Preparation Example

[0041] Preparation Example 1: A precipitant was prepared according to the following method:

[0042] 10g of polyethylene glycol (average molecular weight 6000g / mol) was added to water and stirred to dissolve. The temperature was raised to 80℃, and 0.8g of γ-glycidyl etheroxypropyltrimethoxysilane was added. After stirring for 3 hours, a pre-reaction solution was obtained. 2.5g of polyethyleneimine (average molecular weight 600g / mol) was dissolved in water and mixed with the pre-reaction solution. The mixture was stirred for 20 hours and then dried and ground to obtain the polyethylene glycol-grafted polyethyleneimine composite.

[0043] Preparation Example 2, a precipitant, differs from Preparation Example 1 only in that the amount of polyethyleneimine added is 2g.

[0044] Preparation Example 3, a precipitant, differs from Preparation Example 1 only in that the amount of polyethyleneimine added is 3g.

[0045] Preparation Example 4, a precipitant, differs from Preparation Example 1 only in that the amount of polyethyleneimine added is 1g.

[0046] Preparation Example 5, a precipitant, differs from Preparation Example 1 only in that the amount of polyethyleneimine added is 4g.

[0047] Example

[0048] Example 1: A mulberry leaf extract for controlling blood sugar and weight was prepared according to the following process steps:

[0049] S1. Mulberry leaves were extracted with water, including two extractions with a material-to-liquid ratio of 1g:20mL. The extraction was carried out under reflux at 90℃ for 2 hours. The extract was then passed through a 100-mesh sieve to obtain mulberry leaf water extract and mulberry leaf residue.

[0050] S2. Disperse the mulberry leaf residue in water, control the material-to-liquid ratio to 1g:30mL, sonicate for 15min, raise the temperature to 40℃, heat for 40min, and centrifuge to obtain crude mulberry leaf protein extract;

[0051] S3. Mix the aqueous extract of mulberry leaves and the crude protein extract of mulberry leaves to obtain the crude extract of mulberry leaves;

[0052] S4. Add the precipitant prepared in Preparation Example 1 to the crude mulberry leaf extract, control the mass fraction of the precipitant in the crude mulberry leaf extract to be 15%, stir and mix, and separate to obtain solids.

[0053] S5. Continue to add the precipitant prepared in Preparation Example 1 to the crude mulberry leaf extract, control the mass fraction of the precipitant in the crude mulberry leaf extract to be 35%, adjust the pH of the solution to 6, centrifuge to obtain mulberry leaf extract;

[0054] S6. The solids are dissolved in mulberry leaf extract, and then subjected to microfiltration (12000 r / min) using a tubular separation device, ultrafiltration (37 pores, 100 nm separation accuracy, 3.5 mm pore size, 1 m membrane length) using a ceramic membrane separation device, concentration and spray drying to obtain mulberry leaf extract.

[0055] Example 2, a mulberry leaf extract for controlling blood sugar and weight, differs from Example 1 only in that an equal amount of the precipitant prepared in Preparation Example 2 is used instead of the precipitant prepared in Preparation Example 1.

[0056] Example 3, a mulberry leaf extract for controlling blood sugar and weight, differs from Example 1 only in that an equal amount of the precipitant prepared in Example 3 is used instead of the precipitant prepared in Example 1.

[0057] Example 4, a mulberry leaf extract for controlling blood sugar and weight, differs from Example 1 only in that, in step S4, the mass fraction of the precipitant in the crude mulberry leaf extract is controlled at 20%; and in step S5, the mass fraction of the precipitant in the crude mulberry leaf extract is controlled at 40%.

[0058] Example 5, a mulberry leaf extract for controlling blood sugar and weight, differs from Example 1 only in that, in step S4, the mass fraction of the precipitant in the crude mulberry leaf extract is controlled to be 35%.

[0059] Example 6, a mulberry leaf extract for controlling blood sugar and weight, differs from Example 1 only in that, in step S5, the mass fraction of the precipitant in the crude mulberry leaf extract is controlled to be 20%.

[0060] Example 7, a mulberry leaf extract for controlling blood sugar and weight, differs from Example 1 only in that, in step S4, the mass fraction of the precipitant in the crude mulberry leaf extract is controlled to be 10%.

[0061] Example 8, a mulberry leaf extract for controlling blood sugar and weight, differs from Example 1 only in that, in step S5, the mass fraction of the precipitant in the crude mulberry leaf extract is controlled to be 50%.

[0062] Example 9, a mulberry leaf extract for controlling blood sugar and weight, differs from Example 1 only in that an equal amount of the precipitant prepared in Preparation Example 4 is used instead of the precipitant prepared in Preparation Example 1.

[0063] Example 10, a mulberry leaf extract for controlling blood sugar and weight, differs from Example 1 only in that an equal amount of the precipitant prepared in Preparation Example 5 is used instead of the precipitant prepared in Preparation Example 1.

[0064] Comparative Example

[0065] Comparative Example 1, a mulberry leaf extract for controlling blood sugar and weight, differs from Example 1 only in that an equal amount of polyethylene glycol (average molecular weight of 6000 g / mol) is used to replace the precipitant prepared in Example 1.

[0066] Comparative Example 2, a mulberry leaf extract for controlling blood sugar and weight, differs from Example 1 only in that step S5 is adjusted, specifically: the pH of the crude mulberry leaf extract after separating the solids is adjusted to 6, and centrifuged to obtain the mulberry leaf extract.

[0067] Comparative Example 3, a mulberry leaf extract for controlling blood sugar and weight, was prepared according to the following process steps:

[0068] S1. Mulberry leaves were extracted with water, including two extractions with a material-to-liquid ratio of 1g:20mL. The extraction was carried out under reflux at 90℃ for 2 hours. The extract was then passed through a 100-mesh sieve to obtain mulberry leaf water extract and mulberry leaf residue.

[0069] S2. Disperse the mulberry leaf residue in water, control the material-to-liquid ratio to 1g:30mL, sonicate for 15min, raise the temperature to 40℃, heat for 40min, and centrifuge to obtain crude mulberry leaf protein extract;

[0070] S3. Mix the aqueous extract of mulberry leaves and the crude protein extract of mulberry leaves to obtain the crude extract of mulberry leaves;

[0071] S4. Add the precipitant prepared in Preparation Example 1 to the crude mulberry leaf extract, control the mass fraction of the precipitant in the crude mulberry leaf extract to be 15%, adjust the pH of the solution to 6, centrifuge to obtain mulberry leaf extract;

[0072] S5. The mulberry leaf extract was subjected to microfiltration (12000 r / min) using a tubular separation device, ultrafiltration (37 pores, 100 nm separation accuracy, 3.5 mm pore size, 1 m membrane length) using a ceramic membrane separation device, concentration and spray drying to obtain the mulberry leaf extract.

[0073] Comparative Example 4: A mulberry leaf extract for controlling blood sugar and weight was prepared according to the following process steps:

[0074] S1. Mulberry leaves were extracted with water, including two extractions with a material-to-liquid ratio of 1g:20mL. The extraction was carried out under reflux at 90℃ for 2 hours. The extract was then passed through a 100-mesh sieve to obtain mulberry leaf water extract and mulberry leaf residue.

[0075] S2. Add the precipitant prepared in Preparation Example 1 to the mulberry leaf aqueous extract, control the mass fraction of the precipitant in the crude mulberry leaf extract to be 15%, stir and mix, and separate to obtain solids;

[0076] S3. Continue to add the precipitant prepared in Preparation Example 1 to the mulberry leaf aqueous extract, control the mass fraction of the precipitant in the crude mulberry leaf extract to be 35%, adjust the pH of the solution to 6, centrifuge to obtain mulberry leaf extract;

[0077] S4. The solids are dissolved in mulberry leaf extract, and then subjected to microfiltration (12000 r / min) using a tubular separation device, ultrafiltration (37 pores, 100 nm separation accuracy, 3.5 mm pore size, 1 m membrane length) using a ceramic membrane separation device, concentration and spray drying to obtain mulberry leaf extract.

[0078] Comparative Example 5, a mulberry leaf extract for controlling blood sugar and weight, was prepared according to the following process steps:

[0079] S1. Mulberry leaves were extracted with water, which was added twice with a material-to-liquid ratio of 1g:20mL. The extraction was carried out by reflux at 90℃ for 2h. After passing through a 100-mesh coarse sieve, mulberry leaf water extract and mulberry leaf residue were obtained.

[0080] S2. Disperse the mulberry leaf residue in water, control the material-to-liquid ratio to 1g:30mL, sonicate for 15min, raise the temperature to 40℃, heat for 40min, and centrifuge to obtain crude mulberry leaf protein extract;

[0081] S3. Mix the aqueous extract of mulberry leaves and the crude protein extract of mulberry leaves to obtain the crude extract of mulberry leaves;

[0082] S4. The crude mulberry leaf extract was subjected to microfiltration (12000 r / min) using a tubular separation device, ultrafiltration (37 pores, 100 nm separation accuracy, 3.5 mm pore size, 1 m membrane length) using a ceramic membrane separation device, concentration and spray drying to obtain the mulberry leaf extract.

[0083] Performance testing

[0084] Thirteen healthy adults aged 18–40 years, without metabolic, digestive, or endocrine diseases, with no history of diabetes, and not using hypoglycemic drugs, were selected for clinical testing.

[0085] Test schedule: For the three days prior to the test, subjects should maintain a regular sleep schedule and normal diet. On the day before the test, avoid high-fiber and high-sugar foods for dinner and refrain from drinking alcohol. Begin eating before 10:00 PM. Avoid strenuous exercise on the morning of the test.

[0086] After arriving at the testing site, the subjects sat quietly for 10 minutes before the food tasting began. The test foods were rice and mulberry leaf extract, with 0.7g of mulberry leaf extract and 66.7g of rice. During the food tasting, fasting blood samples were collected every 5 minutes. After the collection, the subjects began to eat, consuming all the test foods and a 250mL glass of water within 5–12 minutes. The timing was started from the first bite, and blood samples were collected at 15, 30, 45, 60, 90, and 120 minutes post-meal.

[0087] The testing period included one control trial (rice only) and a combination of mulberry leaf extract and rice obtained in the examples and comparative examples, for a total of 15 trials. A randomized design was used, and the interval between each independent trial was ≥72 hours.

[0088] The changes in blood glucose and insulin levels were tested, and the average changes from each independent food intake were taken to obtain the peak values ​​of blood glucose and insulin after the meal. The experimental results are shown in Table 1.

[0089] Table 1 Performance Test Results

[0090]

[0091] According to Table 1, and in conjunction with Examples 1 and 5-8, it can be seen that the peak blood glucose and insulin levels in Examples 5-8 are all increased compared to Example 1, indicating that the mulberry leaf extracts obtained in Examples 5-8 have a decreased ability to lower blood glucose and reduce glucose absorption. This is because the concentrations of the precipitating agent added twice were adjusted during the preparation of the mulberry leaf extracts in Examples 5-8. When the concentration was increased or decreased in the first instance, the extraction effect on mulberry leaf protein decreased. Increased concentration made it difficult for the mulberry leaf protein to undergo steric repulsion with the large molecules, leading to protein aggregation and deposition. Conversely, when the concentration was increased or decreased in the second instance, the extraction effect on mulberry leaf alkaloids decreased. Increased concentration caused the mulberry leaf alkaloids to react with the precipitating agent, resulting in a lower concentration of mulberry leaf alkaloids. A lower concentration made it difficult to adsorb and remove impurities from the mulberry leaf extract, thus affecting its blood glucose control effect.

[0092] Combining Examples 1, 9, 10, and Comparative Example 1, it can be seen that the peak blood glucose and insulin levels in Examples 9, 10, and Comparative Example 1 are all increased compared to Example 1. This is because the grafting rate of polyethyleneimine onto the polyethylene glycol in all examples was adjusted. In Example 9, the polyethyleneimine segments on the polyethylene glycol molecular chain were reduced, decreasing the selective adsorption of impurity molecules by the precipitant. Furthermore, some mulberry leaf alkaloids were also adsorbed and removed by the precipitant, resulting in a decrease in the blood sugar control effect of the final mulberry leaf extract. In Comparative Example 1, the polyethylene glycol was not modified with polyethyleneimine, leading to a more significant performance degradation. In Example 10, the grafting rate of polyethyleneimine segments was increased, reducing the attraction of polyethylene glycol itself to large impurity molecules, thus decreasing the impurity removal rate.

[0093] Based on Examples 1 and Comparative Examples 2-5, it can be seen that the peak blood glucose and peak insulin levels in Comparative Examples 2-5 are all increased compared to Example 1. This is because, in Comparative Example 2, the concentration of the second precipitant was not changed, resulting in a significant decrease in the separation effect on small molecule alkaloids and impurity molecules; in Comparative Example 3, the separation was not performed in two stages, leading to a decrease in mulberry leaf protein content and an increase in impurity molecule content in the final mulberry leaf extract; in Comparative Example 4, the lack of pre-ultrasonic treatment made it difficult to completely dissolve mulberry leaf protein using water extraction, resulting in a decrease in mulberry leaf protein content in the extract; and in Comparative Example 5, the absence of a precipitant reduced both the extraction effect on mulberry leaf protein and the separation effect on impurity molecules, leading to a decrease in the blood sugar control effect of the mulberry leaf extract.

[0094] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0095] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a mulberry leaf extract for controlling blood sugar and weight, characterized in that, The process includes the following steps: S1. Mulberry leaves are extracted with water to obtain mulberry leaf aqueous extract and mulberry leaf residue; S2. Disperse the mulberry leaf residue in water, sonicate, raise the temperature to 35-45℃, heat for 30-50 minutes, and centrifuge to obtain crude mulberry leaf protein extract; S3. Mix the aqueous extract of mulberry leaves and the crude protein extract of mulberry leaves to obtain the crude extract of mulberry leaves; S4. Add a precipitant to the crude mulberry leaf extract, and control the mass fraction of the precipitant in the crude mulberry leaf extract to be 15-20%, and separate the solids. S5. Continue to add a precipitant to the crude mulberry leaf extract, controlling the mass fraction of the precipitant in the crude mulberry leaf extract to be 35-40%, adjust the pH of the solution to 5.5-6.5, centrifuge to obtain the mulberry leaf extract. S6. Dissolve the solids in mulberry leaf extract, and obtain mulberry leaf extract by microfiltration, ultrafiltration, concentration and spray drying; The precipitant is a polyethylene glycol-grafted polyethyleneimine composite; the raw materials of the polyethylene glycol-grafted polyethyleneimine composite include polyethylene glycol and polyethyleneimine in a mass ratio of 1:(0.2-0.3); The polyethylene glycol-grafted polyethyleneimine composite was prepared according to the following method: Polyethylene glycol was added to water and stirred to dissolve it. The temperature was raised to 80-85°C, and an epoxy silane coupling agent was added. After stirring for 2-3 hours, a pre-reaction solution was obtained. Polyethylene imine was dissolved in water and mixed with the pre-reaction solution. The mixture was stirred for 15-20 hours, and finally dried and ground to obtain a polyethylene glycol-grafted polyethylene imine composite.

2. The method for preparing mulberry leaf extract for controlling blood sugar and weight according to claim 1, characterized in that, The molecular weight of the polyethylene glycol is 4000-6000 g / mol.

3. The method for preparing mulberry leaf extract for controlling blood sugar and weight according to claim 1, characterized in that, The amount of epoxy-based silane coupling agent added is 5-10% of the mass of polyethylene glycol.

4. The method for preparing mulberry leaf extract for controlling blood sugar and weight according to claim 1, characterized in that, In step S2, the ratio of mulberry leaf residue to water is 1g:(25-30)mL.

5. The method for preparing mulberry leaf extract for controlling blood sugar and weight according to claim 1, characterized in that, The microfiltration is performed using a tubular separation device with a rotation speed of 10,000 to 12,000 r / min.

6. The method for preparing mulberry leaf extract for controlling blood sugar and weight according to claim 1, characterized in that, The ultrafiltration process utilizes a ceramic membrane separation device with 32–45 pores, a separation accuracy of 90–105 nm, a pore size of 3.3–3.7 mm, and a membrane tube length of 0.85–1.25 m.

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