A hypoglycemic peptide composition of monascus for treating type 2 diabetes and its preparation method and application

Abstract

The present application relates to the technical field of biological medicine, and particularly relates to a douchi hypoglycemic peptide composition for treating type 2 diabetes, and a preparation method and application thereof. The douchi hypoglycemic peptide composition is composed of hypoglycemic peptides from Yangjiang douchi and mulberry leaf alcohol extract. It is found for the first time that the combination of unpeeled douchi hypoglycemic peptides (SK-DPF) and mulberry leaf alcohol extract (ML-Et) has a significant synergistic effect in inhibiting the activities of alpha-glucosidase and DPP-IV, and the optimal mass ratio is 1:0.8. In a HFD / STZ-induced T2DM mouse model, the composition can significantly reduce fasting blood glucose, improve glucose tolerance and regulate blood lipids, and the effect is better than that of using each component or other combinations, so that the core metabolic indicators of the model animals can basically return to normal levels, and the composition has a good application prospect.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a soybean-based hypoglycemic peptide composition for treating type 2 diabetes, its preparation method, and its application. Background Technology

[0002] Type 2 diabetes mellitus (T2DM) is a metabolic disease characterized by persistent hyperglycemia, with pathological mechanisms involving insulin resistance and pancreatic β-cell dysfunction. According to the International Diabetes Federation, there are over 500 million people with diabetes worldwide, with T2DM accounting for over 90%. Currently, commonly used oral hypoglycemic agents include metformin, sulfonylureas, alpha-glucosidase inhibitors, and dipeptidyl peptidase-IV (DPP-IV) inhibitors. While these chemical drugs can effectively control blood sugar, long-term use often results in adverse reactions such as gastrointestinal discomfort, hypoglycemia, and weight gain, limiting their use in prediabetic individuals and patients requiring long-term management. Therefore, finding safe and low-side-effect hypoglycemic active ingredients from natural food resources has become an important direction in the research and development of functional foods and natural medicines.

[0003] Doubanjiang (fermented black soybeans) is a traditional Chinese fermented soybean food, primarily made from black or yellow soybeans through fermentation by microorganisms (such as Aspergillus, Mucor, Rhizopus, or bacteria). Based on regional differences in fermentation strains and processes, doubanjiang can be categorized into four main types: Aspergillus-based, Mucor-based, Rhizopus-based, and bacterial-based. Yangjiang doubanjiang, a specialty product of Yangjiang, Guangdong, belongs to the Aspergillus-based type. It is made from black soybeans through natural koji-making and salting, and is widely used in Chinese cooking due to its unique flavor and rich nutritional value.

[0004] Modern research shows that during the fermentation process of fermented soybeans (douchi), soybean protein is hydrolyzed into polypeptides and amino acids by microbial proteases, while isoflavones are converted into aglycones with higher bioavailability. These bioactive components endow douchi with various physiological functions. Chinese invention patent CN118620976A discloses a method for preparing a douchi polypeptide extract. Using freeze-dried douchi powder as raw material, a neutral protease is added and enzymatically hydrolyzed at 40-50℃ for 2-4 hours. The extract is then obtained through centrifugation, concentration, and dialysis. This patent reports that compared with the traditional ultrasound-assisted water extraction method, the enzymatic hydrolysis method increases the extraction rate by 43.5%, and the polypeptide content increases from 41.3% to 62.7%. In a T2DM mouse model induced by streptozotocin (STZ) combined with a high-fat diet (HFD), fasting blood glucose decreased to 7.15 mmol / L after DPT gavage, while it was 9.07 mmol / L in the conventional water extract DPF group, with a hypoglycemic activity of approximately 21.2%.

[0005] Chinese invention patent CN120866453A discloses a method for preparing multiple bioactive peptides based on soybean residue fermentation. Using soybean residue as a base material, it utilizes *Bacillus amyloliquefaciens* CGMCC No. 29157 derived from fermented soybeans for fermentation. Seven peptides with α-glucosidase inhibitory, angiotensin-converting enzyme inhibitory, and antioxidant activities are isolated and purified from the fermentation products. Their amino acid sequences are CKLLL, APGYSC, FPKYG, FPGPLV, FPRSH, FMAV, and SVCC. Among them, CKLLL exhibits all three bioactivities, with a half-maximal inhibitory concentration (IC50) for α-glucosidase. 50 The value is 6.05 mM.

[0006] In basic research, Guo et al. (2023, Food Chemistry: X, 19:100779) used mass spectrometry-based peptidomics to identify novel α-glucosidase and angiotensin-converting enzyme (ACE) inhibitory peptides from fermented black soybeans. Huang et al. (2025, Journal of Functional Foods, 133:107028) reported the hypoglycemic and lipid-lowering effects of DPF (distilled fermented black soybeans) rich in peptides prepared from Yangjiang fermented black soybeans on HFD / STZ-induced type 2 diabetes mellitus (T2DM) mice. The DPF preparation method in this study included peeling the fermented black soybeans, grinding in an ice bath, low-temperature water extraction, high-speed centrifugation, 500 Da dialysis for desalting, and freeze-drying. The obtained DPF contained 65.0% peptides (dry weight), and 288 reliable peptide sequences were identified by LC-MS / MS. In a type 2 diabetes mellitus (T2DM) mouse model, after 8 weeks of gavage administration of DPF (200 mg / kg), fasting blood glucose, total cholesterol, and triglyceride levels were significantly reduced, while insulin levels were significantly increased. Metagenomic analysis of the gut microbiota showed that DPF intervention could partially reverse the gut microbiota dysbiosis induced by HFD / STZ, improving the richness and diversity of the microbial community. Liver transcriptome analysis identified 821 differentially expressed genes, enriched in signaling pathways such as FoxO, MAPK, and PI3K-Akt. These studies provide a scientific basis for the application of fermented black soybeans in hypoglycemic functional foods.

[0007] Yu et al. (2022, Food & Function, 13(6): 3343-3352) purified and identified the hypoglycemic peptides VY (Val-Tyr) and SFLLR (Ser-Phe-Leu-Leu-Arg) from Yongchuan fermented black soybeans. In vitro experiments showed that these two peptides can promote glucose uptake in L6 myotube cells by activating the AMPK / MAPK / GLUT4 signaling pathway. Further animal experiments confirmed that VY and SFLLR can improve glucose homeostasis and gut microbiota dysbiosis in high-fat diet-induced insulin-resistant mice.

[0008] In summary, existing technologies have confirmed that peptides derived from fermented soybeans have the potential to lower blood sugar. However, the following shortcomings still exist in actual development: it is still unknown whether the hypoglycemic active ingredients of existing fermented soybean extracts are mainly derived from the skin or flesh of the fermented soybeans. Furthermore, research on hypoglycemic active peptides mainly focuses on the hypoglycemic effect of single components, while there are very few reports on whether fermented soybean extracts can produce synergistic effects when combined with other natural hypoglycemic components. Summary of the Invention

[0009] To address the aforementioned technical problems, the present invention aims to provide a soybean blood sugar-lowering peptide composition for treating type 2 diabetes, its preparation method, and its application. The soybean blood sugar-lowering peptide composition can significantly reduce fasting blood glucose, improve glucose tolerance, and regulate blood lipids. Its effects are superior to using any single component or other compound combinations. It can restore the core metabolic indicators of model animals to a near-normal level and has good application prospects.

[0010] To achieve the above-mentioned technical effects, the present invention adopts the following technical solution:

[0011] Firstly, this invention provides a fermented soybean hypoglycemic peptide composition for treating type 2 diabetes. The composition comprises fermented soybean hypoglycemic peptide and mulberry leaf extract, wherein the fermented soybean hypoglycemic peptide is an extract of Yangjiang fermented soybean, and the mulberry leaf extract is an ethanolic extract of mulberry leaves. Through extensive experimental screening, this invention has discovered that combining Yangjiang fermented soybean hypoglycemic peptide prepared using a specific process with mulberry leaf ethanolic extract can produce a significant synergistic effect in inhibiting α-glucosidase and DPP-IV activity. Its actual inhibitory activity far exceeds the theoretical superposition expectation of the components, overcoming the deficiency in existing technologies that only focus on the hypoglycemic effect of a single component and lack research on synergistic combinations.

[0012] As a preferred technical solution, the fermented soybean hypoglycemic peptide is either peeled fermented soybean hypoglycemic peptide or unpeeled fermented soybean hypoglycemic peptide.

[0013] The preparation method of the unpeeled fermented black soybean hypoglycemic peptide is as follows: Yangjiang fermented black soybeans are taken without peeling, freeze-dried and pulverized, then subjected to cellulase for fiber disruption, followed by low-temperature water extraction, centrifugation, dialysis, and freeze-drying. Studies have shown that retaining the outer skin of the fermented black soybeans and using cellulase for fiber disruption pretreatment facilitates the full release of intracellular active peptides. The resulting unpeeled fermented black soybean hypoglycemic peptide (SK-DPF) exhibits significantly better enzyme inhibitory activity than the peeled fermented black soybean hypoglycemic peptide (Q-DPF).

[0014] The preparation method of the peeled fermented black soybean hypoglycemic peptide is as follows: Yangjiang fermented black soybeans are peeled, the soybean flesh is freeze-dried and pulverized, and then extracted with water at low temperature, centrifuged, dialyzed, and freeze-dried. Although the two types of fermented black soybean hypoglycemic peptides differ in their hypoglycemic activity, both can exert a synergistic effect when combined with mulberry leaf alcohol extract.

[0015] As a preferred technical solution, the mass ratio of the fermented soybean hypoglycemic peptide to the mulberry leaf alcohol extract is 1:0.5-1.5. Within this ratio range, the two types of active ingredients can produce an effective synergistic effect, and the enzyme inhibitory activity of the composition is superior to that of each individual group.

[0016] More preferably, the mass ratio of the fermented soybean hypoglycemic peptide to the mulberry leaf alcohol extract is 1:0.7-1.0. Within this ratio range, the synergistic effect is more prominent, and the activity of the composition is significantly enhanced.

[0017] Most preferably, the mass ratio of the fermented soybean hypoglycemic peptide to the mulberry leaf alcohol extract is 1:0.8.

[0018] In vitro screening experiments confirmed that when the ratio of the two components was 1:0.8, the composition achieved peak inhibitory activity against DPP-IV, with an IC50 value of [missing value]. 50 With a concentration of only 51.0 μg / mL, it is significantly superior to other conventional ratios, including 1:1.

[0019] Secondly, the present invention provides the use of the aforementioned fermented soybean hypoglycemic peptide composition in the preparation of a medicament or functional food for improving or treating type 2 diabetes by inhibiting α-glucosidase and / or DPP-IV activity.

[0020] Thirdly, the present invention also provides a pharmaceutical preparation comprising the fermented soybean hypoglycemic peptide composition described in any one of the above claims and pharmaceutically acceptable excipients. The dosage form of the pharmaceutical preparation includes, but is not limited to, oral dosage forms such as capsules, tablets, granules, oral liquids, and powders, and can be prepared using conventional pharmaceutical processes according to clinical needs.

[0021] The pharmaceutically acceptable excipients are selected from one or more of the following: fillers, disintegrants, binders, lubricants, flow aids, wetting agents, solubilizers, suspending agents, emulsifiers, flavoring agents, sweeteners, colorants, preservatives, antioxidants, coating materials, and sustained-release materials.

[0022] The filler may be selected from one or more of starch, pregelatinized starch, microcrystalline cellulose, lactose, mannitol, sorbitol, xylitol, dextrin, sucrose, glucose, dicalcium phosphate, calcium carbonate, and calcium sulfate. The disintegrant may be selected from one or more of sodium carboxymethyl starch, crospovidone, crospovidone carboxymethyl cellulose, low-substituted hydroxypropyl cellulose, starch, and microcrystalline cellulose. The binder may be selected from one or more of povidone, hydroxypropyl methylcellulose, hydroxypropyl cellulose, sodium carboxymethyl cellulose, starch paste, gelatin, gum arabic, and sodium alginate. The lubricant may be selected from one or more of magnesium stearate, calcium stearate, stearic acid, talc, micronized silica gel, polyethylene glycol, and hydrogenated vegetable oil. The flow aid may be selected from one or more of micronized silica gel, talc, and corn starch.

[0023] For capsules, the pharmaceutically acceptable excipients preferably include fillers, disintegrants, lubricants, and flow aids. For tablets, the pharmaceutically acceptable excipients preferably include fillers, disintegrants, binders, and lubricants, with coating treatment as needed. The coating material can be selected from hydroxypropyl methylcellulose, polyethylene glycol, acrylic resin, ethyl cellulose, etc. For granules, the pharmaceutically acceptable excipients preferably include fillers, binders, and flavoring agents. The flavoring agents can be selected from one or more of stevioside, aspartame, sucralose, fructose, peppermint flavor, and fruit flavor. For oral liquids, the pharmaceutically acceptable excipients preferably include solubilizers, suspending agents, preservatives, and flavoring agents. The solubilizers can be selected from one or more of polysorbate, poloxamer, and polyoxyethylene castor oil derivatives. The preservatives can be selected from one or more of sodium benzoate, potassium sorbate, and parabens. For powders, the pharmaceutically acceptable excipients preferably include fillers, flow aids, and flavoring agents.

[0024] Fourthly, the present invention also provides a functional food comprising the fermented soybean hypoglycemic peptide composition described in any of the above-mentioned embodiments and food-grade acceptable excipients. The functional food may take the form of, but is not limited to, solid beverages, meal replacement powders, chewable tablets, nutrition bars, etc., and is suitable for daily dietary supplementation for people with prediabetes and consumers requiring long-term blood sugar management.

[0025] The food-grade acceptable excipients are selected from one or more of the following: fillers, sweeteners, acidulants, flavorings, thickeners, anti-caking agents, emulsifiers, stabilizers, colorants, antioxidants, preservatives, dietary fiber, proteins, vitamins, and minerals.

[0026] The filler may be selected from one or more of maltodextrin, resistant dextrin, inulin, isomaltooligosaccharide, polydextrose, and microcrystalline cellulose. The sweetener is preferably a low glycemic index natural sweetener or functional sugar alcohol, and may be selected from one or more of steviol glycosides, mogrosides, erythritol, xylitol, maltitol, and isomaltitol. The acidulant may be selected from one or more of citric acid, malic acid, tartaric acid, and lactic acid. The flavoring may be selected from one or more of fruit flavorings, plant flavorings, and frankincense. The thickener may be selected from one or more of xanthan gum, guar gum, carrageenan, sodium carboxymethyl cellulose, sodium alginate, and agar. The anti-caking agent may be selected from one or more of silica, tricalcium phosphate, and microcrystalline cellulose.

[0027] For solid beverages, the food-grade acceptable excipients preferably include fillers, sweeteners, acidulants, flavorings, and anti-caking agents. For meal replacement powders, the food-grade acceptable excipients preferably include fillers, dietary fiber, protein, vitamins, and minerals, which can be formulated according to the nutritional needs of the target population. For chewable tablets, the food-grade acceptable excipients preferably include fillers, sweeteners, acidulants, lubricants, and flavorings. For nutrition bars, the food-grade acceptable excipients preferably include fillers, thickeners, dietary fiber, protein, and sweeteners, which are shaped and coated as needed.

[0028] Fifthly, the present invention also provides the application of the aforementioned fermented soybean hypoglycemic peptide composition in the preparation of drugs or functional foods for improving gut microbiota dysbiosis in type 2 diabetes. In a T2DM mouse model, the composition of the present invention can significantly increase the richness and diversity of gut microbiota (Chao1 index and Shannon index), restore the relative abundance of beneficial bacteria Bacteroidota, inhibit the abnormal proliferation of conditionally pathogenic bacteria Escherichia-Shigella, and significantly restore the abundance of beneficial bacteria Phocaeicola vulgatus.

[0029] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0030] First, this invention reveals for the first time a significant synergistic effect between fermented soybean hypoglycemic peptides prepared using specific sources and processes and mulberry leaf alcohol extract. Through systematic in vitro enzyme inhibition activity screening, it was confirmed that the combination of unpeeled fermented soybean hypoglycemic peptides and mulberry leaf alcohol extract exhibits significantly better actual inhibitory activity than the theoretical additive effect when each component is used alone, achieving a synergistic level, in simultaneously inhibiting two key hypoglycemic targets: α-glucosidase and DPP-IV. This discovery breaks through the existing research approach that focuses only on the hypoglycemic effect of a single component of fermented soybean, providing a novel formulation strategy and scientific basis for developing multi-target synergistic natural hypoglycemic products.

[0031] Secondly, through optimized experiments, this invention precisely determined the optimal mass ratio of fermented soybean hypoglycemic peptides to mulberry leaf alcohol extract. Under this optimal ratio, the enzyme inhibitory activity of the composition reached its peak, significantly superior to the conventional ratio, indicating a clear synergistic window for each active ingredient in the composition. This provides a quantitative formulation basis for the standardization and industrial production of the product.

[0032] Furthermore, the composition provided by this invention exhibits excellent and comprehensive metabolic regulatory effects in in vivo animal models. In type 2 diabetes model animals, after intervention with the optimally proportioned composition, fasting blood glucose, oral glucose tolerance, and serum triglyceride levels were significantly improved. The overall effect is not only superior to each single component or other combinations, but also restores the core metabolic indicators of the model animals to near-normal levels. The efficacy is comparable to or even better than that of first-line clinical drugs, showing good potential for clinical application.

[0033] Furthermore, this invention also reveals for the first time that the composition has a positive effect on improving diabetes-related gut microbiota dysbiosis. The composition intervention can significantly increase the richness and diversity of the gut microbiota, restore the relative abundance of beneficial bacteria, inhibit the abnormal proliferation of conditionally pathogenic bacteria, and promote the enrichment of specific beneficial bacteria. In particular, the composition using peeled fermented soybean hypoglycemic peptides and mulberry leaf alcohol extract exhibits unique advantages in improving the gut microbiota, providing a new material basis and application direction for developing diabetes adjunctive intervention products targeting the gut microbiota.

[0034] Finally, the raw materials used in this invention are all natural food resources, and the preparation process is mild and environmentally friendly, without involving the large-scale use of organic solvents and high-temperature treatment. The resulting composition has high safety and few side effects. It can be developed into oral preparations as active pharmaceutical ingredients or as a base for functional foods. It is suitable for long-term conditioning and health maintenance for people with prediabetes and has good industrialization prospects and social benefits. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the description herein is for illustrative purposes only and is not intended to limit the scope of protection of this invention.

[0036] Example 1

[0037] The purpose of this embodiment is to provide a method for preparing unpeeled fermented soybean hypoglycemic peptide (SK-DPF):

[0038] Take Yangjiang fermented black beans, without removing the skin, freeze-dry, and then pulverize to a particle size of less than 20 mesh to obtain whole fermented black bean powder. Weigh 100.0 g of whole fermented black bean powder, add 200 mL of deionized water and stir well. Then add 0.3 g of cellulase (enzyme activity 100,000 U / g) and incubate at 45℃ for 1.5 hours to break down the fiber walls. After enzymatic hydrolysis, cool to 4℃, add pre-cooled deionized water to a total added water volume of 400 mL (i.e., a material-to-liquid ratio of 1:4, g / mL), and then grind into a homogenate in an ice bath. Extract the obtained homogenate at 4℃ with low stirring at 80 rpm for 1 hour. After extraction, centrifuge the homogenate at 4℃, 20,000 × g for 30 minutes, and collect the supernatant. Dialyze the supernatant in deionized water at 4℃ for 48 hours using a dialysis bag with a molecular weight cutoff of 500 Da (changing the water every 6 hours). After dialysis, the retained material in the dialysis bag is collected and freeze-dried to constant weight to obtain unpeeled fermented soybean hypoglycemic peptide, named SK-DPF.

[0039] Example 2

[0040] The purpose of this embodiment is to provide a method for preparing a blood sugar-lowering peptide (Q-DPF) from peeled fermented soybeans:

[0041] The difference between this embodiment and Example 1 is that the outer skin of Yangjiang fermented black beans is first peeled off, and only the bean flesh is taken for freeze-drying and pulverizing to a particle size of less than 20 mesh, resulting in peeled fermented black bean powder. Then, the same steps as in Example 1 are followed, but the cellulase hydrolysis step is omitted (i.e., no cellulase is added, and no 45℃ incubation hydrolysis is performed). Instead, after adding deionized water, pre-cooled deionized water is added at 4℃ to bring the material-to-liquid ratio to 1:4. Then, the mixture is ground into a homogenate in an ice bath. Subsequent extraction, centrifugation, dialysis, and freeze-drying are all the same as in Example 1. The obtained product is named peeled fermented black bean hypoglycemic peptide, or Q-DPF.

[0042] Example 3

[0043] The purpose of this embodiment is to provide a method for preparing mulberry leaf alcohol extract (ML-Et):

[0044] Dried mulberry leaves were crushed and passed through a 40-mesh sieve. 70% ethanol was added at a material-to-liquid ratio of 1:10 (w / v, g / mL), and the mixture was extracted with ultrasound at 60℃ for 1 hour (ultrasound power 100 W). The extract was filtered, and the filtrate was collected. The ethanol was recovered by concentration under reduced pressure at 50℃. The resulting concentrate was freeze-dried to constant weight to obtain the mulberry leaf ethanol extract, named ML-Et.

[0045] Example 4

[0046] The purpose of this embodiment is to provide a method for preparing mulberry leaf aqueous extract (ML-Wa). The only difference from Example 3 is that the extraction solvent is replaced with pure water instead of 70% ethanol by volume, and the final mulberry leaf aqueous extract ML-Wa is obtained.

[0047] Experimental Example 1

[0048] This experiment aims to observe the in vitro enzyme inhibitory activity of each monomer sample and the preliminary compound, and to screen out the combination with the best synergistic effect.

[0049] 1.1 Experimental Groups

[0050] The following groups were set up in this experiment: blank control group (with buffer instead of sample), α-glucosidase positive control group (acarbose), DPP-IV positive control group (sitagliptin phosphate); single group: SK-DPF single group, Q-DPF single group, ML-Et single group, ML-Wa single group; combination group (fixed mass ratio 1:1): SK-DPF+ML-Et combination group, Q-DPF+ML-Et combination group, SK-DPF+ML-Wa combination group, Q-DPF+ML-Wa combination group.

[0051] 1.2 Experimental Methods

[0052] The half-maximal inhibitory concentrations (IC50) of each group of samples against α-glucosidase and DPP-IV were determined using standard methods. 50 For each combination group, the actual IC50 was measured. 50 The value, and the equation obtained through the classical medium-efficiency equation "1 / IC" 50 (Theoretical) = fA / IC 50 (A) +fB / IC 50 (B)” The theoretical IC calculated 50 Compare the values. If the actual IC 50 The value is significantly lower than the theoretical IC. 50 If the value is positive, it is determined to have a synergistic effect.

[0053] 1.3 Experimental Results and Analysis

[0054] The results of the comparison of in vitro enzyme inhibitory activity and synergistic effect of each monomer and the 1:1 combination group are shown in Table 1.

[0055] Table 1. Comparison of in vitro enzyme inhibitory activity and synergistic effect of each monomer and the 1:1 combination group (n=3)

[0056] Note: Different letters in the same column indicate significant differences between groups (P < 0.05).

[0057] Table 1 systematically compares the in vitro inhibitory activities of four monomers—unpeeled fermented soybean hypoglycemic peptide (SK-DPF), peeled fermented soybean hypoglycemic peptide (Q-DPF), mulberry leaf alcohol extract (ML-Et), and mulberry leaf water extract (ML-Wa)—and their 1:1 combinations against α-glucosidase and DPP-IV. Based on monomeric activity, ML-Et exhibits the best inhibitory activity against both target enzymes (α-glucosidase IC50). 50 The concentration was 168.4 μg / mL, and the DPP-IV IC50 concentration was [value missing]. 50 The activity of ML-Wa was the lowest (102.6 μg / mL), followed by SK-DPF, while ML-Wa showed the weakest activity. This indicates that the key hypoglycemic active ingredients in mulberry leaves are more easily extracted with 70% ethanol, and the content or potency of active substances in the water extract is relatively low. Regarding the hypoglycemic peptides from fermented soybeans, SK-DPF (without peel) showed better activity than Q-DPF (with peel), suggesting that the outer skin of fermented soybeans may contain cellulose components beneficial for enzymatic hydrolysis and extraction, or that more active peptides are released through fiber cell disruption.

[0058] After combining the monomers at a 1:1 mass ratio, the measured IC values ​​for all combined groups were... 50 The values ​​were all significantly lower than the theoretical superposition value, indicating a synergistic effect in all four combinations. The combination of SK-DPF and ML-Et showed the most significant synergistic effect. The measured IC50 values ​​for this combination against α-glucosidase were [data missing]. 50 The actual concentration was only 89.7 μg / mL, far lower than the theoretical value of 175.1 μg / mL; the measured IC50 for DPP-IV was... 50 The concentration was 62.3 μg / mL, significantly lower than the theoretical value of 113.3 μg / mL, achieving a highly significant synergistic effect on both target enzymes. In contrast, the other three combinations only achieved a significant synergistic effect. This result suggests that SK-DPF and ML-Et have a good complementary or synergistic enhancement effect in their mechanism of action, providing a direct basis for subsequent core compatibility selection.

[0059] Experimental Example 2

[0060] Based on the aforementioned experiments, and having established that SK-DPF and ML-Et have the best synergistic effect, this experimental case aims to further and more precisely observe in vitro whether there is an optimal mass ratio between the two.

[0061] 1.1 Experimental Methods

[0062] A series of compositions were prepared by mixing SK-DPF and ML-Et at mass ratios of 1:0.5, 1:0.7, 1:0.8, 1:0.9, 1:1.0, and 1:1.5, respectively. The DPP-IV inhibitory activity (IC50) of each composition was determined according to the method described in Experimental Example 1. 50 ).

[0063] 2.2 Experimental Results and Analysis

[0064] The results of DPP-IV inhibitory activity of SK-DPF and ML-Et compositions with different ratios are shown in Table 2.

[0065] Table 2. DPP-IV inhibitory activity of SK-DPF and ML-Et compositions with different ratios (n=3)

[0066] Note: Different letters in the same column indicate significant differences between groups (P < 0.05).

[0067] After confirming the optimal synergistic effect of SK-DPF and ML-Et, the optimal mass ratio of the two was further explored. Table 2 shows the overall trend: with increasing ML-Et ratio, the inhibitory activity exhibits a characteristic of first increasing and then decreasing. When the SK-DPF to ML-Et ratio is 1:0.5, the IC50 of the composition against DPP-IV is... 50 The concentration was 78.5 μg / mL; as the ML-Et ratio gradually increased to 1:0.7 and 1:0.8, the inhibitory activity continued to increase, reaching a peak at 1:0.8, with an IC50 concentration of 78.5 μg / mL. 50 The concentration was 51.0 μg / mL; subsequently, further increasing the ML-Et ratio to 1:0.9, 1:1.0, and even 1:1.5 resulted in a decrease in activity and IC50. 50 It rose back to 72.4 μg / mL.

[0068] Statistical comparisons showed that the inhibitory activity at a 1:0.8 ratio was significantly superior to the conventional 1:1 ratio and also superior to all other groups. This phenomenon indicates that higher proportions of each component in the composition do not necessarily lead to better effects; rather, there exists an optimal synergistic window. Within this window, the synergistic effect of the two types of active ingredients is maximized, possibly through complementary inhibition of multiple targets or physicochemical interactions between active components. Conversely, when the proportion of a certain component is too high, the synergistic effect is weakened, possibly due to competition for active sites or interference between components. Based on this, 1:0.8 was determined to be the optimal mass ratio for subsequent in vivo validation experiments.

[0069] Experimental Example 3

[0070] This study aims to comprehensively observe the differences in efficacy of all monomers and combinations in vivo, with a focus on the differences in efficacy after administration of different mass ratios in vivo.

[0071] 3.1 Animal grouping and administration

[0072] Using an HFD / STZ-induced type 2 diabetes mellitus (T2DM) mouse model, successfully modeled mice were randomly divided into 15 groups of 8 mice each, as follows: normal control group, model group, metformin (MF) positive control group, 4 single-drug groups, and 8 combination-drug groups.

[0073] The dosage of metformin (MF) monotherapy was 200 mg / kg.

[0074] The four single-use groups included the SK-DPF group, Q-DPF group, ML-Et group, and ML-Wa group. The dosage for each single-use group was 300 mg / kg.

[0075] The eight combination therapy groups included: SK-DPF+ML-Et (1:1), SK-DPF+ML-Et (1:0.8), Q-DPF+ML-Et (1:1), Q-DPF+ML-Et (1:0.8), SK-DPF+ML-Wa (1:1), SK-DPF+ML-Wa (1:0.8), Q-DPF+ML-Wa (1:1), and Q-DPF+ML-Wa (1:0.8). The total dosage for each group was controlled at 300 mg / kg.

[0076] All groups were administered the medication by gavage for 8 consecutive weeks.

[0077] 3.2 Experimental Results and Analysis

[0078] Serum core biochemical indicators were measured at the end of week 8, and the results are shown in Table 3.

[0079] Table 3 Comparison of key indicators for lowering blood glucose and blood lipids in all groups of mice (n=8, Mean±SD)

[0080] Note: Compared with the normal group, ## P < 0.01, NS indicates no statistical difference; compared with the model group, P < 0.05, P < 0.01; Δ or ΔΔ This indicates a comparison with a 1:1 ratio group of the same group, where P < 0.05 or P < 0.01.

[0081] Table 3 shows the results of a T2DM mouse model induced by HFD / STZ, systematically evaluating the fasting blood glucose, area under the oral glucose tolerance curve (OGTT-AUC), and triglycerides in all 15 groups of mice after 8 weeks of continuous gavage. Compared with the normal group, all three indicators in the model group were significantly elevated (P<0.01), indicating successful model establishment and severe metabolic disorder.

[0082] Overall, all single-use groups showed some hypoglycemic and lipid-lowering effects, but to varying degrees. The fasting blood glucose and OGTT-AUC of the SK-DPF, Q-DPF, and ML-Et single-use groups were significantly improved compared to the model group (P<0.01), while the ML-Wa group only reached a significant level (P<0.05), showing the weakest effect. Regarding multi-component combination therapy, all combination groups using ML-Et showed significantly better hypoglycemic and lipid-lowering effects than their corresponding groups using ML-Wa, indicating the irreplaceable nature of mulberry leaf extract in this combination, which is basically consistent with the in vitro activity screening results.

[0083] Furthermore, the optimized ratio was strongly validated in in vivo experiments. For all combinations, adjusting the mass ratio from the conventional 1:1 to the in vitro optimized 1:0.8 significantly improved all three core indicators (P<0.05 or P<0.01), indicating that this ratio has a universal advantage across combination types. Among them, the combination of SK-DPF and ML-Et at a 1:0.8 ratio achieved the best overall effect. In this group, fasting blood glucose decreased to 5.8 mmol / L, OGTT-AUC decreased to 20.4 mmol·h / L, and triglycerides decreased to 1.03 mmol / L. All three core metabolic indicators significantly approached the levels of the normal group, and the overall improvement effect was better than that of the positive control drug metformin.

[0084] Test Example 4

[0085] This study aims to observe the regulatory effects of different compositions on the gut microbiota composition of T2DM mice.

[0086] 4.1 Experimental Methods

[0087] Building upon Experiment 3, fresh fecal samples were collected from mice in each group at the end of Week 8. Total microbial DNA was extracted from the feces using the CTAB method, and high-throughput sequencing of the V3-V4 region of the 16S rRNA gene was performed using the Illumina NovaSeq 6000 platform. Quality control, OTU clustering, and species annotation were performed on the data, and the alpha diversity of the gut microbiota (Chao1 index, Shannon index) and the relative abundance changes of key genera were analyzed.

[0088] 4.2 Experimental Results and Analysis

[0089] Table 4 shows the comparison results of gut microbiota α diversity and relative abundance of key bacteria in each group of mice. For ease of comparison, Table 4 highlights the data of the normal group, the model group, and the representative combination group. Among them, combination group 1 corresponds to SK-DPF+ML-Et (1:0.8), combination group 2 corresponds to Q-DPF+ML-Et (1:0.8), combination group 3 corresponds to SK-DPF+ML-Wa (1:0.8), and combination group 4 corresponds to Q-DPF+ML-Wa (1:0.8).

[0090] Table 4. Comparison of gut microbiota α diversity and relative abundance of key bacteria in each group of mice (n=6, Mean±SD)

[0091] Note: Compared with the normal group, ## P<0.01; compared with the model group, P<0.05, P<0.01; compared with group 1, Δ P<0.05, ΔΔ P<0.01.

[0092] Table 4 shows the changes in gut microbiota α-diversity (Chao1 index and Shannon index) and the relative abundance of representative bacterial groups in each group of mice. Compared with the normal group, the Chao1 index and Shannon index in the model group were significantly reduced (P<0.01), indicating that the richness and diversity of gut microbiota were severely impaired in diabetic patients. In terms of microbiota structure, the relative abundance of the beneficial bacteria Bacteroidota plummeted from 47.4% to 28.0%, while the abundance of the opportunistic pathogen Escherichia-Shigella increased sharply from 0.8% to 37.5%. At the same time, the abundance of the beneficial bacteria Phocaeicola vulgatus decreased significantly (from 2.1% to 0.8%), indicating that the gut microbiota in the model group had undergone significant pathological remodeling.

[0093] After intervention in each compound group, the aforementioned gut microbiota dysbiosis was improved to varying degrees. The Chao1 and Shannon indices in all compound groups were significantly higher than those in the model group (P<0.05 or P<0.01), the abundance of Bacteroidota significantly decreased, the abnormal proliferation of Escherichia-Shigella was effectively inhibited, and the abundance of Phocaeicola vulgatus significantly increased, indicating that the combination of fermented soybean hypoglycemic peptide and mulberry leaf extract has a genuine ability to regulate gut microbiota.

[0094] Notably, contrary to the results of the hypoglycemic drug efficacy, the best performance in gut microbiota regulation was observed in combination group 2 (Q-DPF + ML-Et in a 1:0.8 ratio). In this group, the Chao1 index recovered to 1585, significantly higher than that of combination group 1 (SK-DPF + ML-Et), which showed the best hypoglycemic effect (P<0.05); the relative abundance of Bacteroidota recovered to 50.2%, also significantly higher than that of combination group 1 (P<0.05) and even slightly exceeding the normal group level; in particular, the abundance of Phocaeicola vulgatus significantly decreased to 2.4%, close to the normal group level and significantly better than the 1.8% of combination group 1 (P<0.05). This indicates that although SK-DPF is more advantageous in hypoglycemic indicators, Q-DPF may produce specific peptides or metabolites during fermentation that are more conducive to the proliferation of beneficial gut bacteria, exhibiting unique advantages in improving the gut microbiota.

[0095] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications and substitutions should be covered within the scope of the claims of the present invention. Technical aspects, shapes, and structures not described in detail in this invention are all well-known technologies.

Claims

1. A soybean-based hypoglycemic peptide composition for treating type 2 diabetes, characterized in that, It contains fermented soybean hypoglycemic peptide and mulberry leaf extract; the fermented soybean hypoglycemic peptide is an extract of Yangjiang fermented soybean, and the mulberry leaf extract is an alcoholic extract of mulberry leaves.

2. The soybean-based hypoglycemic peptide composition according to claim 1, characterized in that, The blood sugar-lowering peptides mentioned are either skinless or unskinned fermented soybeans.

3. The fermented soybean hypoglycemic peptide composition according to claim 2, characterized in that, The preparation method of the unpeeled fermented black soybean hypoglycemic peptide is as follows: take Yangjiang fermented black soybeans without peeling, freeze-dry and pulverize them, first treat them with cellulase to break the cell wall, and then extract them by low temperature water extraction, centrifugation, dialysis and freeze drying.

4. The fermented soybean hypoglycemic peptide composition according to claim 2, characterized in that, The preparation method of the peeled fermented black soybean hypoglycemic peptide is as follows: take Yangjiang fermented black soybeans, peel off the outer skin, freeze-dry and pulverize the soybean flesh, and then extract it by low temperature water extraction, centrifugation, dialysis and freeze-drying.

5. The fermented soybean hypoglycemic peptide composition according to claim 1, characterized in that, The mass ratio of the fermented soybean hypoglycemic peptide to the mulberry leaf alcohol extract is 1:0.5-1.

5.

6. The fermented soybean hypoglycemic peptide composition according to claim 5, characterized in that, The mass ratio of the fermented soybean hypoglycemic peptide to the mulberry leaf alcohol extract is 1:0.7-1.

0.

7. The fermented soybean hypoglycemic peptide composition according to claim 5, characterized in that, The mass ratio of the fermented soybean hypoglycemic peptide to the mulberry leaf alcohol extract is 1:0.

8.

8. The fermented soybean hypoglycemic peptide composition according to any one of claims 1-7, characterized in that, The soybean blood sugar-lowering peptide composition is used in the preparation of pharmaceuticals or functional foods for improving or treating type 2 diabetes by inhibiting α-glucosidase and / or DPP-IV activity.

9. A pharmaceutical preparation comprising the soybean hypoglycemic peptide composition according to any one of claims 1-7 and pharmaceutically acceptable excipients.

10. A functional food comprising the soybean blood sugar-lowering peptide composition according to any one of claims 1-7 and food science acceptable excipients.

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