Hypoglycemic peptide based on hippophae rhamnoides seed meal protein as well as preparation method and application of hypoglycemic peptide

Abstract

The invention discloses a hippophae rhamnoides seed meal protein-based hypoglycemic peptide and a preparation method thereof, and the preparation method comprises the following steps: mixing hippophae rhamnoides seed meal powder with water, adjusting the pH value with sodium hydroxide, stirring, and centrifuging to obtain a supernatant; adjusting the pH value of the supernatant with hydrochloric acid, standing, centrifuging, washing the obtained precipitate with distilled water until the precipitate is neutral, pre-freeze-drying, and freeze-drying to obtain SSP powder; the preparation method comprises the following steps: mixing SSP powder with water to obtain an SSP solution, carrying out water bath preheating, adding protease, carrying out enzymolysis, carrying out water bath heating on the solution, adjusting the pH value of the solution to be neutral, collecting a centrifugal supernatant, and carrying out low-temperature dialysis to obtain the hypoglycemic peptide based on the sea buckthorn seed meal protein, wherein the protease comprises alkaline protease, compound protease, neutral protease, bromelain and papain, and the invention aims to excavate the development prospect of the sea-buckthorn seed meal active peptide and expand the application potential of the sea-buckthorn seed meal active peptide in different disease fields.

Description

Technical Field

[0001] The present invention belongs to the technical field of protein peptides, and in particular relates to a blood sugar-lowering peptide based on seabuckthorn seed meal protein, and a preparation method and application thereof. Background Art

[0002] With the continuous improvement of living standards around the world, the incidence of chronic diseases has increased dramatically, and has become a serious challenge in today's public health field. Among them, diabetes, as a common chronic metabolic disease, is listed as one of the most pressing health problems in the 21st century. At present, the treatment of diabetes mainly relies on drug intervention, combined with a scientific and healthy lifestyle. However, existing hypoglycemic drugs are not only expensive, but may also be accompanied by a series of side effects and potential risks, such as liver damage, indigestion or gastrointestinal discomfort. Bioactive peptides have shown broad application prospects in the field of hypoglycemic due to their high biological activity, low toxicity and diverse functional properties. A large number of studies have found that bioactive peptides can effectively regulate blood sugar levels by inhibiting the activity of key blood sugar metabolic enzymes such as α-Glu, α-Amy or DPP-Ⅳ, providing new ideas for the prevention and treatment of diabetes.

[0003] Seabuckthorn seed meal, a high-quality plant protein resource, is currently used only as animal feed, and its potential value has not yet been fully explored. Although the extraction and enzymatic hydrolysis technologies of seabuckthorn seed meal protein are relatively mature, research on the bioactivity of its hydrolysates remains limited, and the mechanism of action is not well understood, which seriously restricts its further development and application.

[0004] Based on this, the implementation of this study will not only help to reveal the functional properties of bioactive peptides from seabuckthorn seed meal, but will also provide a scientific basis for the high-value utilization of seabuckthorn by-products, and provide experimental basis and technical support for their development and application in the field of functional foods and medicines. Summary of the Invention

[0005] The purpose of this section is to summarize some aspects of the embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the abstract and title of this application to avoid obscuring the purpose of this section, the abstract and the title of the invention, and such simplifications or omissions should not be used to limit the scope of the present invention.

[0006] In view of the above problems and / or the problems existing in the prior art, the present invention is proposed.

[0007] Therefore, the purpose of the present invention is to overcome the deficiencies in the prior art and provide a hypoglycemic peptide based on seabuckthorn seed meal protein and a preparation method and application thereof.

[0008] In order to solve the above technical problems, the present invention provides the following technical solution: a method for preparing a hypoglycemic peptide based on seabuckthorn seed meal protein, characterized in that: it comprises:

[0009] After mixing sea buckthorn seed meal powder with water, adjusting the pH value with sodium hydroxide, stirring, and centrifuging to obtain a supernatant; adjusting the pH value of the supernatant with hydrochloric acid, letting it stand and then centrifuging; the obtained precipitate is washed with distilled water until neutral, pre-lyophilized, and freeze-dried to obtain SSP powder;

[0010] The SSP solution obtained by mixing SSP powder with water is preheated in a water bath, protease is added, and after enzymatic hydrolysis, the solution is heated in a water bath, and the pH of the solution is adjusted to neutral. The supernatant is collected by centrifugation and dialyzed at low temperature to obtain the sea buckthorn seed meal protein-based hypoglycemic peptide;

[0011] The protease includes one or more of alkaline protease, composite protease, neutral protease, bromelain or papain.

[0012] As a preferred solution of the preparation method of the present invention, the concentration of the SSP solution is 3-6%.

[0013] As a preferred embodiment of the preparation method of the present invention, the amount of protease added is 10,000 to 14,000 U.

[0014] As a preferred embodiment of the preparation method of the present invention, the enzymatic hydrolysis time is 120 to 180 minutes.

[0015] As a preferred embodiment of the preparation method of the present invention, the concentration of the SSP solution is 4%.

[0016] As a preferred embodiment of the preparation method of the present invention, the amount of protease added is 12,000 U.

[0017] As a preferred embodiment of the preparation method of the present invention, the enzymatic hydrolysis time is 150 minutes.

[0018] Another object of the present invention is to overcome the deficiencies in the prior art and provide a hypoglycemic peptide based on seabuckthorn seed meal protein prepared by a preparation method.

[0019] As a preferred embodiment of the hypoglycemic peptide of the present invention, wherein: the IC of α-Glu and DPP-IV 50 It can reach 3.45±0.18mg / mL and 5.01±0.21mg / mL.

[0020] As a preferred embodiment of the hypoglycemic peptide of the present invention, the inhibitory activity against α-Glu after simulated gastrointestinal digestion is 45.14%; the inhibitory activity against DPP-IV is 48.76%.

[0021] Beneficial effects of the present invention:

[0022] (1) The present invention successfully prepared five seabuckthorn seed meal peptides, SSPHA, SSPHC, SSPHD, SSPHB, and SSPHP, and they exhibited multiple biological activities. Among them, SSPHD showed excellent development prospects for hypoglycemic peptides; SSPHB showed excellent development prospects for lipid-lowering peptides; SSPHP showed excellent development prospects for blood pressure-lowering peptides; and SSPHA showed excellent development prospects for uric acid-lowering peptides.

[0023] (2) The present invention optimized the enzymatic hydrolysis process using single-factor and BBD experiments, determining the optimal conditions to be: substrate concentration 4%, enzyme dosage 12,300 U, and enzymatic hydrolysis time 140 min. Under these conditions, the IC50 values ​​of the resulting SSPHD for α-Glu and DPP-IV were 3.45 ± 0.18 mg / mL and 5.01 ± 0.21 mg / mL, respectively.

[0024] (3) The peptide omics analysis of the present invention found that SSPHD contains 16 reported ACE inhibitory peptides, 8 antioxidant peptides, 2 DPP-IV inhibitory peptides, and 1 angiotensin II degradation-promoting peptide, HMG-CoA reductase inhibitory peptide, antihypertensive peptide, anti-inflammatory peptide, and stimulatory peptide. Through peptide sequence screening and molecular docking verification, a total of 3 potential α-Glu inhibitory peptides (FHF, FFI, FGI) and 3 potential DPP-IV inhibitory peptides (IYF, IGF, LFF) were screened out. The results of in vitro synthesis and activity assays showed that these potential peptides had varying degrees of inhibitory effects on α-Glu or DPP-IV. Among them, FHF had the lowest IC50 for α-Glu (3.98±0.16mM). The next was FFI (8.21±0.21mM) and FGI (11.57±0.20mM). IYF had the lowest IC50 for DPP-IV (5.32±0.15 mM), followed by IGF (7.17±0.14 mM) and LFF (7.62±0.19 mM).

[0025] (4) The present invention simulated gastrointestinal digestion in vitro and found that SSPHD would be degraded during the digestion process, but its hypoglycemic activity could still retain 45.14% (α-Glu) and 48.76% (DPP-IV). In the db / db diabetic mouse model, after 5 weeks of SSPHD gavage treatment, the symptoms of diabetic mice such as obesity, hyperphagia, impaired glucose tolerance, and liver atrophy were significantly improved, and the blood glucose level and glycated serum protein content were significantly reduced. In addition, after SSPHD intervention, the TC and LDL-C levels of mice decreased, the HDL-C level increased, and lipid metabolism disorders were improved; the levels of proinflammatory cytokines TNF-α, IL-1β and IL-6 decreased, and the inflammatory response was alleviated; the ALT and AST levels decreased, and liver function damage was reduced; the CAT and SOD activities were enhanced, and the liver oxidative stress state was alleviated; the UREA and CREA levels decreased, and renal function damage was alleviated. The results of the mechanism study showed that after SSPHD intervention, the activity of the key enzyme PEPCK in gluconeogenesis was weakened, and the hepatic glucose output was reduced. At the same time, the activity of GK, a key enzyme in glycogen synthesis, is enhanced, promoting the synthesis of glycogen from excess glucose. By regulating gluconeogenesis and glycogen synthesis, SSPHD effectively alleviates hyperglycemia symptoms and improves glucose metabolism disorders. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. Those skilled in the art can also derive other drawings based on these drawings without inventive effort. Among them:

[0027] Figure 1 It is the hydrolysis degree of the crude protein of seabuckthorn seed meal in the embodiment of the present invention.

[0028] Figure 2 This is the molecular weight distribution of the seabuckthorn seed meal protein hydrolyzate in the embodiment of the present invention.

[0029] Figure 3 This is the infrared spectrum of seabuckthorn seed meal protein and its hydrolyzate according to the embodiment of the present invention.

[0030] Figure 4 This is an endogenous fluorescence spectrum of seabuckthorn seed meal protein and its hydrolyzate according to an embodiment of the present invention.

[0031] Figure 5 The effects of seabuckthorn seed meal protein hydrolysate on the activities of α-Glu, α-Amy and DPP-Ⅳ are shown in the examples of the present invention, wherein: Figure 5 (a) is α-Glu, Figure 5 (b) α-Amy, Figure 5 It is (c)DPP-Ⅳ.

[0032] Figure 6 The effect of seabuckthorn seed meal protein hydrolysate on PL and CEase activities in the embodiment of the present invention is shown in FIG. Figure 6 (a) is PL, Figure 6 (b) is CEase.

[0033] Figure 7 This is the effect of seabuckthorn seed meal protein hydrolyzate on ACE activity in the examples of the present invention.

[0034] Figure 8 This is the effect of seabuckthorn seed meal protein hydrolyzate on XO activity in the examples of the present invention.

[0035] Figure 9 This is the single factor experimental result of the embodiment of the present invention, wherein, Figure 9 (a) is a single factor of substrate concentration; Figure 9 (b) single factor of enzymatic hydrolysis time; Figure 9 (c) Single factor of enzyme addition.

[0036] Figure 10 The in vitro gastrointestinal digestion effect of the embodiment of the present invention, wherein, Figure 10 (a) is α-Glu inhibitory activity, Figure 10 (b) DPP-IV inhibitory activity.

[0037] Figure 11 This is the effect of SSPHD on the body weight of db / db mice in the present invention.

[0038] Figure 12 The effects of SSPHD on food intake and water intake in db / db mice according to the present invention are shown in FIG. Figure 12 (a) is the amount of food intake, Figure 12 (b) is the amount of water consumed.

[0039] Figure 13 The effects of SSPHD on liver index, kidney index, pancreas index and spleen index of db / db mice in the embodiment of the present invention are shown in FIG. Figure 13 (a) is the liver index, Figure 13 (b) is the renal index, Figure 13 (c) is the pancreatic index, Figure 13 (d) is the spleen index.

[0040] Figure 14 This is the effect of SSPHD on fasting blood glucose in db / db mice according to the present invention.

[0041] Figure 15 The effects of SSPHD on the OGTT curve (a) and AUC (b) of db / db mice according to the present invention are shown.

[0042] Figure 16 This is the effect of SSPHD on GSP in db / db mice according to the present invention.

[0043] Figure 17 The effect of SSPHD on blood lipids in db / db mice according to the present invention is shown in FIG. Figure 17 (a) is TC, Figure 17 (b) is TG, Figure 17 (c) is LDL-C, Figure 17 (d) is HDL-C.

[0044] Figure 18 The effects of SSPHD on inflammatory factors TNF-α, IL-1β and IL-6 in the liver of db / db mice are shown in the examples of the present invention. Figure 18 (a) is TNF-α, Figure 18 (b) is IL-1β, Figure 18 (c) is IL-6.

[0045] Figure 19 The effects of SSPHD on ALT and AST in db / db mice are shown in the examples of the present invention, wherein: Figure 19 (a) is ALT, Figure 19 (b) is AST.

[0046] Figure 20 The effects of SSPHD on CAT activity, SOD activity, GSH-Px activity and MDA content in the liver of db / db mice are shown in the examples of the present invention, wherein: Figure 20 (a) is CAT activity, Figure 20 (b) is SOD activity, Figure 20 (c) is GSH-Px activity, Figure 20 (d) is the MDA content.

[0047] Figure 21 The effects of SSPHD on the UREA and CREA contents in db / db mice according to the present invention are shown in FIG. Figure 21 (a) is the UREA content, Figure 21 (b) is the CREA content.

[0048] Figure 22 This is the effect of SSPHD on liver histopathology in db / db mice according to the present invention.

[0049] Figure 23 The effects of SSPHD on G6Pase and PEPCK in the liver of db / db mice are shown in the examples of the present invention, wherein: Figure 23 (a) is G6Pase, Figure 23 (b) is PEPCK.

[0050] Figure 24 This is the effect of SSPHD on PAS staining in the liver of db / db mice according to the present invention.

[0051] Figure 25 This is the effect of SSPHD on GK activity in the liver of db / db mice according to the present invention. DETAILED DESCRIPTION

[0052] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the embodiments of the specification.

[0053] In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention may also be implemented in other ways different from those described herein. Those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0054] Secondly, the term "one embodiment" or "embodiment" herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in various places throughout this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive of other embodiments.

[0055] Unless otherwise specified, the raw materials used in the examples of the present invention are all commercially available, among which seabuckthorn seed meal was purchased from Aheqi County, Kizilsu Kirgiz Autonomous Prefecture, Xinjiang Uygur Autonomous Region, China; Coomassie Brilliant Blue G250 was purchased from Beijing Inokai Technology Co., Ltd.; CEase was purchased from Beijing Solaibao Technology Co., Ltd.; PL and xanthine were purchased from Beijing Bailingwei Technology Co., Ltd.; 4-nitrophenyl butyrate (p-NPB) and soluble starch were purchased from Shanghai Titan Technology Co., Ltd.; α-Amy, ACE, DPP-IV, trypsin, and pepsin were purchased from Sigma-Aldrich; alkaline protease, complex protease, neutral protease, bromelain, papain, α-Glu, p-nitrophenyl-α-D-pyranose p-NPG, DNS reagent (NY / T method), XO, N-[3-(2-furyl)acryloyl]-L-phenylalanyl-glycyl-glycine (FAPGG), N-glycylprolyl-p-nitroanilide hydrochloride (Gly-Pro-pNA), acarbose, dipeptidyl peptidase A (IPI), metformin hydrochloride (Met), 4% paraformaldehyde, D(+)-anhydrous glucose, and normal saline were purchased from Shanghai Yuanye Biotechnology Co., Ltd.; synthetic peptides (purity 95%) were purchased from Shanghai Shenggong Biotechnology Co., Ltd.; and INSELISA kits were purchased from Cloud-Clone, USA. Corp.; IL-6, IL-1β, and TNF-α ELISA kits were purchased from Thermo Fisher Scientific, USA; PEPCK, GK, and G6Pase ELISA kits were purchased from Shanghai ELISA Biotechnology Co., Ltd.; BCA protein quantification kit and CAT, SOD, GSH-Px, and MDA ELISA kits were purchased from Wuhan Sevier Biotechnology Co., Ltd.; all other reagents were of analytical grade.

[0056] SPF level db / db(BSK-Lep rem2Cd479 Forty 6-week-old male C57BLKS / Gpt mice (C57BLKS / JGpt background) and eight wild-type wt / wt littermates were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. under license number SCXK(Su)2023-0009. The SPF mice use license number is SYXK(Su)2021-0056 from the Experimental Animal Center of Jiangnan University.

[0057] α-Glu inhibition assay: Prepare a 0.2 U / mL α-Glu solution and a 10 mM p-NPG substrate solution in PBS buffer (0.2 M, pH 6.8). Add 100 μL of SSPHs sample solution and 50 μL of α-Glu to a 96-well plate and incubate at 37°C for 15 minutes. Then, add 50 μL of p-NPG and incubate at 37°C for 30 minutes. Finally, measure absorbance at 405 nm.

[0058] α-Amy inhibition rate determination method: Prepare a 1U / mL α-Amy solution in PBS buffer (0.1M, pH 7.0). Mix 100μL of SSPHs sample solution with 100μL of α-Amy and incubate at 37°C for 10 minutes. Then, add 200μL of soluble starch solution (1%, w / v) and incubate at 37°C for 10 minutes. After the reaction is completed, add 500μL of DNS reagent, place in a boiling water bath for 5 minutes, and then cool in an ice bath for 5 minutes. Finally, measure the absorbance at a wavelength of 540nm.

[0059] DPP-IV inhibition assay: Prepare a 10 U / mL DPP-IV solution and a 1.6 mM Gly-Pro-pNA substrate solution in Tris-HCl buffer (0.1 M, pH 8.0). Add 50 μL of the SSPHs sample solution and 50 μL of Gly-Pro-pNA to a 96-well plate and incubate at 37°C for 10 minutes. Then, add 50 μL of DPP-IV and incubate at 37°C for 60 minutes. Finally, measure absorbance at 405 nm.

[0060] PL inhibition assay: Prepare a 21 U / mL PL solution in Tris-HCl buffer (0.1 M, pH 8.2). Prepare a 4.69 mM p-NPB substrate solution in dimethyl sulfoxide (5%). Add 50 μL of 3-fold diluted SSPHs and 50 μL of PL to a 96-well plate and incubate at 37°C for 10 minutes. Then, add 50 μL of p-NPB and incubate at 37°C for 20 minutes. Finally, measure absorbance at 405 nm.

[0061] CEase inhibition assay: Prepare a 6.98 U / mL CEase solution in PBS buffer (0.1 M, pH 7.0) containing sodium taurocholate (5.16 mM) and NaCl (0.1 M). Prepare a 3.84 mM p-NPB substrate solution in 5% dimethyl sulfoxide (w / v). Add 130 μL PBS, 50 μL SSPHs, and 50 μL CEase to a 96-well plate and incubate at 25°C for 5 minutes. Then, add 20 μL p-NPB and incubate at 25°C for 20 minutes. Finally, measure absorbance at 405 nm.

[0062] Evaluation of the antihypertensive activity of seabuckthorn seed meal protein peptides: 100 U / L ACE solution and 1 mM FAPGG solution were prepared in sodium borate buffer (0.05 M, pH 8.3) containing 0.3 M NaCl. 10 μL ACE, 50 μL FAPGG, and 40 μL of 3-fold diluted SSPHs were added to a 96-well plate and incubated at 37°C for 5 minutes. The absorbance was measured at 340 nm. The plate was incubated at 37°C for 30 minutes, and the absorbance was measured again at 340 nm.

[0063] Evaluation of the uric acid-lowering activity of seabuckthorn seed meal peptides: PBS buffer (0.2 M, pH 7.4) was used to prepare a 0.5 U / mL XO solution and a 0.48 mM xanthine substrate solution. The xanthine solution was initially solubilized with an appropriate amount of 1 M NaOH. 50 μL of the 3-fold diluted SSPHs sample solution was mixed with 50 μL of XO in a 96-well plate and incubated at 25°C for 5 minutes. Then, 50 μL of xanthine was added and incubated at 25°C for 25 minutes. Finally, the absorbance was measured at 290 nm.

[0064] Example 1

[0065] (1) Pretreatment of seabuckthorn seed meal: The seabuckthorn seed meal was crushed by a multifunctional grinder, passed through a 50-mesh sieve, and sealed and stored at -20°C for later use.

[0066] (2) Extraction of sea buckthorn seed meal protein (SSP): SSP was extracted by alkali dissolution and acid precipitation. Take an appropriate amount of sea buckthorn seed meal powder and prepare an aqueous solution at a material-liquid ratio of 1:15 (w / v). Use 1M NaOH to maintain the pH value at 10.0. Stir at 40℃ for 1 hour, then centrifuge at 4000 rpm for 20 minutes and collect the supernatant. Adjust the pH value of the supernatant to 4.0 with 1M HCl solution, let it stand for 30 minutes, and then centrifuge again. The resulting precipitate was washed with distilled water until neutral, placed at -20℃ for pre-freeze-drying, and finally freeze-dried to obtain SSP powder, which was stored at 4℃ for later use.

[0067] (3) Preparation of spinach seed meal protein peptides: Before enzymatic hydrolysis, the SSP solution (4%, w / v) was preheated in an 85°C water bath for 30 minutes to promote protein denaturation and thereby improve the hydrolysis efficiency. Subsequently, neutral protease was added at an enzyme-substrate ratio of 8400 U / g, and enzymatic hydrolysis was carried out under the optimal conditions of protease (temperature 40°C, pH 7.5). After 3 hours of enzymatic hydrolysis, the solution was placed in an 85°C water bath for 15 minutes to inactivate the protease, and the pH of the solution was adjusted to neutral. The supernatant was collected by centrifugation (20 minutes, 4000 rpm) and dialyzed at 4°C for 3 days to remove small molecular salt substances. Finally, a portion of the solution was freeze-dried to obtain SSPHD, which was stored at -20°C for later use.

[0068] Comparative Example 1

[0069] The difference from Example 1 is that step (3) is the preparation of thorn seed meal protein peptide: before enzymatic hydrolysis, the SSP solution (4%, w / v) is placed in an 85°C water bath for 30 minutes to preheat to promote protein denaturation and thereby improve the hydrolysis efficiency. Subsequently, alkaline protease is added according to an enzyme-substrate ratio of 8400U / g, and enzymatic hydrolysis is carried out under the optimal conditions of the protease (temperature 40°C, pH 10). After 3 hours of enzymatic hydrolysis, the solution is placed in an 85°C water bath for 15 minutes to inactivate the protease, and the pH of the solution is adjusted to neutral. The supernatant is collected by centrifugation (20 minutes, 4000rpm) and dialyzed at 4°C for 3 days to remove small molecular salt substances. Finally, a portion of the solution is freeze-dried to obtain SSPHA, which is stored at -20°C for later use. The rest is consistent with Example 1.

[0070] Comparative Example 2

[0071] The difference from Example 1 is that in step (3), the preparation of spinosad meal protein peptide is as follows: before enzymatic hydrolysis, the SSP solution (4%, w / v) is placed in a water bath at 85°C for 30 minutes to promote protein denaturation and thereby improve the hydrolysis efficiency. Subsequently, a composite protease is added according to an enzyme-substrate ratio of 8400U / g, and enzymatic hydrolysis is carried out under the optimal conditions of the protease (temperature 50°C, pH 7.5). After 3 hours of enzymatic hydrolysis, the solution is placed in a water bath at 85°C for 15 minutes to inactivate the protease, and the pH of the solution is adjusted to neutral. The supernatant is collected by centrifugation (20 minutes, 4000 rpm) and dialyzed at 4°C for 3 days to remove small molecular salt substances. Finally, a portion of the solution is freeze-dried to obtain SSPHC, which is stored at -20°C for later use. The rest is consistent with Example 1.

[0072] Comparative Example 3

[0073] The difference from Example 1 is that step (3) is the preparation of spinach seed meal protein peptide: before enzymatic hydrolysis, the SSP solution (4%, w / v) is placed in an 85°C water bath for 30 minutes to preheat to promote protein denaturation and thereby improve the hydrolysis efficiency. Subsequently, bromelain is added according to an enzyme-substrate ratio of 8400U / g, and enzymatic hydrolysis is carried out under the optimal conditions of the protease (temperature 50°C, pH 7.5). After 3 hours of enzymatic hydrolysis, the solution is placed in an 85°C water bath for 15 minutes to inactivate the protease, and the pH of the solution is adjusted to neutral. The supernatant is collected by centrifugation (20 minutes, 4000rpm) and dialyzed at 4°C for 3 days to remove small molecular salt substances. Finally, a portion of the solution is freeze-dried to obtain SSPHB, which is stored at -20°C for later use. The rest is consistent with Example 1.

[0074] Comparative Example 4

[0075] The difference from Example 1 is that step (3) is the preparation of thorn seed meal protein peptide: before enzymatic hydrolysis, the SSP solution (4%, w / v) is placed in an 85°C water bath for 30 minutes to preheat to promote protein denaturation and thereby improve the hydrolysis efficiency. Subsequently, papain is added according to an enzyme-substrate ratio of 8400U / g, and enzymatic hydrolysis is carried out under the optimal conditions of the protease (temperature 40°C, pH 7.5). After 3 hours of enzymatic hydrolysis, the solution is placed in an 85°C water bath for 15 minutes to inactivate the protease, and the pH of the solution is adjusted to neutral. The supernatant is collected by centrifugation (20 minutes, 4000rpm) and dialyzed at 4°C for 3 days to remove small molecule salt substances. Finally, a portion of the solution is freeze-dried to obtain SSPHP, which is stored at -20°C for later use. The rest is consistent with Example 1.

[0076] Comparative Example 5

[0077] The difference from Example 1 is that step (3) is not performed, and sea buckthorn seed meal protein (SSP) is obtained.

[0078] The protein peptides prepared in Example 1 and Comparative Examples 1-4 were tested for the degree of protein hydrolysis (DH) using the pH-state method:

[0079] like Figure 1 As shown, the hydrolysis efficiency of each protease for SSP under the optimal conditions showed significant differences. The hydrolysis degree from high to low was SSPHD (27.69±0.79%), SSPHC (24.45±0.70%), SSPHA (15.78±0.47%), SSPHP (12.18±0.43%), and SSPHB (4.85±0.46%). Among them, the neutral protease of Example 1 had the best hydrolysis effect on SSP, which may be related to its specific effect on aliphatic amino acids and aromatic amino acids.

[0080] Example 1 and Comparative Examples 1-4 are carried out to molecular weight distribution determination, and 100 mg of sample is weighed and placed in a 10 ml volumetric flask. Add appropriate amount of deionized water, after being settled to the scale mark, the solution is placed in an ultrasonic cleaning instrument and processed for 5 min. After centrifugation, the gained supernatant is filtered through a 0.22 μm microporous membrane and used for high performance liquid chromatograph sample injection, and with cytochrome C (12384 Da), aprotinin (6500 Da), bacitracin (1422 Da), tyrosine-tyrosine-arginine (451 Da), and tyrosine-tyrosine-tyrosine (189 Da) as standard specimens.

[0081] Molecular weight distribution characteristics such as Figure 2As shown in Table 1. The analysis results show that the main components of SSPHA, SSPHC and SSPHD are all small molecular weight peptides, of which the 0-1kDa range accounts for 58.63%, 57.96% and 56.49% respectively, showing a significant hydrolysis effect. This distribution feature confirms that compared with bromelain and papain, these three proteases can more effectively hydrolyze large molecular proteins into small molecular peptides. Studies have found that low molecular weight peptides (<1kDa) generally exhibit superior biological activity, which is mainly attributed to their easier absorption by the human body. However, it should be emphasized that the biological activity of peptides does not simply depend on the molecular weight, but is also closely related to their characteristic amino acid composition.

[0082] Table 1

[0083]

[0084] Figure 3 The infrared spectrum scanning results show that the seabuckthorn seed meal protein and its hydrolyzate have a wavelength of 3200 cm -1 to 3500cm -1 The absorption bands at 2800 cm-1 correspond to the stretching vibrations of OH and NH bonds. -1 Up to 3000cm -1 The absorption bands in the range reflect the stretching vibration of the CH bond. It is particularly noteworthy that in the amide I band (1600 cm -1 to 1700cm -1 ) area, all protein hydrolysis products appeared 3cm -1 -4cm -1 This change indicates that the enzymatic hydrolysis of protease significantly changed the secondary structure of seabuckthorn seed meal protein.

[0085] Figure 4 The intrinsic fluorescence spectra of seabuckthorn seed meal protein and its hydrolyzate are shown in the figure. As can be seen from the figure, compared with SSP (Comparative Example 5), the maximum intrinsic fluorescence peaks of SSPHs (Example 1 and Comparative Examples 1-4) all show a red shift, from 336nm to 340nm~346nm. This red shift phenomenon may be due to the change in protein structure caused by enzymatic hydrolysis, which exposes aromatic amino acid residues from the inside of the protein, thereby changing the polar environment of the tryptophan residues. Further analysis found that the fluorescence intensity of SSPHs is generally higher than that of SSP, indicating that enzymatic hydrolysis causes the protein structure to unfold and more tryptophan residues to be exposed.

[0086] The inhibitory activities of Example 1 and Comparative Examples 1-4 on α-Glu, α-Amy and DPP-Ⅳ are as follows Figure 5The results showed significant differences in the inhibitory activity of the three enzymes among the different samples. Specifically, SSPHD exhibited the strongest inhibitory effect on α-Glu, with an inhibition rate of 53.60±0.98%. For α-Amy, SSPHC exhibited the greatest inhibitory effect (21.22±0.97%), followed closely by SSPHD (21.01±0.38%). For DPP-IV, SSPHA exhibited the greatest inhibitory effect (74.44±0.44%), followed by SSPHD (70.98±2.74%). Overall, SSPHD exhibited significant inhibitory effects on α-Glu, α-Amy, and DPP-IV, suggesting that neutral proteases may be a high-quality enzyme source for the development of hypoglycemic peptides from seabuckthorn seed meal, worthy of further optimization of the enzymatic hydrolysis process and in-depth study. Further analysis revealed that the hydrolysis degree of SSPHD was significantly higher than that of the other groups (P<0.05), which may account for its outstanding hypoglycemic activity. From the perspective of enzymatic hydrolysis mechanism, a higher degree of hydrolysis can promote the formation of polypeptide sequence diversity, and then enhance the inhibitory effect on related enzymes through multi-site synergistic effects.

[0087] The inhibitory activities of Example 1 and Comparative Examples 1-4 on PL and CEase are as follows Figure 6 As shown. The results indicate that bromelain may be a high-quality enzyme source for the development of lipid-lowering peptides from seabuckthorn seed meal. Among them, SSPHB exhibited an inhibition rate of 34.75±4.10% against PL, second only to SSPHD (54.55±4.41%) and SSPHC (53.77±3.55%). Furthermore, SSPHB exhibited an inhibition rate of 24.27±0.90% against CEase, second only to SSPHP (39.19±2.41%), making it the only SSPHs with significant inhibitory effects on both PL and CEase. This finding suggests that the biological activity of peptides is not solely determined by the degree of hydrolysis but is also closely related to the protease cleavage site, peptide sequence, and structure.

[0088] The inhibitory activity of SSPHs on ACE is as follows Figure 7 Compared with other hydrolysates, SSPHP showed a significant inhibitory effect on ACE, with an inhibition rate of 46.06±2.78%.

[0089] The inhibitory activity of SSPHs on XO is as follows Figure 8 Compared with other hydrolysates, SSPHA exhibited a significant inhibitory effect on XO, with an inhibition rate of 81.01±1.37%. Therefore, alkaline protease may be a high-quality enzyme source for the development of antihypertensive peptides derived from seabuckthorn seed meal.

[0090] Example 2

[0091] The difference from Example 1 is that the SSP solution in step (3) is 3%, w / v, and the rest is the same as Example 1.

[0092] Example 3

[0093] The difference from Example 1 is that the SSP solution in step (3) is 5%, w / v, and the rest is the same as Example 1.

[0094] Example 4

[0095] The difference from Example 1 is that the amount of enzyme added in step (3) is 10,000 U, and the rest is the same as Example 1.

[0096] Example 5

[0097] The difference from Example 1 is that the amount of enzyme added in step (3) is 12000U, and the rest is the same as Example 1.

[0098] Example 6

[0099] The difference from Example 1 is that the amount of enzyme added in step (3) is 14000U, and the rest is the same as Example 1.

[0100] Example 7

[0101] The difference from Example 1 is that the enzymatic hydrolysis time in step (3) is 120 min, and the rest is the same as Example 1.

[0102] Example 8

[0103] The difference from Example 1 is that the enzymatic hydrolysis time in step (3) is 150 min, and the rest is the same as Example 1.

[0104] Example 9

[0105] The difference from Example 1 is that the enzymatic hydrolysis time in step (3) is 180 min, and the rest is the same as Example 1.

[0106] Effect of substrate concentration on the preparation of SSPHD hypoglycemic peptides Figure 9 (a) Under the conditions of 8,400 U of enzyme and 180 min of enzymatic digestion, substrate concentrations ranging from 1% to 5% were tested. The results showed that increasing the substrate concentration from 1% to 4% significantly increased the inhibition of SSPHD against α-Glu and DPP-IV, reaching 53.60±0.98% and 70.98±2.74%, respectively. However, when the concentration was further increased to 5%, the inhibition rate showed a downward trend. Therefore, 4% was the optimal substrate concentration determined experimentally.

[0107] Effect of enzymatic hydrolysis time on the preparation of SSPHD hypoglycemic peptides Figure 9(b) The inhibition rate was measured at 30-min intervals from 60 min to 180 min. The results showed that as the hydrolysis time increased from 60 min to 150 min, the inhibition rate of SSPHD against α-Glu and DPP-IV significantly increased, reaching 56.13 ± 2.32% and 74.67 ± 2.40%, respectively. However, when the hydrolysis time exceeded 150 min, the inhibition rate decreased slightly, which may be due to further degradation or denaturation of the active peptide. Therefore, 150 min was the optimal enzymatic hydrolysis time determined experimentally.

[0108] Effect of enzyme addition on the preparation of SSPHD hypoglycemic peptide Figure 9 (c) Experiments were conducted within a range of 6,000 U to 14,000 U. When the enzyme dosage increased from 6,000 U to 12,000 U, SSPHD significantly increased its inhibition of α-Glu and DPP-IV, reaching 58.17 ± 1.93% and 83.15 ± 0.70%, respectively. This improvement was primarily attributed to the fact that the increased enzyme dosage accelerated the enzymatic reaction, resulting in more complete and rapid protein hydrolysis. However, when the enzyme dosage exceeded 12,000 U, the inhibition rate gradually stabilized and slightly decreased. This may be due to excessive enzyme dosage, resulting in excessive hydrolysis of SSP to free amino acids. Therefore, 12,000 U was the optimal enzyme dosage determined experimentally.

[0109] In vitro simulation of gastrointestinal digestion:

[0110] An appropriate amount of SSPHD was dissolved in pepsin hydrochloric acid solution (2000 U / mL, pH 3.0) and incubated at 37°C in a thermostatted shaker for 2 hours to simulate gastric digestion. Subsequently, the pH of the reaction system was adjusted to 7.0, and a portion of the sample was removed, inactivated, and cooled. Trypsin (100 U / mL) was added to the remaining sample to simulate intestinal digestion. The reaction was continued for 2 hours, with samples removed every 30 minutes, inactivated, and cooled. The lyophilized samples were assayed for α-Glu inhibitory activity and DPP-IV inhibitory activity. A hydrolyzate sample that had not undergone simulated in vitro digestion served as a control. Figure 10The results show the inhibitory activity of SSPHD against α-Glu and DPP-IV after simulated in vitro gastrointestinal digestion. After simulated in vitro gastric digestion, the inhibitory rate of SSPHD against α-Glu decreased from 57.44±0.63% to 44.28±0.87%, with an activity retention of 77.09%. The inhibitory rate of SSPHD against DPP-IV decreased from 48.67±1.87% to 40.12±1.31%, with an activity retention of 82.43%. Further, after simulated in vitro intestinal digestion, the inhibitory rate of SSPHD against α-Glu decreased from 44.28±0.87% to 25.93±0.74%, with an activity retention of 67.59%. The inhibitory rate of SSPHD against DPP-IV decreased from 40.12±1.31% to 23.73±1.57%, with an activity retention of 71.61%. These simulation results indicate that gastrointestinal digestion reduces SSPHD's inhibitory activity against α-Glu and DPP-IV, with the effect being more pronounced. After in vitro gastrointestinal digestion simulation, SSPHD retained 45.14% and 48.76% of its inhibitory activity against α-Glu and DPP-IV, respectively.

[0111] Animal husbandry and intervention:

[0112] The animal experiment protocol was approved by the Jiangnan University Laboratory Animal Care and Animal Welfare Ethics Committee (Ethics Number: JN.No.20240930t0481202). Experimental mice (8 wt / wt and 40 db / db, for a total of 48) were housed in SPF-protected enclosures at a temperature of 20°C–24°C, a humidity of 50%–60%, and a 12 h:12 h lighting cycle.

[0113] After a one-week acclimation period, mice were randomly assigned to five groups of eight (wt / wt) mice, each designated a blank control group, and db / db mice were randomly assigned to five groups for a five-week gavage intervention. The detailed schedule is shown in Tables 2-5. During the intervention period, mice had free access to food and water, with water and food added as appropriate, and bedding changed. Body weight, food intake, and water consumption were recorded regularly. After a 12-hour fast (without water withdrawal) at a fixed time each week, tail vein blood was collected for fasting blood glucose (FBG) measurement.

[0114] Table 2

[0115]

[0116] After 5 weeks of intervention, all mice were fasted for 12 hours without food or water deprivation, and at least 200 μL of blood was collected from their eyeballs. Subsequently, the mice were sacrificed by cervical dislocation. The liver, pancreas, kidneys, and spleen were promptly harvested and accurately weighed to calculate organ indices. Part of the liver tissue was fixed with 4% paraformaldehyde for subsequent liver histomorphological analysis; the remaining tissue was snap-frozen in liquid nitrogen and stored at -80°C until further use.

[0117] After 5 weeks of intervention, an oral glucose tolerance test (OGTT) was performed on the mice. After fasting for 12 hours but not water, the mice were gavaged with 1.0 g / kg·bw glucose. Blood glucose levels were measured at 0, 30, 60, 90, 120, and 180 minutes. OGTT curves were drawn and the area under the curves was calculated.

[0118] like Figure 11 As shown, compared with the NC group, the MC group exhibited a significant obesity phenotype, with an abnormally increased rate of weight gain. Met and SSPHD intervention differentially inhibited the weight gain of db / db mice. A clear dose-dependent trend was observed in weight regulation, with increasing doses increasing the inhibitory effect on weight gain. These results suggest that SSPHD can effectively downregulate the weight gain rate of db / db mice.

[0119] like Figure 12 As shown in Figures 12a and 12b, the average daily food and water intake of mice in the MC group was significantly increased compared with the NC group. Among the intervention groups, the SSPHD-L group had no significant effect on the mice's food intake; however, the food intake of mice in the Met, SSPHD-M, and SSPHD-H groups decreased. This result suggests that SSPHD can alleviate hyperglycemia-induced hyperphagia (excessive eating) in db / db mice. In contrast, water intake in db / db mice increased after SSPHD intervention.

[0120] like Figure 13 As shown in the results, compared with the NC group, the characteristic organ mass index of the mice in the MC group was abnormal: the liver index was significantly increased (P<0.0001), which may be related to liver glycogen deposition and fatty degeneration

[124] ; while the kidney index, pancreas index and spleen index were significantly decreased (P<0.0001). These changes are closely related to obesity and organ atrophy in db / db mice. In the intervention group, the kidney index of the Met group was not statistically different from that of the MC group; while the kidney index of the low, medium and high dose SSPHD groups was significantly increased (P<0.001), and the growth difference between the groups was small, with an average growth level of 29.62±3.19%. In addition, compared with the MC group, the liver index, pancreas index and spleen index of db / db mice in the SSPHD dose groups and the Met group did not show significant changes. The above results indicate that SSPHD has a protective effect on the kidneys of db / db mice and can effectively alleviate kidney atrophy, but has no significant effect on the liver, pancreas and spleen.

[0121] like Figure 14As shown, FBG levels in the NC group remained stable over the 6-week experimental period (6.17±1.26mmol / L), whereas those in the MC group significantly increased at the beginning of the experiment (18.85±4.84mmol / L, approximately three times that of the NC group) and further climbed to 25.93±4.44mmol / L at the end of the experiment. Intervention results revealed that the SSPHD-L and SSPHD-M groups had no significant regulatory effect on FBG levels during the experimental period. However, the rate of FBG increase in the Met and SSPHD-H groups slowed significantly after the initiation of intervention, with a downward trend beginning in the third and fourth weeks, respectively. By the fifth week, FBG levels in the Met and SSPHD-H groups were 35.31% and 33.22% lower than those in the MC group, respectively. These results demonstrate that SSPHD can effectively regulate hyperglycemia in db / db mice.

[0122] like Figure 15 As shown in Figure a, mice in the NC group exhibited a typical physiological response to glucose metabolism after receiving a glucose load of 1.0 g / kg / bw: blood glucose concentrations rose rapidly within a short period of time, peaked at 30 minutes, and then began to decline, returning to fasting baseline levels by around 120 minutes. In contrast, after peaking, db / db mice experienced a slow rate of glucose clearance, maintaining pathologically high blood glucose levels at 120 minutes. Therefore, the experiment also measured blood glucose levels at 180 minutes. The results showed that blood glucose levels in db / db mice still struggled to return to normal, indicating severely impaired glucose tolerance.

[0123] In the intervention study, the SSPHD-L and SSPHD-M groups showed no significant differences in their OGTT curves compared with the MC group, indicating no improvement in glucose tolerance. However, the Met and SSPHD-H groups showed some therapeutic effects, with a significant increase in the rate of post-peak blood glucose clearance. By 180 minutes, blood glucose concentrations had recovered to 85.35% and 74.64% of their peak values, respectively.

[0124] like Figure 15 As shown in Figure b, the AUC calculation results were highly consistent with the OGTT curves. Compared with the MC group, the AUC values ​​of the SSPHD-L and SSPHD-M groups showed no significant differences. However, the AUC values ​​of the Met and SSPHD-H groups were significantly reduced (P < 0.01), by 15.15% and 13.47%, respectively. Overall, SSPHD effectively improved glycemic control in db / db mice.

[0125] like Figure 16As shown, compared with the NC group, the GSP concentration in the MC group was significantly increased (P < 0.0001), indicating severe glucose metabolism disorders and persistent hyperglycemia in the mice. Among the intervention groups, GSP levels in the SSPHD-L and SSPHD-M groups did not significantly improve, while GSP levels in the Met and SSPH-H groups were significantly decreased (P < 0.05), by 6.88% and 6.51%, respectively, compared with the MC group. These results indicate that SSPHD can reduce GSP levels in db / db mice, effectively regulating blood sugar and improving glucose metabolism disorders.

[0126] like Figure 17 As shown, compared with the NC group, the MC group showed abnormally elevated TC and LDL-C levels (P < 0.001), while HDL-C levels decreased (P < 0.05), suggesting lipid metabolism disorders in db / db mice. Met and SSPHD intervention significantly improved TC, LDL-C, and HDL-C levels in the mice (P < 0.05). All SSPHD dose groups showed comparable effects in improving TC and LDL-C levels. Compared with the MC group, TC and LDL-C concentrations decreased by an average of 42.89 ± 2.66% and 40.78 ± 2.59% in all SSPHD dose groups. The increase in HDL-C concentration was dose-dependent. Compared with the MC group, the SSPHD-H group increased HDL-C concentration by 53.30%, a superior effect compared to the Met group (30.95%). These results indicate that SSPHD can effectively improve lipid metabolism disorders in db / db mice by reducing TC and LDL-C levels and increasing HDL-C levels.

[0127] like Figure 18 As shown in the results, compared with the NC group, the expression levels of TNF-α, IL-1β, and IL-6 in the liver of mice in the MC group were significantly increased (P < 0.0001), indicating a significant inflammatory response in the mice. After intervention with Met and SSPHD, the expression levels of these inflammatory factors were significantly reduced (P < 0.05), showing a dose-dependent inhibitory trend. Compared with the MC group, the concentrations of TNF-α, IL-1β, and IL-6 in the liver of mice in the SSPHD-H group were reduced by 48.78%, 43.38%, and 47.83%, respectively (P < 0.01). These results indicate that SSPHD can effectively reduce the levels of inflammatory factors in the liver of db / db mice, alleviate the inflammatory response in vivo, and thus alleviate the insulin resistance of T2DM.

[0128] like Figure 19As shown, compared with the NC group, the serum AST and ALT activities of mice in the MC group were significantly elevated (P < 0.0001), indicating severe liver damage. Following Met and SSPHD intervention, both ALT and AST activities in the mice's serum showed a decreasing trend. The reduction in ALT activity was dose-dependent, with the SSPHD-H group reducing ALT activity by 40.84%. However, changes in AST activity showed varying patterns, with only the SSPHD-L group showing a significant effect (a decrease of 33.68%, P < 0.01). Although the SSPHD-M and SSPHD-H groups showed some decreases compared with the MC group, the differences were not statistically significant. These results suggest that SSPHD can effectively improve liver function in db / db mice by regulating transaminase (ALT and AST) metabolism, thereby exerting a protective effect on the liver.

[0129] like Figure 20 As shown, compared with the NC group, the MC group showed significantly decreased CAT and SOD activities in the liver, while MDA content was significantly increased (P < 0.01), indicating severe oxidative stress in the mice liver. Furthermore, GSH-Px activity also decreased, but the difference was not significant. After SSPHD treatment, both CAT and SOD activities in the liver of mice were enhanced, while MDA content was decreased. The SSPHD-L group showed a significant effect on CAT activity (P < 0.05), while the SSPHD-M and SSPHD-H groups showed some improvement compared with the MC group, but the differences were not statistically significant. The increase in SOD activity and the decrease in MDA content showed a dose-dependent inhibitory trend. Compared with the MC group, the SSPHD-H group showed a significant difference in SOD activity (P < 0.01), while no significant difference was observed among the SSPHD dose groups on MDA content. Overall, SSPHD may ameliorate oxidative stress in the liver of db / db mice by upregulating CAT and SOD activities, effectively enhancing the liver's antioxidant capacity.

[0130] like Figure 21As shown, compared with the NC group, serum UREA and CREA concentrations in the MC group were significantly elevated (P < 0.001), suggesting severe impairment of renal clearance. Following Met and SSPHD intervention, serum UREA concentrations improved in a dose-dependent manner. The SSPHD-H group significantly downregulated UREA, with a 20.93% decrease. While the SSPHD-L and SSPHD-M groups showed a downward trend, it was not statistically significant. Furthermore, all SSPHD doses demonstrated a significant effect in downregulating CREA concentrations, with no significant differences between groups. The average decrease reached 55.52 ± 1.20%. These results suggest that SSPHD can effectively improve renal function in db / db mice by downregulating UREA and CREA metabolism, thereby exerting a protective effect on the kidneys.

[0131] like Figure 22 As shown, hepatocytes in the NC group were arranged in an orderly radial pattern, with uniform cytoplasmic staining and no lipid droplets or vacuoles. In the MC group, typical pathological features of steatohepatitis were observed, including disorganized hepatic cord structure, disrupted hepatocyte membrane integrity, and numerous lipid vacuoles surrounding the liver. Treatment with Met and SSPHD significantly improved overall liver damage and fatty lesions. The improvement with SSPHD was dose-dependent. The improvement was particularly significant in the Met and SSPHD-H groups, with liver tissue architecture essentially returning to normal. These results demonstrate that SSPHD effectively alleviates liver damage and fatty lesions in db / db mice, exerting a protective effect on the liver.

[0132] like Figure 23 As shown, compared with the NC group, the activities of G6Pase and PEPCK in the liver of mice in the MC group were significantly increased (P < 0.001), indicating abnormal gluconeogenesis. After intervention with Met and SSPHD, both G6Pase and PEPCK activities in the liver of mice showed a downward trend, with a dose-dependent effect on SSPHD. Of note, PERCK activity decreased in the low- and medium-dose SSPHD groups, as well as G6Pase activity across all dose groups, but the differences were not statistically significant. Furthermore, PEPCK activity in the SPPHD-H group decreased significantly by 26.80% (P < 0.001), comparable to the effect of Met intervention (a 26.59% decrease). These results suggest that SSPHD can alleviate abnormal hepatic gluconeogenesis, reduce hepatic glucose output, and alleviate hyperglycemia symptoms in db / db mice by inhibiting the activity of PEPCK, a key gluconeogenic enzyme.

[0133] like Figure 24As shown, in normal liver tissue (NC group), PAS staining clearly revealed evenly distributed purple glycogen granules. However, in the diabetic model (MC group), PAS staining revealed a lighter color of glycogen granules and the presence of numerous fat vacuoles in the liver tissue, indicating a significant decrease in glycogen content. Intervention with Met and SSPHD improved liver glycogen content, and this improvement was dose-dependent. These results demonstrate that SSPHD can effectively improve abnormal liver glycogen content in db / db mice.

[0134] like Figure 25 As shown, compared with the NC group, GK activity in mice in the MC group was significantly decreased (P < 0.01), suggesting that hepatic glycogen synthesis was inhibited. After intervention with Met and SSPHD, GK activity increased significantly, and this was dose-dependent for SSPHD. The SSPHD-H group showed a significant upregulation of GK activity, while GK activity in the medium and low-dose groups increased, but without statistically significant differences. Compared with the MC group, GK activity in the SSPHD-H and Met groups increased by 25.35% and 34.54%, respectively. These results suggest that SSPHD promotes glycogen synthesis and improves glucose metabolism disorders by enhancing the activity of GK, a key enzyme in glycogen synthesis.

[0135] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, and all of these should be included in the scope of the present invention.

Claims

1. A method for preparing a hypoglycemic peptide based on seabuckthorn seed meal protein, characterized by: include, After mixing sea buckthorn seed meal powder with water, adjusting the pH value with sodium hydroxide, stirring, and centrifuging to obtain a supernatant; adjusting the pH value of the supernatant with hydrochloric acid, letting it stand and then centrifuging; the obtained precipitate is washed with distilled water until neutral, pre-lyophilized, and freeze-dried to obtain SSP powder; The SSP solution obtained by mixing SSP powder with water is preheated in a water bath, protease is added, and after enzymatic hydrolysis, the solution is heated in a water bath, and the pH of the solution is adjusted to neutral. The supernatant is collected by centrifugation and dialyzed at low temperature to obtain the sea buckthorn seed meal protein-based hypoglycemic peptide; The protease includes one or more of alkaline protease, composite protease, neutral protease, bromelain or papain.

2. The preparation method according to claim 1, wherein: The concentration of the SSP solution is 3-6%.

3. The preparation method according to claim 1, wherein: The added amount of the protease is 10,000 to 14,000 U.

4. The preparation method according to claim 1, wherein: The enzymatic hydrolysis time is 120 to 180 minutes.

5. The preparation method according to claim 2, wherein: The concentration of the SSP solution is 4%.

6. The preparation method according to claim 5, wherein: The added amount of protease was 12,000 U.

7. The preparation method according to claim 6, wherein: The enzymatic hydrolysis time is 150 min.

8. The blood sugar lowering peptide based on seabuckthorn seed meal protein obtained by the preparation method according to claims 1 to 7.

9. The blood sugar-lowering peptide based on seabuckthorn seed meal protein according to claim 8, characterized in that: IC for α-Glu and DPP-IV 50 Reaching 3.45±0.18mg / mL and 5.01±0.21mg / mL respectively.

10. The blood sugar-lowering peptide based on seabuckthorn seed meal protein according to claim 9, characterized in that: After simulated gastrointestinal digestion, the inhibitory activity against α-Glu was 45.14%; the inhibitory activity against DPP-IV was 48.76%.

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