A modified analgesic peptide AP6 derived from Bufo bufo gargarizans and its application

Abstract

The present invention discloses a modified analgesic polypeptide AP6 derived from Bufo bufo gargarizans and its application. The amino acid sequence of the modified analgesic polypeptide AP6 is shown in SEQ ID NO.1, and is obtained by modifying the amino acid sequence of the analgesic peptide AR1-6 shown in SEQ ID No.2. The modified analgesic polypeptide AP6 has obvious anti-inflammatory activity, simple structure, and convenient acquisition. It can be used in the preparation of drugs for treating or relieving inflammation, and therefore has good clinical application prospects.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and specifically relates to a modified analgesic polypeptide AP6 derived from Bufo bufo gargarizans and an application thereof. Background Art

[0002] CI5 is a peptide with good anti-inflammatory and analgesic functions that was identified and screened from Huachansu injection. It can significantly inhibit the protein expression levels of IL-6, IL-1β, TNF-α and PGE2 in the tissues of inflammatory pain model mice and reduce the expression of COX-2 and P-erk. Some researchers used the carrageenan mouse foot swelling model as the research basis and used mRNA-seq sequencing technology to perform bioinformatics analysis on the mouse feet of the model group and CI5 group. The results showed that CI5 may relieve inflammatory pain through the MAPK signaling pathway.

[0003] At present, peptides, as a class of biologically active molecules, have received widespread attention and application in medical fields such as disease treatment and tissue repair. In addition, artificially synthesized peptides are used for biomaterial modification, avoiding the immune risks of biological proteins, with low toxic side effects, high biological activity and good compatibility. The development of new peptide analgesics has become a new trend. Summary of the invention

[0004] The purpose of the present invention is to provide a modified analgesic polypeptide AP6 derived from Bufo bufo gargarizans and its application. The analgesic polypeptide AP6 has significant anti-inflammatory activity, a simple structure and convenient preparation.

[0005] In order to achieve the above-mentioned purpose, the present invention adopts the following technical solutions:

[0006] The present invention provides a modified analgesic polypeptide AP6 derived from Bufo bufo gargarizans, the amino acid sequence of which is shown in SEQ ID NO.1.

[0007] Furthermore, the modified analgesic peptide AP6 is obtained by modifying the amino acid sequence of the analgesic peptide AR1-6; and the analgesic peptide AR1-6 is obtained by modifying the amino acid sequence of the analgesic peptide CI5.

[0008] Furthermore, the amino acid sequence of the analgesic peptide CI5 is shown as SEQ ID No.3; the amino acid sequence of the analgesic peptide AR1-6 is shown as SEQ ID No.2.

[0009] Furthermore, the glycine at position 2 and the leucine at position 3 of the amino acid sequence of the analgesic peptide AR1-6 were simultaneously replaced with hydrophobic alanine to obtain a modified analgesic peptide AP6 with an amino acid sequence of AAAQFP.

[0010] Furthermore, the modified analgesic polypeptide AP6 has significant anti-inflammatory activity.

[0011] The present invention provides the use of the modified analgesic polypeptide AP6 in the preparation of anti-inflammatory drugs.

[0012] Furthermore, the anti-inflammatory drug contains the modified analgesic polypeptide AP6 at a concentration of 5 mg / ml-10 mg / ml.

[0013] Furthermore, the modified analgesic polypeptide AP6 can significantly inhibit inflammation.

[0014] The present invention provides an anti-inflammatory drug, wherein the active ingredient in the drug is a modified analgesic polypeptide AP6 having an amino acid sequence as shown in SEQ ID No. 1.

[0015] Furthermore, the dosage forms of the drug include injectable dosage forms and oral dosage forms; the dosage forms include powders, tablets, granules, capsules, solutions, emulsions, suspensions, injections, transdermal preparations or sprays.

[0016] Compared with the prior art, the advantages and technical effects of the present invention are:

[0017] The present invention is a novel analgesic peptide AP6 obtained by modifying analgesic peptide CI5. The amino acid sequence of the peptide AP6 is searched and compared in a protein database, and no identical peptide is found. The peptide AP6 has the advantages of simple structure and good anti-inflammatory effect. The present invention verifies through experiments that the peptide AP6 has significant anti-inflammatory activity, and can be used to prepare anti-inflammatory drugs. Compared with other anti-inflammatory analgesic peptides, the analgesic peptide AP6 of the present invention has a simple structure, is easy to obtain, and has broad clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The results are the first writhing time of mice after injection of analgesic peptide AR1-6 and the number of acetic acid writhing times of mice within 20 minutes.

[0019] Figure 2 These are the results of phase I and phase II in formalin experimental mice after injection of analgesic peptide AR1-6.

[0020] Figure 3 The results show the swelling of mouse toes after injection of analgesic peptide AR1-6.

[0021] Figure 4 The results of mouse paw swelling after injection of analgesic peptides G2A, L3A, Q4A, F5A and P6A.

[0022] Figure 5 The results show the swelling of mouse toes after injection of the analgesic peptide AP6. DETAILED DESCRIPTION

[0023] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. The experimental methods in the following embodiments without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer.

[0024] Example 1

[0025] 1. Obtaining analgesic peptides

[0026] According to the amino acid sequence (AGLQFPVGR) of the analgesic peptide CI5, it was modified and then synthesized by solid phase synthesis. The anti-inflammatory and analgesic activities were screened using the acetic acid writhing test, formalin model test and carrageenan model test, respectively, to obtain the analgesic peptide CI5 modified analgesic peptide AR1-6 with anti-inflammatory and analgesic activities. After further modification and synthesis, the anti-inflammatory activity was screened using the carrageenan model, and finally the modified analgesic peptide AP6 was obtained.

[0027] 2. Molecular identification of analgesic peptides

[0028] (1) Determination of amino acid sequence:

[0029] The crude product of artificial solid phase synthesized polypeptide was purified by high performance liquid chromatography, and the purified analgesic peptides AR1-6, G2A, L3A, Q4A, F5A, P6A and AP6 were determined by Edman degradation method on a fully automatic protein sequencer to determine the complete amino acid sequences of the analgesic peptides AR1-6, G2A, L3A, Q4A, F5A, P6A and AP6. The results showed that the amino acid sequences of the analgesic peptides AR1-6, G2A, L3A, Q4A, F5A, P6A and AP6 were AGLQFP, AALQFP, AGAQFP, AGLAFP, AGLQAP, AGLQFA and AAAQFP (SEQ ID NO.1), respectively.

[0030] (2) Mass spectrometry molecular weight determination:

[0031] The precise molecular weights of the purified analgesic peptides AR1-6, G2A, L3A, Q4A, F5A, P6A and AP6 were detected by matrix-assisted laser desorption time-of-flight mass spectrometry, and the instrument used was Bruker's AutoflexIIITOF / TOF mass spectrometer. The results showed that the molecular weights of the analgesic peptides AR1-6, G2A, L3A, Q4A, F5A, P6A and AP6 determined by mass spectrometry were 631.73Da, 645.76Da, 589.65Da, 574.68Da, 555.63Da, 605.69Da and 645.76Da, respectively.

[0032] Example 2: Determination of anti-inflammatory and analgesic activity of analgesic peptides

[0033] In order to gain a deeper understanding of the anti-inflammatory and analgesic activities of analgesic peptides AR1-6, G2A, L3A, Q4A, F5A, P6A and AP6, the present invention conducted a study using the classic acetic acid writhing animal pain model, the formalin animal pain model and the carrageenan animal pain model. The results showed that analgesic peptide AR1-6 showed good anti-inflammatory and analgesic activities in the above three animal pain models, analgesic peptides G2A, L3A and AP6 showed significant anti-inflammatory activities in the carrageenan pain model, and the anti-inflammatory effects of analgesic peptides Q4A, F5A and P6A were not as good as the anti-inflammatory effects of analgesic peptide AR1-6, as described below:

[0034] 1. Analgesia experiment of acetic acid writhing pain model in mice

[0035] The experimental animals used were 4-week-old Kunming mice (weight 18-22g) of SPF grade. The mice were randomly divided into a control group (sterile saline), a positive drug group (50mg / kg aspirin), and an AR1-6 group (50mg / kg analgesic peptide AR1-6), with 10 mice in each group. The analgesic peptide AR1-6 was dissolved in sterile saline to a dilution concentration of 5mg / ml. The mice in the AR1-6 group were injected with 200μl of 5mg / ml analgesic peptide AR1-6 dilution into the left peritoneum; the mice in the positive drug group were injected with an equal volume of 50mg / kg aspirin into the left peritoneum; and the mice in the control group were injected with an equal volume of sterile saline into the left peritoneum. 30 minutes later, 250μl of 0.7% acetic acid solution was injected into the right peritoneum of the three groups of mice, and the time of the first writhing of the mice after the injection of acetic acid and the number of writhings within 20 minutes were recorded respectively.

[0036] like Figure 1 As shown, the analgesic peptide AR1-6 can prolong the time before the first writhing of mice and reduce the number of acetic acid writhing of mice, showing that the analgesic peptide AR1-6 has a significant analgesic effect on the pain caused by acetic acid in animals.

[0037] 2. Formalin-induced pain experiment in mice

[0038] Mice were randomly divided into a control group (sterile saline), a positive drug group (50 mg / kg aspirin), and an AR1-6 group (50 mg / kg analgesic peptide AR1-6), with 10 mice in each group. The analgesic peptide AR1-6 was dissolved in sterile saline to a dilution concentration of 5 mg / ml. Mice in the AR1-6 group were intraperitoneally injected with 200 μl of analgesic peptide AR1-6 at a concentration of 5 mg / ml; mice in the positive drug group were intraperitoneally injected with an equal volume of 50 mg / kg aspirin; and mice in the control group were intraperitoneally injected with an equal volume of sterile saline. 30 minutes after the intraperitoneal injection, 20 μL of 3% formalin solution was injected into the right hind foot of the mouse, and the licking time of phase I (0 5min) and phase II (15 30min) was recorded.

[0039] like Figure 2 As shown, the experimental results showed that the analgesic peptide AR1-6 can significantly reduce the paw licking time of mice in phase I and phase II, and both are lower than that of the aspirin group, indicating that the analgesic peptide AR1-6 not only has a significant analgesic effect on acute pain, but also has a better analgesic effect on tonic pain.

[0040] 3. Mouse carrageenan foot swelling experiment

[0041] The anti-inflammatory effects of analgesic peptides AR1-6, G2A, L3A, Q4A, F5A, P6A and AP6 were evaluated by carrageenan-induced mouse foot swelling experiment. The mice were randomly divided into a normal group, a model group (carrageenan), a positive group (10 mg / kg indomethacin), and analgesic peptide group (50 mg / kg analgesic peptide AR1-6 / G2A / L3A / Q4A / F5A / P6A / AP6), with 6 mice in each group. The analgesic peptides were dissolved in sterile saline to a dilution concentration of 5 mg / ml. The mice in the normal group and the model group were intraperitoneally injected with 200 μl of sterile saline, the mice in the analgesic peptide group were injected with 200 μl of 5 mg / ml analgesic peptide, and the mice in the positive group were injected with 200 μl of 10 mg / kg indomethacin; 30 minutes after the intraperitoneal injection of the drugs, the right hind paws of the mice in the normal group were injected with 30 μl of saline, the model group were injected with 30 μl of 1% carrageenan, and the mice in the positive group, AR1-6 group, G2A group, L3A group, Q4A group, F5A group, P6A group and AP6 group were injected with 30 μl of 1% carrageenan, respectively. The thickness of the right hind toes of the mice was measured every hour after 1 hour.

[0042] like Figure 3-5As shown in the figure, comparison of the data of the control group and the model group shows that the carrageenan mouse model was successfully established. Both the model group and the positive group reached the peak of swelling within 3 hours. Compared with the model group, the analgesic peptides had different degrees of inhibitory effects on inflammation at 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, and 6 hours. Among them, the anti-inflammatory effects of the analgesic peptides Q4A, F5A, and P6A were not as good as those of the analgesic peptides AR1-6, while the analgesic peptides G2A, L3A, and AP6 showed stronger anti-inflammatory effects.

[0043] In summary, the present invention obtains a new analgesic peptide AP6 by modifying the amino acid sequence of the analgesic peptide CI5 derived from the Chinese giant toad, and verifies through various mouse pain experimental animal model experiments that the analgesic peptide AP6 has a significant anti-inflammatory effect and a wide range of applications. It can be used to treat or relieve inflammation or can be used in the preparation of drugs for treating or inhibiting inflammation.

[0044] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for a person skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to replace some of the technical features therein by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions claimed to be protected by the present invention.

Claims

1. A modified analgesic polypeptide AP6 derived from Bufo bufo gargarizans, characterized in that: The amino acid sequence of the modified analgesic polypeptide AP6 is shown in SEQ ID NO.

1.

2. Use of the modified analgesic polypeptide AP6 according to claim 1 in the preparation of anti-inflammatory drugs.

3. The use according to claim 2, characterized in that: The anti-inflammatory drug contains the modified analgesic polypeptide AP6 at a concentration of 5 mg / ml-10 mg / ml.

4. An anti-inflammatory drug, characterized in that: The active ingredient in the drug is a modified analgesic polypeptide AP6 with an amino acid sequence as shown in SEQ ID No.1.

Images