A zinc ion chelating modified peptide derived from the traditional Chinese medicine American cockroach and its application

Abstract

The invention provides a zinc ion chelating modified peptide derived from the traditional Chinese medicine American cockroach and its application. The sequence of the zinc ion chelating modified peptide is: GHCSPPFAPRL, the three amino acids GHC from the 1st to the 3rd position have a coordination chelating effect on divalent metal zinc ions, and the eight amino acids SPPFAPRL from the 4th to the 11th position are natural polypeptide sequences derived from American cockroach. The metal zinc ion chelating modified peptide adopts a polypeptide solid phase synthesis technology to prepare a linear polypeptide GHCSPPFAPRL, and after purification, a microwave synthesis method is used to chelate the metal zinc ions with the three amino acid residues of GHC; and then an ethanol precipitation method is used to precipitate it from the reaction system. The zinc ion chelating modified peptide has the functions of anti-oxidation, promoting cell migration and / or proliferation, promoting wound healing, etc., and also has low cytotoxicity to red blood cells, and the preparation process is simple and easy to be industrialized on a large scale.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to a zinc ion chelating modified peptide derived from the traditional Chinese medicine American cockroach and application thereof. Background Art

[0002] The American cockroach (Periplaneta americana, English name American cockroach), also known as cockroach, belongs to the Blattaria family. It is an important animal-based Chinese medicine in traditional Chinese medicine. The earliest record of it in traditional medical books can be traced back to the Shennong's Herbal Classic in the Han Dynasty: its efficacy is described as "blood stasis, hard symptoms, cold and heat, breaking up accumulation, throat and pharynx closure, internal cold and infertility." With the development of traditional Chinese medicine, in the Ming Dynasty, Li Shizhen's Compendium of Materia Medica also recorded this unique medicine, describing it as "a body like a silkworm moth, with a red belly and back, and two wings that can fly."

[0003] Although the American cockroach has not yet been included in the Chinese Pharmacopoeia, it has been recorded in the Hunan Provincial Traditional Chinese Medicine Standard (2009 Edition): This product is the dried whole body of the American cockroach Periplaneta americana (Linnaeus), an insect of the Blattidae family. It is caught in summer or raised artificially. It is killed by soaking in ethanol or scalding in boiling water, and then dried in the sun or dried at 60℃. Nature and flavor and meridians: salty, cold, enters the heart, liver, spleen, and kidney meridians. This product has the effects of strengthening the spleen and eliminating malnutrition, promoting blood circulation and unblocking meridians, promoting diuresis and reducing swelling, and astringing sores and promoting tissue regeneration. It is used for hypochondriac pain, lumps, malnutrition, palpitations, asthma, and edema. It is used externally to treat burns caused by water or fire, various wounds, and ulcers.

[0004] Modern medical and pharmaceutical research shows that the American cockroach is rich in amino acids, polysaccharides, cytochromes, various enzymes, as well as mineral elements (among which zinc, copper, and potassium have relatively high contents, 96.11, 103.25, and 14.39 mg / kg, respectively), vitamins, fats, fatty acids, and other ingredients. The effective medicinal ingredients mainly include polypeptides, polyols, and mucosine. In addition, for mineral elements, metallic zinc (Zinc) plays an important role in the efficacy of the American cockroach, and zinc ions participate in many enzymatic reactions related to human health. With the in-depth research on zinc in recent years, it has been found that zinc ions participate in the enzymatic catalytic reactions of more than 300 enzymes, as well as several transcription factors that play their roles. For example, during wound healing, the demand for zinc ions will increase significantly. Studies have shown that Zn 2+ The ion has the following physiological functions: ① Participate in epithelial tissue differentiation; ② Participate in the transport and storage of metallothionein; ③ It has anti-inflammatory effects; ④ Can reduce ultraviolet-induced cell and gene damage; ⑤ Can improve the tolerance of skin fibroblasts to oxidative stress; ⑥ Can improve skin elasticity and reduce wrinkles. Metallic zinc is mainly present in the body as divalent zinc ions Zn2+ It exists in the form of metal-binding enzymes or proteins / peptides. More interestingly, there are many proteins in the human body that require Zn 2+ Ions are involved in maintaining their three-dimensional spatial structure (such as NDM-1 hydrolase and zinc finger proteins), allowing these proteins to exert high catalytic activity or regulate DNA replication or RNA transcription.

[0005] Kangfuxin Liquid, a drug currently on the market, is mainly composed of dried extracts of American cockroaches. It has the effects of promoting blood circulation, nourishing yin and promoting muscle growth. It can be taken orally or applied externally. For oral administration: used for blood stasis, stomach pain and bleeding, gastric and duodenal ulcers; and auxiliary treatment of yin deficiency pulmonary tuberculosis. For external use: used for wounds of wounds, trauma, ulcers, fistulas, burns, scalds, and bedsores. Since its main ingredient is extracts of insect bodies, there are some urgent problems to be solved in the actual application of patients: ① The extracts are complex and easily lead to adverse reactions. Monitoring data show that the common adverse reactions of Kangfuxin Liquid are mainly: adverse reactions of the digestive system, nausea, vomiting, abdominal distension, abdominal pain, diarrhea, etc. Adverse reactions at the site of application, such as redness, swelling, pain, etc. Adverse skin reactions, such as rashes and itching, etc. Other adverse reactions, such as dizziness, headache, chest tightness, palpitations, flushing, dyspnea, allergic reactions, etc., have also been reported in individual cases of local numbness and anaphylactic shock. ② The quality stability of drug batches is difficult to control. The quality of Chinese patent medicines (extracts) is affected by the quality of medicinal materials, and there are many uncontrollable factors. Although powerful pharmaceutical companies have built their own medicinal material raw material bases that meet GAP requirements in the origin of authentic medicinal materials, there are still unstable medicinal material quality in actual work. ③ The quality standards are difficult to meet the requirements of quality control. Relevant studies have shown that the main active ingredient in Kangfuxin Liquid is polypeptide, and the quality standard uses the total protein content as an indicator, which cannot well reflect the content of the active ingredient.

[0006] Therefore, in view of the bottlenecks and shortcomings of the existing technology: the American cockroach body is not completely dried and deteriorates; the total protein content of the extract is unstable; the impurities in the extract are not completely removed and adverse reactions occur; although the prepared Chinese patent medicine meets the quality standards, there are large differences between batches and other problems, which affect the treatment effect. It is necessary to develop a new type of safe and effective skin repair product that can improve the treatment effect and better control the product quality. Summary of the invention

[0007] The present invention aims to provide a zinc ion chelating modified peptide derived from the traditional Chinese medicine American cockroach and its application, based on the modification of the natural polypeptide SPPFAPRL derived from the traditional Chinese medicine American cockroach, three amino acid residues GHC are introduced at its amino end (N-terminus), glycine G, histidine H, cysteine ​​C can bind to divalent zinc ion Zn through their electron-rich atoms or groups 2+The peptide is modified by coordination and chelation to form a zinc ion chelate peptide derived from the traditional Chinese medicine American cockroach. It has multiple pharmacological functions such as anti-oxidation, promoting cell migration or proliferation, and promoting wound healing.

[0008] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0009] In the first aspect of the present invention, a zinc ion chelating modified peptide derived from the traditional Chinese medicine American cockroach is provided, wherein the amino acid sequence of the zinc ion chelating modified peptide derived from the traditional Chinese medicine American cockroach is shown in SEQ ID NO.1, and the amino acid GHC residue in the peptide chelates the divalent zinc ion Zn 2+ Make modifications.

[0010] In the second aspect of the present invention, there is provided the use of the zinc ion chelating modified peptide derived from the traditional Chinese medicine American cockroach in the preparation of a product for anti-oxidation.

[0011] In the third aspect of the present invention, provided is the use of the zinc ion chelating modified peptide derived from the traditional Chinese medicine American cockroach in the preparation of a product for promoting cell migration and / or proliferation.

[0012] In a fourth aspect of the present invention, provided is the use of the zinc ion chelating modified peptide derived from the traditional Chinese medicine American cockroach in the preparation of a product for promoting wound repair and / or healing.

[0013] Furthermore, the product includes at least one of medicine, cosmetics or daily necessities.

[0014] In the fifth aspect of the present invention, a product for anti-oxidation or promoting wound repair and healing is provided, comprising the zinc ion chelating modified peptide derived from the traditional Chinese medicine American cockroach.

[0015] Furthermore, the product also includes salts and / or excipients acceptable in medicine or daily chemicals.

[0016] Furthermore, the product can be prepared into various pharmaceutical dosage forms or / and daily chemical products such as injections, sterile powders for injection, powders, ointments, creams, gels, microneedles, etc. according to conventional preparation methods.

[0017] Furthermore, the product can be introduced into muscle, endothelium, subcutaneous or mucosal tissue by injection, smearing, application, acupuncture, or other physical or chemical mediated methods, or can be introduced into the body after being mixed and / or wrapped with other substances.

[0018] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0019] 1. The present invention provides a zinc ion chelating modified peptide derived from the traditional Chinese medicine American cockroach and its application. The present invention chelates divalent metal zinc ions by adding three amino acid residues GHC through amide bonds before the amino end of the natural polypeptide sequence SPPFAPRL. The modified peptide has the following advantages:

[0020] (1) Divalent metal zinc ion Zn 2+ The physiological effects in the body are combined and coordinated with the functions of active polypeptides, making it a multifunctional molecule. The molecular weight of the modified peptide is 1181.38Da, and its properties are similar to those of small molecules, which can avoid the immunogenicity caused by large molecules.

[0021] (2) Compared with inorganic substances (such as zinc oxide and zinc carbonate in calamine), peptides chelate zinc ions through coordination bonds, which have the advantages of stable structure, controllable quality, and easy absorption.

[0022] (3) Different from the neurohormone effect of the natural polypeptide sequence SPPFAPRL of American cockroach, the zinc ion chelating modified peptide designed and prepared by the present invention has the functions of anti-oxidation, promoting cell migration and / or proliferation, and promoting wound healing. However, the experiment showed that the natural polypeptide had no effect of promoting cell migration and / or proliferation, no effect of promoting wound healing, and only had a weak antioxidant effect (and there was no statistically significant difference compared with the blank control group).

[0023] 2. The preparation method of the zinc ion chelate modified peptide derived from the traditional Chinese medicine American cockroach provided by the present invention has cheap and easy-to-obtain raw materials, and the preparation process is mature and simple, which can meet the requirements of industrial large-scale production of modern enterprises. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0025] Figure 1 The mass spectrum of the peptide sequence GHCSPPFAPRL prepared by solid phase synthesis;

[0026] Figure 2 This is the HPLC graph of the prepared zinc ion chelated modified peptide from the traditional Chinese medicine American cockroach;

[0027] Figure 3 This is a computer-simulated spatial structure diagram of the zinc ion chelating modified peptide derived from the traditional Chinese medicine American cockroach;

[0028] Figure 4The results are for the hemolytic activity of zinc ion chelated modified peptides derived from the traditional Chinese medicine American cockroach;

[0029] Figure 5 The zinc ion chelate modified peptide derived from the traditional Chinese medicine American cockroach resists the damage of H2O2 to NIH-3T3 cells;

[0030] Figure 6 The zinc ion chelating modified peptide derived from the traditional Chinese medicine American cockroach promotes the proliferation of endothelial cell line HUVEC;

[0031] Figure 7 The zinc ion chelating modified peptide derived from the traditional Chinese medicine American cockroach promotes the migration of fibroblast cell line NIH-3T3;

[0032] Figure 8 Zinc ion chelate modified peptide derived from the traditional Chinese medicine American cockroach promotes wound healing in mice. DETAILED DESCRIPTION

[0033] The present invention will be described in detail below in conjunction with specific implementations and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific implementations and examples are used to illustrate the present invention, rather than to limit the present invention.

[0034] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.

[0035] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or obtained by existing methods.

[0036] The embodiment of the present invention provides a zinc ion chelating modified peptide derived from the traditional Chinese medicine American cockroach and its application, and the overall idea is as follows:

[0037] The natural polypeptide involved in the present invention is derived from the natural polypeptide isolated and identified from the American cockroach Periplaneta americana, a traditional Chinese medicine. The natural polypeptide consists of 8 amino terminal residues, and the sequence of its amino acid residues is: SPPFAPRL. (References: Predel R, Kellner R, Kaufmann R, Penzlin H, G.Isolationand structural elucidation of two pyrokinins from the retrocerebral complex of the American cockroach.Peptides.1997,18(4):473-8.doi:10.1016 / s0196-9781(97)00067-3.)

[0038] The present invention introduces three amino acid residues, namely glycine G, histidine H and cysteine ​​C, before the amino terminus (N terminus) of the natural polypeptide sequence SPPFAPRL isolated from American cockroaches through amide bond linkage. The sequence of the complete zinc ion chelating modified peptide is GHCSPPFAPRL. The spatial structure of the molecule simulated by calculation and the design idea of ​​the modified peptide molecule are as follows:

[0039]

[0040] As a specific embodiment, the preparation method of the zinc ion chelating modified peptide derived from the traditional Chinese medicine American cockroach comprises:

[0041] The peptide GHCSPPFAPRL was synthesized by solid phase peptide synthesis technology; then the three amino acid residues GHC introduced were reacted with divalent zinc ions Zn by microwave reaction. 2+ Chelating to form a metal zinc ion chelate modified peptide; finally, precipitating the final product zinc ion chelate modified peptide in a certain concentration of ethanol solution, filtering and freeze-drying to obtain the final product;

[0042] The zinc ion chelating modified peptide derived from the traditional Chinese medicine American cockroach was prepared by peptide solid phase synthesis technology. The peptide is composed of 11 amino acid residues, has a theoretical molecular weight of 1181.38Da, and a theoretical isoelectric point pI of 8.26.

[0043] The modified peptide has a strong antioxidant effect on mouse fibroblasts NIH-3T3, and can also promote the migration of fibroblasts NIH-3T3 and the proliferation of human vascular endothelial cells HUVEC; animal level experiments show that it has the effect of accelerating wound healing. Hemolytic activity studies have shown that it also has low cytotoxicity to red blood cells, and the preparation process is simple, which meets the requirements of industrial large-scale production. It has good development value and application prospects, and can be used as a candidate drug for in-depth research.

[0044] The following will describe in detail a zinc ion chelating modified peptide derived from the traditional Chinese medicine American cockroach and its application in combination with examples and experimental data.

[0045] Example 1: Zinc ion chelating modified peptide derived from the traditional Chinese medicine American cockroach

[0046] 1. Preparation of the linear polypeptide sequence GHCSPPFAPRL derived from the zinc ion chelating modified peptide intermediate of the traditional Chinese medicine American cockroach designed by the present invention

[0047] In the synthesis of this linear peptide segment, 9-fluorenylmethyloxycarbonyl (FMOC) was used as the protecting group of the amino terminal, 4-methyl-benzhydrylamine resin (4-methyl-benzhydrylamine resin·HCl, MBHAresin) was selected as the solid phase carrier for peptide synthesis, and HOBt / DCC was used as the condensing agent for the reaction to extend the peptide chain from the carboxyl terminal to the amino terminal (C terminal → N terminal). The peptide sequence GHCSPPFAPRL containing 11 amino acid residues was cleaved from the MBHA resin using a mixture of 90.0% trifluoroacetic acid, 4.0% water and 6.0% triisopropylsilane (TIA) (mass percentage). After repeated precipitation with ether, it was purified by preparative reverse HPLC RP-HPLC.

[0048] Purification process: Use C18 reverse phase preparation column (250mm×20mm, 5μm); mobile phase: 1‰ trifluoroacetic acid, 0%~65% (volume percentage) acetonitrile as mobile phase, gradient elution at a flow rate of 1.5mL / min. Collect the main peak eluate, freeze it at -80℃ for 6 hours, put it into a freeze dryer, freeze it into loose solid powder and place it in a -20℃ refrigerator for use. ESI-Q-TOF-MS identification shows that its molecular weight is consistent with the theoretical molecular weight of the peptide intermediate to be prepared, m / z 1182.68[M+H] + ,m / z 591.15[M+2H] 2+ ,m / z 395.42[M+3H] 3+ , ( Figure 1 ).

[0049] 2. Preparation of zinc ion chelated modified peptide derived from the traditional Chinese medicine American cockroach according to the present invention

[0050] The polypeptide prepared in Example 1 was prepared into a polypeptide solution with a concentration of 5 mg / mL using Milli-Q ultrapure water. 2+=1:3 molar ratio, add concentrated zinc chloride aqueous solution, adjust the solution pH to about 6.5 with hydrochloric acid, and evacuate the reaction system. In the ultrasonic instrument, set the ultrasonic water bath temperature to 65℃-70℃, use low ultrasonic power to promote the combination of polypeptide and zinc ion, react for 4 hours, slowly add 3-5 volumes (v / v) of ethanol, centrifuge at 4℃8000rpm, and discard the supernatant (remove unbound metal ions and unbound peptide segments). Wash the precipitate with a small amount of anhydrous ethanol several times, and after freeze-drying, the peptide-metal zinc ion conjugate of the present invention is obtained, that is, the zinc ion chelated modified peptide derived from the traditional Chinese medicine American cockroach, and its HPLC chart is as follows Figure 2 As shown in FIG. 1 , the retention time is 10.31 min and the purity is >99%. The spatial structure of the metal zinc ion chelate modified peptide simulated by computer is shown in FIG. Figure 3 The above results show that the present invention successfully prepared the zinc ion chelating modified peptide derived from the traditional Chinese medicine American cockroach.

[0051] Example 2: Hemolytic activity of the zinc ion chelated modified peptide derived from the Chinese medicine American cockroach according to the present invention

[0052] Fresh blood was obtained from healthy male volunteers, and human red blood cells were separated and prepared into a suspension. Sterile PBS buffer was used as a negative control and sterile Milli-Q ultrapure water was used as a positive control to determine the hemolytic activity of the zinc ion chelated modified peptide.

[0053] The specific steps are as follows: collect whole blood with a heparin anticoagulation tube, gently invert the blood to fully anticoagulate, centrifuge at room temperature at 1000rpm for 10min, absorb the upper plasma and retain the lower red blood cells; add 3 to 5 times the volume of sterile PBS buffer, gently blow and suck with a pipette to suspend the red blood cells precipitated at the bottom of the centrifuge tube, centrifuge at room temperature at 1000rpm for 6min, discard the supernatant and retain the precipitated red blood cells, repeat the above operation 3 to 5 times until the supernatant becomes colorless; use sterile PBS buffer to make a cell suspension with a concentration of 2% to 3% (v / v) of red blood cells; use sterile physiological saline to configure the zinc ion chelated modified peptide derived from the traditional Chinese medicine American cockroach into solutions with concentrations of 32μmol / L, 16μmol / L, 8μmol / L, 4μmol / L and 2μmol / L; add 100μL of the zinc ion The chelated peptide solution and 100 μL of red blood cell suspension were mixed and added to a 96-well plate, so that the final concentrations of the peptides were 16 μmol / L, 8 μmol / L, 4 μmol / L, 2 μmol / L and 1 μmol / L, respectively; the negative control group was red blood cells suspended in sterile PBS buffer, and the positive control was red blood cells suspended in sterile Milli-Q ultrapure water; the sample was placed in a constant temperature shaker and incubated at 80 rpm to 120 rpm for 40 min at (36.5±0.5)°C; the sample was centrifuged at 5000 rpm for 5 min at room temperature, and 150 μL of supernatant was taken from each well and transferred to another clean 96-well plate; the absorbance at a wavelength of 490 nm was measured with a full-wavelength microplate reader, and the hemolysis percentage was calculated by the following formula, where H is the absorbance at a wavelength of 490 nm: Hemolysis rate (%) = (H sample -H negative ) / (H positive -H negative )×100%, such as Figure 4 shown.

[0054] Figure 4 The results showed that the zinc ion chelated modified peptide derived from the traditional Chinese medicine American cockroach described in the present invention did not show obvious hemolysis (hemolysis rate ≤ 4%) when the final concentration was 16 μmol / L.

[0055] Example 3: The zinc ion chelating modified peptide of the present invention resists H2O2 damage to NIH-3T3 cells

[0056] Select NIH-3T3 cells with good growth status, change the medium without serum the night before, starve overnight, remove the cells the next morning, digest the cells with trypsin, centrifuge at 1000rpm for 5min, remove the cells, discard the supernatant, wash the cells with DMEM medium, and set the cells at about 6×10 3The cells were inoculated into two 96-well plates at a density of 100 cells / well and placed in a constant temperature incubator at 37°C and 5% CO2 for overnight culture. After the cells adhered to the wall, they were taken out and replaced with fresh culture medium. H2O2 and zinc ion chelating peptide derived from American cockroach were filtered with a 0.22 μm filter and divided into two groups. In the first group, different concentrations of H2O2 were added into the 96-well plates, and the final concentrations of H2O2 were 62.5, 125, 250, 500, and 1000 μmol / L, respectively. A group without H2O2 was set as a blank control group. After culturing for 2 hours, zinc ion chelating peptide derived from American cockroach was added to make the final concentration of 16 μmol / L, and the culture was continued for 24 hours. The next day, the culture medium was discarded and the wells were washed twice with sterile PBS buffer. 100 μL of 5 mg / mL MTT solution was added to each well, and the wells were cultured for 4 hours, then aspirated and discarded. 150 μL of DMSO was added to dissolve the formazan, and the wells were placed in a 37°C constant temperature shaker and shaken at a low speed of 80-100 rpm for 10-15 min to completely dissolve the crystals. The absorbance at 490 nm was measured with an enzyme marker.

[0057] The zinc ion chelating modified peptides designed by the present invention and derived from American cockroaches had no obvious toxicity to NIH-3T3 cells at different concentrations (≤16 μmol / L). Figure 5 A).

[0058] After adding different concentrations of H2O2, cell viability decreased to varying degrees, and the cell viability decreased more significantly with increasing concentrations, showing a dose-dependency. After adding the zinc ion chelating modified peptide derived from American cockroaches described in the present invention at a final concentration of 16 μmol / L, the viability of cells damaged by H2O2 oxidative damage at different concentrations was restored, and the difference was statistically significant (P<0.01). In particular, after the cells damaged by low concentrations of H2O2 (62.5 μmol / L H2O2) were treated with the zinc ion chelating modified peptide, their cell viability could be restored to the level of the control group ( Figure 5 B) It shows that the zinc ion chelating modified peptide derived from the traditional Chinese medicine American cockroach of the present invention has cellular antioxidant effect.

[0059] Example 4: The zinc ion chelating modified peptide of the present invention promotes the proliferation of endothelial cell line HUVEC

[0060] Take out the cell cryopreservation tube from liquid nitrogen and immediately put it in a 37℃ water bath to thaw until a little ice remains in the tube. After wiping the cryopreservation tube with 75% alcohol, gently blow and aspirate, and transfer the cell suspension to a cell culture dish, add 10mL of Gibco M200 medium (containing 10% FBS and 10% low serum growth factor vascular growth factor S00310) pre-warmed to 37℃. Since HUVEC cells are limited lines, they should be discarded after passage to the 10th generation, and the cells should be revived for experiments.

[0061] The cell passaging operation is as follows: when the fusion rate of HUVEC cells is about 80% to 85%, discard the culture medium, rinse with sterile PBS buffer for 3 times and then suck out the residual liquid, add an appropriate amount of 0.25% trypsin solution to soak the bottom of the culture dish, place it in a 37°C cell culture incubator for 30 to 45 seconds, and add 2 times the volume of complete culture medium to terminate digestion after most cells become round under a microscope, gently blow and aspirate the cells, and pipette an appropriate amount of cell suspension into another new culture dish, and culture it in a 37°C, 5% CO2 constant temperature cell culture incubator.

[0062] The CCK-8 method was used to determine the effect of the zinc ion chelating modified peptide of the present invention on the proliferation of the endothelial cell line HUVEC. The specific operation was as follows:

[0063] 1. Collect cells in logarithmic growth phase, dilute the cell suspension to an appropriate concentration, add 100 μl to each well of a 96-well plate to make the cell density 6,000 cells / well; 2. Culture cells at 5% CO2 and 37°C for 6 to 8 hours; 3. Add 10 ul of the zinc ion chelating modified peptide of the present invention at different concentrations to each well to make the final concentrations 1 μM, 2 μM, 4 μM, 8 μM, and 16 μM, and incubate the culture plate in an incubator for 6 hours; 4. Add 10 μl of CCK-8 solution to each well; 5. Continue to incubate in the cell culture incubator for 1 to 4 hours (take out of the incubator first and visually observe the degree of staining. If color development is difficult, put the culture plate back into the incubator and continue to culture before measuring); 6. Use an enzyme marker to measure the absorbance of each well at a wavelength of 450 nm.

[0064] The results are as follows Figure 6 As shown, it is shown that the zinc ion chelating modified peptide of the present invention has no proliferation effect (nor toxicity) on HUVEC cells when the final concentration is 1-4 μM; it has the effect of promoting cell proliferation ( Figure 6 ), compared with the control group, the difference was statistically significant (P < 0.05); when the concentration increased to 16 μM, the difference was statistically significant (P < 0.01).

[0065] Example 5: The zinc ion chelating modified peptide of the present invention promotes the migration of fibroblast cell line NIH-3T3

[0066] Take mouse fibroblast cell line NIH-3T3 cells cultured to a fusion rate of 70% to 80%, aspirate the culture medium and discard, wash 2 to 3 times with sterile PBS buffer incubated at 37°C; add an appropriate amount of 0.25% trypsin and digest at 37°C for 30 to 45 seconds, gently tap the culture dish or culture bottle to make the cells fall off the bottom of the culture container, add an appropriate amount of DMEM culture medium containing 10% fetal bovine serum (FBS) and gently blow the cells to make a single cell suspension; dilute the cells with the culture medium to adjust the cell concentration to about (3 to 5) × 10 5 cells / mL, inoculated in a 6-well plate, 2mL per well. When the cell fusion rate is 85% to 90%, scratch with a sterilized 200μL gun tip (the scratch width is about 600μm); discard the culture medium, and gently wash the 6-well plate with incubated sterilized PBS buffer to remove the cells peeled off by the gun tip scratch as much as possible. Add 2mL DMEM culture medium to the control group, and add 2mL DMEM culture medium containing the zinc ion chelating modified peptide described in the present invention (the final concentration of the modified peptide is 16μmol / L) to the experimental group. Place the 6-well plate in a cell culture incubator at 37°C and 5% CO2, culture for 12 to 18 hours to observe the migration of cells, and calculate the migration rate of the cells using the following formula:

[0067] Mobility % = (L0-L)÷L0×100%.

[0068] Wherein L0 is the distance between cells after the tip of the gun scratches, and L is the distance between cells after culturing the cells for a period of time (in this example, after 14 hours of cell migration).

[0069] The results are as follows Figure 7 As shown, compared with the control group, the cell migration rate in the experimental group (DMEM complete medium containing 16 μmol / L modified peptide) was higher ( Figure 7 A), the difference was statistically significant P < 0.001 ( Figure 7 B).

[0070] Example 6: The zinc ion chelated modified peptide of the present invention promotes the repair of mouse skin wounds

[0071] Adult 8-week-old male BALB / c mice weighing 20-25 g were divided into two groups (control group and experimental group), with 6 mice in each group, and anesthetized by isoflurane inhalation. After the mice were anesthetized, a biopsy sampler with a diameter of 6 mm was used to artificially create a skin injury in the middle of the back of the mice.

[0072] 10 μL of sterile sodium chloride solution for injection was dripped onto the wound of the control group, and 10 μL of the zinc ion chelating modified peptide of the present invention with a concentration of 16 μmol / L was dripped onto the wound of the experimental group, and the liquid was basically absorbed after 15-30 min. Finally, the wound was covered with a sterile dressing and covered and fixed with a small piece of sterile adhesive tape. The drug was administered once every other day for a total of 5 times for a total of 10 days.

[0073] The results are as follows Figure 8 As shown in Figure 2, the wound healing speed in the experimental group was significantly faster than that in the control group. The representative wounds on the 1st, 5th and 10th days are shown in Figure 2. Figure 8 As shown (scale is 5 mm).

[0074] Example 7. Preparation of different dosage forms

[0075] 1. Tablets

[0076] 1 g of the zinc ion chelated modified peptide derived from the traditional Chinese medicine American cockroach of the present invention, 20 g of starch and 5 g of dextrin are mixed, 50% of pharmaceutical grade polyvinyl pyrrolidone (PVP) is used as a binder, granulated, granulated and tableted to obtain tablets.

[0077] 2. Cream

[0078] Take 0.5 g of the zinc ion chelated modified peptide derived from the traditional Chinese medicine American cockroach, 1.0 g of glyceryl monostearate, 2.5 g of stearic acid, 0.2 g of white vaseline, 1.2 g of liquid paraffin, 1.0 g of lanolin, 0.1 g of triethanolamine, and 15 mL of distilled water, and mix well to obtain the mixture.

[0079] 3. Gel

[0080] Take 5 mL of distilled water, sprinkle an appropriate amount of carbomer (about 0.2 g) on ​​the liquid surface, warm water bath at 45°C and stir to fully swell. Add propylene glycol and mix evenly, then add triethanolamine while stirring to form a gel matrix; dissolve 0.2 g of the zinc ion chelating modified peptide derived from the traditional Chinese medicine American cockroach in PBS buffer (pH=7.2) with a concentration of 0.15 M, and slowly add it to the gel matrix under continuous stirring. Finally, add the remaining distilled water to obtain 10.0 g of the modified peptide gel.

[0081] 4. Lyophilized powder for injection

[0082] Take 3g of the zinc ion chelated modified peptide derived from the Chinese medicine American cockroach and 15g of mannitol described in the present invention, place them in a container, add an appropriate amount of PBS buffer (0.15M, pH7.4) to dissolve, add water for injection to 250mL, shake well, add 8-10g of activated carbon for injection, stir gently at room temperature for about 60 minutes, coarse filter, filter and sterilize with a 0.22μm sterilizing filter membrane, and pack them into 2.0mL bottles. Use a quick freezing method to cool down 10-15℃ per minute to -45-50℃, maintain for 1 hour, and evacuate. After freeze drying for 12-18 hours, slowly heat up under vacuum, with a heating rate of 4-5℃ per hour, stop heating when the temperature rises to room temperature (25℃), take out after the temperature is close to room temperature, cover and seal, and obtain a freeze-dried powder injection.

[0083] Finally, it should be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements that are inherent to such process, method, article, or apparatus.

[0084] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0085] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A zinc ion chelating modified peptide derived from the traditional Chinese medicine American cockroach, characterized in that: The amino acid sequence of the zinc ion chelating modified peptide derived from the traditional Chinese medicine American cockroach is shown in SEQ ID NO.1, and the amino acid GHC residue in the zinc ion chelating modified peptide chelates the divalent zinc ion Zn 2+ Make modifications.

2. Use of the zinc ion chelating modified peptide derived from the traditional Chinese medicine American cockroach as claimed in claim 1 in the preparation of products for antioxidant purposes.

3. Use of the zinc ion chelating modified peptide derived from the traditional Chinese medicine American cockroach as claimed in claim 1 in the preparation of a product for promoting wound repair and / or healing.

4. The use according to claims 2-3, characterized in that: The product includes at least one of medicine, cosmetics or daily necessities.

5. A product for anti-oxidation or promoting wound repair or healing, characterized in that: The invention comprises the zinc ion chelating modified peptide derived from the traditional Chinese medicine American cockroach as claimed in claim 1.

6. The product according to claim 5, characterized in that The product also includes salts and / or excipients acceptable in medicine or daily chemicals.

7. A method for preparing the zinc ion chelated modified peptide derived from the traditional Chinese medicine American cockroach according to claim 1, characterized in that: The method comprises: A polypeptide with a sequence of GHCSPPFAPRL was prepared by using a peptide solid phase synthesis technique and separated and purified by using a semi-preparative high performance liquid chromatography technique to obtain a purified polypeptide; The metal zinc ion is chelated with the three amino acid residues of GHC in the purified polypeptide by microwave synthesis method, and then the metal zinc ion chelated modified peptide is precipitated from the reaction system by ethanol precipitation method to obtain the zinc ion chelated modified peptide derived from the traditional Chinese medicine American cockroach.

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