Hippophae rhamnoides protein peptide with blood sugar lowering activity and application thereof

Abstract

This invention relates to a seabuckthorn protein peptide with hypoglycemic activity and its applications, belonging to the field of polypeptide technology. This invention provides a seabuckthorn protein peptide, the amino acid sequence of which is selected from SEQ ID NO.1 and / or SEQ ID NO.2. Animal experiments have shown that the seabuckthorn protein peptide has the effect of lowering blood glucose levels and improving insulin resistance, and can be used in the preparation of products that improve blood glucose levels. This invention also discovered two seabuckthorn protein peptides with hypoglycemic effects through enzymatic hydrolysis of seabuckthorn seed protein. Experiments have confirmed that the seabuckthorn protein peptide can reverse insulin resistance in HepG2 cells, reduce blood glucose levels in a zebrafish hyperglycemia model, and improve insulin resistance in a diabetic mouse model.

Description

Technical Field

[0001] This invention relates to the field of polypeptide technology, and in particular to a sea buckthorn protein peptide with hypoglycemic activity and its applications. Background Technology

[0002] Diabetes mellitus is a chronic metabolic disease characterized by insulin secretion defects and / or impaired function (insulin resistance), leading to disorders in carbohydrate, fat, and protein metabolism. In recent years, influenced by multiple factors including genetics, environment, dietary structure, and lifestyle, its global incidence has shown a rapid increase, developing into a major public health problem that seriously threatens human health. Latest data shows that the number of adults with diabetes worldwide has surged from 200 million in 1990 to 828 million in 2022, with approximately 148 million adults in my country, accounting for nearly one-fifth of the global total, resulting in an extremely heavy disease burden. Sustained hyperglycemia can trigger various life-threatening complications, such as retinopathy, nephropathy, neuropathy, and cardiovascular and cerebrovascular diseases, which are major causes of disability and death in patients. Therefore, exploring safe and effective strategies for the prevention and treatment of diabetes is of paramount importance.

[0003] Currently, the clinical management of diabetes mainly relies on exogenous insulin injections and oral hypoglycemic agents (such as metformin and sulfonylureas). While these traditional methods can effectively control blood sugar, long-term use may be accompanied by a series of problems, including the risk of hypoglycemia, weight gain, gastrointestinal adverse reactions, and potential liver and kidney dysfunction, often challenging patient adherence. Furthermore, lifestyle interventions (such as diet control and exercise) as foundational therapies depend on long-term patient self-discipline, which is difficult to sustain in practice. Based on these limitations, the development of novel functional components derived from natural products has become a current research hotspot. Among them, bioactive peptides, due to their good digestibility and absorption properties, diverse physiological regulatory functions (such as mimicking insulin action, improving insulin resistance, and protecting pancreatic β-cells), and high safety, show broad application prospects, providing new directions for nutritional intervention and adjuvant therapy for diabetes.

[0004] Therefore, there is an urgent need to develop a bioactive peptide that can improve blood sugar levels and has a high safety profile. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a sea buckthorn protein peptide and its application.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a sea buckthorn protein peptide with hypoglycemic activity, wherein the amino acid sequence of the sea buckthorn protein peptide is selected from SEQ ID NO.1 and / or SEQ ID NO.2.

[0007] This invention has shown through animal experiments that sea buckthorn protein peptides have the effect of lowering blood sugar levels and improving insulin resistance, and can be used in the preparation of blood sugar improvement products.

[0008] As a preferred embodiment of the seabuckthorn protein peptide of the present invention, the seabuckthorn protein peptide is prepared by solid-phase synthesis.

[0009] As a preferred embodiment of the seabuckthorn protein peptide of the present invention, the seabuckthorn protein peptide is obtained by enzymatic hydrolysis and purification of seabuckthorn seed protein.

[0010] In a preferred embodiment of the seabuckthorn protein peptide of the present invention, the seabuckthorn protein peptide is prepared by the following operation: S1. Using sea buckthorn seed powder as raw material, crude protein from sea buckthorn seeds is extracted by treatment with alkaline solution and cellulase. S2. The crude protein from sea buckthorn seeds obtained in step S1 is mixed with trypsin to form a reaction system for enzymatic hydrolysis. After the enzymatic hydrolysis is completed, the reaction system is subjected to enzyme inactivation treatment, concentrated, and dried to obtain sea buckthorn protein hydrolysate. S3. The seabuckthorn protein hydrolysate obtained in step S2 is purified by ultrafiltration to obtain the ultrafiltration fraction. S4. The ultrafiltration fraction obtained in step S3 is purified by gel filtration chromatography to obtain sea buckthorn protein peptides.

[0011] In a preferred embodiment of the sea buckthorn protein peptides of the present invention, the specific operation of extracting crude sea buckthorn protein by alkaline solution and cellulase treatment in step S1 is as follows: Disperse sea buckthorn seed powder in water at a weight ratio of 1:10, adjust the pH to 11, and extract at 43-47℃ for 4 hours, controlling the pH to 10.5-11 during the extraction. After extraction, let stand for 30 minutes, centrifuge, and collect the supernatant. Adjust the pH of the obtained supernatant to 5.0-5.5, add cellulase, mix, adjust the pH to 4.0, let stand for 2 hours, remove the supernatant, and obtain the precipitate; The precipitate was dispersed in water at a weight ratio of precipitate:water = 1:3, kept at 85℃ for 15 min, cooled to 50℃, and then the pH was adjusted to 9.0 and kept for 30 min to obtain crude protein from sea buckthorn seeds.

[0012] In a preferred embodiment of the seabuckthorn protein peptide of the present invention, the cellulase accounts for 0.5 wt% of the total weight of the supernatant.

[0013] As a preferred embodiment of the seabuckthorn protein peptide of the present invention, in step S2, the mass ratio of seabuckthorn seed crude protein to pancreatic enzyme is seabuckthorn seed crude protein: pancreatic enzyme = (48-50):1.

[0014] In a preferred embodiment of the seabuckthorn protein peptide of the present invention, in step S2, the mass ratio of seabuckthorn seed crude protein to trypsin is seabuckthorn seed crude protein: trypsin = 49:1.

[0015] As a preferred embodiment of the seabuckthorn protein peptide of the present invention, in step S2, the enzymatic hydrolysis reaction is carried out at pH=9 and 50°C for 4 hours.

[0016] As a preferred embodiment of the seabuckthorn protein peptide of the present invention, in step S2, the enzyme inactivation treatment is to heat the reaction system at 85°C for 30 min.

[0017] As a preferred embodiment of the sea buckthorn protein peptide of the present invention, in step S3, the ultrafiltration membrane used for ultrafiltration purification has a molecular weight cutoff of 1-10 kDa.

[0018] As a preferred embodiment of the sea buckthorn protein peptide of the present invention, in step S3, the molecular weight cutoff of the ultrafiltration membrane used for ultrafiltration purification includes 1kDa, 3kDa, 5kDa and 10kDa.

[0019] As a preferred embodiment of the sea buckthorn protein peptide of the present invention, in step S3, the molecular weight of the ultrafiltration fraction includes at least one of >10kDa, 5-10kDa, 3-5kDa, 1-3kDa and <1kDa.

[0020] As a preferred embodiment of the sea buckthorn protein peptide of the present invention, in step S4, the specific operation of the gel filtration chromatography is as follows: purification is performed using a Sephadex G-25 gel filtration column, elution is performed using ultrapure water as the mobile phase, the eluent is monitored by the BCA method, the eluents corresponding to the main protein peaks are combined and concentrated, sterilized and dried to obtain the sea buckthorn protein peptide.

[0021] As a preferred embodiment of the seabuckthorn protein peptide of the present invention, the concentration includes, but is not limited to, concentration using at least one of nanofiltration, rotary evaporation, and ultrafiltration.

[0022] As a preferred embodiment of the sea buckthorn protein peptide of the present invention, the sterilization operation includes, but is not limited to, at least one of the following: filtration sterilization using a filter membrane with a pore size of 0.22 μm, instantaneous sterilization at 115-135°C, irradiation sterilization, and ethylene oxide sterilization.

[0023] As a preferred embodiment of the seabuckthorn protein peptide of the present invention, the drying includes, but is not limited to, at least one of freeze drying, spray drying, and vacuum drying.

[0024] Secondly, the present invention provides the application of the above-mentioned sea buckthorn protein peptide in the preparation of blood sugar-improving products.

[0025] As a preferred embodiment of the application described in this invention, the blood sugar improving product includes at least one of food, pharmaceuticals, and cosmetics.

[0026] Thirdly, the present invention provides the application of the above-mentioned sea buckthorn protein peptide in the preparation of health food that helps maintain healthy blood sugar levels.

[0027] Fourthly, the present invention provides a product comprising the above-mentioned sea buckthorn protein peptide.

[0028] In a preferred embodiment of the product described in this invention, the product further includes auxiliary materials.

[0029] In a preferred embodiment of the product described in this invention, the excipients include, but are not limited to, at least one of flavor enhancers, emulsifiers, thickeners, and preservatives.

[0030] As a preferred embodiment of the product described in this invention, the flavor enhancer includes, but is not limited to, at least one of yeast extract, citric acid, malic acid, lactic acid, fructose syrup, and erythritol.

[0031] In a preferred embodiment of the product described in this invention, the emulsifier includes, but is not limited to, at least one of glyceryl monostearate, sucrose fatty acid ester, etc.

[0032] As a preferred embodiment of the product described in this invention, the thickener includes, but is not limited to, at least one of carrageenan, sodium carboxymethyl cellulose, soybean lecithin, Span 60, Tween 80, and polyglycerol fatty acid esters.

[0033] As a preferred embodiment of the product described in this invention, the preservative includes, but is not limited to, at least one of sodium benzoate, sodium D-isoascorbate, potassium sorbate, and sodium dehydroacetate.

[0034] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention discovers two seabuckthorn protein peptides with blood sugar-improving effects through enzymatic hydrolysis of seabuckthorn seed protein. Experiments have confirmed that seabuckthorn protein peptides can reverse insulin resistance in HepG2 cells, reduce blood sugar levels in a zebrafish hyperglycemia model, and improve dyslipidemia, liver biochemical imbalance, and insulin resistance in a diabetic mouse model. Attached Figure Description

[0035] Figure 1 The α-amylase standard curve and the α-amylase inhibitory activity of the ultrafiltration fraction in Experimental Example 1 of this invention; Figure 2The gel permeation chromatograms (BCA peaks) of the UF4 and UF5 fractions in Experimental Example 1 of this invention are shown. Figure 3 The results of the HepG2 cytotoxicity test of different sea buckthorn protein peptides in Experiment Example 1 of this invention; Figure 4 The results of glucose uptake tests on HepG2 cells with different sea buckthorn protein peptides in Experiment Example 1 of this invention; Figure 5 This illustrates the effect of different sea buckthorn protein peptides at a concentration of 200 μM on blood glucose levels in a zebrafish hyperglycemia model, as described in Experimental Example 1 of this invention. Figure 6 This refers to the effect of different sea buckthorn protein peptides at a concentration of 200 μM on the exercise capacity of a zebrafish hyperglycemia model in Experiment Example 1 of this invention. Figure 7 This invention illustrates the effects of different doses and different sea buckthorn protein peptides on glucose tolerance and blood biochemical indicators in diabetic mice in Example 1, where A represents the oral glucose tolerance test (OGTT) and B represents the area under the blood glucose curve during the OGTT. Figure 8 Example 1 of the present invention illustrates the effects of different doses and different sea buckthorn protein peptides on serum circulating hormones and enzyme indicators in diabetic mice, where A is serum α-amylase, B is serum α-glucosidase, C is insulin, D is glucagon, E is DPP-4, and F is GLP-1. In the above diagram, the two groups are marked with " " " " "Indicates a significant difference between the two groups" p<0.05 p<0.01 p<0.001 p < 0.0001, with "ns" indicating no significant difference between the two groups. Detailed Implementation

[0036] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0037] In this article, the amino acid sequence “A” refers to alanine (Ala), “C” refers to cysteine ​​(Cys), “D” refers to aspartic acid (Asp), “E” refers to glutamic acid (Glu), “F” refers to phenylalanine (Phe), “G” refers to glycine (Gly), “H” refers to histidine (His), “I” refers to isoleucine (Ile), “K” refers to lysine (Lys), “L” refers to leucine (Leu), “M” refers to methionine (Met), “N” refers to asparagine (Asn), “P” refers to proline (Pro), “Q” refers to glutamine (Gln), “R” refers to arginine (Arg), “S” refers to serine (Ser), “T” refers to threonine (Thr), “V” refers to valine (Val), “W” refers to tryptophan (Trp), and “Y” refers to tyrosine (Tyr).

[0038] The terms “comprising,” “including,” “having,” “containing,” or any other variations thereof, as used herein, are intended to cover a non-exclusive inclusion. For example, a composition, step, method, article, or apparatus that includes the listed elements is not necessarily limited to those elements, but may include other elements not expressly listed or elements inherent to such composition, step, method, article, or apparatus.

[0039] When a quantity, concentration, or other value or parameter is expressed as a range, a preferred range, or a range defined by a series of upper and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pair of any upper or preferred value with any lower or preferred value, regardless of whether the range is disclosed individually. For example, when the range “1-5” is disclosed, the described range should be interpreted as including ranges “1-4”, “1-3”, “1-2”, “1-2 and 4-5”, “1-3 and 5”, etc. When numerical ranges are described herein, unless otherwise stated, the range is intended to include its endpoints and all integers and fractions within that range.

[0040] "Parts by mass" refers to the basic unit of measurement that expresses the mass ratio of multiple components. One part can represent any unit mass, such as 1g or 3.1456g. If we say that component A has "a" parts by mass and component B has "b" parts by mass, it means the ratio of the mass of component A to the mass of component B is a:b. Alternatively, it can mean that the mass of component A is aK and the mass of component B is bK (where K is any number representing a multiplier). It is important to understand that, unlike parts by mass, the sum of the mass parts of all components is not limited to 100 parts.

[0041] "And / or" is used to indicate that one or both of the described situations may occur, for example, A and / or B includes (A and B) and (A or B).

[0042] Unless otherwise specified, all other materials and reagents used in the experimental examples, implementation examples, comparative examples, and effect examples are commercially available.

[0043] The α-amylase inhibition rate described below was performed according to the instructions of the α-amylase inhibitor screening test kit, which was purchased from Shanghai Zhenke Biotechnology Co., Ltd., product number ZK-0510W.

[0044] The biuret method (BCA method) described below is a method for quantitative detection of proteins. It can be operated with reference to common knowledge such as "Biochemistry Experiments" (Science Press, 2003) and "Biochemistry Experiments" (Chemical Industry Press, 2009).

[0045] The glucose assay kit was purchased from Nanjing Jiancheng Biotechnology Research Institute Co., Ltd., catalog number A154-1-1.

[0046] The following nanofiltration techniques were performed with reference to the following literature: Guo Honghui, Lin Xiefeng, Hong Zhuan, Xie Quanling, Chen Sijin, Sun Jipeng. Optimization of multi-stage membrane separation and purification process for fish skin collagen oligopeptides. Food Industry, 2015, 36(2):174-177; The specific operation includes: using a multi-stage membrane separation device for nanofiltration, with nanofiltration membrane parameters of 200U molecular weight cutoff and 0.25m² membrane area. 2 Operating pressure: 1.4 MPa; Temperature: 30-40℃.

[0047] Experimental Example 1 The specific procedure for isolating, purifying, and identifying sea buckthorn protein peptides is as follows: 1. Isolation and purification of sea buckthorn protein peptides.

[0048] (1) After crushing the sea buckthorn seed meal, pass it through a 20-mesh sieve to obtain sea buckthorn seed powder. Disperse the sea buckthorn seed powder in water at a weight ratio of sea buckthorn seed powder: water = 1:10. Adjust the pH value to 11 with sodium hydroxide aqueous solution. Extract at 45±2℃ for 4 hours, during which the pH value is controlled at 10.5-11. After extraction, let stand for 30 minutes, centrifuge, and collect the supernatant. (2) Adjust the pH of the supernatant obtained in step (1) to 5.0-5.5, add cellulase, stir for 10 min, adjust the pH to 4.0 with hydrochloric acid, let stand for 2 h, remove the supernatant, and obtain the precipitate. The weight of cellulase accounts for 0.5 wt% of the total weight of the supernatant. (3) Disperse the precipitate in water at a weight ratio of precipitate:water = 1:3, keep warm at 85℃ for 15 min, cool to 50℃ and adjust the pH to 9.0 and keep for 30 min to obtain sea buckthorn seed crude protein; (4) The seabuckthorn crude protein obtained in step (3) was mixed with the trypsin in a mass ratio of 49:1. The reaction system was enzymatically hydrolyzed at pH 9 and 50℃ for 4 hours. After the enzymatic hydrolysis was completed, the reaction system was heated at 85℃ for 30 minutes to inactivate the enzyme and obtain the hydrolysate. The hydrolysate was concentrated using nanofiltration technology to obtain the concentrated hydrolysate. The concentrated hydrolysate was subjected to ultra-high temperature instantaneous sterilization at 115-135℃ and spray-dried to obtain the seabuckthorn protein hydrolysate. (5) Take the sea buckthorn seed protein hydrolysate obtained in step (4), dissolve it in ultrapure water, and use a tangential flow ultrafiltration system to sequentially use ultrafiltration membranes with molecular weight cutoffs of 10kDa, 5kDa, 3kDa and 1kDa to separate the fractions and obtain five ultrafiltration fractions with molecular weights >10kDa (UF1), 5-10kDa (UF2), 3-5kDa (UF3), 1-3kDa (UF4) and <1kDa (UF5), which are then freeze-dried for later use. (6) The α-amylase inhibitory activity of the ultrafiltration fraction obtained in step (5) was determined, with metformin as a positive control. The results are as follows: Figure 1 As shown, the UF4 and UF5 fractions exhibited the highest inhibitory activity. The UF4 and UF5 fractions were purified using a Sephadex G-25 gel filtration column with ultrapure water as the mobile phase. The eluent was monitored using the BCA method. The eluents corresponding to the main protein peak of the UF4 fraction were combined and lyophilized to obtain the subfractions GF1 and GF2. The gel chromatograms of the UF4 and UF5 fractions are shown below. Figure 2 ; The elution parameters for purification using a Sephadex G-25 gel filtration column were as follows: sample concentration 100 mg / mL, column size 1.5 cm × 120 cm, injection volume 100 μL, flow rate 0.9 mL / min, and eluent collection, with each 1 mL of eluent counted as one tube. A total of 330 tubes of eluent were collected for the UF4 fraction, and a total of 300 tubes of eluent were collected for the UF5 fraction.

[0049] 2. Identification of sea buckthorn protein peptides.

[0050] The UF5, GF1, and GF2 fractions obtained above were subjected to peptide sequence analysis using reversed-phase nanoliquid chromatography-tandem mass spectrometry (RP-MS / MS), yielding multiple peptides with different amino acid sequences, which were then numbered. The antidiabetic activity (including inhibition of DPP-4, α-glucosidase, and α-amylase) of the identified peptides was assessed using the BIOPEP-UWM database. Based on relative content and specific α-amylase inhibitory motifs, candidate peptides (numbered Pep4 and Pep75) were screened, with Pep4 having the amino acid sequence REGDIIAIPA (SEQ ID NO.1) and Pep75 having the amino acid sequence TSVLRALPDEV (SEQ ID NO.2). These candidate peptides were synthesized by Shanghai Xinpeptide Biotechnology Co., Ltd. using a solid-phase synthesis method.

[0051] 3. The hypoglycemic effect of the candidate peptides was evaluated using the HepG2 cell model. The specific method is as follows: (1) Cell viability assay: Cell viability was determined using the 3-(4,5-dimethylthiazolyl-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. Cells were cultured at 1 × 10⁶ cells per well. 4 Cells were seeded at a density of 100 μL of culture medium in each well of a 96-well plate and cultured for 12 h to allow adhesion. Subsequently, cells were treated with 100 μL of peptide solutions of different concentrations (3.125, 6.25, 12.5, 25, 50, 100, 200, and 400 μM) for 24 h. Control cells were cultured in peptide-free medium. The next day, 10 μL of 5 mg / mL MTT solution was added to each well and incubated for 4 h. After 4 h, the culture medium was discarded, and 100 μL of DMSO was added to each well to dissolve the generated formazan blue. Absorbance was measured at 570 nm (absorbance of untreated cells was 100%). Figure 3 As shown, after treating HepG2 cells with sea buckthorn peptides at a concentration as high as 100 μM for 24 h, the cell viability was not significantly reduced compared with the untreated group, indicating that at the selected concentration, these peptides had no cytotoxic effect on HepG2 cells.

[0052] (2) Inducing an IR model and treating with peptide drugs: Cells were spaced at 1.25 × 10⁶ cells per well. 6 Cells were seeded at a density of 3 mL per well in 24-well plates. The cells were cultured for 48 hours until confluence reached 80-90% (approximately 5 × 10⁶ cells / well). 6 (cells). After 48 hours, the culture medium was aspirated, and the cells were gently washed twice with PBS. The control group was cultured in DMEM medium without FBS for 24 hours. The insulin resistance (IR) model group was cultured in a medium containing 25 mmol / L glucose and 10⁻ 6After culturing in DMEM medium containing mol / L insulin for 24 h, DMEM medium containing 100 µM metformin or peptide sample was added, and culturing continued for another 24 h. The groups with added Pep4 or Pep75 were designated as experimental groups, while metformin (positive control group) and standard peptide LPLLR (standard peptide control group) were set up as controls, and an equal amount of DMEM was added as a blank control.

[0053] Intracellular glucose consumption was measured according to the glucose assay kit to evaluate the promoting effect of Pep4 and Pep75 on glucose uptake in insulin-resistant HepG2 cells. Specifically, the intracellular glucose content of the experimental group and the model group was compared, and the rate of glucose uptake promotion in different treatment groups was calculated to verify their hypoglycemic activity. The results are as follows: Figure 4 As shown, compared with the control group, the IR model group had reduced cellular glucose uptake, indicating that the IR model had been successfully established. The metformin group showed improved glucose uptake and higher glucose bioactivity compared with the model group. Furthermore, the Pep4 and Pep75 groups showed significantly increased glucose content, reversing insulin resistance in HepG2 cells.

[0054] 4. The hypoglycemic efficacy of the candidate peptides was evaluated using a zebrafish hyperglycemia model. The specific method is as follows: Wild-type AB strain zebrafish were used. A hyperglycemic model was established by exposing zebrafish embryos 1 day post-fertilization (1 dpf) to a 2.5 wt% glucose solution for 2 consecutive days.

[0055] After the model was built (3dpf), the zebrafish embryos were randomly grouped, including: Normal control group: healthy zebrafish that have not undergone any treatment; The control group, zebrafish with hyperglycemia, did not receive drug treatment; Positive control group: zebrafish with hyperglycemia, treated with metformin, a known antidiabetic drug; Standard peptide control group, hyperglycemic model zebrafish, treated with standard peptide LPLLR; The experimental group consisted of zebrafish, a hyperglycemic model, treated with a candidate peptide at a concentration of 200 μM.

[0056] Zebrafish embryos were treated with a 2.5 wt% glucose solution from 1 day old (dpf) to 3 days old (dpf) to establish a hyperglycemic model. Subsequently, diabetic zebrafish models were treated with sea buckthorn peptide dissolved in a 2.5% glucose solution from 3 days old (dpf) to 5 days old (dpf), and blood glucose levels in zebrafish homogenates were measured at 5 days old (dpf).

[0057] After treatment (5 days post-processing), zebrafish from each group were collected. The supernatant was obtained by grinding and centrifuging the zebrafish, and blood glucose levels were measured using glucose test strips or a glucose assay kit. The hypoglycemic effect of the candidate peptide was evaluated by comparing the blood glucose values ​​of the experimental group and the model control group.

[0058] The results are as follows Figure 5 As shown, compared with the model group, the blood glucose levels of the Pep4 and Pep75 experimental groups were closer to those of the normal control group, indicating that both 200 μM concentrations of Pep4 and Pep75 have the effect of improving blood glucose levels in zebrafish with hyperglycemia.

[0059] 5. A zebrafish hyperglycemia model was used to evaluate the effect of peptides on improving motor dysfunction caused by hyperglycemia. The specific methods are as follows: From day 5 to day 9 post-fertilization (dpf), zebrafish embryos were treated day and night with alternating doses of 2.5% glucose solution and 0.15% yolk solution. Simultaneously, from day 5 to day 9, the embryos were treated with a 200 μM peptide. Zebrafish larvae were placed in multi-well plates, and their movement was recorded using a behavioral tracking system under light-dark stimulation cycles (5s light → 25s darkness, repeated 8 times). Movement distances were calculated after 5s of light and after 25s of darkness. The efficacy of Pep4 and Pep75 in improving the movement ability of a hyperglycemic model was evaluated by analyzing the total movement distance of the zebrafish within specific time periods, with the same group settings as in Experiment 4 above.

[0060] Table 1. Motion patterns of different treatment groups under light and dark stimulation cycles (unit: cm) The results are as follows Figure 6 As shown in Table 1, compared with the model group, 200 μM concentrations of Pep75 and Pep4 tended to increase the movement distance of zebrafish.

[0061] In summary, Pep75 (TSVLRALPDEV) and Pep4 (REGDIIAIPA) have the effects of reversing insulin resistance in HepG2 cells, reducing blood glucose levels in a zebrafish hyperglycemia model, and improving the decline in exercise capacity caused by hyperglycemia.

[0062] Example 1 Example 1 provides a seabuckthorn protein peptide, the amino acid sequence of which is REGDIIAIPA (SEQ ID NO.1).

[0063] Example 2 Example 2 provides a sea buckthorn protein peptide, the amino acid sequence of which is TSVLRALPDEV (SEQ ID NO.2).

[0064] Example 1 To verify whether the sea buckthorn protein peptides in Examples 1-2 have the effect of improving blood sugar, a diabetic mouse model was constructed for the experiment. The specific protocol is as follows: (1) Establishment of a diabetic animal model (T2DM model) and peptide treatment Mice were acclimatized to a standard control diet for one week, and their serum glucose levels were measured after a 12-hour fast to determine their baseline blood glucose levels. The glucose-balanced mice were divided into 6 groups (n=8 per group), including the following groups: Normal control group (standard feed, product number MD17121: protein ≥18.0%, fat ≥4.0%, protein ≤5.0%, purchased from Jiangsu Medison Biomedical Co., Ltd.); HFD model group (type 2 diabetes; high-fat diet (HF) with blue dye, catalog number D12492: rodent diet containing 60 kcal% fat; purchased from Research Diets, USA). P4-L (high-fat diet, supplemented with a low dose of sea buckthorn protein peptide obtained in Example 1, 0.5 mmol / kg·BW). P4-H (high-fat diet, with high dose of sea buckthorn protein peptide obtained in Example 1 added, 1 mmol / kg·BW). P75-L (high-fat diet, supplemented with a low dose of sea buckthorn protein peptide obtained in Example 2, 0.5 mmol / kg·BW). P75-H (high-fat diet, with high dose of sea buckthorn protein peptide obtained in Example 2 added, 1 mmol / kg·BW). P4-L, P4-H, P75-L, and P75-H were the experimental groups.

[0065] After the adaptation period, mice in the normal control group were fed a normal diet for one week, followed by a standard diet, while mice in the model and treatment groups were fed a high-fat diet for four weeks. Four weeks later, mice in the treatment group were administered peptide drugs via gavage. After 35 days of treatment, mice in the high-fat diet and treatment groups were intraperitoneally injected with 50 mg / kg streptozotocin (STZ) dissolved in 0.1 M citrate buffer (pH=4.5) for five consecutive days. Mice in the control and model groups were injected with the same volume of citrate buffer. Two weeks after STZ injection, mice with a fasting blood glucose level ≥16 mM were diagnosed with type 2 diabetes mellitus (T2DM). Three days before dissection, mice fasted for 12 hours underwent an oral glucose tolerance test (OGTT). During the experiment, animal weight and feed intake were measured twice weekly. Gavage administration of peptides continued until the day before dissection. On the day of dissection, animals were fasted for 12 hours, blood samples were collected by enucleation for biochemical analysis, and the animals were then euthanized by cervical dislocation.

[0066] The entire experiment lasted for several weeks, during which the mice's weight and food intake were monitored regularly.

[0067] (2) Oral glucose tolerance test At the end of the experiment, all fasted mice underwent an oral glucose tolerance test. Blood glucose levels were measured at different time points after fasting and oral glucose administration, and blood glucose change curves were plotted and the area under the curve was calculated to evaluate the mice's glucose tolerance and the effect of peptide intervention.

[0068] (3) Sample collection and indicator detection After the experiment, the mice were euthanized and blood samples were collected.

[0069] Hormone and enzyme activity assays: Serum levels of hormones such as insulin, glucagon, glucagon-like peptide-1, dipeptidyl peptidase-4, and related enzymes (α-amylase, α-glucosidase) were detected using enzyme-linked immunosorbent assay (ELISA).

[0070] The results are as follows Figure 7 As shown, compared with the high-fat diet group (HFD group), the P4 group (Example 1) and the P75 group (Example 2) showed lower blood glucose levels at both doses (at all time points). Area under the curve (AUC) plot ( Figure 7 B) shows that the P75-L group (low-dose group of Example 2) and the P75-H group (high-dose group of Example 2) had a more significant effect on reducing blood glucose levels.

[0071] like Figure 8 As shown, both peptides significantly reduced serum α-amylase levels at both doses, but only P75-L significantly reduced α-glucosidase levels and significantly increased GLP-1 levels. Furthermore, both peptides reduced insulin levels at both doses, and both doses of P4 significantly reduced glucagon and DPP-4 levels, while these two indicators were significantly reduced in P75-H and P75-L, respectively.

[0072] In summary, the sea buckthorn protein peptide of the present invention has the effect of improving blood glucose levels and insulin resistance in diabetic animal models, and can be used in products that improve blood glucose and health foods that help maintain healthy blood glucose levels.

[0073] Finally, 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 scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A sea buckthorn protein peptide with hypoglycemic activity, characterized in that, The amino acid sequence of the sea buckthorn protein peptide is selected from SEQ ID NO.1 and / or SEQ ID NO.

2.

2. The sea buckthorn protein peptide as described in claim 1, characterized in that, Includes at least one of the following (I)-(II): (I) The seabuckthorn protein peptides were prepared by solid-phase synthesis. (II) The seabuckthorn protein peptide is obtained by enzymatic hydrolysis and purification of seabuckthorn seed protein.

3. The sea buckthorn protein peptide as described in claim 2, characterized in that, The sea buckthorn protein peptides were prepared by the following steps: S1. Using sea buckthorn seed powder as raw material, crude protein from sea buckthorn seeds is extracted by treatment with alkaline solution and cellulase. S2. The crude protein from sea buckthorn seeds obtained in step S1 is mixed with trypsin to form a reaction system for enzymatic hydrolysis. After the enzymatic hydrolysis is completed, the reaction system is subjected to enzyme inactivation treatment, concentrated, and dried to obtain sea buckthorn protein hydrolysate. S3. The seabuckthorn protein hydrolysate obtained in step S2 is purified by ultrafiltration to obtain the ultrafiltration fraction. S4. The ultrafiltration fraction obtained in step S3 is purified by gel filtration chromatography to obtain sea buckthorn protein peptides.

4. The sea buckthorn protein peptide as described in claim 3, characterized in that, Includes at least one of the following (I)-(III): (I) In step S2, the mass ratio of sea buckthorn seed crude protein to trypsin is sea buckthorn seed crude protein: trypsin = (48-50):1; (II) In step S2, the enzymatic hydrolysis reaction is carried out at pH=9 and 50℃ for 4 hours; (III) In step S2, the enzyme inactivation treatment is to heat the reaction system at 85°C for 30 min.

5. The sea buckthorn protein peptide as described in claim 3, characterized in that, In step S3, the ultrafiltration membrane used for ultrafiltration purification has a molecular weight cutoff of 1-10 kDa.

6. The sea buckthorn protein peptide as described in claim 3, characterized in that, In step S4, the specific operation of the gel filtration chromatography is as follows: purification is performed using a Sephadex G-25 gel filtration column, elution is performed using ultrapure water as the mobile phase, the eluent is monitored by the BCA method, the eluents corresponding to the main protein peaks are combined and lyophilized to obtain sea buckthorn protein peptides.

7. The use of the sea buckthorn protein peptide as described in any one of claims 1-6 in the preparation of blood glucose-improving products.

8. The application as described in claim 7, characterized in that, The blood sugar-improving products include at least one of food, medicine, and cosmetics.

9. The use of the sea buckthorn protein peptide as described in any one of claims 1-6 in the preparation of health food products that help maintain healthy blood sugar levels.

10. A product characterized in that, Includes the sea buckthorn protein peptide according to any one of claims 1-6.

Images