Dateplum persimmon polypeptide and application of dateplum persimmon polypeptide in synergic antithrombosis with polyphenol

Abstract

The invention relates to black date polypeptide and application thereof in synergic antithrombosis with polyphenol, belongs to the technical field of biology, and particularly relates to thrombin inhibitory peptide as well as a preparation method and application thereof. The method comprises the following steps: extracting protein by taking black date wine residues as a raw material, preparing active polypeptide by a biological enzymolysis method, analyzing and screening thrombin inhibitory peptide PPHG by utilizing bioinformatics, and achieving an IC50 value of 30.69 + / -0.54 mg / mL to thrombin. The inhibition effect of the combination of the thrombin inhibitory peptide PPHG and the gallic acid GA on the thrombin activity is further explored, when the concentration ratio of the PPHG to the GA is 5: 2, the synergistic inhibition rate of the PPHG / GA on the thrombin is 38.76% + / -2.05%, and the synergistic score of the ZIP is the highest and is 13.50. The invention further provides a beta-cyclodextrin inclusion compound PPHG / GA-beta-CD embedded with PPHG / GA, the synergic inhibition thrombin activity of PPHG / GA can be well preserved under the low-temperature and dark condition, it is shown that the beta-cyclodextrin inclusion compound PPHG / GA-beta-CD can better control and slowly release bioactive substances compared with monomeric substances, and the beta-cyclodextrin inclusion compound PPHG / GA-beta-CD can be applied to preparation of products for inhibiting thrombin activity and has good application prospects. Or the traditional Chinese medicine composition can be used for preparing products for treating thrombus diseases.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a thrombin inhibitory peptide as well as a preparation method and application thereof. BACKGROUND

[0002] Thrombosis is a group of diseases caused by abnormal blood coagulation function in the body, leading to thrombosis and causing corresponding symptoms, mainly including deep vein thrombosis, pulmonary embolism, atrial fibrillation, etc. As a common cardiovascular disease, thrombosis poses a serious threat to human health.

[0003] Thrombin, as a key enzyme in the coagulation system, plays an important role in hemostasis and thrombosis. It is an important enzyme in all thrombus formation processes, including physiological hemostasis and pathological thrombosis, and is essential for the transformation of fibrinogen into fibrin. By inhibiting the activity of thrombin, the formation of thrombus can be effectively delayed. At present, some drugs prevent the formation of thrombus by inhibiting the aggregation of platelets, such as aspirin enteric-coated tablets, but such therapeutic drugs can have adverse effects on the human body, leading to indigestion, gastrointestinal and abdominal pain, etc., limiting their application in special populations. The occurrence of these side effects has prompted the research on natural enzyme activity inhibitors to find effective products for treating thrombosis.

[0004] Diospyros lotus L., also known as black jujube, belongs to the family Ebenaceae and the genus Diospyros. The fruit is spherical and turns yellow or black when mature. As a kind of edible fruit, black jujube has high nutritional and medicinal value, such as antioxidant, anti-hemolysis, anti-inflammatory and other biological activities. Black jujube wine lees is a by-product generated during the brewing process of black jujube, which contains rich organic matter such as cellulose, starch and protein. The wine lees has high water content and is prone to spoilage, resulting in a large amount of waste of high-quality resources and environmental pollution. Gallic acid (GA) is the most abundant polyphenol monomer in black jujube, which has various physiological activities such as antioxidant and antibacterial activities. In this application, bioactive peptides are extracted from black jujube wine lees in order to obtain natural anticoagulant active ingredients. The synergistic anticoagulant effect between the bioactive peptides and gallic acid is explored, and the stability of the bioactive peptides and gallic acid is improved by embedding them with β-cyclodextrin (β-CD). SUMMARY

[0005] In order to solve the above technical problems, the present application extracts protein from black date wine residue as raw material, prepares black date active peptide by biological enzymatic hydrolysis, screens thrombin inhibitory peptide by bioinformatics analysis, and further explores the inhibitory effect of thrombin inhibitory peptide and black date gallic acid on thrombin activity, calculates the synergistic effect score by zero interaction potency (ZIP) using SynergyFinder online software (https: / / synergyfinder.fimm.fi). The interaction between thrombin inhibitory peptide and gallic acid is explored by molecular docking technology, and the interaction between thrombin inhibitory peptide and gallic acid and the synergistic antithrombotic effect are explored by characterizing the structure, and the black date active peptide / gallic acid-β-cyclodextrin inclusion compound is prepared to improve its stability.

[0006] One of the technical solutions provided by the present application is a thrombin inhibitory peptide, and the amino acid sequence of the inhibitory peptide is Pro-Pro-His-Gly, abbreviated as PPHG. The thrombin inhibitory peptide PPHG is prepared by biological enzymatic hydrolysis of black date wine residue as raw material to extract protein, and the IC50 value of the thrombin inhibitory peptide PPHG reaches 30.69±0.54 mg / mL.

[0007] The second technical solution provided by the present application is the application of the thrombin inhibitory peptide PPHG in the first technical solution, particularly in the application of inhibiting thrombin activity, more particularly in the application of preparing a product for inhibiting thrombin activity, or in the application of preparing an antithrombotic product.

[0008] The product includes but is not limited to food, health care product or medicine.

[0009] The third technical solution provided by the present application is a composition containing the thrombin inhibitory peptide PPHG.

[0010] Further, the composition takes the inhibitory peptide PPHG as an active ingredient.

[0011] Further, the composition contains the inhibitory peptide PPHG and GA.

[0012] Further, the mass ratio of the inhibitory peptide PPHG and GA in the composition is 1-100:1.

[0013] Preferably, when the concentration ratio of the inhibitory peptide PPHG and GA in the composition is 5:2, the synergistic inhibitory rate of PPHG / GA on thrombin is 38.76%±2.05%, the synergistic anticoagulation effect is the best, and the ZIP synergistic score is 13.50.

[0014] The fourth technical solution provided by the present application is to obtain the composition by β-CD embedding according to the third technical solution.

[0015] Further, the preparation method is as follows:

[0016] PPHG / GA and β-CD (CAS: 7585-39-9) were mixed and diluted in 50 mL distilled water, stirred at room temperature in the dark for 5 h, and then placed in the dark for 12 h. Centrifugal freeze-drying obtained PPHG / GA-β-CD inclusion complex.

[0017] The fifth technical solution provided by the present application is the application of the composition of the third technical solution and the fourth technical solution, particularly the application in inhibiting thrombin activity, more particularly the application in preparing a product for inhibiting thrombin activity, or the application in preparing an antithrombotic product.

[0018] The product includes but is not limited to food, health care products or pharmaceuticals.

[0019] Beneficial effects:

[0020] (1) The present application uses black date wine residue as raw material, and prepares and separates thrombin inhibitory peptide PPHG by biological enzymolysis method, and the IC50 value of the thrombin is 30.69±0.54 mg / mL.

[0021] (2) The present application explores the interaction of thrombin inhibitory peptide PPHG and GA on thrombin. The IC50 values of PPHG and GA on thrombin are 30.69±0.54 mg / mL and 45.08±0.08 mg / mL, respectively. When the concentration ratio of PPHG and GA is 5:2, the synergistic inhibition rate of PPHG / GA on thrombin is 38.76%±2.05%, the synergistic anticoagulation effect is the best, and the ZIP synergistic score is 13.50.

[0022] (3) The present application provides a new type of thrombin inhibitory peptide and gallic acid composition inclusion complex, which improves the stability of the composition, and uses black date wine residue as raw material, so the production cost is low and the economic benefit is high. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 The inhibition of different molecular weight components after ultrafiltration on thrombin.

[0024] Figure 2 LC-MS / MS total ion chromatogram of the polypeptide.

[0025] Figure 3 Mass spectrum analysis diagram of PPHG.

[0026] Figure 4 Molecular docking diagram of PPHG and GA.

[0027] Figure 5 Inhibition rate curve of PPHG on thrombin.

[0028] Figure 6 Inhibition rate curve of GA to thrombin.

[0029] Figure 7 Synergy plot of PPHG and GA.

[0030] Figure 8 PPHG / GA-β-CD lyophilized powder plot.

[0031] Figure 9 Encapsulation efficiency of PPHG and GA.

[0032] Figure 10 Fourier infrared analysis of PPHG, GA, β-CD, PPHG / GA-β-CD.

[0033] Figure 11 Effect of temperature on stability of PPHG / GA-β-CD.

[0034] Figure 12 Effect of light on stability of PPHG / GA-β-CD. DETAILED DESCRIPTION

[0035] In order to make the purpose, technical scheme and advantages of the patent more clear, the patent will be further described in detail below in combination with specific examples. It should be understood that the specific examples described herein are only used to explain the patent and do not limit the invention.

[0036] The thrombin inhibitory activity determination method involved in the present application is as follows:

[0037] The principle of using the product nitroaniline (pNA) after the hydrolysis of chromogenic substrate H-D-Phe-Pip-Arg-pNA acetate (S-2238) by thrombin has a specific absorption peak at 405 nm, the inhibitory effect of the sample to be tested on the activity of thrombin is evaluated, the OD value is measured at 405 nm, and the inhibition rate of the sample is calculated. Each sample is tested in parallel at least 3 times. The specific experimental steps are as follows:

[0038] The polypeptides were dissolved in 50 mM imidazole buffer with 7.5 mM EDTA and 150 mM NaCl at pH 7.4, and thrombin and S-2238 were dissolved in the same buffer. In a 96-well plate, 50 μL of polypeptide solution at different concentrations (5, 10, 15, 20, 25 mg / mL) and 90 μL of imidazole buffer were first added and incubated at 37°C for 2 min. After incubation, 20 μL of thrombin at a concentration of 10 U / mL was added and incubated at 37°C for 1 min. Then, 20 μL of S-2238 at a concentration of 1.5 mM was added and incubated for 2 min. The absorbance value was measured at 405 nm using a microplate reader. The control group used imidazole buffer instead of polypeptide solution. The absorbance value of the imidazole buffer was set as A0. Then, the imidazole buffer was replaced with polypeptide solution at different concentrations, and the absorbance was measured in the same way and set as A. Each sample was measured in triplicate.

[0039] The inhibition rate was calculated according to the following formula:

[0040]

[0041] In the formula, A is the absorbance of the sample group, and A0 is the absorbance of the control group.

[0042] Table 1 Thrombin inhibition activity assay system

[0043]

[0044] The application will be further explained and described through specific examples below.

[0045] Example 1 Preparation of polypeptide PPHG

[0046] (1) Pretreatment of black date wine lees

[0047] The black date wine lees were dried in an oven at 50°C, crushed in a crusher, sieved through a 60-mesh sieve, placed in a sealed bag, and stored in a desiccator for use.

[0048] (2) Extraction of black date wine lees protein by alkali dissolution and acid precipitation

[0049] The black date wine lees powder was dissolved in distilled water at a ratio of 1:30 (w / v), stirred uniformly, and the pH was adjusted to 12.0 with 1 mol / L NaOH solution. The extraction was carried out at 53°C for 90 min, and the lower precipitate was removed by centrifugation at 6000 rpm for 20 min. The pH of the collected supernatant was adjusted to 4.0 using 0.1 mol / L hydrochloric acid solution, and the protein was precipitated by standing for 30 min. The precipitate was collected by centrifugation at 6000 rpm for 15 min. All the extracted proteins were vacuum freeze-dried for 48 h to obtain dry powder of wine lees protein.

[0050] (3) Protease enzyme hydrolysis to obtain black date wine lees polypeptide

[0051] The freeze-dried black date wine lees protein was mixed with distilled water at a ratio of 1:20 (w / v). Under the conditions of 37°C and pH 3.0, the amount of added pepsin (8000 U / g) was 2% (w / w) of the substrate mass, and the reaction was carried out for 4 h. After enzyme inactivation treatment at 100°C, the pH value was adjusted to 4.5 to precipitate the large molecular weight proteins. The supernatant was obtained by centrifugation at 8000 rpm for 15 min, and the pH value was adjusted to 7.0. The black date wine lees polypeptide hydrolysate was obtained.

[0052] (4) Ultrafiltration

[0053] The black date wine lees polypeptide hydrolysate was centrifuged at 8000 rpm for 15 min to obtain the supernatant, which was filtered through a 0.45 μm microfiltration membrane, and then separated using a 5 KDa ultrafiltration membrane to obtain the residue and filtrate. The filtrate was further separated using a 3 KDa ultrafiltration membrane to obtain the residue and filtrate. Finally, black date wine lees polypeptide solutions with different molecular weights (<3 KDa, 3-5 KDa, and >5 KDa) were obtained. The three polypeptide solutions with different molecular weights were vacuum freeze-dried and stored. The effects of the three polypeptide components (1 mg / mL) with different molecular weights on the inhibition of thrombin activity were determined.

[0054] The results are shown in Figure 1 From the results, it can be seen that the effects of the polypeptide components on thrombin activity are related to the molecular weight. The polypeptide components with different molecular weights all have inhibitory effects on thrombin activity, but the smaller the molecular weight, the stronger the inhibitory effect on thrombin activity. The thrombin inhibition rate of the polypeptide component with a molecular weight of <3 KDa was 20.68% ± 0.51%, which was significantly higher than that of the polypeptide components with molecular weights of 3-5 KDa and >5 KDa.

[0055] Therefore, the black date wine lees polypeptide component with a molecular weight of <3 KDa was further separated and purified to screen for a peptide segment with antithrombotic activity for subsequent experiments.

[0056] (5) LC-MS / MS identification

[0057] The black date wine lees polypeptide component with a molecular weight of <3 KDa was subjected to chromatographic separation and mass spectrometric identification according to the relevant operations, and the results were analyzed and identified using the MaxQuant database to obtain the corresponding polypeptide sequences. A total of 266 polypeptide sequences were obtained by liquid chromatography-mass spectrometry identification analysis, and the total ion chromatogram is shown in Figure 2 .

[0058] (6) Bioinformatics analysis of non-toxic and non-allergenic active peptides

[0059] 266 polypeptide sequences were obtained by LC-MS / MS sequencing. The target peptide segments were screened using the NCBI website to download the black date protein sequence, and the peptide segments derived from black date were obtained; the target peptide segments with a score greater than 0.5 were evaluated on the Peptide Ranker online software; whether the peptide segment was non-toxic and non-allergen was confirmed by analyzing the ToxinPred, AllerTOP v.2.0 online website. According to the polypeptide amino acid composition analysis, database comparison and computer analysis results, 14 polypeptides without toxicity, allergen and with an activity score of more than 0.5 were finally screened. The peptide segment PPHG was compared with the amino acid sequences of existing functional peptides in the BIOPEP-UWM data to ensure the novelty of the research peptide segment.

[0060] (7) Molecular docking simulation screening of inhibitory peptides

[0061] ① Preparation of peptide ligands

[0062] According to the Peptide Ranker evaluation, the hydrophilicity and hydrophobicity of the peptide segment and whether it is an allergen, the appropriate peptide segment was selected for molecular docking. Peptide segments with 3-9 amino acids were selected. First, the polypeptide structure plate was prepared on the yinfotek.com platform to obtain the 3D structure of the required peptide segment, which was saved as a ligand. The PDBOT file was output by using AutoDock software.

[0063] ② Preparation of thrombin receptor

[0064] The 3D structure of thrombin derived from human body (PDBID: 2BVR) was searched and downloaded from the PDB database (www.rcsb.org). It was pretreated: remove crystal water, non-standard peptide chain, combine non-polar hydrogen, combine lone pair electrons, remove solvent molecules, add all hydrogen atoms, recalculate Gasteiger charges and adjust atom types, and convert it to pdbqt format for standby.

[0065] ③ Molecular docking method and evaluation

[0066] AutoDock was used to dock the ligand peptide segment and the receptor thrombin protein. The PDBOT files of thrombin protein and ligand peptide were opened respectively, and the specific parameters such as GridBox binding site, docking algorithm and docking parameters were set according to the steps of molecular docking. The final docking result was obtained by running AutoDock. According to the binding energy of the docking result, the best conformation of the peptide segment and thrombin was screened, and the 3D image of the best conformation was viewed using PyMol software and the best conformation was further analyzed.

[0067] The results are as follows:

[0068] The computer molecular simulation docking shows that the peptide segment under study can dock with thrombin. The negative binding energy indicates that the target bioactive peptide can form a stable complex with thrombin. The lowest binding energy is PPHG (4-peptide). The relevant properties of the polypeptide PPHG are shown in Table 2, and the mass spectrum of PPHG is shown in Figure 3 .

[0069] Table 2 Relevant properties of polypeptide PPHG

[0070]

[0071] The 3D image of the optimal conformation by computer molecular simulation is shown in Figure 4 . Blue represents PPHG and green represents GA. It can be seen from the figure that hydrogen bonds are generated when PPHG and GA interact, and the bond lengths of the hydrogen bonds are . Generally, the shorter the length of the hydrogen bond, the greater the force. The docking results show that the binding energy of PPHG and thrombin is the lowest, and the binding ability is the highest, so PPHG is selected for artificial synthesis and further study.

[0072] Example 2 Determination of thrombin inhibitory activity of polypeptide PPHG

[0073] (1) According to the amino acid sequence of PPHG, a polypeptide PPHG was synthesized by a commissioned synthesis company (Nanjing Source Peptide Biotechnology Co., Ltd.).

[0074] (2) According to the aforementioned method for determining thrombin inhibitory activity, the effect of thrombin inhibitory peptide PPHG on the activity of thrombin was determined at concentrations of 5, 10, 15, 20, and 25 mg / mL, respectively.

[0075] The results are shown in Figure 5 . It can be seen that the inhibitory effect of PPHG on thrombin increases with increasing concentration. The logarithm of the added concentration (X) of PPHG is taken, and a linear regression equation Y = 20.6895lnX - 22.7213 between the inhibitory rate (Y) and the logarithm of the added concentration (X) is established, R 2 = 0.9901. The IC50 value is calculated. The IC50 value of PPHG for thrombin is 30.69 ± 0.54 mg / mL.

[0076] Example 3 Determination of thrombin inhibitory activity of gallic acid

[0077] According to the aforementioned method for determining thrombin inhibitory activity, the effect of GA on the activity of thrombin was determined at concentrations of 3, 4, 5, 6, and 7 mg / mL, respectively.

[0078] The results are shown in Figure 6As shown, it can be seen that the inhibitory effect of GA on thrombin increases with increasing concentration. The logarithm of the added GA concentration (X) is taken, and a linear regression equation Y = 16.8183lnX - 16.4328 is established between the inhibition rate (Y), R 2 = 0.9913, and the IC50value is calculated. The IC50value of GA for thrombin is 45.08 ± 0.08 mg / mL.

[0079] Example 4 Determination of the inhibitory activity of polypeptide PPHG and GA in combination on thrombin

[0080] (1) PPHG and GA in combination, PPHG concentrations were 5, 10, 15, 20, 25 mg / mL, and GA concentrations were 3, 4, 5, 6, 7 mg / mL. Different concentration combinations of PPHG and GA were set up, a total of 25 groups, as shown in Table 3, and the effect of different concentration combinations of PPHG and GA in combination on thrombin activity was determined according to the method described above.

[0081] (2) The SynergyFinder website was used to evaluate the interaction between the inhibitory peptide and GA according to the zero-interaction potency (ZIP) algorithm. The ZIP synergy score of PPHG and GA for thrombin inhibition was calculated by Synergyfinder 3.0 software. ZIP synergy scores greater than 0 are considered to be synergistic (red area), and scores greater than 10 are considered to be strong synergistic. In ZIP heat map analysis, the synergistic effect of drug combination can be analyzed by the depth of color. The deeper the color (red), the stronger the synergistic effect, and the lighter the color (blue), the more obvious the antagonistic effect. The synergy plot is shown in Figure 7 .

[0082] The ZIP model of different concentration combinations of PPHG and GA shown in Table 3 shows that the inhibitory effect of PPHG and GA on thrombin is synergistic. Among them, when the concentration ratio of PPHG and GA is 5:2, the synergistic inhibition rate of PPHG / GA on thrombin is 38.76% ± 2.05%, and the synergy score of this combination is the highest, with a ZIP of 13.50, indicating that the synergistic effect between the two is the best at this combination concentration.

[0083] Table 3 Effect of polypeptide PPHG and GA in combination on thrombin activity

[0084]

[0085]

[0086] Example 5 Black date active peptide / gallic acid-β-cyclodextrin inclusion complex

[0087] (1) Preparation of PPHG / GA-β-CD inclusion complex

[0088] Mix 10 mg / mL PPHG and 4 mg / mL GA, then mix PPHG / GA with β-CD at 1:2 (w / w), dilute the mixture of PPHG / GA and β-CD in 50 mL water, stir at room temperature in the dark for 5 h, then place in the dark for 12 h. Place the resulting solution in a 1 kDa ultrafiltration centrifuge tube, centrifuge at 3500 rpm for 20 min, take the cut-off liquid and freeze at -80°C, then dry in a vacuum freeze dryer for 24 h to obtain PPHG / GA-β-CD inclusion complex, as shown in Figure 8 .

[0089] (2) Determination of the encapsulation efficiency of PPHG / GA-β-CD inclusion complex

[0090] Draw the standard curve with PPHG and GA concentrations as the horizontal axis and the absorbance value as the vertical axis, respectively, to obtain the standard curve equation of PPHG y = 6.0752x + 0.0371, R 2 = 0.9935 and the standard curve equation of GA y = 0.1061x + 0.0006, R 2 = 0.9926. When determining the concentration of PPHG / GA not embedded on the β-CD inclusion complex liquid, take a certain amount of inclusion complex liquid in a 1 kDa ultrafiltration centrifuge tube, centrifuge at 3500 rpm for 20 min; take 1 mL of supernatant and centrifuge to obtain the liquid containing unembedded PPHG / GA in a 25 mL brown colorimetric tube, the rest of the steps are the same as determining the standard curve, and the concentration is calculated according to the standard curve equation. Calculate the encapsulation efficiency of PPHG / GA according to the formula.

[0091]

[0092] In the formula, C PPHG1 — the concentration of PPHG added during the preparation process, mg / mL; C PPHG2 — the concentration of PPHG in the filtrate after ultrafiltration centrifugation, mg / mL.

[0093]

[0094] In the formula, C GA1 — the concentration of GA added during the preparation process, mg / mL; C GA2 — the concentration of GA in the filtrate after ultrafiltration centrifugation, mg / mL.

[0095] As Figure 9As shown in the figure, with the increase of the molecular weight ratio of β-CD to PPHG / GA, the encapsulation efficiency showed a trend of first increasing and then decreasing. When the mass ratio was 1:2, the encapsulation efficiency was the largest, at which time the PPHG encapsulation efficiency was 81.15%±1.68% and the GA encapsulation efficiency was 80.52%±0.11%.

[0096] (3) Fourier transform infrared spectroscopy analysis of PPHG / GA-β-CD inclusion complex

[0097] The infrared spectra of PPHG, GA, and the PPHG / GA-β-CD inclusion complex were measured at room temperature using a Fourier transform infrared spectrometer. The samples were mixed and ground with potassium bromide at a ratio of 1:100. The mixed sample powder was placed in a tableting mold and pressed into tablets, ensuring that the surface of the sample tablets was flat, transparent, and free of cracks and bubbles. The infrared spectra of β-CD and the PPHG / GA-β-CD inclusion complex were measured in the wavelength range of 400-4000 cm -1 .

[0098] like Figure 10 As shown in the inclusion complex spectrum, PPHG is detected at 3423 cm -1 The peak red-shifted to 3386 cm -1 , at 2919cm -1 The peak is blue-shifted to 2929 cm -1 , at 1670cm -1 The peak is blue-shifted to 1673 cm -1 ; The five characteristic peaks of GA are at 3282, 1714, and 1622 cm -1 The three characteristic peaks disappeared, 3370cm -1 、1022cm -1 The characteristic peaks at the two sites were blue-shifted respectively; the results of Fourier transform infrared spectroscopy also verified the results of the docking of PPHG and GA molecules, indicating that the PPHG-GA complex was formed through non-covalent interactions.

[0099] Compared with β-CD, the peak position of β-CD inclusion complex changed from 3355.76 cm -1 and 2926.2cm -1 blue shifted to 3386.40 cm -1 and 2928.78cm -1 Compared with the characteristic curves of PPHG, GA, and β-CD, the curve of the β-CD inclusion complex shows that the characteristic peaks of PPHG and GA either disappear or shift, and the morphology of the absorption peak is basically consistent with that of the β-CD curve, indicating that PPHG and GA have successfully penetrated into the cavity of β-CD to form an inclusion complex.

[0100] (4) Stability determination of PPHG / GA-β-CD inclusion complex

[0101] ① The temperature stability of the prepared PPHG / GA-β-CD inclusion complex was determined. The inclusion complex was dissolved in distilled water to prepare a 1 mg / mL solution, which was heated in a water bath at 0, 4, 20, 40, 60, 80, and 100°C for 20 min, and then cooled immediately. The retention rate of PPHG and GA was determined. The retention rate of polypeptide PPHG and GA was calculated according to the formula.

[0102]

[0103] M PPHG1 The content of PPHG dissolved after treatment of the PPHG / GA-β-CD inclusion complex, mg / mL; M PPHG2 The total content of PPHG in the PPHG / GA-β-cyclodextrin inclusion complex before treatment, mg / mL.

[0104]

[0105] M GA1 The content of GA dissolved after treatment of the PPHG / GA-β-CD inclusion complex, mg / mL; M GA2 The total content of GA in the PPHG / GA-β-cyclodextrin inclusion complex before treatment, mg / mL.

[0106] As shown in Figure 11 , with the increase of temperature, the retention rate of PPHG and GA in the inclusion complex showed a continuous decrease, and the higher the temperature, the poorer the stability of the inclusion complex. At 0°C and 4°C, PPHG and GA in the inclusion complex hardly escaped, and the PPHG / GA-β-CD inclusion complex could be better preserved.

[0107] ② The light stability of the prepared PPHG / GA-β-CD inclusion complex was determined. The PPHG / GA-β-CD inclusion complex was dissolved in distilled water to prepare a 1 mg / mL solution, which was then placed in the dark, indoor natural light, and sunlight for 10 h, and then the absorbance was measured again to calculate the retention rate of PPHG and GA. As shown in Figure 12 , under the light-free condition, the retention rate of PPHG and GA was the highest, and the active substances could be retained to a greater extent.

[0108] The above examples only express several embodiments of the present application, which are described in more detail and in detail, but cannot be understood as limiting the scope of the patent. It should be noted that for ordinary skilled persons in the art, the above embodiments can be deformed, combined, and improved without departing from the concept of the present patent, and these all belong to the protection scope of the present patent. Therefore, the protection scope of the present patent should be subject to the claims.

Claims

1. A black date polypeptide, characterized in that: The black date polypeptide is PPHG, and the amino acid sequence is: Pro-Pro-His-Gly.

2. Use of the inhibitory peptide PPHG according to claim 1 in inhibiting thrombin activity, or in preparing a product for inhibiting thrombin activity, or in preparing an antithrombotic product.

3. A composition comprising a thrombin inhibitory peptide PPHG, characterized in that: The composition uses inhibitory peptides PPHG and GA as active ingredients. The composition contains PPHG and GA, and the mass ratio of PPHG to GA in the composition is 1-100:

1.

4. The method for preparing the β-CD inclusion compound of the PPHG / GA composition according to claim 3, characterized in that: The steps are as follows: 10 mg / mL PPHG and 4 mg / mL GA were mixed. The PPHG / GA mixture was then mixed with β-CD at a 1:2 (w / w) ratio. The mixture was diluted in 50 mL of distilled water and stirred in the dark at room temperature for 5 hours, followed by 12 hours in the dark. The solution was placed in a 1 kDa ultrafiltration centrifuge tube and centrifuged at 3500 rpm for 20 minutes. The retained liquid was vacuum freeze-dried for 24 hours to obtain the PPHG / GA-β-CD inclusion complex.

5. Use of the PPHG / GA-β-CD inclusion compound according to claim 3 in inhibiting thrombin activity, or in preparing a product for inhibiting thrombin activity, or in preparing an antithrombotic product.

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