Pumpkin polysaccharide-milk compound for resisting diabetes as well as preparation method and application of pumpkin polysaccharide-milk compound

Abstract

The invention discloses an antidiabetic pumpkin polysaccharide-milk compound and a preparation method and application thereof.The preparation method comprises the steps that firstly, pumpkin pulp serves as a raw material and is subjected to superfine pretreatment, pumpkin polysaccharide is extracted in a free radical mediation mode, the polysaccharide yield and activity are effectively improved, then the pumpkin polysaccharide is compounded with different brands of milk, and the antidiabetic pumpkin polysaccharide-milk compound is obtained. Active oxygen free radicals and active nitrogen free radicals are respectively used as indexes to preferably select the pumpkin polysaccharide-milk compound with the optimal synergistic interaction ratio. The pumpkin polysaccharide-milk compound prepared by the method disclosed by the invention not only has the nutritional values of polysaccharide and milk, but also synergistically enhances the free radical scavenging and T2DM resisting capabilities, and generates synergistic effects of reducing blood glucose, improving insulin resistance and protecting target organs. The compound possibly has stronger universality and efficacy in the aspect of protecting the functions of pancreatic beta cells, shows a more comprehensive and balanced anti-inflammatory spectrum, and realizes'strong combination 'and'advantage complementation' of the anti-inflammatory effect.

Description

(I) Technical Field

[0001] This invention relates to a pumpkin polysaccharide-milk compound for anti-diabetic purposes, its preparation method, and its application. (II) Background Technology

[0002] Type 2 diabetes mellitus (T2DM) is a complex metabolic disease characterized by insulin resistance and impaired β-cell function, and its incidence continues to rise globally. Current first-line treatments, such as metformin and insulin sensitizers, while effectively lowering blood sugar, have long-term side effects (e.g., gastrointestinal reactions, weight gain, and risk of hypoglycemia) and are insufficient in addressing diabetes-related oxidative stress complications. Therefore, developing novel drugs that can target diabetes and its complications at multiple points is a persistent goal in this field. Unlike ordinary physiological oxidative stress, oxidative stress in diabetes has unique roots. Persistent hyperglycemia and lipid metabolism disorders generate excessive superoxide in the mitochondrial electron transport chain, activating multiple biochemical pathways leading to excessive reactive oxygen species production (e.g., polyol pathway, hexosamine pathway, protein kinase C activation, and formation of advanced glycation end products). This 'metabolic oxidative stress' directly driven by metabolic disorders is a core component leading to impaired insulin signaling, β-cell apoptosis, and diabetic complications such as cardiovascular disease and nephropathy.

[0003] Against this backdrop, the search for safe and effective natural anti-T2DM ingredients has become a research hotspot in the fields of food science and nutrition. Polysaccharides have attracted much attention due to their unique biological activity, biocompatibility, bioadhesion, and other special functions, as well as their lack of toxic side effects. For example, polysaccharides extracted from pumpkin pulp are increasingly coming into focus.

[0004] pumpkin( Cucurbita moschata Cucurbita is cultivated and consumed worldwide as a herbaceous plant of the genus Cucurbita. Its pulp is rich in carbohydrates, with pumpkin polysaccharides being its main bioactive component. These polysaccharides possess a wide range of biological functions, including antioxidant, immunomodulatory, and hypoglycemic properties. Studies have found that the molecular weight of pumpkin polysaccharides is mainly concentrated in the range of 16-200 kDa, primarily composed of rhamnose, arabinose, glucose, and galactose, containing various glycosidic bond types such as β-1→4, β-1→3, β-1→2, and α-1→6. However, existing hot water extraction or enzymatic extraction methods either have high extraction yields but high energy consumption, or are costly.

[0005] Meanwhile, dairy products, as an important part of the daily diet, are a nutrient-rich food and a valuable source of minerals, fats, amino acids, and vitamins, helping to meet the recommended daily intake of essential nutrients. Studies have shown that consuming 0.5 L of milk daily can provide many of the essential nutrients needed for daily nutrition. However, completely eliminating dairy products may increase the risk of nutritional complications and deficiencies (such as calcium, vitamin D, or long-chain Omega-3 fatty acids). Furthermore, dairy products not only provide high-quality protein and calcium, but their whey protein, lactoferrin, and other components also exhibit potential antioxidant activity. However, the antioxidant capacity of ordinary dairy products is limited, making it difficult to meet the health needs of specific populations.

[0006] Therefore, it is necessary to explore how to enhance the functional value of polysaccharides and dairy products through scientific means and to find safe and effective natural anti-T2DM ingredients. (III) Summary of the Invention

[0007] The purpose of this invention is to provide a pumpkin polysaccharide-milk compound for anti-diabetic purposes, its preparation method, and its application. This invention first uses pumpkin pulp as raw material, pre-treating it with ultrafine particles, and then extracts pumpkin polysaccharides using a free radical-mediated extraction method, effectively improving the polysaccharide yield and activity. Next, it is compounded with different brands of milk through a mixing and blending process. The optimal synergistic effect of the pumpkin polysaccharide-milk compound is selected using reactive oxygen species (·OH) and reactive nitrogen species (DPPH) as indicators. The pumpkin polysaccharide-milk compound prepared by this invention not only possesses the nutritional value of both polysaccharides and milk, but also synergistically enhances free radical scavenging and anti-T2DM capabilities, producing synergistic effects in lowering blood sugar, improving insulin resistance, and protecting target organs.

[0008] The technical solution adopted in this invention is:

[0009] In a first aspect, the present invention provides a pumpkin polysaccharide-milk compound for anti-diabetic purposes, the compound being a mixture of pumpkin polysaccharide aqueous solution and milk; the concentration of the pumpkin polysaccharide aqueous solution is 1.6-4.8 mg / mL.

[0010] Furthermore, the volume ratio of the pumpkin polysaccharide aqueous solution to milk is 1:1.

[0011] Furthermore, the milk includes pure milk with a protein content of 3.0-3.6 g / 100mL, preferably 3.2 g / 100mL.

[0012] Furthermore, the pure milk is selected from brands such as Mengniu, Yili, Jin Dian, Shu Hua, or Telunsu.

[0013] Furthermore, the milk is Mengniu pure milk or lactose-free milk, with solid content of 142.10 mg / mL and 109.07 mg / mL, respectively, and protein content of 3.2 g / 100 mL and 3.0 g / 100 mL, respectively.

[0014] Furthermore, the compound is prepared by mixing a 1.6 mg / mL pumpkin polysaccharide aqueous solution with pure milk containing 1.6 mg / mL of solids at a volume ratio of 1:1.

[0015] Further, the pumpkin polysaccharide is prepared as follows: Pumpkin powder (passed through a 200-mesh sieve) is extracted in a water bath shaker with deionized water and 30% hydrogen peroxide as the extraction solvent at a speed of 100-150 rpm (preferably 125 rpm) for 50-70 min (preferably 61 min) at a temperature of 80-100℃ (preferably 90℃). The mixture is then centrifuged at 8000 rpm for 15 min, and the supernatant is collected and concentrated to 1 / 5 of its original volume. Subsequently, Sevag reagent (chloroform:n-butanol = 4:1, volume ratio) is added to the concentrate at a volume ratio of 1:5, and the mixture is vigorously shaken for 20 min. It is then centrifuged at 3000 rpm for 10 min to form an organic solvent layer, a protein layer, and an aqueous layer. The aqueous layer is concentrated to remove residual organic solvent, and then 95% ethanol is added at a ratio of 1:4 (v / v). The mixture is then allowed to stand overnight at 4℃. Finally, the mixture is centrifuged at 1000 rpm for 5 minutes. The precipitate was collected, redissolved in distilled water, and residual organic reagents were removed by rotary evaporation to obtain a purified pumpkin polysaccharide solution. The solution was then freeze-dried (preferably at -55°C) to obtain pumpkin polysaccharide (PPe).

[0016] Furthermore, the hydrogen peroxide concentration in the extractant is 0.6%-1.8% (preferably 1.13%), and the volumetric amount of the extractant is 30-120 mL / g (preferably 61 mL / g) based on the weight of the pumpkin powder. The pumpkin powder is obtained by washing, peeling, removing the seeds and pulp from a pumpkin, shredding it, drying it with hot air (65℃) until the moisture content is below 10%, pulverizing it using a BO-250S2 high-speed multi-functional crusher, and passing it through a 200-mesh sieve.

[0017] Secondly, the present invention provides an application of the aforementioned pumpkin polysaccharide-milk compound in the preparation of antioxidant products, wherein the antioxidant products are products that scavenge reactive nitrogen (such as DPPH) free radicals and / or reactive oxygen (such as ·OH) free radicals.

[0018] Thirdly, the present invention provides the application of the pumpkin polysaccharide-milk compound in the preparation of a drug for treating diabetes.

[0019] Furthermore, the drug is a medication for treating or improving type 2 diabetes.

[0020] Furthermore, the drug is for treating type 2 diabetes mellitus induced by a high-fat diet combined with a low dose of streptourea (STZ).

[0021] Furthermore, the drug can alleviate weight loss and fasting blood glucose increase caused by type 2 diabetes mellitus (T2DM); slow down the decrease in the activity of antioxidant enzymes SOD and CAT, the decrease in antioxidant GSH levels, the decrease in glucose transporter (GLUT2), liver glycogen and insulin levels, and the decrease in lipid peroxide MDA, lipopolysaccharide (LPS) and sodium levels caused by T2DM. + Increased levels of glucose-dependent transporter 1 (SGLT1) and decreased activity of glucose metabolism-related enzymes such as pyruvate kinase (PK), while increased activity of glucose-6-phosphatase G-6-pase (G6P) and phosphoenolpyruvate carboxykinase (PEPCK) were observed.

[0022] Compared with existing technologies, the pumpkin polysaccharide-milk compound provided by this invention exhibits synergistic and multi-target therapeutic advantages in a type 2 diabetic mouse model, and its beneficial effects are mainly reflected in:

[0023] 1. This invention involves pre-treating pumpkin pulp with ultrafine particles and then extracting pumpkin polysaccharides using a free radical-mediated method. This improves the polysaccharide yield and activity. The process is green and environmentally friendly, simple and easy to operate, and the polysaccharide dissolution rate can reach over 32%. The yield is 7.596 times higher than that obtained by traditional hot water extraction, 1.541 times higher than that obtained by traditional mechanical grinding, 1.802 times higher than that obtained by cellulase extraction, and 1.161 times, 1.184 times, 1.243 times, and 1.174 times higher than that obtained by adding 1 / 2, 1 / 4, 1 / 8, and 1 / 16 of the mass of hydrogen peroxide and vitamin C, respectively. It is also 2.534 times, 2.250 times, 2.442 times, and 2.898 times higher than that obtained by adding 1 / 2, 1 / 4, 1 / 8, and 1 / 16 of the mass of hydrogen peroxide and FeSO4, respectively.

[0024] 2. This invention employs a mixture and compounding method as a screening tool, ensuring from a mechanistic perspective that the compound does not simply add up the effects, but rather produces a true "synergistic effect." Using reactive oxygen species (·OH) and reactive nitrogen species (DPPH) as indicators, different natural sources of active ingredients (pumpkin polysaccharide and milk components) are compounded, and the pumpkin polysaccharide-milk compound with the best synergistic ratio is selected. Verification using both indicators (·OH and DPPH) confirms that the compound of this invention significantly enhances the scavenging ability of the strongest reactive oxygen species (·OH) while also comprehensively enhancing the total antioxidant capacity, indicating a more reliable and broader antioxidant potential in complex physiological environments. Furthermore, it not only ensures the safety of the raw materials but also offers numerous advantages such as no impurities introduced during the entire operation process, no pollution, low equipment requirements, and low energy consumption.

[0025] 3. Compared to PPE alone, the pumpkin polysaccharide-milk (PPe-Milk) compound exhibits comprehensive advantages in the following aspects: ① More comprehensive therapeutic effect: In improving pancreatic islet structural damage, only high-dose PPE showed a significant effect, while different doses of 1,6 PPe-Milk A and B significantly alleviated pancreatic islet lesions, indicating that the compound may have stronger universality and efficacy in protecting pancreatic β-cell function. ② Stronger effect on key targets: In inhibiting the important hypoglycemic target of the intestinal glucose transporter SGLT1, PPE's effect is comparable to that of the classic drug acarbose, while the effects of both 1,6 PPe-Milk compounds are significantly better than acarbose, indicating that the compound achieves enhanced efficacy at the key mechanism. ③ The compound exhibits a more comprehensive and balanced anti-inflammatory spectrum, achieving a "powerful synergy" and "complementary advantages" in anti-inflammatory effects: Ppe itself is a powerful "specialist" in reducing inflammatory factors in colonic tissue (particularly adept at inhibiting IL-6 / IL-1β), while the compound has evolved into a more outstanding "generalist." Building upon the advantages of Ppe, it compensates for its relative deficiency in TNF-α inhibition, thus providing a broader, more balanced, and more robust comprehensive anti-inflammatory efficacy at certain targets. This indicates that the compounding strategy successfully integrates the synergistic effects of different components, resulting in a more comprehensive inflammatory regulatory capacity that a single component cannot possess. (iv) Description of the attached drawings

[0026] Figure 1DPPH· scavenging capacity of PPe, milk, and PPe-milk complex (A. DPPH· scavenging capacity of five types of milk; B. DPPH· scavenging capacity of PPe alone and PPe-milk complex at concentrations of 1.6 and 3.2 mg / mL; C. DPPH· scavenging capacity of PPe alone and PPe-milk complex at a concentration of 4.8 mg / mL; D. IC50 of milk and different concentrations of PPe-milk complex for DPPH· scavenging) 50 Value; E. Synergistic effect of different concentrations of PPe-milk compound in scavenging DPPH·).

[0027] Figure 2 ·OH scavenging capacity of PPe, milk, and PPe-milk complex (A. ·OH scavenging capacity of five types of milk; B. ·OH scavenging capacity of PPe alone and PPe-milk complex at concentrations of 1.6 and 3.2 mg / mL; C. ·OH scavenging capacity of PPe alone and PPe-milk complex at a concentration of 4.8 mg / mL; D. IC50 of milk and different concentrations of PPe-milk complex for ·OH scavenging) 50 Value; E. Synergistic effect of different concentrations of PPe-milk complex in scavenging ·OH).

[0028] Figure 3 Effects of different doses of PPe, 1.6 PPe-Milk A and 1.6 PPe-Milk B on body weight and fasting blood glucose in mice.

[0029] Figure 4 Effects of different doses of PPe, 1.6 PPe-Milk A, and 1.6 PPe-Milk B on the morphology of mouse pancreatic islet tissue (first row from left to right: K group, N group, P group and low-dose PPe group; second row from left to right: medium-dose PPe group, high-dose PPe group, low-dose PPe-Milk A group and medium-dose PPe-Milk A group; third row from left to right: high-dose PPe-Milk A group, low-dose 1.6 PPe-Milk B group, medium-dose 1.6 PPe-Milk B group and high-dose 1.6 PPe-Milk B group).

[0030] Figure 5 Effects of different doses of PPe, 1.6 PPe-Milk A and 1.6 PPe-Milk B on serum insulin and GLP-1 levels in mice.

[0031] Figure 6Effects of different doses of PPe, 1.6 PPe-Milk A, and 1.6 PPe-Milk B on hepatic glycogen content and activities of enzymes related to glucose metabolism (PK, G-6-P, and PEPCK) in mice.

[0032] Figure 7 Effects of different doses of PPe, 1.6 PPe-Milk A, and 1.6 PPe-Milk B on the level of SGLT1 in the colon tissue of mice.

[0033] Figure 8 Effects of different doses of PPe, 1.6 PPe-Milk A, and 1.6 PPe-Milk B on antioxidant-related indexes (activities of SOD and CAT, levels of GSH and MDA) in the liver of mice.

[0034] Figure 9 Effects of different doses of PPe, 1.6 PPe-Milk A, and 1.6 PPe-Milk B on the levels of LSP in the serum and inflammatory factors (TNF-α, IL-6, and IL-1β) in the colon tissue of mice. (V) Specific implementation manners

[0035] The following further describes the present invention in combination with specific embodiments, but the protection scope of the present invention is not limited thereto: The water used in the embodiments of the present invention is deionized water, and other reagents are commercially available and are all of analytical grade.

[0036] Assay kits for catalase, malondialdehyde, glutathione, and total superoxide dismutase (T-SOD), total protein quantification assay, insulin test kit, hepatic glycogen, glucokinase, glucose-6-phosphatase, pyruvate kinase, phosphoenolpyruvate carboxykinase, sodium / glucose cotransporter 1 enzyme-linked immunosorbent assay kit, glucose transporter 2 enzyme-linked immunosorbent assay kit, and glucagon-like peptide-1 assay kit were all purchased from Nanjing Jiancheng Bioengineering Institute.

[0037] C57BL / 6J male mice, db / m mice, and db / db mice were all purchased from Shanghai Slake Experimental Animal Co., Ltd., license number: SCXK (Shanghai) 2022-0004. All animal experiments complied with the "Guide for the Care and Use of Laboratory Animals" of the National Research Council, and this study has obtained the experimental animal ethics review approval of the school ethics committee.

[0038] Experimental data were expressed as Means ± SD, and one-way analysis of variance (ANOVA) in IBM SPSS Statistics 25 software was used for homogeneity of variance test and LSD multiple comparison analysis for significant difference analysis, and p < 0.05 was considered significantly different.

[0039] Example 1: Pumpkin polysaccharide extraction

[0040] 1. Pumpkin powder

[0041] The pumpkin was purchased from Beibei Pumpkin on Taobao. After washing the pumpkin, peeling, removing the seeds and pulp, and shredding it, the pumpkin was dried with hot air (65℃) until the moisture content was below 10%. It was then crushed using a BO-250S2 high-speed multi-functional crusher and passed through a 200-mesh sieve to obtain pumpkin powder, which was then set aside.

[0042] 2. Extraction of pumpkin polysaccharides using a free radical-mediated method

[0043] Take 1.0 g of pumpkin powder, add 58.7 mL of deionized water, and then add 2.3 mL of 30% hydrogen peroxide (deionized water and hydrogen peroxide constitute the extractant, and the mass concentration of hydrogen peroxide in the extractant is 1.13%). Mix well, with a solid-liquid ratio of 1:61 (g / mL). Extract in a water bath at 125 rpm for 61 min at 100℃, and then centrifuge at 8000 rpm for 15 min. Collect the supernatant and concentrate it to 1 / 5 of the original volume. Then, add Sevag reagent (chloroform:n-butanol = 4:1, volume ratio) to the concentrate at a volume ratio of 1:5, shake vigorously for 20 min, and centrifuge at 3000 rpm for 10 min to form an organic solvent layer, a protein layer, and an aqueous layer. The aqueous layer is concentrated to remove residual organic solvent, and then 95% ethanol is added at a ratio of 1:4 (v / v). Let it stand overnight at 4℃. Finally, the precipitate was collected by centrifugation at 1000 rpm for 5 min, redissolved in distilled water, and residual organic reagents were removed by rotary evaporation to obtain a purified pumpkin polysaccharide solution. After freeze-drying at -55℃, 0.36 g of pumpkin polysaccharide was obtained, denoted as PPe.

[0044] 3. Detection of the yield of pumpkin polysaccharides

[0045] Take 1.0 mL of the polysaccharide supernatant from step 2 as the test solution, dilute it with deionized water to the detection range, and use the phenol-sulfuric acid method to detect the absorbance at 480 nm. Calculate the polysaccharide concentration according to the glucose standard curve, and convert it into the extraction yield of pumpkin polysaccharide in the crude polysaccharide extract according to formula (1), which is 32.89%.

[0046] Polysaccharide extraction yield (%) = (C×V×f) / m ×100% Formula (1)

[0047] In formula (1), C is the polysaccharide concentration (mg / mL) calculated by the standard equation; V is the volume of the test liquid (mL); f is the dilution factor; and m is the mass of the pumpkin powder sample (mg).

[0048] Glucose standard curve: A standard curve is plotted with the concentration of glucose aqueous solution on the x-axis and the absorbance at 480 nm on the y-axis. The equation of the standard curve is y = 7.9807x - 0.014, R0 2 =0.9983, where y represents the absorbance value and x represents the polysaccharide concentration.

[0049] Example 2: Preparation and Antioxidant Capacity Detection of Pumpkin Polysaccharide-Milk Complex

[0050] 1. Preparation of pumpkin polysaccharide-milk compound

[0051] The pumpkin polysaccharide prepared by the method in Example 1 was prepared into polysaccharide solutions of different concentrations (1.6 mg / mL, 3.2 mg / mL, 4.8 mg / mL) using deionized water. Each concentration of polysaccharide solution was mixed with five types of commercially available milk (milk A, milk B, milk C, milk D, and milk E) from Table 1 at a volume ratio of 1:1 to obtain 15 pumpkin polysaccharide-milk complexes, denoted as 1.6PPe-MilkA, 1.6PPe-MilkB, 1.6PPe-MilkC, 1.6PPe-MilkD, 1.6PPe-MilkE; 3.2PPe-MilkA, 3.2PPe-MilkB, 3.2PPe-MilkC, 3.2PPe-MilkD, 3.2PPe-MilkE; and 4.8PPe-MilkA, 4.8PPe-MilkB, 4.8PPe-MilkC, 4.8PPe-MilkD, 4.8PPe-MilkE.

[0052] Table 1. Solid content of various milk products

[0053]

[0054] 2. Calculation of Synergistic Ratio (SR)

[0055] The total content of polysaccharides and milk solids in each compound from step 1 is denoted as C1. For determination, each C1 is serially diluted with deionized water to prepare 0.5C1, 0.25C1, 0.125C1, 0.0625C1, 0.03175C1, and 0.015875C1, respectively, and denoted as C2, C3, C4, C5, C6, and C7. The free radical scavenging rate is tested using methods from steps 3 and 4, and the IC50(A) values ​​are obtained. 50 And IC(B) 50 .

[0056] The combined theoretical efficacy IC of compound 50 (The theory) is:

[0057] Formula (2)

[0058] IC(A) 50 IC(B) represents the polysaccharide concentration at which the free radical scavenging rate is 50%. 50 The value represents the milk concentration when the free radical scavenging rate is 50%, a represents the mass of polysaccharides in the compound, and b represents the mass of milk solids in the compound.

[0059] IC of compound solution 50 (In practice) the results were obtained through experiments in steps 3 and 4, and the synergistic ratio SR was calculated according to formula (3).

[0060] SR = IC 50 (Theory) / IC 50 (Actual) Formula (3)

[0061] When 0.5 < SR < 1.5, it is an additive effect; when SR > 1.5, it is a synergistic effect; when SR < 0.5, it is an antagonistic effect.

[0062] 3. Determination of DPPH free radical scavenging rate and synergistic ratio (SR)

[0063] DPPH (1,1-diphenyl-2-pyridyl-hydrazinoyl) is a stable free radical with one unpaired valence electron on one atom of the nitrogen bridge. As a stable free radical, DPPH is stable in organic solvents, its alcoholic solution is purple, and it requires low-temperature, light-protected storage. Having a single electron, it can accept one electron or a hydrogen ion, and it has maximum absorption at a wavelength of 517 nm. In the presence of free radical scavengers, the single electron of DPPH is captured, causing its color to lighten and the absorbance at the maximum absorption wavelength to decrease linearly. The decrease in absorbance level indicates an increase in antioxidant activity, thus evaluating the antioxidant capacity of the test sample. This antioxidant capacity is expressed as an inhibition rate; the higher the inhibition rate, the stronger the antioxidant activity.

[0064] Sample 1 solution: Pumpkin polysaccharide aqueous solutions of different concentrations (0.0375, 0.075, 0.15, 0.3, 0.6, 1.2, 2.4, 4.8 mg / mL), denoted as PPe;

[0065] Sample 2 solution: aqueous solutions of milk (milk A, milk B, milk C, milk D, milk E) with different solid contents (0.025, 0.05, 0.1, 0.2, 0.4, 0.6, 0.8, 1.6 mg / mL), denoted as MilkA, MilkB, MilkC, MilkD, and MilkE;

[0066] Sample 3 solution: A polysaccharide solution of 1.6 mg / mL was prepared with deionized water and mixed with five types of milk with a solid content of 1.6 mg / mL, and then reconstituted at a volume ratio of 1:1 as the stock solution C1; then it was serially diluted with deionized water to C2 to C8 (total polysaccharide and solid content of 0.025, 0.05, 0.1, 0.2, 0.4, 0.8, and 1.6 mg / mL, respectively), and denoted as 1.6PPe-MilkA, 1.6PPe-MilkB, 1.6PPe-MilkC, 1.6PPe-MilkD, and 1.6PPe-MilkE;

[0067] Sample 4 solution: A polysaccharide solution of 3.2 mg / mL was mixed with five types of milk with a solid content of 3.2 mg / mL at a volume ratio of 1:1 as stock solution C1. Then, it was serially diluted with deionized water to C2 to C8 (total polysaccharide and solid content of 0.05, 0.1, 0.2, 0.4, 0.8, 1.6, and 3.2 mg / mL, respectively), and denoted as 3.2PPe-MilkA, 3.2PPe-MilkB, 3.2PPe-MilkC, 3.2PPe-MilkD, and 3.2PPe-MilkE.

[0068] Sample 5 solution: A 4.8 mg / mL polysaccharide solution was mixed with five types of milk with a solid content of 4.8 mg / mL at a volume ratio of 1:1 as stock solution C1. Then, it was serially diluted with deionized water to C2 to C8 (the total polysaccharide and solid content were 0.075, 0.15, 0.3, 0.6, 1.2, 2.4, and 4.8 mg / mL, respectively), and denoted as 4.8PPe-MilkA, 4.8PPe-MilkB, 4.8PPe-MilkC, 4.8PPe-MilkD, and 4.8PPe-MilkE.

[0069] 0.1 mmol / L DPPH solution: Weigh 3.94 mg of DPPH, dissolve it in anhydrous ethanol and bring the volume to 100 mL to obtain a 0.1 mmol / L DPPH ethanol solution.

[0070] V C Solution: As a control, prepare gradient concentrations, each with the same concentration as the sample.

[0071] Mix the test solutions evenly according to the following groups, and incubate at room temperature for 30 min under light-protected conditions. Measure the absorbance at 517 nm, with three replicates for each group. Calculate the DPPH scavenging rate according to formula (4). Calculate the synergistic ratio SR according to the method in step 2.

[0072] Sample group: 2 mL sample solution + 2 mL 0.1 mmol / L DPPH solution

[0073] Positive group: 2 mL V C Solution + 2 mL 0.1 mmol / L DPPH solution

[0074] Control group: 2 mL sample solution + 2 mL anhydrous ethanol

[0075] Blank group: 2 mL H2O + 2 mL 0.1 mmol / L DPPH solution

[0076] DPPH removal rate (%) = [1-(A 样 -A 对 ) / A 空 ]×100% formula (4)

[0077] See results Figure 1 As shown, within the experimental concentration range, the positive control V C The scavenging rate remained consistently high, and all experimental components (PPe alone, milk alone, and PPE-milk compound) exhibited concentration-dependent scavenging effects, with their scavenging rates consistently lower than those of V at the same concentration. C The clearance rates of milk alone were generally below 50%, with the highest clearance rate of Milk C at 4 mg / mL being only 49.18%; Ppe alone achieved a maximum clearance rate of 63.25% at a concentration of 4.8 mg / mL. When milk was combined with polysaccharides, the clearance ability was significantly enhanced. For example, 3.2 PPe-Milk B (i.e., a combination of 3.2 mg / mL Ppe and 3.2 mg / mL Milk B) achieved a clearance rate as high as 87.89% at a concentration of 1.6 mg / mL. The IC50 of the combined system... 50 The values ​​show that different brands and concentrations of milk, when combined with PPE, result in different IC values. 50 All decreased significantly, with the IC50 of milk alone decreasing. 50 All were above 5.0 mg / mL (e.g., Milk A was 14.96 mg / mL), while the IC50 of the compound system was... 50 All were below 2.5 mg / mL, with the IC50 of 1.6 PPe-Milk A (i.e., a mixture of 1.6 mg / mL PPe and 1.6 mg / mL Milk A) being the highest. 50 It was only 0.40 mg / mL. Further analysis of the synergistic ratio showed that the SR of all the compound combinations was greater than 1.5, indicating that the combination of milk and polysaccharides produced a synergistic effect. Among them, the SR of 1.6 PPe-Milk A reached 33.31, and the SR of 4.8 PPe-Milk E was as high as 73.44.

[0078] 4. Determination of hydroxyl radical scavenging rate and synergistic ratio (SR)

[0079] Hydroxyl radicals are highly reactive reactive oxygen species that can damage biological macromolecules such as proteins, lipids, and DNA, potentially leading to cell damage, aging, and various diseases. Therefore, measuring the scavenging ability of antioxidants or compounds against hydroxyl radicals is an important indicator for evaluating their antioxidant activity.

[0080] Sample 1 solution: Pumpkin polysaccharide aqueous solutions of different concentrations (0.002344, 0.004568, 0.009375, 0.01875, 0.0375, 0.075, 0.15, 0.3, 0.6, 1.2, 2.4, 4.8 mg / mL), denoted as PPe;

[0081] Sample 2 solution: aqueous solutions of milk (milk A, milk B, milk C, milk D, milk E) with different solid contents (0.003125, 0.00625, 0.0125, 0.025, 0.05, 0.1, 0.2, 0.4, 0.6, 0.8, 1.6 mg / mL), denoted as MilkA, MilkB, MilkC, MilkD, and MilkE;

[0082] Sample 3 solution: A polysaccharide solution of 1.6 mg / mL was mixed with five types of milk with a solid content of 1.6 mg / mL at a volume ratio of 1:1 as stock solution C1. Then, it was serially diluted with deionized water to C2 to C13 (0.000781, 0.001563, 0.003125, 0.00625, 0.0125, 0.025, 0.05, 0.1, 0.2, 0.4, 0.8, 1.6 mg / mL), and denoted as 1.6PPe-MilkA, 1.6PPe-MilkB, 1.6PPe-MilkC, 1.6PPe-MilkD, and 1.6PPe-MilkE.

[0083] Sample 4 solution: A polysaccharide solution of 3.2 mg / mL was mixed with five types of milk with a solid content of 3.2 mg / mL at a volume ratio of 1:1 as stock solution C1. Then, it was serially diluted with deionized water to C2 to C8 (0.000781, 0.001563, 0.003125, 0.00625, 0.0125, 0.025, 0.05, 0.1, 0.2, 0.4, 0.8, 1.6, 3.2 mg / mL), and designated as 3.2PPe-MilkA, 3.2PPe-MilkB, 3.2PPe-MilkC, 3.2PPe-MilkD, and 3.2PPe-MilkE.

[0084] Sample 5 solution: A 4.8 mg / mL polysaccharide solution was mixed with five types of milk with a solid content of 4.8 mg / mL at a volume ratio of 1:1 as stock solution C1. Then, it was serially diluted with deionized water to C2 to C11 (0.002344, 0.004688, 0.009375, 0.01875, 0.0375, 0.075, 0.15, 0.3, 0.6, 1.2, 2.4, 4.8 mg / mL), and designated as 4.8PPe-MilkA, 4.8PPe-MilkB, 4.8PPe-MilkC, 4.8PPe-MilkD, and 4.8PPe-MilkE.

[0085] 1.8 mmol / L FeSO4: Weigh 0.500 g of FeSO4. ·7 Dissolve O in water and bring the volume to 1 L.

[0086] 1.8 mmol / L salicylic acid solution: Weigh 0.249 g of salicylic acid, dissolve it in anhydrous ethanol and bring the volume to 1 L.

[0087] 0.03% H2O2: Take 10 mL of 30% hydrogen peroxide, add it to 80 mL of distilled water, mix well, and then make up to 100 mL.

[0088] V C Solution: As a control, prepare gradient concentrations, each with the same concentration as the sample.

[0089] Mix the test solutions evenly according to the following groups, incubate in a 37°C water bath for 30 min, and measure the absorbance at 510 nm under light-protected conditions. Set up 3 replicates for each group. Calculate the hydroxyl radical scavenging rate according to formula (5). Calculate the synergistic ratio SR according to the method in step 2.

[0090] Sample group: 1 mL sample solution + 2 mL 1.8 mmol / L FeSO4 + 1.5 mL 1.8 mmol / L salicylic acid solution + 0.1 mL 0.03% H2O2

[0091] Positive group: 1 mL V C Solution + 2 mL 1.8 mmol / L FeSO4 + 1.5 mL 1.8 mmol / L salicylic acid solution + 0.1 mL 0.03% H2O2

[0092] Control group: 1 mL sample solution + 2 mL 1.8 mmol / L FeSO4 + 1.5 mL 1.8 mmol / L salicylic acid solution + 0.1 mL H2O2

[0093] Blank group: 1 mL H2O + 2 mL 1.8 mmol / L FeSO4 + 1.5 mL 1.8 mmol / L salicylic acid solution + 0.1 mL 0.03% H2O2

[0094] Hydroxyl radical scavenging rate (%) = [1-(A 样 -A 对 ) / A 空 ]×100% formula (5)

[0095] See results Figure 2 Within the experimental concentration range, the positive control V C The scavenging ability against hydroxyl radicals remained consistently high, and all experimental components (PPe alone, milk alone, and PPE-milk compound) exhibited concentration-dependent scavenging effects, with their scavenging rates consistently lower than those of V at the same concentration. C The scavenging rates of milk alone were all below 66%. At a concentration of 4 mg / mL, Milk C showed the highest scavenging rate among the five milk samples, but even that was only 66.17%. Ppe alone, at low concentrations (<0.1 mg / mL), showed a scavenging rate of less than 60% for hydroxyl radicals. When the Ppe concentration increased to 1.6 mg / mL, its scavenging rate for hydroxyl radicals was comparable to that of the milk-polysaccharide complex and positively charged substance V. C There were no significant differences, all exceeding 95%. Except for a few experimental points (such as 1.6 PPe-Milk A at 0.1 and 0.2 mg / mL, 3.2 PPe-Milk B at 0.2 mg / mL, 3.2 PPe-Milk E at 0.25 mg / mL and below, and 4.8 PPe-Milk E at 0.0375 mg / mL and below), at the three concentrations of 1.6, 3.2, and 4.8 mg / mL, when PPe was mixed with the five Milks in a 1:1 ratio, the scavenging rate of hydroxyl radicals was higher than that of PPe alone in the range of 0.00625-1.6 mg / mL.

[0096] ICs from composite systems 50 The values ​​show that, compared to milk alone, different brands and concentrations of milk, when blended with PPE, result in higher IC50 levels. 50 The values ​​were all significantly reduced (p < 0.05), indicating that the free radical scavenging ability was enhanced after compounding. Except for the IC50 value of milk E... 50 Except for the value of 0.274 mg / mL, the IC50 values ​​of the other four types of milk were... 50 All values ​​were greater than 1.2 mg / mL. Among them, the IC50 value of Milk A was... 50 The value was 1.49 mg / mL, while the IC50 value for 1.6 PPe-Milk A was [missing value]. 50 The value dropped to 0.00638 mg / mL; the IC50 of Milk B...50 The value was 1.65 mg / mL, while the IC50 value for the 1.6PPe-Milk B combination was 1.65 mg / mL. 50 The value decreased to 0.00639 mg / mL. Further analysis of the synergistic ratio showed that the SR of all compound combinations was greater than 1.5, indicating that milk and PPe compounding produced a synergistic effect. Among them, the RIE of 1,6 PPe-Milk A compounding was as high as 120.54, and the RIE of 1,6 PPe-Milk B compounding also reached 109.97.

[0097] Based on the DPPH· and ·OH scavenging abilities of different concentrations of pumpkin polysaccharide (1.6, 3.2, and 4.8 mg / mL)-milk complexes, the preferred formulations are a 1.6 mg / mL pumpkin polysaccharide aqueous solution mixed with 1.6 mg / mL milk A (1.6 PPe-Milk A) and a 1.6 mg / mL pumpkin polysaccharide aqueous solution mixed with 1.6 mg / mL milk B (1.6 PPe-Milk B), prepared at a 1:1 volume ratio. Since the optimal ratio for scavenging DPPH and OH free radicals selected through mixture formulation does not guarantee its effectiveness in complex mammalian models, mice will be further selected as a model organism to investigate its in vivo anti-T2DM ability.

[0098] Example 3: Anti-T2DM ability of pumpkin polysaccharide-milk A complex

[0099] 1. Construction and grouping of a mouse model of type 2 diabetes

[0100] High-fat diet plus low-dose STZ, where STZ has a specific killing effect on pancreatic β cells in animals, has been widely used to prepare experimental type 2 diabetes models.

[0101] Healthy male C57BL / 6 clean-grade mice aged 5-6 weeks with uniform weight were selected. The rearing temperature was 20 ℃ ± 2℃, and the relative humidity was 55-60%. After purchase, mice were allowed free access to food and water during the acclimatization period. One week later, the mice were randomly divided into 12 groups of 7 mice each, including a normal control group (K group, standard diet), a negative control group (N group, high-fat diet), a positive control group (P group, high-fat diet, deionized water containing 30 mg / kg acarbose), a high-dose sample group (high-fat diet, deionized water containing 300 mg / kg PPe, 1.6 PPe-Milk A, and 1.6 PPe-Milk B), a medium-dose sample group (high-fat diet, deionized water containing 200 mg / kg PPe, 1.6 PPe-Milk A, and 1.6 PPe-Milk B), and a low-dose sample group (high-fat diet, deionized water containing 100 mg / kg PPe, 1.6 PPe-Milk A, and 1.6 PPe-Milk B). Mice in the normal control group were fed a standard diet throughout the experiment, while mice in the other groups were fed a high-fat diet (ResearchDiets D12492 60 kcal% Fat) containing different drugs for each group. After 11 weeks of high-fat diet feeding, mice were intraperitoneally injected with a small dose of streptozocin (STZ) for 3 consecutive days at a daily dose of 20 mg / kg body weight; three days later, a second injection of 40 mg / kg body weight of STZ was administered to establish a type 2 diabetes model. Mice were fasted for 12 hours before each injection, but water was allowed. During this period, mice continued to be fed both the standard diet and the high-fat diet for each group until the end of week 16 of the experiment.

[0102] 2. Dynamic changes in mouse body weight and blood glucose levels

[0103] Mice were fasted overnight but allowed free access to water for 12 hours. They were fed at 9:00 AM the following day, and the feed was removed at 3:00 PM. Initial weight was recorded, and blood was collected from the tail tip using a glucometer to measure initial blood glucose levels. During the intervention period, mouse weight and fasting blood glucose were dynamically monitored periodically using this fasting method. Results are shown below. Figure 3 .

[0104] like Figure 3As shown, with the extension of the feeding time, the average weight of mice in each group showed a gradual increasing trend. The average weight of mice in the negative control (N) group was consistently higher than that of the normal control (K) group, while the average weight of mice in the PPe group, 1.6 PPe-Milk A group, 1.6 PPe-Milk B group, and positive control (P) group was consistently lower than that of mice in the N group. By week 11, the average weight of mice in the N group increased from the initial 21.37 g to 31.76 g, which was higher than the average weight of mice in other groups. This is because the high-fat diet has a high energy density, which easily leads to excess energy and fat accumulation in mice, resulting in a significant increase in weight. Starting from week 12, the weight of mice in all groups decreased. This was because the mice were injected intraperitoneally with a small dose of STZ at week 11, which destroyed the insulin-secreting β cells, leading to an absolute or relative deficiency of insulin. Insulin is a key anabolic hormone that promotes the entry of glucose, amino acids, and fatty acids into cells and the synthesis of glycogen, protein, and fat. When insulin levels decrease significantly, the body's anabolic metabolism is inhibited, and catabolism is enhanced, leading to the consumption of muscle and fat tissue, and thus a decrease in weight. Compared to their initial body weight, at week 11, the average body weight of mice in group K increased by 34.84%, group P by 31.07%, and the high, medium, and low dose PPe groups by 40.17%, 51.49%, and 42.58%, respectively; the high, medium, and low dose 1.6 PPe-Milk A groups by 40.71%, 46.28%, and 48.71%, respectively; and the high, medium, and low dose 1.6 PPe-Milk B groups by 43.57%, 48.64%, and 44.70%, respectively. Except for the medium dose PPe group, the low dose 1.6 PPe-Milk A group, and the medium-low dose 1.6 PPe-Milk B group, the body weight gain rate in other sample groups was lower than that of group N (48.60%), indicating that a high-fat diet can promote body weight gain in mice, while the PPe and PPe-Milk combination can slow down body weight gain.

[0105] When the high-fat diet was continued for 16 weeks, the body weight of mice in group K increased by 2.86%, that of mice in group P increased by 2.56%, while that of mice in group N decreased by 3.37%. The decrease in body weight in group N is a typical manifestation of the acute toxicity of STZ. STZ has certain systemic toxicity, which may lead to loss of appetite, poor mental state, and metabolic disorders in mice, resulting in weight loss. Except for the high-dose PPe group and the low-dose 1.6 PPe-Milk A group, all other sample groups alleviated the weight loss in mice. The high-dose 1.6 PPe-Milk A group showed the most significant increase in body weight (5.44%), followed by the high-dose 1.6 PPe-Milk B group and the medium-dose 1.6 PPe-Milk A group, at 3.87% and 3.47%, respectively. Therefore, compared with group N, the high-dose 1.6 PPe-Milk A group and the -Milk B group can alleviate the weight loss induced by HFD+STZ in mice to some extent.

[0106] like Figure 3As shown, the fasting blood glucose level of mice in group K remained relatively stable at 6-9 mmol / L throughout the experiment, while the blood glucose levels of mice in other groups increased to varying degrees. Before week 12, the blood glucose levels of mice in group N remained high. This is because a long-term high-fat diet induces insulin resistance, and the mice are in a transitional state from "compensatory hyperinsulinemia" to "decompensation," resulting in continuously rising blood glucose. At week 12, except for group K, the blood glucose levels of mice in other groups showed a downward trend. One possible reason is the rebound effect after the initial hyperglycemic effect of STZ. High-dose STZ can rapidly cause severe hyperglycemia, but low-dose STZ may involve a complex physiological process in the initial period after injection. While damaging β cells, STZ may stimulate them to release a large amount of stored insulin temporarily, leading to a transient decrease in blood glucose. Another possible reason is decreased appetite and insufficient intake. The toxicity of STZ can cause mice to exhibit significant anorexia and reduced food intake in the days following injection. Without exogenous glucose intake, blood glucose levels will decrease. From week 12 to week 16, the mice in group N showed a steady increase in blood glucose, a result of permanent damage to pancreatic function. The β-cell death caused by STZ is irreversible; after the double blow of a high-fat diet and STZ, the number of surviving, functional β-cells in the mice was far lower than at week 11. Although the body attempted to compensate, insulin secretion was significantly reduced, making it unable to effectively control blood glucose and promote anabolism as before. At this point, the mice had transitioned from a simple "insulin resistance" state to a "insulin resistance + insulin secretion deficiency" state, indicating damage to their blood glucose regulation system. At week 16, the average blood glucose level in group K was 7.27 mmol / L, in group P it was 10.14 mmol / L, while in group N it was 12.30 mmol / L, higher than all other groups. Both PPe and the PPe-Milk compound can alleviate the rise in blood glucose in mice to some extent. Specifically, the high- and medium-dose PPe groups and the high-dose 1.6PPe-Milk A group had blood glucose levels below 9.5 mmol / L, while other sample groups had blood glucose levels between 9.5 and 11 mmol / L. Therefore, we hypothesize that intervention with the PPe and PPe-Milk compound can alleviate the abnormal rise in blood glucose induced by HFD + STZ in mice to a certain extent.

[0107] 3. Mouse pancreatic islet tissue sections

[0108] The pancreatic tails of mice fed for 16 weeks were fixed in 10% neutral formaldehyde solution for 48 hours, embedded in paraffin, sectioned, stained with basic fuchsin, counterstained with orange-yellow G-brilliant green solution, and observed under a 100x microscope. Results are shown below. Figure 4 .

[0109] The experiment, through observation of the morphology of pancreatic islet tissues from different mice, revealed that in the normal (K) group, the islets were numerous, plump, and rounded, with regular and neat outlines, dense arrangement, and uniform distribution of islet cells. β-cells were light purplish-red, with full cytoplasm and clear cell boundaries. In the negative (N) group, the islets were damaged, becoming shriveled, with blurred boundaries. The islets appeared loose and disordered, with β-cell granule loss, cytoplasmic vacuolation, edema, and increased water content in the cytoplasmic matrix, resulting in pale staining or no staining. After intervention with the positive control drug acarbose, the looseness, shriveling, blurred boundaries, and granule loss of the islets were significantly alleviated. Different doses of PPe, 1.6 PPe-Milk A, and 1.6 PPe-Milk B all had varying degrees of repair effects on damaged islet cells, significantly improving the islet cell outline, restoring the islet morphology to near normal, and showing relatively full cytoplasm. In particular, high-dose PPe, low-high-dose PPe-Milk A, and low-medium-high-dose 1.6 PPe-Milk B showed no significant difference in the number of islets compared to the normal group, with clear cell boundaries and no cell necrosis observed within the islets.

[0110] 4. Biochemical indicator testing

[0111] (1) Measurement of glucose metabolism-related indicators

[0112] After 16 weeks of animal experiments, mice were fasted but allowed free access to water for 12 hours. Blood was collected from the ophthalmic plexus vein and placed in 1.5 mL centrifuge tubes. The tubes were incubated at room temperature for 30 min, then centrifuged at 3500 rpm for 20 min at 4 °C. The supernatant serum was collected and stored at -80 °C for later use. The levels of insulin and glucagon-like peptide-1 (GLP-1) in mouse serum were measured using a biochemical assay kit (Nanjing Jiancheng Bioengineering Institute). After blood collection, the livers were dissected, rinsed with pre-cooled physiological saline, dried, weighed, and placed in 1.5 mL centrifuge tubes. The tubes were labeled, flash-frozen in liquid nitrogen, and stored at -80 °C. A suitable amount of liver was weighed and added to 9 times its weight of pre-cooled physiological saline. The mixture was homogenized using a handheld high-speed homogenizer. After centrifugation, the supernatant was collected to obtain a 10% liver tissue homogenate. The levels of liver glycogen, pyruvate kinase (PK), glucose-6-phosphatase (G-6-P), and phosphoenolpyruvate carboxykinase (PEPCK) activity in the liver were detected using a kit from Nanjing Jiancheng Bioengineering Institute. After blood collection from mice, colon tissue was dissected and prepared into a 10% homogenate using a disperser at 15000 rpm. The homogenate medium was physiological saline. After centrifugation, the precipitate was discarded, and the supernatant was collected. The level of sodium / glucose cotransporter 1 (SGLT1) was determined according to the kit instructions.

[0113] Insulin is a hormone secreted by pancreatic β cells that has a hypoglycemic effect. Figure 5 Serum insulin levels in each group of mice are shown. Figure 5It was found that the serum insulin level in group N mice (83.95 mIU / L) was significantly lower than that in group K (130.56 mIU / L, p < 0.05). This is because the cumulative toxicity of low-dose STZ caused irreversible damage to pancreatic β cells that were already "fatigued" by the high-fat diet, leading to massive β cell apoptosis. The number and function of insulin-secreting "factories" were severely reduced, resulting in a significant decrease in plasma insulin levels. Under acarbose intervention, the serum insulin level in the positive group mice reached 97.33 mIU / L, significantly higher than that in group N mice (p < 0.05). Compared with group N, high, medium, and low doses of PPe and PPe-Milk compound all significantly increased the serum insulin level in mice (p < 0.05), and the increase in serum insulin levels by the three doses of PPe and PPe-Milk compound was not significantly different from that in group P (p > 0.05). This indicates that both PPe and PPe-Milk compound can exert a hypoglycemic effect by promoting insulin secretion in mice, and their effects are similar to those of acarbose.

[0114] Glucagon-like peptide-1 (GLP-1) not only stimulates pancreatic β-cells to secrete insulin and lower blood sugar, but also inhibits gastric emptying and gastric acid secretion, reduces food intake and glucagon secretion, and stimulates β-cell proliferation. Figure 5 As shown, the serum GLP-1 level in group K mice was 2.499 pmol / L, while that in group N mice was 0.399 pmol / L, which was significantly lower than that in group K (p < 0.05). Compared with group N, intervention with acarbose and three concentrations of PPe and PPe-Milk compound significantly increased the serum GLP-1 level in mice (p < 0.05).

[0115] The liver is a core organ for maintaining blood glucose homeostasis; in the absence of insulin, its metabolic patterns are fundamentally reversed. Insulin is a key signal for activating glycogen synthase; in insulin deficiency, the liver glycogen synthesis pathway is inhibited. Simultaneously, insulin deficiency removes the antagonistic effect of glucose-raising hormones such as glucagon, which activate glycogen phosphorylase, accelerating glycogen breakdown. This leads to depletion of liver glycogen reserves. Figure 6The liver glycogen content of mice in group K was 3.416 mg / g, significantly higher than that of mice in group N (2.651 mg / g) (p < 0.05). Compared with group N, intervention with acarbose and three concentrations of PPe and PPe-Milk compound significantly increased liver glycogen content in mice (p < 0.05); and the increase in liver glycogen content was not significantly different from that in group P (p > 0.05). Among them, the ability of medium doses of 1.6PPe-Milk A and 1.6PPe-Milk B, as well as low doses of 1.6PPe-Milk B, to increase liver glycogen content in mice was not significantly different from that in group K (p > 0.05), indicating that the PPe-Milk compound is more effective in preventing liver glycogenolysis and stabilizing blood glucose levels.

[0116] Pyruvate kinase (PK) is a key enzyme and one of the main rate-limiting enzymes in the glycolysis pathway, catalyzing the production of pyruvate and ATP from phosphoenolpyruvate. Figure 6 The liver PK activity in group K mice was 37.02 U / gprot, significantly higher than that in group N mice (24.90 U / gprot, p < 0.05). This is because insulin upregulates PK gene expression and activity through transcription factors (such as ChREBP), thereby promoting glucose breakdown and utilization. In group N mice, under insulin-deficient diabetic conditions, the hepatic glycolysis pathway was inhibited, resulting in decreased PK activity. Compared with group N, intervention with acarbose and three concentrations of PPE and PPE-Milk complexes significantly increased liver PK activity in mice (p < 0.05).

[0117] Phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G-6-Pase) are both important kinases in gluconeogenesis, with G-6-Pase being the final step in catalyzing glycogenolysis. Figure 6In group K mice, the hepatic PEPCK and G-6-Pase activities were 273.27 U / g and 0.0284 ng / mL, respectively, significantly lower than those in group N mice (649.35 U / g, p < 0.05) and 0.394 ng / mL, p < 0.05. The significantly elevated PEPCK and G-6-Pase activities are the most critical molecular mechanism leading to fasting hyperglycemia. G-6-Pase catalyzes the hydrolysis of glucose-6-phosphate into free glucose, the final step in both glycogenolysis and gluconeogenesis; PEPCK is the rate-limiting enzyme in the gluconeogenesis pathway, catalyzing the conversion of oxaloacetate to phosphoenolpyruvate. Insulin strongly inhibits the transcription of these two enzymes, while glucagon and glucocorticoids promote their expression. Decreased insulin levels in group N mice led to a sharp upregulation of G-6-Pase and PEPCK mRNA and protein expression levels in the liver, causing excessive gluconeogenesis and glycogenolysis in the liver, releasing glucose into the bloodstream and resulting in fasting hyperglycemia. Compared with group N, acarbose and interventions with three concentrations of PPe and PPe-Milk compound significantly reduced the activity of PEPCK and G-6-Pase in mouse liver (p < 0.05). Furthermore, in reducing PEPCK activity in mouse liver, high and medium doses of PPe and medium and low doses of 1,6-PPe-MilkA were more effective than the positive control drug acarbose (p < 0.05).

[0118] Sodium-glucose cotransporter 1 (SGLT1) is primarily responsible for the active absorption of glucose in the intestine. It utilizes the electrochemical potential gradient of sodium ions to transport glucose against its concentration gradient and plays a crucial role in the homeostasis of blood glucose levels in the human body. For example... Figure 7The SGLT1 content in the intestines of mice in group K was 2.162 ng / mL, significantly lower than that in group N (3.235 ng / mL, p < 0.05). The elevated SGLT1 content in the intestines of mice in group N was due to two main factors: firstly, low-dose STZ damaged some pancreatic β-cells, and secondly, high-fat-induced insulin resistance led to persistent hyperglycemia. To cope with the persistently high glucose concentration in the lumen, the intestines adaptively upregulated SGLT1 expression and activity to maximize glucose absorption. Secondly, the increased intestinal SGLT1 level led to an increased rate and total amount of postprandial glucose absorption, which in turn exacerbated postprandial hyperglycemia, creating a vicious cycle. This is an increasingly important aspect of the pathogenesis of T2DM—"gut-derived" hyperglycemia. Compared with group N, interventions with three concentrations of PPe, 1.6 PPe-Milk A, and a medium dose of 1.6 PPe-Milk B all significantly reduced the SGLT1 content in the mouse intestines (p < 0.05). The efficacy of PPe was not significantly different from that of the positive control drug acarbose (p > 0.05), while the efficacy of 1,6 PPe-Milk A and 1,6 PPe-Milk B was better than that of the positive control drug acarbose (p < 0.05), indicating that the combination of PPe and Milk was more effective than PPe alone.

[0119] (2) Measurement of oxidative stress-related indicators

[0120] The 10% liver tissue homogenate obtained in step (1) was used to detect the total superoxide dismutase (T-SOD) and catalase (CAT) activities, as well as the malondialdehyde (MDA) and reduced glutathione (GSH) content in the liver using a kit from Nanjing Jiancheng Bioengineering Institute.

[0121] like Figure 8 Compared with group K, the serum SOD, CAT activities and GSH levels of mice in group N were significantly reduced. p < 0.05. The activities of SOD and CAT and the level of GSH in group P were significantly higher than those in the negative control group ( p < 0.05 indicates that the positive control drug can effectively activate the synthetic pathways of SOD, CAT activity and GSH levels, thereby enhancing the body's antioxidant defense function. High, medium, and low doses of PPe, the 1.6 PPe-Milk A group, and the 1.6 PPe-Milk B group all significantly enhanced SOD, CAT activity, and GSH levels. p < 0.05); and the effects of the three doses of 1.6 PPe-Milk A group on SOD, CAT activity and GSH levels were not significantly different from those of the positive control drug acarbose ( p > 0.05), indicating enhanced antioxidant defense capabilities after compounding. Compared with group K, the serum MDA level in group N mice was significantly increased (p < 0.05). The MDA level in group P was significantly lower than that in group N ( p < 0.05). Compared with group N, the high, medium, and low dose PPe groups, the 1.6 PPe-Milk A group, and the 1.6 PPe-Milk B group all significantly reduced serum MDA levels. p < 0.05), and the effect was essentially the same as that of the positive control drug acarbose ( p > 0.05).

[0122] (3) Measurement of inflammatory factor-related indicators

[0123] Blood was collected from the ophthalmic plexus vein, and the serum was separated and stored at -80℃ for later use. The level of lipopolysaccharide (LPS) in mouse serum was measured using a biochemical assay kit. After blood collection from mice, colon tissue was dissected and prepared into a 10% homogenate using a disperser at 15000 rpm in physiological saline. After centrifugation, the precipitate was discarded, and the supernatant was used to determine the levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) according to the kit instructions.

[0124] Serum LPS levels in mice reflect intestinal permeability. Figure 9 It was found that the serum LPS level in group K mice was at a low physiological level, which is closely related to the intact normal intestinal barrier function, effectively preventing LPS translocation from the intestine into the bloodstream. The serum LPS level in group N mice was significantly higher than that in group K (p < 0.05), indicating that a high-fat diet reduces tight junction proteins (such as occludin), and colonic oxidative stress in the later stages of T2DM further damages the barrier; in addition, the increase in Gram-negative bacteria due to dysbiosis in the later stages of diabetes leads to increased LPS production, coupled with barrier leakage, resulting in elevated serum LPS. Compared with group N, intervention with acarbose and three concentrations of PPe and PPe-Milk compound significantly reduced serum LPS levels in mice (p < 0.05), indicating that both PPe and PPe-Milk compound can effectively reduce LPS levels by repairing intestinal barrier function and inhibiting bacterial translocation, thereby alleviating inflammatory damage in T2DM.

[0125] Interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor (TNF-α) are key pro-inflammatory cytokines. Figure 9In group N mice, the levels of IL-1β, IL-6, and TNF-α in the colon were significantly higher than those in group K (p < 0.05). This is because the intestinal barrier of T2DM becomes "leaky," leading to elevated serum LPS levels and "metabolic endotoxemia," which activates pattern recognition receptors such as TLR4, initiating powerful downstream inflammatory signaling pathways such as NF-κB and MAPK. TNF-α is one of the earliest released "core commanders" of the inflammatory response; it can further disrupt tight junctions, forming a positive feedback loop and inducing the production of other inflammatory factors. IL-6, produced by various cells including macrophages, is an important pro-inflammatory factor and a key mediator leading to systemic insulin resistance. The production of IL-1β requires the activation of inflammasomes (such as NLRP3). High-fat diets, LPS, and mitochondrial reactive oxygen species (mtROS) induced by hyperglycemia are all potent activators of the NLRP3 inflammasome; IL-1β is a very potent pro-inflammatory factor with direct toxic effects on pancreatic β cells. Figure 9 The levels of IL-1β, IL-6, and TNF-α in the colon tissue of mice in the P group were significantly lower than those in the P group (p < 0.05), indicating that the positive control drug acarbose can effectively reduce the levels of IL-1β, IL-6, and TNF-α in the colon tissue. Compared with group N, intervention with all three concentrations of PPe and the PPe-Milk compound significantly reduced the levels of IL-1β, IL-6, and TNF-α in mouse colon tissue (p < 0.05). Among them, the medium dose of 1,6 PPe-Milk A was more effective than the positive control drug in reducing TNF-α levels in mouse colon tissue (p < 0.05), the high doses of 1,6 PPe-Milk A and 1,6 PPe-Milk B were more effective than the positive control drug in reducing IL-6 levels in mouse colon tissue (p < 0.05), and the high-medium dose of 1,6 PPe-Milk B was more effective than the positive control drug in reducing IL-1β levels in mouse colon tissue (p < 0.05). This indicates that the combination of PPe and Milk can alleviate systemic chronic low-grade inflammation caused by inflammatory factors produced locally in the colon and reduce insulin resistance.

[0126] Based on the above data, both 1.6 PPe-Milk A and 1.6 PPe-Milk B significantly alleviated high-fat diet-induced weight gain in mice. After entering the HFD+STZ induction phase, high doses of 1.6 PPe-Milk A and 1.6 PPe-Milk B more effectively slowed the resulting weight loss, showing better effects than PPe intervention alone. Regarding blood glucose and islet protection, high doses of PPe and 1.6 PPe-Milk A significantly inhibited HFD+STZ-induced hyperglycemia. Histological analysis further showed that high doses of PPe, as well as different doses of 1.6 PPe-Milk A and 1.6 PPe-Milk B, significantly improved typical islet pathological damage in diabetic mice, including islet atrophy, loose structure, blurred boundaries, and granule loss. At the level of glucose metabolism regulation, all PPe and its compound treatments significantly increased serum GLP-1 levels and hepatic pyruvate kinase (PK) activity, while significantly decreasing hepatic phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G-6-Pase) activity (p < 0.05). Furthermore, all intervention groups significantly increased hepatic glycogen reserves (p < 0.05), with no statistically significant difference compared to the positive control group (P group). The effects of the medium-dose 1.6 PPe-Milk A, 1.6 PPe-Milk B, and low-dose 1.6 PPe-Milk B groups were even comparable to the normal control group (K group). Regarding intestinal and systemic responses, PPe and its compound significantly reduced serum LPS levels and intestinal SGLT1 content (p < 0.05). In terms of SGLT1 inhibition, PPe was comparable to the positive control drug acarbose, while the two 1.6 PPe-Milk compound treatments were significantly more effective than acarbose (p < 0.05). Simultaneously, all intervention groups showed significantly enhanced antioxidant capacity (SOD, CAT activity, and GSH levels), with the effects of each dose group of 1.6PPe-Milk A showing no difference from acarbose. Finally, regarding anti-inflammatory effects, all treatment groups significantly reduced the levels of pro-inflammatory cytokines in the colon (IL-1β, IL-6, TNF-α). Notably, high and medium doses of PPe already showed superior effects compared to positive control agents in reducing IL-6 and IL-1β (p < 0.05); and the 1.6PPe-Milk combination further broadened and strengthened this advantage: medium doses of 1.6PPe-Milk A were more effective in reducing TNF-α; high doses of 1.6PPe-Milk A and 1.6PPe-Milk B were more effective in reducing IL-6; and high and medium doses of 1.6PPe-Milk B were more effective in reducing IL-1β.

[0127] Comparative Example 1: Direct Hot Water Extraction

[0128] Take 1.0 g of pumpkin powder (passed through a 200-mesh sieve), add 61 mL of deionized water and mix well. The material-to-liquid ratio is 1:61 (g / mL). Extract in a water bath shaker at 125 rpm for 61 min at 100℃. Centrifuge at 8000 rpm for 20 min, take 1.0 mL of supernatant, and use the phenol-sulfuric acid method to detect the polysaccharide content in the supernatant. The content is then converted into the extraction yield. The results are shown in Table 2.

[0129] Table 2 Effect of different extraction processes on the extraction yield of pumpkin polysaccharides

[0130]

[0131] As shown in Table 2, different extraction processes have a significant impact on the extraction yield of pumpkin polysaccharides. The free radical extraction method can significantly improve the extraction yield of pumpkin polysaccharides, which is 7.596 times higher than that of pumpkin polysaccharides extracted by traditional hot water extraction.

[0132] Comparative Example 2: Effect of Pumpkin Flour Mesh Size on Pumpkin Polysaccharide Yield

[0133] In Example 1, the pumpkin powder was replaced with a 20-mesh coarse sieve, and other operations were the same as in Example 1. The extraction yield of pumpkin polysaccharides was detected and calculated using the same method as in Example 1. The results are shown in Table 3.

[0134] Table 3. Effect of different pumpkin powder particle sizes on the extraction yield of pumpkin polysaccharides

[0135]

[0136] As shown in Table 3, the particle size of the crushed pumpkin has a great influence on the extraction yield of pumpkin polysaccharides. The pretreatment method of crushing and passing through a 200-mesh sieve can increase the extraction yield of pumpkin polysaccharides by 1.541 times compared with the coarse powder produced by traditional mechanical crushing.

[0137] Comparative Example 3: Effect of cellulase extraction on the yield of pumpkin polysaccharides

[0138] Take 1.0 g of pumpkin powder (passed through a 200-mesh sieve), add 0.08 g of cellulase (enzyme activity 50 U / mg, the amount added is 1.5% of the weight of pumpkin powder), add 60 mL of deionized water, and extract for 3.0 h at pH 4.8 and temperature 55℃; centrifuge at 8000 rpm for 20 min, take 1.0 mL of supernatant, and use the phenol-sulfuric acid method to detect the polysaccharide content in the supernatant and convert it into the extraction yield. The results are shown in Table 4.

[0139] Table 4. Effects of different extraction processes on the extraction yield of pumpkin polysaccharides

[0140]

[0141] As shown in Table 4, the extraction process of pumpkin has a great influence on the extraction yield of pumpkin polysaccharides. The free radical extraction method can increase the extraction yield of pumpkin polysaccharides by 1.802 times compared with cellulase extraction.

[0142] Comparative Example 4: Effect of hydrogen peroxide-vitamin C extraction on the yield of pumpkin polysaccharides

[0143] The hydrogen peroxide used in this invention generates free radicals in the presence of vitamin C, which cleaves the glycosidic bonds of pumpkin pulp dietary fiber and promotes the release of pumpkin polysaccharides. Therefore, this reagent was selected for comparison. 1.0 g of pumpkin powder (passed through a 200-mesh sieve) was added to 58.7 mL of deionized water, followed by 2.3 mL of 30% hydrogen peroxide (deionized water and hydrogen peroxide constitute the extraction solvent, with a hydrogen peroxide concentration of 1.13%). The mixture was then stirred, with a solid-liquid ratio of 1:61 (g / mL). Then, 1 / 2, 1 / 4, 1 / 8, and 1 / 16 of the mass of vitamin C were added, respectively. Extraction was performed in a water bath shaker at 125 rpm at 100℃ for 61 min, followed by centrifugation at 8000 rpm for 20 min. 1.0 mL of the supernatant was collected, and the polysaccharide content in the supernatant was determined using the phenol-sulfuric acid method and converted to the extraction yield. The results are shown in Table 5.

[0144] Table 5. Effects of different extraction processes on the extraction yield of pumpkin polysaccharides

[0145]

[0146] Table 5 shows that the ratio of hydrogen peroxide to vitamin C has a significant impact on the extraction yield of pumpkin polysaccharides. The extraction method without adding vitamin C can actually increase the extraction yield of pumpkin polysaccharides by 1.161 times, 1.184 times, 1.243 times, and 1.174 times, respectively, compared with the extraction methods that added 1 / 2, 1 / 4, 1 / 8, and 1 / 16 of hydrogen peroxide and vitamin C.

[0147] Comparative Example 5: Effect of hydrogen peroxide-FeSO4 extraction on the yield of pumpkin polysaccharides

[0148] The hydrogen peroxide used in this invention generates free radicals in the presence of FeSO4, which cleaves the glycosidic bonds of pumpkin pulp dietary fiber and promotes the release of pumpkin polysaccharides. Therefore, this reagent was selected for comparison. 1.0 g of pumpkin powder (passed through a 200-mesh sieve) was added to 58.7 mL of deionized water, followed by 2.3 mL of 30% hydrogen peroxide (deionized water and hydrogen peroxide constitute the extraction solvent, with a hydrogen peroxide concentration of 1.13%). The mixture was then stirred, with a solid-liquid ratio of 1:61 (g / mL). Then, 1 / 2, 1 / 4, 1 / 8, and 1 / 16 of the mass of hydrogen peroxide in FeSO4 were added, respectively. Extraction was performed in a water bath shaker at 125 rpm at 100℃ for 61 min, followed by centrifugation at 8000 rpm for 20 min. 1.0 mL of the supernatant was collected, and the polysaccharide content in the supernatant was determined using the phenol-sulfuric acid method and converted to the extraction yield. The results are shown in Table 6.

[0149] Table 6. Effects of different extraction processes on the extraction yield of pumpkin polysaccharides

[0150]

[0151] As shown in Table 6, the ratio of hydrogen peroxide to FeSO4 has a significant impact on the extraction yield of pumpkin polysaccharides. The extraction method without adding FeSO4 can significantly improve the extraction yield of pumpkin polysaccharides, which is 2.534 times, 2.250 times, 2.442 times, and 2.898 times higher than that extracted with 1 / 2, 1 / 4, 1 / 8, and 1 / 16 of the mass of hydrogen peroxide and FeSO4, respectively.

Claims

1. A pumpkin polysaccharide-milk compound for anti-diabetes, characterized in that, The compound is prepared by mixing the water solution of pumpkin polysaccharide with milk; the concentration of the water solution of pumpkin polysaccharide is 1.6-4.8 mg / mL.

2. The pumpkin polysaccharide-milk compound of claim 1, characterized in that, The volume ratio of the water solution of pumpkin polysaccharide to milk is 1:

1.

3. The pumpkin polysaccharide-milk compound of claim 1, characterized in that, The milk includes pure milk with a protein content of 3.0-3.6 g / 100 mL.

4. The pumpkin polysaccharide-milk compound of claim 1, characterized in that, The compound is prepared by mixing the water solution of pumpkin polysaccharide with pure milk with a solid content of 1.6 mg / mL at a volume ratio of 1:

1.

5. The pumpkin polysaccharide-milk compound of claim 1, wherein, The pumpkin polysaccharide is prepared as follows: pumpkin powder is extracted with deionized water and hydrogen peroxide with a mass concentration of 30% as an extracting agent in a water bath shaker at a rotation speed of 100-150 rpm at a temperature of 80-100 ℃ for 50-70 min, and then centrifuged at 8000 rpm for 15 min; the supernatant is collected and concentrated to 1 / 5 of the original volume; then Sevag reagent is added to the concentrated solution at a volume ratio of 1:5, and shaken vigorously for 20 min, and centrifuged at 3000 rpm for 10 min to form an organic solvent layer, a protein layer and a water layer; the water layer is concentrated to remove residual organic solvent, and then 95% ethanol is added at a volume ratio of 1:4, and the mixture is placed at 4 ℃ overnight; finally, the mixture is centrifuged at 1000 rpm for 5 min to collect the precipitate, which is redissolved in distilled water, and the residual organic reagent is removed by rotary evaporation to obtain a purified pumpkin polysaccharide solution, which is freeze-dried to obtain the pumpkin polysaccharide.

6. The squash-powder-milk compound of claim 5, wherein, The mass concentration of hydrogen peroxide in the extracting agent is 0.6%-1.8%, and the volume of the extracting agent is 30-120 mL / g of the pumpkin powder.

7. Use of the pumpkin polysaccharide-milk compound of claim 1 in the preparation of an antioxidant product.

8. Use of the pumpkin polysaccharide-milk compound of claim 1 in the preparation of a drug for treating diabetes.

9. Use according to claim 8, wherein the compound is ###0002### The drug is a drug for treating or improving type 2 diabetes.

10. The use according to claim 8, wherein the compound is ###00002### The drug is a drug for treating high-fat diet plus low-dose streptozotocin-induced type 2 diabetes.

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