Hippophae rhamnoides leaf active peptide with blood sugar reducing function as well as preparation method and application thereof

Abstract

The invention relates to the technical field of bioactive peptides, in particular to a hippophae rhamnoides leaf bioactive peptide with a blood sugar reducing function as well as a preparation method and application of the hippophae rhamnoides leaf bioactive peptide. The amino acid sequence of the hippophae rhamnoides leaf active peptide is shown as any one of SEQ ID No.1 to SEQ ID No.2. The sea-buckthorn leaf protein is extracted through an alkali-solution and acid-isolation method, the sea-buckthorn leaf polypeptide is obtained through an enzymolysis method, the hypoglycemic activity is detected, the effective extraction of the sea-buckthorn leaf protein is improved by adopting the method provided by the invention, and a technical support is provided for the development of the sea-buckthorn leaf polypeptide.

Description

Technical Field

[0001] The present invention relates to the technical field of bioactive peptides, in particular to a seabuckthorn leaf active peptide with blood sugar lowering function, and a preparation method and application thereof. Background Art

[0002] Sea buckthorn (also known as vinegar willow, blackthorn, or sourthorn), belonging to the genus Hippophae rhamnoides of the Elaeagnaceae family, is a natural deciduous shrub with both medicinal and edible properties. Due to its tolerance to cold, extreme heat, wind, sand, and drought, it is widely cultivated for greening in Northwest my country, primarily in North, Northwest, and Southwest China. Both its leaves and fruits contain numerous bioactive substances, including flavonoids, polyphenols, fatty acids, vitamins, and amino acids. Sea buckthorn boasts numerous benefits, including anti-tumor, anti-photoaging, antioxidant, and weight-loss benefits, earning it the folk reputation of being the "longevity fruit." Studies have shown that the bioactive substances in sea buckthorn are beneficial for blood sugar regulation, but current research on the physiological functions of sea buckthorn leaf components primarily focuses on flavonoids, polysaccharides, and polyphenols, while relatively little research has been conducted on peptides. Therefore, studying the inhibitory effects of sea buckthorn leaf protein peptides on α-glucosidase and DPP-IV is of great clinical significance. On this basis, the separation and purification of hypoglycemic peptides and the identification of peptide sequences provided a new strategy for screening α-glucosidase and DPP-IV inhibitory peptides, and also provided a scientific basis for the research and development of sea buckthorn leaf hypoglycemic health foods. Summary of the Invention

[0003] In order to solve the above problems, the present invention provides a sea buckthorn leaf active peptide with hypoglycemic function, as well as a preparation method and application thereof. The present invention extracts sea buckthorn leaf protein by alkali dissolution and acid precipitation method, and then obtains sea buckthorn leaf polypeptide by enzymatic hydrolysis and detects the hypoglycemic activity. The method provided by the present invention improves the effective extraction of sea buckthorn leaf protein and provides technical support for the development of sea buckthorn leaf polypeptide.

[0004] In order to achieve the above object, the present invention provides the following technical solutions:

[0005] The present invention provides a seabuckthorn leaf active peptide with blood sugar lowering function. The amino acid sequence of the seabuckthorn leaf active peptide is shown in any one of SEQ ID Nos. 1 to 4.

[0006] The present invention also provides a method for preparing the seabuckthorn leaf active peptide described in the above technical solution, comprising the following steps:

[0007] 1) Extracting seabuckthorn leaf protein using alkali dissolution and acid precipitation method;

[0008] 2) hydrolyzing the seabuckthorn leaf protein obtained in step 1) with an enzyme to obtain an enzymatic hydrolyzate;

[0009] 3) Purifying the enzymatic hydrolysate obtained in step 2) by gel chromatography to obtain seabuckthorn leaf active peptides.

[0010] Preferably, the method of step 1) alkali dissolution and acid precipitation comprises the following steps:

[0011] A. Mix seabuckthorn leaves and deionized water, and dissolve them in alkali to obtain an alkaline solution;

[0012] B. Centrifuging the alkaline solution obtained in step A at a speed of 4000-6000 rpm for 20 min to obtain a supernatant;

[0013] C. acid precipitating the supernatant obtained in step B, and centrifuging at a speed of 4000-6000 rpm for 30 min to obtain a precipitate;

[0014] D. Wash the precipitate obtained in step C with water and centrifuge at a speed of 4000-6000 rpm for 20 min. Freeze-dry the obtained supernatant at -60°C in a vacuum freeze-drying process to obtain seabuckthorn leaf protein.

[0015] Preferably, in step A, the mass ratio of sea buckthorn leaves to deionized water is 1:20, and the sea buckthorn leaves are processed into sea buckthorn leaf powder and then ultrasonically mixed with deionized water for 20 minutes.

[0016] Preferably, the conditions for alkaline dissolution in step A include: stirring at a pH of 12.0 and a temperature of 85° C. for 1 hour.

[0017] Preferably, the acid precipitation conditions in step C include: standing for 1 hour at a pH value of 3.5.

[0018] Preferably, the enzyme in step 2) comprises papain, flavor protease, alkaline protease or trypsin.

[0019] Preferably, the enzymatic hydrolysis conditions of the papain include: enzymatic hydrolysis at 45° C. for 5 h, a pH value of 7.0, a mass ratio of the seabuckthorn leaf protein to the papain of 1:0.025, and an enzymatic activity of the papain of 800 U / mg;

[0020] The enzymatic hydrolysis conditions of the flavor protease include: enzymatic hydrolysis at 50° C. for 5 hours, a pH value of 7.0, a mass ratio of the seabuckthorn leaf protein to the flavor protease of 1:0.02, and an enzyme activity of the flavor protease of 1100 U / mg;

[0021] The enzymatic hydrolysis conditions of the alkaline protease include: enzymatic hydrolysis at 50° C. for 5 h, a pH value of 9.0, a mass ratio of the seabuckthorn leaf protein to the alkaline protease of 1:0.1, and an enzymatic activity of the alkaline protease of 200 U / g;

[0022] The enzymatic hydrolysis conditions of the trypsin include: enzymatic hydrolysis at 37° C. for 5 hours, a pH value of 8.0, a mass ratio of the seabuckthorn leaf protein to trypsin of 1:0.08, and an enzymatic activity of the trypsin of 250 U / mg.

[0023] Preferably, the conditions for gel chromatography purification in step 3) include: using Sephadex G-50, deionized water: pH=7.0, gel column: 30×2 cm, gel height: 20 cm; number of sampling tubes: 50 tubes; solution in each tube: 0.6 mL.

[0024] The present invention also provides the use of the seabuckthorn leaf active peptide described in the above technical solution in the preparation of a blood sugar-lowering product.

[0025] Beneficial effects of the present invention:

[0026] The present invention extracts seabuckthorn leaf protein by alkali dissolution and acid precipitation, obtains seabuckthorn leaf polypeptide by enzymatic hydrolysis and detects its hypoglycemic activity. The method provided by the present invention improves the effective extraction of seabuckthorn leaf protein and provides technical support for the development of seabuckthorn leaf polypeptide. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments are briefly introduced below.

[0028] Figure 1 The present invention is a flow chart of the preparation method of seabuckthorn leaf protein;

[0029] Figure 2 This is a flow chart of enzymatic hydrolysis and analysis of seabuckthorn leaf protein;

[0030] Figure 3 Complete flow chart for the preparation and analysis of seabuckthorn leaf hypoglycemic peptides;

[0031] Figure 4 is the inhibition rate of α-glucosidase by the enzymatic hydrolysis products of different enzymes within 0-4 hours, Papin: papain; Flavor protease: flavor protease; Alkaline protease: alkaline protease; trypsin: trypsin; No add enzyme: no enzyme added;

[0032] Figure 5 The inhibition rate of DPP-IV enzyme by the enzymatic products of different enzymes within 0-4 hours, Papin: papain; Flavorprotease: flavor protease; Alkaline protease: alkaline protease; trypsin: trypsin; No addenzyme: no enzyme added;

[0033] Figure 6The inhibition rates of seabuckthorn leaf protein and enzymatic peptides at different concentrations on α-glucosidase are shown in Table 1. Protein: protein; Papin hydrolyzed polypeptide: papain hydrolyzed polypeptide; Trypsin-digested peptides: trypsin hydrolyzed polypeptide;

[0034] Figure 7 The inhibition rates of seabuckthorn leaf protein and enzymatic peptides at different concentrations on DPP-IV enzyme are shown in Table 1. Protein: protein; Papinhydrolyzed polypeptide: papain hydrolyzed polypeptide; Trypsin-digested peptides: trypsin hydrolyzed polypeptide;

[0035] Figure 8 The inhibition rate of papain hydrolyzed peptide on α-glucosidase in different tube numbers;

[0036] Figure 9 The inhibition rate of trypsin-hydrolyzed peptides on α-glucosidase in different tube numbers;

[0037] Figure 10 The inhibition rate of papain hydrolyzed peptide on DPP-IV enzyme in different tube numbers;

[0038] Figure 11 The inhibition rate of trypsin-hydrolyzed peptides on DPP-IV enzyme in different tube numbers;

[0039] Figure 12 is the MS / MS spectrum of the relevant peptides;

[0040] Figure 13 This is a diagram showing the molecular docking of peptide TQDWVSLPGVLPVA with α-glucosidase;

[0041] Figure 14 Schematic diagram of molecular docking of peptide TQDWVSLPGVLPVA with DPP-IV (dipeptidyl peptidase-4);

[0042] Figure 15 Schematic diagram of molecular docking of peptide TAIDIGILR and α-glucosidase;

[0043] Figure 16 Schematic diagram of molecular docking of peptide TAIDIGILR and DPP-IV (dipeptidyl peptidase-4);

[0044] Figure 17 Schematic diagram of molecular docking between peptide DLFEEGSVTN and α-glucosidase;

[0045] Figure 18Schematic diagram of molecular docking of peptide DLFEEGSVTN and DPP-IV (dipeptidyl peptidase-4);

[0046] Figure 19 Schematic diagram of molecular docking of peptide VAYPLDLFEEG with α-glucosidase;

[0047] Figure 20 Schematic diagram of the molecular docking of the peptide VAYPLDLFEEG and DPP-IV (dipeptidyl peptidase-4). DETAILED DESCRIPTION

[0048] The present invention provides a seabuckthorn leaf active peptide with blood sugar lowering function. The amino acid sequence of the seabuckthorn leaf active peptide is shown in any one of SEQ ID Nos. 1 to 4. The product of seabuckthorn leaf protein hydrolyzed by trypsin has the best effect. The seabuckthorn leaf active peptides are all hydrolyzed by trypsin, as shown below:

[0049] SEQ ID No. 1:

[0050] TQDWVSLPGVLPVA;

[0051] SEQ ID No. 2:

[0052] TAIDIGILR;

[0053] SEQ ID No.3:

[0054] DLFEEGSVTN;

[0055] SEQ ID No.4:

[0056] VAYPLDLFEEG.

[0057] The present invention provides a method for preparing the active peptide from seabuckthorn leaves as described in the above technical solution, comprising the following steps:

[0058] 1) Extracting seabuckthorn leaf protein using alkali dissolution and acid precipitation method;

[0059] 2) hydrolyzing the seabuckthorn leaf protein obtained in step 1) with an enzyme to obtain an enzymatic hydrolyzate;

[0060] 3) Purifying the enzymatic hydrolysate obtained in step 2) by gel chromatography to obtain seabuckthorn leaf active peptides.

[0061] The invention adopts an alkali dissolution and acid precipitation method to extract seabuckthorn leaf protein.

[0062] In the present invention, the alkali dissolution and acid precipitation method preferably comprises the following steps: A. mixing sea buckthorn leaves with deionized water for alkali dissolution to obtain an alkaline solution; B. centrifuging the alkaline solution obtained in step A at 4000-6000 rpm for 20 minutes to obtain a supernatant; C. subjecting the supernatant obtained in step B to acid precipitation and centrifuging at 4000-6000 rpm for 30 minutes to obtain a precipitate; D. washing the precipitate obtained in step C with water, centrifuging at 4000-6000 rpm for 20 minutes, and freeze-drying the resulting supernatant at -60°C to obtain sea buckthorn leaf protein. In the present invention, the mass ratio of sea buckthorn leaves to deionized water is preferably 1:20. After the sea buckthorn leaves are processed into sea buckthorn leaf powder, they are preferably ultrasonically mixed with deionized water for 20 minutes. In the present invention, the alkali dissolution conditions preferably include stirring at a pH of 12.0 and a temperature of 85°C for 1 hour. In the present invention, the acid precipitation conditions preferably include: standing for 1 hour at a pH value of 3.5.

[0063] The present invention hydrolyzes the obtained seabuckthorn leaf protein with an enzyme to obtain an enzymatic hydrolyzate. In the present invention, the enzyme preferably includes papain, flavor protease, alkaline protease or trypsin.

[0064] In the present invention, the enzymatic hydrolysis conditions of papain preferably include: enzymatic hydrolysis at 45°C for 5 hours, a pH of 7.0, a mass ratio of seabuckthorn leaf protein to papain of 1:0.025, and an enzymatic activity of papain of 800 U / mg. In the present invention, the enzymatic hydrolysis conditions of flavor protease preferably include: enzymatic hydrolysis at 50°C for 5 hours, a pH of 7.0, a mass ratio of seabuckthorn leaf protein to flavor protease of 1:0.02, and an enzymatic activity of flavor protease of 1100 U / mg. In the present invention, the enzymatic hydrolysis conditions of alkaline protease preferably include: enzymatic hydrolysis at 50°C for 5 hours, a pH of 9.0, a mass ratio of seabuckthorn leaf protein to alkaline protease of 0.2:0.020, and an enzymatic activity of alkaline protease of 200 U / mg. In the present invention, the enzymatic hydrolysis conditions of the trypsin preferably include: enzymatic hydrolysis at 37° C. for 5 h, a pH value of 8.0, a mass ratio of the seabuckthorn leaf protein to trypsin of 1:0.08, and an enzymatic activity of the trypsin of 250 U / mg.

[0065] The enzymatic hydrolysate is purified by gel chromatography to obtain active peptides from seabuckthorn leaves. In the present invention, the gel chromatography purification conditions preferably include: Sephadex G-50, deionized water, pH = 7.0, a gel column of 30 x 2 cm, a gel height of 20 cm, 50 sampling tubes, and 0.6 mL of solution per tube.

[0066] The present invention also provides the use of the seabuckthorn leaf active peptide described in the above technical solution in the preparation of a blood sugar-lowering product.

[0067] In order to further illustrate the present invention, the present invention is described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0068] Example 1

[0069] Extraction of seabuckthorn leaf protein by alkali dissolution and acid precipitation

[0070] Figure 1 The figure is a flow chart of the preparation method of seabuckthorn leaf protein.

[0071] Weigh 30g of seabuckthorn leaves, wash, dry and grind them to obtain seabuckthorn leaf powder, add 600mL of deionized water and ultrasonicate for 20min, and then add 1mol·L -1 Adjust the pH to 12.0 with NaOH. Stir at 85℃ for 1h and dissolve the alkali. -1 Centrifuge for 20 min at a speed of 1 mol / L and take the supernatant. -1 Adjust the pH to 3.5 with HCl, let it stand for 1 hour, and rotate at 5000 r·min -1 After centrifugation for 30 min, the precipitate was collected and washed with water and then centrifuged at 6000 r·min. -1 The supernatant was centrifuged for 30 minutes and freeze-dried to obtain freeze-dried seabuckthorn leaf protein powder for later use.

[0072] Example 2 Enzymatic hydrolysis of seabuckthorn leaf protein

[0073] Figure 2 This is the enzymatic hydrolysis and analysis flow chart of seabuckthorn leaf protein. Figure 3 Complete flow chart for the preparation and analysis of seabuckthorn leaf hypoglycemic peptides.

[0074] Weigh 0.20g of sea buckthorn leaf freeze-dried protein powder and dissolve it in 10mL of water (solid-liquid ratio 1:50). Prepare 4 groups of the same protein solution. In the first group, adjust the solution pH to 7.0 and add 0.005g of papain (800U·mg -1 ) and enzymatically hydrolyzed at 45°C for 5 h. In the second group, the solution pH was adjusted to 7.0 and 0.004 g flavor protease (1100 U·mg -1 ) and enzymatically hydrolyzed at 50°C for 5 h. The third group adjusted the pH to 9.0 and added 0.020 g alkaline protease (200 U·mg -1 ) and enzymatically hydrolyzed at 50°C for 5 h. The fourth group adjusted the pH to 8.0 and added 0.0163 g trypsin (250 U·mg -1) and enzymatically digested at 37°C for 5 hours. For all four groups of experiments, 200 μL of the enzymatic hydrolyzate was sterilized in a 90°C water bath for 5 minutes at 1 hour intervals and diluted to 4 mL with water. The hypoglycemic effects of the four enzymes were tested after 0, 1, 2, 3, and 4 hours of enzymatic digestion. The results (Table 1) showed that papain and trypsin were more effective.

[0075] Table 1 Inhibitory activity of proteolytic products

[0076]

[0077] Example 3

[0078] Separation and purification of protein hydrolysates

[0079] Seabuckthorn leaf polypeptides were isolated and purified using gel chromatography (Sephadex G-50). The gel chromatography conditions were: deionized water, pH = 7.0, gel column, 30 × 2 cm, gel height, 20 cm, 50 sampling tubes, 0.6 mL of solution per tube. The isolated fractions were used to measure their hypoglycemic activity, and the results are shown in Table 2.

[0080] Table 2 Inhibitory activity of protein hydrolysate chromatography fractions

[0081]

[0082]

[0083] Example 4 Determination of the blood sugar lowering ability of seabuckthorn leaf protein hydrolysate

[0084] The total experimental system for the α-glucosidase inhibition activity assay is 220 μL, including 40 μL of sample, containing approximately 40 μg of enzymatic peptide. The positive control uses acarbose as an inhibitor, and 40 μL of acarbose solution is used, containing approximately 2 μg of acarbose. 40 μL of enzyme solution (1 U mL -1 ) were mixed with 40uL of different sample solutions / acarbose in a 96-well plate and incubated at 37°C for 10 min in a biochemical incubator. 40uL of 4-nitrophenyl-β-D-pyranoglucopyranoside (PNPG) was added as the reaction substrate and incubated at 37°C for 10 min before adding 100uL of 1 mol·L -1 The reaction was terminated with a Na2CO3 solution. The absorbance was measured at 405 nm using a full-wavelength microplate reader. Deionized water was used as a blank control in place of the sample. A graph was plotted with concentration as the horizontal axis and inhibition rate as the vertical axis. The inhibition rate was calculated using the following formula.

[0085]

[0086] Where: A sample is the absorbance after adding inhibitor and enzyme reaction; A blank is the absorbance after adding inhibitor only without enzyme reaction; A control is the absorbance after adding enzyme reaction only without adding sample.

[0087] The DPP-IV (Dipeptidyl Peptidase-4) inhibitory activity assay was performed in three experimental groups: the experimental group, the 100% active control group, and the blank background group. 30 μL of buffer solution, 10 μL of DPP-IV, and 10 μL of sample solution were added to a 96-well microtiter plate in sequence, and finally 50 μL of DPP substrate (containing 100 μmol·L -1 The reaction was initiated with a 50% H-Gly-Pro (H-Gly-Pro) buffer. The reaction was incubated at 37°C for 30 minutes, and the fluorescence intensity of the sample was measured in a microplate reader at an excitation wavelength of 360 nm and an emission wavelength of 460 nm. The 100% active control group and the blank background group were tested with deionized water instead of the sample solution, while the blank background group was tested with buffer solution instead of the DPP-IV solution. The DPP-IV inhibition rate was calculated as shown in formula (2):

[0088]

[0089] Where: Fcontrol is the fluorescence intensity of the control group; Fblank is the fluorescence intensity of the blank group; Fsample is the fluorescence intensity of the experimental group.

[0090] Figure 4 is the inhibition rate of α-glucosidase by the enzymatic hydrolysis products of different enzymes within 0-4h, Figure 5 The inhibition rates of the enzymatic hydrolysis products of different enzymes on DPP-IV within 0-4 hours are shown in Table 2. The inhibition rates of α-glucosidase and DPP-IV by trypsin and papain after enzymatic hydrolysis are significantly higher than those of the other two enzymes.

[0091] IC50, or half-inhibitory concentration, indicates the concentration of a drug or substance that achieves 50% inhibition of a biological process. IC50 is widely used in assessing cytotoxicity, studying drug interactions, and drug screening and development. A certain weight of sample is weighed to prepare solutions of varying concentrations. The inhibitory activity is measured, and a curve is plotted with concentration as the horizontal axis and inhibitory activity as the vertical axis. All experiments are repeated three times, and the results are expressed as mean ± standard deviation. The IC50 value is then calculated using a fitted equation. Lower IC50 values ​​indicate greater inhibitory potency.

[0092] Figure 6 is the inhibition rate of α-glucosidase by different concentrations of seabuckthorn leaf protein and enzymatic peptide, Figure 7 The inhibition rate of seabuckthorn leaf protein and enzymatic peptide at different concentrations on DPP-IV enzyme can be calculated based on the data.

[0093] Example 5

[0094] In vitro simulated digestion experiment

[0095] To investigate the stability and absorption of seabuckthorn leaf polypeptides during digestion, the inhibition rates of protein and enzymatically hydrolyzed peptides (seabuckthorn leaf polypeptides hydrolyzed by papain) against α-glucosidase were tested before and after simulated gastrointestinal digestion, as shown in Table 3. Compared with the untreated group, both protein and enzymatically hydrolyzed peptides exhibited higher inhibition rates for α-glucosidase after simulated gastric, intestinal, and gastrointestinal digestion, with the overall inhibition rate trend being protein > enzymatically hydrolyzed peptide. The inhibition rate for protein was 69.43% when untreated. After 0 h of gastric treatment, the inhibition rate increased significantly to 99.58%. After subsequent intestinal and gastrointestinal treatments, the inhibition rate remained at a relatively high level, close to that of the positive control. This indicates that the protein's inhibitory activity against α-glucosidase was significantly enhanced after gastric treatment and maintained a high inhibitory activity during subsequent gastrointestinal digestion. The acidic environment of the stomach and the action of pepsin partially hydrolyze the protein, exposing more active sites capable of binding to α-glucosidase, thereby enhancing the inhibitory effect. The inhibition rate was highest after 2 hours of treatment in the gastric environment. Because papain peptides have a certain ability to inhibit α-glucosidase, they can also show high inhibitory activity in the stomach in the early stages. However, as the gastrointestinal digestion process prolongs, the peptides may be further hydrolyzed, resulting in a decrease in their effective inhibitory components, and thus a downward trend in the inhibition rate.

[0096] Table 3 Inhibition rate of seabuckthorn leaf protein and papain hydrolyzed polypeptide on α-glucosidase before and after in vitro digestion

[0097]

[0098]

[0099] Example 6

[0100] Peptide sequencing

[0101] The collection inhibitory activity is high ( Figure 8 、 9, 10, and 11) were used as samples for identification of seabuckthorn leaf polypeptide sequences using high-performance liquid chromatography-tandem mass spectrometry (LC / MS-MS). The samples were desalted using a C18 column. The system employed a Q Exactive tandem HPLC mass spectrometer, with a Column Technology Inc. RP-C18 (0.15 mm x 150 mm) analytical column. Solution A consisted of 0.1% formic acid in water, and solution B consisted of 0.1% formic acid in acetonitrile (84% acetonitrile). The samples were separated over a 60-minute gradient, with an elution gradient of 4% to 50% B (0-50 min); 50% to 100% B (50-54 min); and 100% B (54-60 min). Table 4 shows partial protein sequencing data. The data indicate that some of the detected proteins are not found in the seabuckthorn leaf database, further enriching and supplementing the seabuckthorn leaf protein database.

[0102] Table 4 Protein sequencing

[0103]

[0104]

[0105] The enzymatic digestion products were separated by capillary high-performance liquid chromatography and analyzed by mass spectrometry on a Q Exactive mass spectrometer for 60 minutes. Detection was positive ionization. The mass-to-charge ratios of peptides and peptide fragments were acquired using the following method: 10 fragmentation spectra (MS2 scans) were collected after each full scan. The mass spectrometry raw files were searched against the corresponding database using MaxQuant 1.5.5.1 software, resulting in protein identification and quantification. Table 5 shows the peptide sequencing results from the trypsin digestion, which revealed 15 peptides derived from protein XKT97467.

[0106] Table 5 shows the results of trypsin enzymatic peptide sequencing.

[0107]

[0108]

[0109] Tryptic peptides have good inhibitory effects on both α-glucosidase and DPP-IV, so high performance liquid chromatography-mass spectrometry (LC / MS-MS) was further used to identify peptides with good hypoglycemic effects. After amino acid sequence analysis and database retrieval (MaxQuant 1.5.5.1), it was found that the peptide sequences with higher fitting scores were TQDWVSLPGVLPVA, TAIDIGILR, DLFEEGSVTN, VAYPLDLFEEG and LSLYTPAG. Their m / z were 741.91, 486.80, 556.25, 626.81, and 410.52, respectively. The peptide sequences of these ions were also identified, and the MS / MS spectra of TQDWVSLPGVLPVA, TAIDIGILR, DLFEEGSVTN and VAYPLDLFEEG were displayed, as shown in Figure 3. Figure 12 Table 6 shows the relevant information of each peptide segment.

[0110] Table 6 Related peptide information

[0111]

[0112] Example 7

[0113] Computer simulation of protein docking

[0114] First, download the structure of the receptor protein from the PDB database (the receptor proteins are α-glucosidase and DPP-IV), use the PEP-FOLD tool to predict the peptide structure, then use the GRAMM docking tool to dock the macromolecules, and finally use the PDBePISA tool to analyze the interaction results of the macromolecules, such as Figure 13-20 The results of docking of peptides TQDWVSLPGVLPVA, TAIDIGILR, DLFEEGSVTN, and VAYPLDLFEEG with α-glucosidase and DPP-IV, respectively, show that the peptides can dock well with the target proteins. Indicates the area of ​​the protein interaction surface and the free energy Δ i The lower the free energy, the more stable the structure. Table 7 lists the data of peptide and protein docking. Figures 13-20 Schematic diagram of macromolecular docking.

[0115] Table 7 lists the docking data of active peptides and receptor proteins

[0116]

[0117]

[0118] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A seabuckthorn leaf active peptide with blood sugar lowering function, characterized in that: The amino acid sequence of the seabuckthorn leaf active peptide is shown in any one of SEQ ID No. 1 to 4.

2. A method for preparing the active peptide from seabuckthorn leaves according to claim 1, characterized in that: The following steps are involved: 1) Extracting seabuckthorn leaf protein using alkali dissolution and acid precipitation method; 2) hydrolyzing the seabuckthorn leaf protein obtained in step 1) with an enzyme to obtain an enzymatic hydrolyzate; 3) Purifying the enzymatic hydrolysate obtained in step 2) by gel chromatography to obtain seabuckthorn leaf active peptides.

3. The preparation method according to claim 2, characterized in that The method of step 1) alkali dissolution and acid precipitation comprises the following steps: A. Mix seabuckthorn leaves and deionized water, and dissolve them in alkali to obtain an alkaline solution; B. Centrifuging the alkaline solution obtained in step A at a speed of 4000-6000 rpm for 20 min to obtain a supernatant; C. acid precipitating the supernatant obtained in step B, and centrifuging at a speed of 4000-6000 rpm for 30 min to obtain a precipitate; D. Wash the precipitate obtained in step C with water and centrifuge at a rotation speed of 4000-6000 rpm for 20 minutes. Freeze-dry the obtained supernatant at -60°C to obtain seabuckthorn leaf protein.

4. The preparation method according to claim 3, characterized in that In step A, the mass ratio of sea buckthorn leaves to deionized water is 1:20, and the sea buckthorn leaves are processed into sea buckthorn leaf powder and then ultrasonically mixed with deionized water for 20 minutes.

5. The preparation method according to claim 3, characterized in that The conditions for alkaline dissolution in step A include: stirring at a pH of 12.0 and a temperature of 85° C. for 1 hour.

6. The preparation method according to claim 3, characterized in that The conditions for acid precipitation in step C include: standing for 1 hour at a pH value of 3.

5.

7. The preparation method according to claim 2, characterized in that The enzyme in step 2) includes papain, flavor protease, alkaline protease or trypsin.

8. The preparation method according to claim 7, characterized in that The enzymatic hydrolysis conditions of the papain include: enzymatic hydrolysis at 45° C. for 5 hours, a pH value of 7.0, a mass ratio of the seabuckthorn leaf protein to the papain of 1:0.025, and an enzymatic activity of the papain of 800 U / mg; The enzymatic hydrolysis conditions of the flavor protease include: enzymatic hydrolysis at 50° C. for 5 hours, a pH value of 7.0, a mass ratio of the seabuckthorn leaf protein to the flavor protease of 1:0.02, and an enzyme activity of the flavor protease of 1100 U / mg; The enzymatic hydrolysis conditions of the alkaline protease include: enzymatic hydrolysis at 50° C. for 5 hours, a pH value of 9.0, a mass ratio of the seabuckthorn leaf protein to the flavor protease of 1:0.1, and an enzymatic activity of the alkaline protease of 200 U / mg; The enzymatic hydrolysis conditions of the trypsin include: enzymatic hydrolysis at 37° C. for 5 hours, a pH value of 8.0, a mass ratio of the seabuckthorn leaf protein to trypsin of 1:0.08, and an enzymatic activity of the trypsin of 250 U / mg.

9. The preparation method according to claim 2, characterized in that The conditions for gel chromatography purification in step 3) include: using Sephadex G-50, deionized water: pH=7.0, gel column: 30×2 cm, gel height: 20 cm; number of sampling tubes: 50 tubes; solution in each tube: 0.6 mL.

10. Use of the seabuckthorn leaf active peptide according to claim 1 in the preparation of a blood sugar lowering product.

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