Compound for promoting GLP-1 secretion, fermentation preparation method and application thereof

Abstract

The invention relates to the field of fermentation compounds, in particular to a compound for promoting GLP-1 secretion, a fermentation preparation method and application of the compound. According to the compound, fermented mulberry leaf powder is used as a main material, soybean oligosaccharide and rhizoma kaempferiae rhizome extract are used as synergistic materials, white birch juice concentrate and white kidney bean extract are used as auxiliary materials, the mulberry leaf powder is subjected to compound probiotic fermentation and multi-stage directional enzymolysis to enrich active components, and meanwhile, the soybean oligosaccharide is extracted and purified by adopting ultrasonic-assisted extraction in combination with a gradient alcohol precipitation method. Supercritical CO2 extraction is assisted to obtain a high-purity rhizoma kaempferiae rhizome active extract; finally, all the extracts are compounded in proportion and embedded through sodium alginate-chitosan wall material microcapsules, and the stability is improved with the assistance of a vitrification carrier and an antioxidant; the composition provided by the invention has a multi-target synergistic effect, and can significantly promote the secretion of GLP-1.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of fermentation complex, in particular to a complex for promoting GLP-1 secretion, a fermentation preparation method and application thereof. BACKGROUND

[0002] Glucagon-like peptide-1 (GLP-1) is a gut-secreted incretin hormone that plays a key role in maintaining blood glucose homeostasis and controlling appetite. GLP-1 can promote insulin secretion, inhibit glucagon release, delay gastric emptying and act on the central nervous system to produce satiety, and has important significance for the prevention and treatment of type II diabetes and obesity. Currently, GLP-1 receptor agonist drugs are used in the clinic for the treatment of diabetes and weight loss, but such drugs are expensive and require injection, and patient compliance is poor. Therefore, developing nutritional interventions that can promote endogenous GLP-1 secretion in vivo has become a research hotspot.

[0003] Many traditional plant extracts have been found to have the effects of reducing blood sugar and controlling weight. For example, mulberry leaves, as a commonly used Chinese herbal medicine, are recorded in "Compendium of Materia Medica" as being able to "treat diabetes". Modern pharmacological studies have confirmed that mulberry leaves contain active ingredients such as flavonoids and alkaloids, which can inhibit small intestinal disaccharidase activity, reduce glucose absorption, and stimulate small intestinal L cell secretion of GLP-1, thereby synergistically lowering blood sugar through multiple pathways. Among them, 1-deoxynojirimycin (1-DNJ) in mulberry leaves is a potent alpha-glucosidase inhibitor and has also been shown to exert a hypoglycemic effect through the GLP-1 pathway. However, there are significant limitations in the utilization of active ingredients in mulberry leaves: flavonoids such as rutin and quercetin have poor water solubility and are difficult to absorb, resulting in low oral bioavailability; while mulberry leaf 1-DNJ is easily soluble in water but has a very short half-life in the human body, making it difficult to maintain a sustained hypoglycemic effect. In addition, the content of 1-DNJ in mulberry leaves is low, and the extraction and utilization efficiency is limited; these factors often result in actual efficacy of traditional mulberry leaf extracts that is not as expected, and special processes are needed to improve the content and stability of active ingredients.

[0004] In addition to mulberry leaves, other plant-derived ingredients also have their own effects in assisting in blood sugar control and weight loss. Although each plant ingredient has the potential to lower blood sugar or reduce weight, direct and simple mixing of them often has limited effect; some existing technologies attempt to improve the performance of plant extracts through inclusion, nano-carriers, etc., but still have the disadvantages of complex process, high cost, or only targeting a single ingredient.

[0005] Therefore, there is an urgent need to provide an innovative composition and process that can organically integrate multiple plant active ingredients, improve the content and stability of key ingredients, and achieve the effect of synergistic blood sugar control and weight loss through multiple pathways. SUMMARY

[0006] The present application aims to provide a complex for promoting GLP-1 secretion, a fermentation preparation method and application thereof, and overcomes the technical problems of low activity, poor stability, low bioavailability and poor compatibility synergy of plant extracts in regulating blood sugar and weight in the prior art, and provides a plant-derived complex for promoting GLP-1 secretion and a fermentation preparation method thereof, and the complex is applied to a sugar-controlling and weight-reducing preparation.

[0007] The specific technical scheme is as follows:

[0008] A complex for promoting GLP-1 secretion, comprising the following components in mass percentage: 40-45% of fermented mulberry leaf powder, 25-30% of soybean oligosaccharide, 15-20% of kaempferia galantha root extract, 8-12% of birch juice concentrate, and 5-8% of white kidney bean extract; and each component is mixed and then embedded by microcapsules.

[0009] Further, the fermented mulberry leaf powder is obtained by inoculating mixed bacteria of Lactobacillus rhamnosus, Bifidobacterium adolescentis and Clostridium butyricum on mulberry leaves as raw materials for solid-state fermentation for 72 hours, and then refined by multi-stage directional enzymolysis and membrane separation.

[0010] Further, the soybean oligosaccharide mainly comprises mixed oligosaccharides of stachyose and raffinose, which is prepared by ultrasonic-assisted water extraction and step-by-step ethanol precipitation of defatted soybean powder, and then desalted by nanofiltration and purified by low-temperature crystallization.

[0011] Further, the kaempferia galantha root extract is prepared by supercritical CO2 extraction technology, and is rich in volatile oil and flavonoid active ingredients.

[0012] Further, the embedding by microcapsules is to embed the complex by sodium alginate and chitosan as wall materials to form microcapsules, and further add 5wt% of inulin as a glass transition carrier in the microcapsules to improve the glass transition temperature of the microcapsule wall, improve the stability of the active ingredients, and further add 0.05wt% of rosemary acid and 0.02wt% of alpha-tocopherol as antioxidant protective agents.

[0013] The present application also provides a preparation method of the complex for promoting GLP-1 secretion, which is prepared by the following steps:

[0014] S1: After the fresh mulberry leaves are washed, dried, crushed, sieved, and mulberry leaf powder is obtained, pure water is added to adjust the moisture content to 70%, and after sterilization, the mixed bacterial strains of Lactobacillus rhamnosus, Bifidobacterium adolescentis and Clostridium butyricum are inoculated, and the fermentation culture is maintained at a temperature and relative humidity for 72 hours; after the fermentation is completed, the fermented mulberry leaf powder is suspended in phosphate buffer, and then egg white lysozyme, natto kinase and papain are added in sequence for enzymolysis, and after the enzymolysis is completed, the powder is sieved and filtered with an ultrafiltration membrane with a molecular weight cutoff of 3000 Da, and the filtrate is concentrated to a solid content of 20%, and finally spray dried to obtain fermented mulberry leaf powder;

[0015] S2: Add deionized water to the defatted soybean powder, and extract with ultrasonic waves under the condition of water bath at 25°C. After completion, filter and add anhydrous ethanol to the extract to a solution ethanol volume fraction of 30%, stand for 1 hour, centrifuge to remove the precipitate, and then add anhydrous ethanol to the supernatant to a final concentration of 60%. After standing overnight at 4°C, centrifuge to collect the precipitate, dissolve the precipitate in pure water and transfer it into a dialysis bag, and desalt by nanofiltration with a membrane with a molecular weight cutoff of 500 Da. Stop dialysis when the dialysate has no reducing sugar reaction. Concentrate the dialysate and stand at 4°C for 24 hours to crystallize. Filter out the crystals and vacuum dry to obtain soybean oligosaccharides.

[0016] S3: Crush the sliced kaempferia galanga rhizome and place it in a supercritical CO2 extraction device. Adjust the pressure and temperature for extraction, which lasts for 2 hours. Collect the product every 15 minutes during the extraction process and combine the products. After the first extraction is complete, add 10% volume fraction of ethanol and continue the extraction at 28 MPa and 38°C for 1 hour. Finally, mix the two extracts together to obtain the kaempferia galanga rhizome extract.

[0017] S4: Mix the fermented mulberry leaf extract obtained in S1, the soybean oligosaccharides obtained in S2, the kaempferia extract obtained in S3, commercially available white birch juice freeze-dried powder, and commercially available white kidney bean extract powder. Dissolve in pure water and stir to form a mixed solution. Add the mixed solution to a sodium alginate solution and stir to form a suspension. Add inulin, rosemary acid, and alpha-tocopherol during stirring. Then perform high-voltage electrostatic spraying on the suspension. After completion, stand for 30 minutes to solidify. Filter out the solidified gel beads and place them in a chitosan solution. Stir for 15 minutes, filter and rinse to obtain wet microcapsules. Transfer to a freeze dryer, pre-freeze at -50°C for 2 hours, and then sublimate dry for 24 hours to obtain the compound preparation.

[0018] Further, the mixed bacterial strains of S1 have a mass ratio of Lactobacillus rhamnosus:Bifidobacterium adolescentis:Clostridium butyricum = 6:2:1.

[0019] Further, the temperature and relative humidity for fermentation culture in S1 are specifically set as follows: fermentation temperature 33-37°C, relative humidity 68-72%.

[0020] Further, the ultrasonic extraction in S2 is set as follows: extraction time 25-35 minutes, extraction power 280-320 W, and frequency 35-45 kHz; the extraction in S3 is set as follows: extraction pressure 30-35 MPa and temperature 40-45 DEG C; the high-voltage electrostatic spraying in S4 is set as follows: voltage 10 kV, spraying rate 1-2 mL / min, and coagulation solution 2% CaCl2 solution.

[0021] The application also provides a complex for promoting GLP-1 secretion.

[0022] Compared with the prior art, the application has the following beneficial effects:

[0023] (1) The application significantly improves the content and easy absorbability of active ingredients in mulberry leaves through fermentation and directional enzymolysis, so that the potential of mulberry leaves for reducing blood sugar is fully exerted; the high-content prebiotic intake is ensured through the purification process of soybean oligosaccharides, so that the production of short-chain fatty acids by intestinal flora is promoted and GLP-1 secretion is enhanced; the introduction of kaempferia extract provides a different pathway for reducing blood sugar than flavonoids in mulberry leaves; white kidney bean extract blocks the absorption of carbohydrates, so that more undigested carbohydrates reach the ileum and colon to stimulate L cells to release GLP-1, and the absorption of heat is reduced; white birch juice has anti-inflammatory effects, is rich in nutrients, and improves the taste.

[0024] (2) The components in the application produce a multi-target synergistic effect through scientific proportioning: on the one hand, the absorption of carbohydrates is inhibited, and blood sugar production is reduced; on the other hand, endogenous hormone secretion is promoted, and the utilization of glucose by the body is improved, so that blood sugar is regulated from the source and the end.

[0025] (3) The application uses a microcapsule embedding and antioxidant protection technical solution to stably release active ingredients in the gastrointestinal environment, and the bioavailability is obviously improved. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 The application also provides a complex for promoting GLP-1 secretion.

[0027] Figure 2 The application also provides a complex for promoting GLP-1 secretion.

[0028] Figure 3 The application also provides a complex for promoting GLP-1 secretion.

[0029] Figure 4The high performance liquid chromatography (HPLC) test data optimization result graph of the effective component content of soybean oligosaccharide in the experimental example 1 of the present application is as follows:

[0030] Figure 5 The high performance liquid chromatography (HPLC) test data optimization result graph of the volatile oil content in the experimental example 1 of the present application is as follows:

[0031] Figure 6 The high performance liquid chromatography (HPLC) test data optimization result graph of the total flavonoid content in the experimental example 1 of the present application is as follows:

[0032] Figure 7 The experimental result comparison graph of GLP-1 peak value and body weight growth rate parameters in the experimental example 4 of the present application is as follows:

[0033] Figure 8 The experimental result trend comparison graph of fasting blood glucose and 2h postprandial blood glucose parameters in the experimental example 4 of the present application is as follows. DETAILED DESCRIPTION

[0034] The following examples further explain and illustrate the technical solutions of the present application. It is particularly pointed out that each specific embodiment is a specific embodiment and explanation of the technical solutions, and should not be regarded as a limitation on the protection scope of the present application. Those skilled in the art still have the right to modify the technical solutions of these examples, to equivalently replace part or all of the technical features, and these modifications or replacements do not change the essence of the corresponding technical solutions, and do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions described in the present application.

[0035] The present application proposes a compound for promoting the secretion of glucagon-like peptide-1 (GLP-1), a fermentation preparation method and its application, as shown in the accompanying Figure 1 The preparation flow chart of the compound for promoting the secretion of GLP-1 of the present application is as follows, the preparation of the compound of the present application includes fermentation of mulberry leaf powder preparation, soybean oligosaccharide extraction and purification, preparation of kaempferia extract, and composite mixing and microcapsule embedding, the specific technical solution design and general preparation process are as follows:

[0036] 1. Plant source component system construction

[0037] The compound of the present application is composed of multiple medicinal and edible plant ingredients, including main material fermented mulberry leaf powder, synergistic material soybean oligosaccharide and kaempferia galanga rhizome extract, auxiliary material white birch juice concentrate and white kidney bean extract. Each component is in the following mass percentage: fermented mulberry leaf powder 40-45%, soybean oligosaccharide 25-30%, kaempferia galanga rhizome extract 15-20%, white birch juice concentrate 8-12%, and white kidney bean extract 5-8%. Each component plays a corresponding role and can have synergistic effect: the fermented mulberry leaf powder provides the main hypoglycemic component 1-deoxynojirimycin (1-DNJ) and high-activity flavonoids, the white kidney bean extract blocks starch absorption to reduce blood glucose production, the soybean oligosaccharide acts as a prebiotic to promote GLP-1 release, the kaempferia galanga extract enhances insulin sensitivity and assists in reducing blood glucose, and the white birch juice supplies trace nutrients and has antioxidant effect. After the multiple components are compounded, they maintain blood glucose balance and reduce fat accumulation through different mechanisms.

[0038] 2. Plant components are extracted respectively

[0039] (1) Preparation of main material fermented mulberry leaf powder: The mulberry leaves are treated by probiotic fermentation coupled with multi-stage enzymolysis to improve the content and accessibility of active ingredients. First, the mulberry leaf powder is inoculated with a specific proportion of complex probiotic strains and subjected to solid-state fermentation under suitable temperature and humidity. During the fermentation process, the β-glucosidase produced by the microorganisms can convert flavonoid aglycone precursors in the mulberry leaves into free flavonoid aglycones, increasing the content of effective components such as quercetin by 1-1.5 times. At the same time, the fermentation products are rich in vitamins and organic acids produced by microbial metabolism, which helps to improve the nutritional value. Then, the fermented mulberry leaf material is subjected to multi-stage directional enzymolysis: egg white lysozyme is added to lyse the cell wall and release water-soluble components such as 1-DNJ in the cells, increasing the extraction rate of 1-DNJ; natto kinase is then added to degrade plant residual proteins into easily absorbed small peptides; subsequently, papain is added to decompose fiber impurities and reduce the viscosity of the sample. After the above-mentioned step-by-step enzymolysis treatment, the clarity and active substance content of the mulberry leaf fermentation broth can be greatly improved. Finally, the enzymolysis liquid is filtered through a screen to remove undissolved substances, and an ultrafiltration membrane is used to separate and enrich the target product. The macromolecular impurities in the retentate are removed, and the permeate contains the main active small molecular components. Finally, the permeate is spray dried to obtain refined fermented mulberry leaf powder, in which the mulberry alkaloid 1-DNJ is highly preserved. Compared with the mulberry leaf powder without the above-mentioned fermentation and enzymolysis treatment, the method of the present application makes the active ingredients in the mulberry leaves more enriched and easily absorbed, and fully realizes the hypoglycemic potential of the mulberry leaves.

[0040] (2) Synergistic material soybean oligosaccharide extraction and purification: ultrasonic-assisted extraction combined with gradient alcohol precipitation process is used to efficiently obtain soybean oligosaccharide. Specifically, defatted soybean powder is added into deionized water at a ratio of 1:5, and the soybean powder is treated under the action of ultrasonic waves, which significantly improves the leaching rate of oligosaccharide; the extract is first added with ethanol to a volume fraction of 30%, and high-molecular polysaccharide impurities are precipitated and removed; the supernatant is then added with anhydrous ethanol to a volume fraction of 60%, at which time the main target oligosaccharide is selectively precipitated, in which the oligosaccharide is mainly composed of raffinose and stachyose with a molecular weight of about 500; the second precipitate is dissolved in a small amount of pure water, and then the impurities such as monosaccharide and inorganic salt are removed through nanofiltration membrane to obtain a purified oligosaccharide solution. The oligosaccharide concentrate is placed at 4°C for 24 hours to crystallize, and needle-shaped crystals are precipitated, which are then filtered and dried to obtain high-purity soybean oligosaccharide powder.

[0041] (3) Synergistic material Kaempferia galanga rhizome extract preparation: supercritical CO2 extraction technology is used to obtain active essence from Kaempferia galanga rhizome. Dry Kaempferia galanga rhizome powder is placed in a supercritical extraction device, and CO2 fluid is passed through for extraction. On the one hand, volatile oil components of Kaempferia galanga are obtained, and on the other hand, part of the polar flavonoids such as kaempferol and quercetin can also be carried out under high pressure. After the extraction is completed, the oil or resin-like extract obtained by separation under reduced pressure is the Kaempferia galanga rhizome extract. This method has no organic solvent residue, and the product purity and biological activity are significantly better than those of traditional solvent reflux extraction.

[0042] (4) Auxiliary material extraction and preparation: birch sap concentrate preparation, fresh birch sap is collected, sterilized by 0.22 μm microfiltration, and concentrated to 1 / 5-1 / 10 of the original volume under vacuum and low temperature conditions to obtain birch sap concentrate; the present application uses commercially available birch sap cold-dried powder as a substitute, and the birch sap concentrate mainly provides trace nutrients and synergistic active substances, which can be directly used for subsequent mixing. White kidney bean extract preparation, white kidney bean extract can be obtained by water extraction and alcohol precipitation, preferably, commercially available white kidney bean extract powder with high content of α-amylase inhibitor is used, and the effective ingredient phase amylase content is ≥20%; in the present application, the white kidney bean extract is added as an auxiliary functional ingredient to block carbohydrates.

[0043] 3. Mixing and stabilization of the complex

[0044] The fermented mulberry leaf powder, soy oligosaccharide, kaempferol extract, birch juice concentrate and white kidney bean extract prepared above are mixed according to a pre-designed ratio to obtain an extract mixture. The mixture is further treated by using a microcapsule embedding technique to improve the stability and controlled release performance. Specifically, sodium alginate is used as a capsule material to prepare a 2% concentration solution, and chitosan is used as a complex capsule material to prepare a 1% concentration solution; the mixture powder is dissolved and dispersed in the sodium alginate solution to form a uniform suspension, and then the suspension is atomized and dropped into a solidification bath containing calcium chloride through an electrostatic spraying device to form gel microbeads instantaneously; then the microbeads are transferred to the chitosan solution to coat a second layer of film, forming microcapsules with a core-shell structure; the microcapsules are centrifuged and washed to remove surface salt, and then freeze-dried or spray-dried to obtain microcapsule powder with good fluidity. Preferably, when preparing the wall material solution, 5% of the total mass of the extract mixture is added to the inulin, which is used to improve the glass transition temperature of the microcapsule wall by using its glassy properties, so that the structure can still be stable at a higher temperature, reducing the leakage of active ingredients during storage; at the same time, 0.05wt% of rosemary acid and 0.02wt% of α-tocopherol are added as antioxidants, which can effectively inhibit the oxidative degradation of plant extracts during processing and storage.

[0045] Example 1

[0046] A complex for promoting GLP-1 secretion, and a preparation method specifically as follows:

[0047] Table 1 Detailed information table of main raw materials

[0048]

[0049]

[0050]

[0051]

[0052] S1: Take fresh mulberry leaves, wash, dry, and crush through a 40-mesh sieve to obtain mulberry leaf powder; take 1000 g of the mulberry leaf powder, add pure water to adjust the moisture content to 70%, sterilize, and inoculate with mixed bacteria of Lactobacillus rhamnosus, Bifidobacterium adolescentis, and Clostridium butyricum at a mass ratio of 6:2:1, mix thoroughly, and place in a fermentation tray; place the tray in a temperature-controlled fermentation chamber, maintain the temperature at 35°C and the relative humidity at 70%, and culture for 72 hours; after fermentation, collect the fermented mulberry leaf material, then suspend the fermented material in 10 times the mass of 0.05 mol / L phosphate buffer (pH 6.5), and then sequentially add the following enzyme preparations for enzymolysis: first, add 8 g of egg white lysozyme, stir at 45°C for 1.5 hours; then adjust the pH to 7.2, add 5 g of natto kinase, and act at 50°C for 2 hours; then adjust the pH to 6.0, add 10 g of papain, and act at 60°C for 1 hour; stir uniformly during the enzymolysis process, and after the enzymolysis is completed, heat the reaction solution in a 95°C water bath for 5 minutes to inactivate the enzymes; filter the obtained enzymolysis solution through a 200-mesh sieve to remove undissolved residues, then filter using an ultrafiltration membrane with a molecular weight cutoff of 3000 Da, collect the permeate, which is a filtrate rich in small-molecule active ingredients, concentrate the filtrate to a solid content of 20%, and finally perform spray drying, with an inlet temperature of 160°C and an outlet temperature of 60°C, to obtain a light brown powder of fermented mulberry leaf extract powder.

[0053] S2: Take 500 g of defatted soybean powder, add 2500 mL of deionized water in a container, and use ultrasonic extraction at 25°C for 30 minutes, with an extraction power of 300 W and a frequency of 40 kHz; after extraction, filter to obtain 2400 mL of soybean water extract, place the extract in a stirring tank, and slowly add anhydrous ethanol to a solution ethanol volume fraction of 30%, stand for 1 hour, during which time some impurities precipitate; after centrifugation to remove the precipitate, continue to add anhydrous ethanol to a final concentration of 60%, at which time a large amount of white precipitate is produced; stand at 4°C overnight to fully settle, centrifuge to collect the precipitate, dissolve the precipitate in pure water and transfer to a dialysis bag, use a nanofiltration membrane with a molecular weight cutoff of 500 Da to dialyze and desalt, until the dialysate has no reducing sugar reaction; concentrate the dialysate to 100 mL, transfer to a clean container, and stand at 4°C for 24 hours to crystallize, then filter out the crystals and dry in a vacuum dryer to obtain high-purity soybean oligosaccharide crystals.

[0054] S3: Take 200 g of dried Morus alba root stem slices, crush to 60 mesh, and place in an extraction tank of a 5 L supercritical CO2 extraction device; after pre-cooling, CO2 fluid is pumped into the extraction tank by a high-pressure pump, the pressure is adjusted to 32 MPa and the temperature is 43℃, the extraction process is started, and the duration is 2 hours; during the extraction process, the product in the separation tank is collected every 15 minutes, and the combined light yellow oily paste is obtained, 10% volume fraction of ethanol is added to the extraction tank as an entraining agent, and the extraction is continued at 28 MPa and 38℃ for 1 hour to improve the extraction of polar components, and the two obtained extracts are mixed to obtain the Morus alba root stem extract.

[0055] S4: Mix 84 g of fermented mulberry leaf extract powder obtained in S1, 56 g of soybean oligosaccharide powder obtained in S2, 36 g of Morus alba extract obtained in S3, 20 g of commercially available white birch juice freeze-dried powder, and 14 g of commercially available white kidney bean extract powder; first mix the above powder components, dissolve in pure water and stir thoroughly to form a uniform mixture; weigh 8 g of sodium alginate into 392 mL of pure water, and take 2 g of chitosan into 198 mL of pH 5.5 acetic acid buffer; add the mixture to the sodium alginate solution, and add pure water to make the total volume 500 mL, slowly stir until a stable suspension is formed, and add inulin 10 g, rosemary acid 0.1 g and α-tocopherol 0.04 g dissolved in the system; load the suspension into a high-voltage electrostatic sprayer with a 0.5 mm nozzle, place a 500 mL, 2% mass concentration CaCl2 cross-linking solution under the nozzle, turn on the high-voltage power supply (voltage 10 kV) and the pump, and spray the suspension into the CaCl2 solution at a speed of 1.5 mL / min; after spraying, the obtained calcium alginate gel beads are allowed to solidify for 30 minutes, then filtered out and placed into the prepared chitosan solution, and stirred gently for 15 minutes to form chitosan-calcium alginate composite film microcapsules; filter out the microcapsules, rinse with distilled water 3 times to remove unreacted substances on the surface, and finally transfer the wet microcapsules into a freeze dryer, pre-freeze at -50℃ for 2 hours, and sublimate dry for 24 hours to obtain dry powder of microencapsulated composite, which is the composite preparation of the application.

[0056] Example 2

[0057] Referring to the preparation method of Example 1, the difference is that:

[0058] In S1, the fermentation temperature is set to 33℃ and the relative humidity is set to 68%;

[0059] In S2, the ultrasonic extraction time is set to 25 minutes, the extraction power is 280 W, and the frequency is 35 kHz;

[0060] In S3, the supercritical CO2 extraction pressure is 30 MPa and the temperature is 40℃;

[0061] In S4, the suspension in the high-voltage electrostatic spraying is sprayed at a speed of 1 mL / min;

[0062] The other steps are the same.

[0063] Example 3

[0064] The preparation method of Example 1 is referred to, except that:

[0065] The fermentation temperature in S1 is set to 37℃, and the relative humidity is set to 72%;

[0066] The ultrasonic extraction time in S2 is set to 35 minutes, the extraction power is 320W, and the frequency is 45kHz;

[0067] The supercritical CO2 extraction pressure in S3 is 35MPa, and the temperature is 45℃;

[0068] The suspension spraying speed in the high-voltage electrostatic spraying in S4 is 2mL / min;

[0069] The other steps are the same.

[0070] Example 4

[0071] The preparation method of Example 1 is referred to, except that:

[0072] In S4, 80g of fermented mulberry leaf extract powder, 50g of soybean oligosaccharide powder, 30g of kaempferol extract, 24g of commercially available white birch juice cold-dried powder, and 16g of commercially available white kidney bean extract powder are mixed; the other steps are the same.

[0073] Example 5

[0074] In S4, 90g of fermented mulberry leaf extract powder, 52g of soybean oligosaccharide powder, 32g of kaempferol extract, 16g of commercially available white birch juice cold-dried powder, and 10g of commercially available white kidney bean extract powder are mixed; the other steps are the same.

[0075] Example 6

[0076] In S4, 82g of fermented mulberry leaf extract powder, 60g of soybean oligosaccharide powder, 32g of kaempferol extract, 16g of commercially available white birch juice cold-dried powder, and 10g of commercially available white kidney bean extract powder are mixed; the other steps are the same.

[0077] Example 7

[0078] In S4, 82g of fermented mulberry leaf extract powder, 52g of soybean oligosaccharide powder, 40g of kaempferol extract, 16g of commercially available white birch juice cold-dried powder, and 10g of commercially available white kidney bean extract powder are mixed; the other steps are the same.

[0079] Comparative Example 1

[0080] The preparation method of Example 1 is referred to, but the mulberry leaf raw material is not subjected to probiotic fermentation and multi-stage enzymatic treatment, and only the mulberry leaf is crushed and sieved to make powder, and other ingredients and preparation steps are the same as the present application.

[0081] Comparative Example 2

[0082] The preparation method of Example 1 is referred to, but the micro-encapsulation step is omitted, and the extracts are directly mixed and dried into a mixed powder. Other steps are the same.

[0083] Comparative Example 3

[0084] The preparation method of Example 1 is referred to, but the soybean oligosaccharide powder, kaempferol extract, commercially available white birch juice cold-dried powder, and commercially available white kidney bean extract powder are removed respectively to prepare four groups of preparations A, B, C, and D lacking components.

[0085] Experimental Example 1

[0086] The fermented mulberry leaf extract powder prepared in S1, the high-purity soybean oligosaccharide crystal prepared in S2, and the kaempferol root extract prepared in S3 in Example 1 are tested for key substance content using a high-performance liquid chromatograph (HPLC) instrument, with the specific test settings as follows:

[0087] 1. Test of fermented mulberry leaf extract powder

[0088] (1) Test target components: 1-deoxynojirimycin (DNJ) and total flavonoids (calculated as rutin).

[0089] (2) Sample preparation and pretreatment: For DNJ determination, the sample is weighed and extracted with 0.05 mol / L hydrochloric acid solution, and the two extraction solutions are combined. To enhance detection sensitivity, an appropriate amount of the extraction solution is subjected to column pre-derivation with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) reagent in a pH 8.5 boric acid buffer. The derived solution is filtered through a 0.45 μm filter membrane for HPLC analysis.

[0090] For total flavonoid determination, the sample is weighed and ultrasonically extracted with 70% ethanol solution to extract total flavonoid components. The extraction solution is filtered and made up to volume, and the extraction solution is added with 2 mol / L concentrated hydrochloric acid and placed in a water bath at 80°C for 1 hour to convert flavonoid glycosides to free flavonoid aglycones. The hydrolysis solution is extracted with an equal volume of ethyl acetate, and the organic phase is collected and concentrated under reduced pressure. The residue is dissolved in methanol and made up to volume, and then filtered through a 0.45 μm filter membrane for testing.

[0091] (3) HPLC chromatographic conditions: Due to the differences in the properties of DNJ and flavonoids, HPLC detection methods are established respectively, with the specific conditions shown in Table 2:

[0092] Table 2 HPLC chromatographic conditions for fermented mulberry leaf extract powder

[0093]

[0094] 2. Test of high purity soybean oligosaccharide crystal

[0095] (1) Test target components: stachyose and raffinose.

[0096] (2) Sample preparation and pretreatment: high purity soybean oligosaccharide crystal was weighed, dissolved in pure water, and the sample of the present application was very high in purity, so no impurity removal step was needed, only water dissolution and filtration were needed; the sample liquid after the above treatment was filtered through a 0.45 μm filter membrane and then injected.

[0097] (3) The HPLC chromatographic conditions are shown in Table 3:

[0098] Table 3 HPLC chromatographic conditions of high purity soybean oligosaccharide crystal

[0099]

[0100] 3. Test of kaempferia galanga rhizome extract

[0101] (1) Test target components: volatile oil (calculated as ethyl p-methoxycinnamate) and total flavonoids (calculated as kaempferol).

[0102] (2) Sample preparation and pretreatment: for volatile oil determination, 0.1 g was accurately weighed, dissolved in methanol and made up to 10 mL in a volumetric flask, the obtained solution was allowed to stand to allow the insoluble matter to settle, the supernatant was filtered through a 0.45 μm filter membrane for HPLC analysis.

[0103] For total flavonoid determination, the extract was taken and 50% ethanol solution was added to extract flavonoids by heating and refluxing, insoluble matter was filtered out, the filtrate was added to 2 mol / L concentrated hydrochloric acid, hydrolyzed at 80°C water bath for 1 hour to hydrolyze flavonoid glycosides to free flavonoid aglycone, after cooling, the flavonoid aglycone was extracted with ethyl acetate, the organic phase residue was evaporated to dryness and dissolved in methanol, filtered through a 0.45 μm filter membrane and then subjected to HPLC determination.

[0104] (3) The HPLC chromatographic conditions are shown in Table 4:

[0105] Table 4 HPLC chromatographic conditions of kaempferia galanga rhizome extract

[0106]

[0107] The test results are shown in Figures 4, 5 and 6, which are data optimization charts of test results, and the peak position and peak shape of the target substance can be clearly shown, and the content of the key target substance is calculated based on 100 g of fermented mulberry leaf extract powder, high purity soybean oligosaccharide crystal and kaempferia galanga rhizome extract, and the statistics are shown in Table 5: Figure 2 , 3 ,

[0108] Table 5 Content statistics of key targets

[0109]

[0110]

[0111] In combination with the charts, the contents of the key effective components contained in the fermented mulberry leaf extract powder, high-purity soybean oligosaccharide crystals and kaempferia galanga extract extracted by the method of the present application are higher, and the extraction efficiency of the key effective components is increased by 1.5-2 times compared with the traditional extraction and purification method.

[0112] Experimental Example 2

[0113] The final compound preparations obtained in Example 1 and Comparative Example 2 were respectively tested for in-vitro release effect in simulated gastric juice and intestinal juice, and the reference release substance was soybean oligosaccharide (the total of raffinose and stachyose), and the content was tested by the same method as in Experimental Example 1. The artificial gastric juice and artificial intestinal juice were prepared according to the provisions in the Chinese Pharmacopoeia, and the specific preparation was as follows:

[0114] Preparation of artificial gastric juice: 234 mL of concentrated hydrochloric acid was diluted to 1000 mL to obtain solution A. Then, 16.4 mL of solution A was diluted to 1000 mL to obtain solution B. 100 mg of pepsin was diluted with solution B to a final concentration of 4 g / L to obtain the gastric juice;

[0115] Preparation of artificial intestinal juice: 1.36 g of potassium dihydrogen phosphate was weighed into 100 mL of distilled water, and the pH value was adjusted to 6.8 using 0.2 mol / L NaOH solution for standby use. 1 g of trypsin was placed in a 100 mL volumetric flask, and the volume was adjusted with the above standby solution and shaken well;

[0116] Release effect test in artificial gastric juice: 0.1 g of sample was placed in 100 mL of simulated gastric juice, and the reaction was carried out at 37°C. Every half hour, 1 mL was taken out and diluted to 100 mL, and the total content of soybean oligosaccharide in the gastric juice was tested using high performance liquid chromatography (HPLC) instrument, and the cumulative release amount was calculated. The cumulative release time was set to 2 hours, and every half hour was set as a sampling point. Note that after each time, an equal amount of simulated gastric juice of the same concentration was supplemented. The release effect test conditions in the intestinal juice were basically the same as those in the gastric juice, except that the artificial gastric juice was replaced by artificial intestinal juice, and the cumulative release time was 12 hours, and the sampling points were 3 hours, 4 hours, 6 hours, 8 hours, 10 hours and 12 hours.

[0117] The release effect test results in artificial gastric juice are shown in Table 6, and the release effect test results in artificial intestinal juice are shown in Table 7.

[0118] Table 6 Comparison table of release effect determination results in artificial gastric juice of experimental example 2

[0119]

[0120] Table 7 Comparison table of release effect determination results in artificial intestinal juice of experimental example 2

[0121]

[0122] From the above results, it can be seen that in the release effect determination in artificial gastric juice, example 1 maintains a low release amount, and in the artificial intestinal juice, it is released in stages and in an appropriate amount, which meets the purpose of structural design; by contrast, comparative example 2 is not microencapsulated, has no protection in the artificial gastric juice stage, and cannot achieve protection in the gastric juice and appropriate long-acting sustained release in the intestinal juice.

[0123] Experimental example 3

[0124] The final compound preparations obtained from examples 1 and comparative example 2 were subjected to accelerated stability experiments, specifically, the compound preparations were placed in an accelerated stability test box (1x1x1m) at 40°C and a relative humidity of 65% for 30 days, and the residual rate of DNJ was determined. The test method of DNJ was the same as that in experimental example 1, and the test results are shown in Table 8.

[0125] Table 8 Comparison table of accelerated stability experiment results of experimental example 3

[0126]

[0127] From the above results, it can be seen that example 1 has a physical oxygen / humidity barrier of sodium alginate-chitosan microcapsules, and a double antioxidant barrier of rosmarinic acid and α-tocopherol synergistically providing, directly inhibiting the oxidation of DNJ; by contrast, comparative example 2 is not microencapsulated, and the active ingredients are easily lost.

[0128] Experimental example 4

[0129] The final preparations obtained from examples 1-7 and comparative examples 1-3 were subjected to animal model pharmacodynamics experiments for comparison, and the specific experimental process design is as follows:

[0130] The experimental animals were derived from Sanye (Suzhou) Biotechnology Co., Ltd., license number: SCXK (Su) 2025-0008. The test design is as follows:

[0131] 120 healthy male rats were selected and randomly divided into 15 groups, 8 rats in each group; among them, the normal control group was fed with ordinary feed without any treatment; the rest groups were fed with high-fat and high-sugar feed for 4 weeks to establish a metabolic syndrome model, and after the model was successfully established, the groups were intervened for 8 weeks: the model control group was given the same volume of normal saline by gavage; the rest groups were given the preparation by gavage, and the daily dose was equivalent to 500 mg / kg of body weight of the total amount of active ingredients, and the above gavage was once a day; during the whole intervention period, the animals in each group were normally fed, the body weight change was recorded, 24 hours after the last administration, after fasting for 12 hours, glucose solution 2 g / kg was given by gavage to simulate postprandial feeding, blood was collected from the tail vein 30 minutes after gavage, the plasma GLP-1 concentration was determined by ELISA method, and the fasting and 2-hour postprandial blood glucose values were determined, and the specific parameter settings are shown in Table 9.

[0132] Table 9 Specific parameter setting table of animal model pharmacodynamics experiment of experimental example 4

[0133]

[0134]

[0135] The experimental results are shown in Tables 10, Appendix Figure 7 and Appendix Figure 8 .

[0136] Table 10 Comparison table of animal model pharmacodynamics experiment results of experimental example 4

[0137]

[0138]

[0139] It can be seen from the chart results that Example 1 improves insulin sensitivity significantly, and GLP-1 secretion is restored to near normal level; Example 2 protects heat-sensitive DNJ by low-temperature fermentation, but the insufficient generation of fermentation metabolites leads to weakened prebiotic effect; Example 3 degrades flavonoids in mulberry leaves due to slightly high fermentation temperature, which weakens the antioxidant chain and reduces blood glucose control ability; Example 4 strengthens anti-inflammatory effect by strengthening betulin, which improves insulin resistance in adipose tissue; Example 5 has low white kidney bean extract, and the insufficient α-amylase inhibitor leads to postprandial hyperglycemia; Example 6 has high soybean oligosaccharides, and the prebiotic effect promotes the colonization of bifidobacterium, but the lack of anti-inflammatory components makes the overall effect slightly weak; Example 7 has high kaempferol extract, and kaempferol activates the PPARγ pathway, which significantly promotes lipid oxidation and blood glucose regulation. Compared with the above, Comparative Example 1 is not fermented mulberry leaves, which lacks active DNJ converted by fermentation, and the bioavailability is reduced by 50%, and the efficacy is lost; Comparative Example 2 is not embedded powder, and the gastric acid destroys probiotics and peptides, and the failure of sustained release leads to inactivation of components; Comparative Example 3A lacks soybean oligosaccharides, and the lack of prebiotics leads to the failure of probiotic colonization, and the stimulation of GLP-1 secretion is interrupted; Comparative Example 3B lacks kaempferol extract, which breaks the antioxidant chain and blocks the activation of the insulin signaling pathway; Comparative Example 3C lacks white birch juice, which weakens the anti-inflammatory effect and exacerbates insulin resistance; Comparative Example 3D lacks white kidney bean extract, and the lack of α-amylase inhibitor leads to postprandial hyperglycemia, and high blood glucose toxicity damages L cells. From the above results, it can be seen that microencapsulation is necessary, and the lack of any core component leads to the failure of metabolic improvement.

Claims

1. A complex for promoting GLP-1 secretion, characterized by, The compound comprises the following components in percentage of mass: 40-45% of fermented mulberry leaf powder, 25-30% of soybean oligosaccharide, 15-20% of kaempferia galantha rhizome extract, 8-12% of birch juice concentrate, and 5-8% of white kidney bean extract; and each component is mixed and then embedded in microcapsules.

2. The complex for facilitating GLP-1 secretion according to claim 1, wherein The fermented mulberry leaf powder is prepared by inoculating mulberry leaves with mixed bacteria of Lactobacillus rhamnosus, Bifidobacterium adolescentis and Clostridium butyricum for solid-state fermentation for 72 hours, and then refined by multi-stage directional enzymolysis and membrane separation.

3. The complex for facilitating GLP-1 secretion according to claim 1, wherein the GLP-1 is a GLP-1 (7-37) or a GLP-1 (7-36) amide. The soybean oligosaccharide is mainly composed of mixed oligosaccharides of stachyose and raffinose, prepared by ultrasonic-assisted water extraction and stepwise ethanol precipitation of defatted soybean powder, and then desalted by nanofiltration and purified by low-temperature crystallization.

4. The complex for facilitating GLP-1 secretion according to claim 1, wherein The kaempferia galantha rhizome extract is prepared by supercritical CO2 extraction technology, and is rich in volatile oil and flavonoid active ingredients.

5. The complex for facilitating GLP-1 secretion according to claim 1, wherein the GLP-1 is a GLP-1 (7-37) or a GLP-1 (7-36) amide. The embedding in microcapsules is achieved by embedding the compound in sodium alginate and chitosan as wall materials to form microcapsules, further adding 5wt% of inulin as a glass transition carrier in the microcapsules to improve the glass transition temperature of the microcapsule wall, improve the stability of the active ingredients, and further compounding 0.05wt% of rosemary acid and 0.02wt% of α-tocopherol as antioxidant protective agents.

6. A method for preparing a complex that promotes GLP-1 secretion as described in claim 1, characterized in that, The preparation is carried out by the following steps: S1: fresh mulberry leaves are washed, dried, crushed, sieved to obtain mulberry leaf powder, and then added with pure water to adjust the moisture content to 70%, sterilized, inoculated with mixed bacteria of Lactobacillus rhamnosus, Bifidobacterium adolescentis and Clostridium butyricum, and then fermented and cultured for 72 hours under the conditions of maintaining temperature and relative humidity; after fermentation, the fermented mulberry leaf powder is obtained by adding phosphate buffer solution, suspending, adding egg white lysozyme, natto kinase and papain in sequence for enzymolysis, sieving after enzymolysis, filtering with an ultrafiltration membrane with a molecular weight cut-off of 3000 Da, concentrating the filtrate to a solid content of 20%, and finally spray drying; S2: defatted soybean powder is added with deionized water, and ultrasonic extraction is carried out under the condition of water bath at 25℃, and then filtered and added with anhydrous ethanol to the extraction liquid to a volume fraction of 30%, and then placed for 1 hour, and then centrifuged to remove the precipitate, and then the supernatant is added with anhydrous ethanol to a final concentration of 60%, and then placed at 4℃ overnight, and then centrifuged to collect the precipitate, and then the precipitate is dissolved in pure water and transferred into a dialysis bag, and then desalted by nanofiltration membrane with a molecular weight cut-off of 500 Da, until the dialysate has no reducing sugar reaction, and then the dialysate is concentrated and placed at 4℃ for 24 hours to crystallize, and then the crystals are filtered out and vacuum dried to obtain soybean oligosaccharide; S3: kaempferia galantha rhizome is sliced and crushed, and then placed in a supercritical CO2 extraction device, and then extracted by adjusting pressure and temperature, and then the product is collected every 15 minutes during the extraction process and then combined, and then 10% volume fraction of ethanol is added after the first extraction is completed, and then the extraction is continued under the condition of 28MPa and 38℃ for 1 hour, and then the two extraction products are combined and mixed to obtain the kaempferia galantha rhizome extract; S4: the fermented mulberry leaf extract powder obtained in S1, the soy oligosaccharide obtained in S2, the kaempferol extract obtained in S3, commercially available white birch juice cold-dried powder and commercially available white kidney bean extract powder are mixed uniformly, and then dissolved in pure water and stirred to form a mixed solution; the mixed solution is added to the sodium alginate solution, and stirred to form a suspension, during which inulin, rosemary acid and alpha-tocopherol are added; the suspension is then subjected to high-pressure electrostatic spraying, and after completion, it is allowed to stand for solidification for 30 minutes; the solidified gel beads are filtered out and put into a chitosan solution, and stirred for coating for 15 minutes; the wet microcapsules are filtered and rinsed, and then transferred into a freeze-drying machine, and subjected to sublimation drying for 24 hours after pre-freezing at-50℃ for 2 hours, to obtain the compound preparation.

7. The method of claim 6, wherein the complex is prepared by the steps of: a) providing a GLP-1 compound; b) providing a compound of formula (I) or a pharmaceutically acceptable salt thereof; and c) combining the GLP-1 compound and the compound of formula (I) or a pharmaceutically acceptable salt thereof. The mixed bacteria in S1 are added in a mass ratio of Lactobacillus rhamnosus:Bifidobacterium adolescentis:Clostridium butyricum = 6:2:

1.

8. The method of claim 6, wherein the complex is prepared by the steps of: a) providing a GLP-1 compound; b) providing a compound of formula (I) or a pharmaceutically acceptable salt thereof; and c) combining the GLP-1 compound and the compound of formula (I) or a pharmaceutically acceptable salt thereof. The fermentation culture in S1 is maintained at a temperature and relative humidity, and the temperature and relative humidity are specifically set as follows: fermentation temperature 33℃-37℃, relative humidity 68%-72%.

9. A method for preparing a complex that promotes GLP-1 secretion as described in claim 6, characterized in that, The ultrasonic extraction in S2 is performed with the following parameters: extraction time 25-35 minutes, extraction power 280W-320W, and frequency 35kHz-45kHz; the extraction in S3 is performed by adjusting the pressure and temperature, and the parameters are set as follows: extraction pressure 30MPa-35MPa, and temperature 40℃-45℃; the high-pressure electrostatic spraying in S4 is performed with the following parameters: voltage 10kV, spraying rate 1mL / min-2mL / min, and coagulation solution 2% CaCl2 solution.

10. Use of a complex for promoting GLP-1 secretion according to any one of claims 1 to 9, characterized in that, The compound is applied to promote the endogenous secretion of glucagon-like peptide-1 in the intestinal tract.

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