Frog bradykinin and application thereof
By discovering the bradykinin RPARPPGFTP of the black spot side frog, the existing bradykinin lack of biological activity and major side effects were solved, and a new bradykinin drug with high biological activity was developed to control inflammatory response, hypertension and cardiac protection.
Patent Information
- Application Number
- CN202510331034.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-20
- Publication Date
- 2025-06-17
AI Technical Summary
The existing bradykinin has problems with insufficient biological activity and major side effects in drug development, and it is difficult to effectively use it to control inflammatory responses, hypertension and cardiac protection.
A black spotted frog bradykinin with an amino acid sequence of RPARPPGFTP was discovered and prepared. By efficiently screening bradykinin from amphibians and other species, a novel bradykinin drug with high biological activity was developed.
The new bradykinin Kinin-PN showed high biological activity in biological activity tests, which can effectively promote ileum contraction, and its activity is stronger than BK previously reported, and has huge drug development potential.
Smart Images
Figure CN120157744A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of biomedical technologies, and particularly relates to a frog bradykinin and its applications. Background Art
[0002] Bradykinin is a powerful endothelium-dependent vasodilator that can induce a decrease in blood pressure and the contraction of non-vascular smooth muscles in the bronchi and ileum, and has a pain mechanism. Bradykinin is also involved in inflammation, allergy, and other physiological reactions. Currently, some bradykinins discovered and artificially designed can be used as drugs for controlling inflammatory reactions, hypertension, and cardiac protection processes. The skin of amphibians contains many types of bioactive compounds, including alkaloids, bioorganic amines, steroids, peptides, and proteins, etc. The bradykinin found in amphibian skin tissues is generally considered to play a key role in defending against predators. However, with the in-depth research, the activity of the bradykinin of amphibians has been developed to be higher and higher, and the side effects have been lower and lower, and it has become a new type of polypeptide with great potential for drug development. Summary of the Invention
[0003] The present invention provides a Rana nigromaculata bradykinin, and the amino acid sequence of the bradykinin is RPARPPGFTP.
[0004] The present invention also provides the application of the above-mentioned Rana nigromaculata bradykinin in the preparation of a product for promoting ileum contraction.
[0005] Preferably, the product is a drug.
[0006] More preferably, the Rana nigromaculata bradykinin in the drug is the only active ingredient.
[0007] More preferably, the drug also contains pharmaceutically acceptable excipients.
[0008] More preferably, the dosage form of the drug is any one of tablets, capsules, oral tincture ointments, pills, granules, and powders.
[0009] More preferably, the tablet is any one of enteric-coated tablets, sugar-coated tablets, plain tablets, film-coated tablets, extract tablets, dispersible tablets, sustained-release tablets, and controlled-release tablets.
[0010] More preferably, the capsule is any one of hard capsules, soft capsules, enteric-coated capsules, sustained-release capsules, and controlled-release capsules.
[0011] More preferably, the oral tincture ointment is any one of oral solutions, oral suspensions, oral emulsions, mucilages, mixtures, tinctures, and drops.
[0012] More preferably, the pill is any one of dripping pills, honey pills, water-honey pills, water pills, concentrated pills, and pellets.
[0013] Compared with the prior art, the present invention has the following beneficial effects:
[0014] The bradykinin Kinin-PN discovered in the present invention has higher biological activity than other bradykinins. It is a novel bradykinin drug with great potential for drug development. The present invention provides a new idea for the efficient screening of bradykinins in amphibians and other species, which is of great significance. BRIEF DESCRIPTION OF THE DRAWINGS
[0015] Figure 1 It is the test result of the biological activity of bradykinin Kinin-PN in Example 1. DETAILED DESCRIPTION OF THE INVENTION
[0016] Example 1
[0017] 1.1 Materials and Methods
[0018] 1.1.1 Experimental materials: Pelophylax nigromaculatus
[0019] 1.1.2 Material collection:
[0020] Anesthetize the frog at low temperature and remove 1 square millimeter of frog skin tissue and freeze it in liquid nitrogen for later use.
[0021] 1.1.3 cDNA sequencing:
[0022] To extract mRNA from the frog skin, use an mRNA isolation kit (Dynal Biotech, UK) according to the manufacturer's instructions. Using Creator TM SMART TM cDNA library construction kit (TaKaRa, Japan), primers were designed, F1 (SEQ ID NO.1: 5′-C-C-A-A-A-G-A-G-A-T-C-A-C-C-(T / A)-(T / C)-3′) and F2 (SEQ ID NO.2: 5′-(T / C)-T-(A / C)-T-(A / C)-G-T-G-A-T-G-T-C-A-3′). CDS III / 3′ PCR primer was used as the antisense primer, and Advantage DNA polymerase (TaKaRa, Japan) was used for the PCR reaction. The PCR reaction protocol was as follows: 95°C (5 minutes), then 30 cycles of 95°C (20 seconds), 51°C (30 seconds) and 72°C (30 seconds), and finally 72°C (10 minutes). Finally, the PCR product was cloned into pGEM- Easy vector (Promega, USA). DNA sequencing was performed using a DNA sequencer (Applied Biosystems, USA).
[0023] 1.1.4 Solid-phase synthesis of peptides:
[0024] The 433A peptide synthesizer was used to synthesize polypeptides. The synthesized peptides were purified on a C18 RP-HPLC (3 × 25 cm, Kromasil, Sweden). After sample loading, the concentration of acetonitrile in the elution solvent was increased from 10% to 100% (v / v) within 25 minutes (22 ml / min, linear gradient). The purity of the peptides was identified by a mass spectrometer.
[0025] 1.1.5 Pharmacological activity analysis:
[0026] In this study, the ileum of Wistar rats was used to test bradykinin activity in vitro. After sacrificing the animals, approximately 10 cm of ileum was immediately isolated. Then, the ileum strips were cut into 1-cm segments and carefully washed with Tyrode's solution to remove intestinal contents. The ileum segments were fixed on a force sensor and loaded with 1 g. The ileum segments were immersed in 2 ml of Tyrode's solution, bubbled with air, and maintained at 37 °C. The system was stabilized for 10 min, and then an equal proportion of diluted bradykinin was continuously added to the bath every 10 min to stimulate the contraction changes of smooth muscle, and the biological signals were recorded after each addition. The negative control samples were treated with 3 × Tyrode's solution. The positive control samples were experimented with commercially available bradykinin.
[0027] 1.2 Experimental results
[0028] 1.2.1 Bradykinin structure
[0029] The bradykinin Kinin-PN found in this study has the same structure as the molecular active center of other types of bradykinins, but there are significant differences in the N-terminus and C-terminus of the polypeptide. The N-terminus has a higher content of proline, and the C-terminus is shorter, which may be an important factor in enhancing the biological activity of the polypeptide. The polypeptide sequence is RPARPPGFTP (SEQ ID NO.3).
[0030] 1.2.2 Contractile effect of bradykinin on isolated rat ileum
[0031] The results of the biological activity test of the bradykinin Kinin-PN found in this study are as Figure 1 shown. The data indicate that bradykinin Kinin-PN can cause concentration-dependent contraction of isolated rat ileum, and its biological activity is stronger than that of BK reported previously. When the concentration reaches -8 M, it exhibits relatively high biological activity.
[0032] The above-described embodiments are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.
Claims
1. A bradykinin from Pelophylax nigromaculata, characterized in that: The amino acid sequence of bradykinin is RPARPPGFTP.
2. Use of the bradykinin of Pelophylax nigromaculata according to claim 1 in the preparation of a product that promotes ileal contraction.
3. The use according to claim 2, characterized in that: The product described is a drug.
4. The use according to claim 3, characterized in that: The bradykinin of Pelophylax nigromaculata is the only active ingredient in the medicine.
5. The use according to claim 4, characterized in that: The medicine also contains medically acceptable auxiliary materials.
6. The use according to claim 5, characterized in that: The dosage form of the medicine is any one of tablets, capsules, oral tinctures, pills, granules and powders.
7. The use according to claim 6, characterized in that: The tablet is any one of enteric-coated tablets, sugar-coated tablets, plain tablets, film-coated tablets, extract tablets, dispersible tablets, sustained-release tablets and controlled-release tablets.
8. The use according to claim 6, characterized in that: The capsule is any one of a hard capsule, a soft capsule, an enteric-coated capsule, a sustained-release capsule, and a controlled-release capsule.
9. The use according to claim 6, characterized in that: The oral tincture ointment is any one of an oral solution, an oral suspension, an oral emulsion, a syrup, a mixture, a tincture, and drops.
10. The use according to claim 6, characterized in that: The pills are any one of dropping pills, honey pills, water-honey pills, water pills, concentrated pills and micro pills.