A truncated modified anti-inflammatory peptide derived from the traditional Chinese medicine Periplaneta americana and its application

Abstract

The present invention provides a truncated modified anti-inflammatory peptide derived from the traditional Chinese medicine Periplaneta americana and its application. The amino acid sequence of the truncated modified anti-inflammatory peptide from Periplaneta americana is shown as SEQ ID NO.1. Compared with the natural sequence, the truncated modified peptide has less impact on cell viability and lower hemolytic toxicity. It is convenient to prepare and has a low cost. After purification by semi-preparative reverse high performance liquid chromatography technology, a chemical entity of the truncated modified peptide with a purity > 95% can be obtained. Studies at the cellular level show that the anti-inflammatory mimetic peptide has no obvious effect on the viability of the macrophage cell line RAW264.7 at a concentration up to 64 μM, and has significant inhibitory effects on the inflammatory mediator NO and the pro-inflammatory cytokines IL-1β, IFN-γ, IL-6 and TNF-α induced by LPS, and all show concentration dependence. The present invention has the characteristics of a convenient preparation process, easy large-scale industrial production, low cost, clear pharmacological activity and low toxicity.

Description

Technical Field

[0001] The invention relates to the field of biomedical technology, and in particular to a truncated modified anti-inflammatory peptide derived from the traditional Chinese medicine American cockroach and an application thereof. Background Art

[0002] The American cockroach (Periplaneta americana in Latin, American cockroach in English), commonly known as cockroach, stove ant, etc., is a medium-to-large insect of the genus Periplaneta in the family Blattidae, and has lived on Earth for 350 million years. In traditional Chinese medicine, medicinal insects are a unique and important part of the treasure house of medicinal resources. Chinese medicine has unique and rich experience in using insect-based Chinese medicine to treat difficult diseases, and it is rich in resources and has great potential. The medical monograph "Famous Doctors' Records" records that it has the effect of "clearing the blood vessels". Monographs on traditional Chinese medicine throughout the dynasties have given detailed descriptions of it, such as: ① "Shennong's Herbal Classic" records that it "tastes salty and cold, and is used to treat blood stasis, cold and heat, break up accumulation, throat and pharyngeal numbness, and internal cold and infertility", and lists it as a medium-grade medicine that can both cure diseases and nourish the body; ② "Compendium of Materia Medica" records that "three types of cockroaches, night crawlers, and locusts are all eaten by the Southwestern Yi people."; ③ "Tang Materia Medica" calls the American cockroach "food that reduces lower qi."

[0003] Modern pharmaceutical research has conducted a relatively detailed analysis of its components. American cockroaches contain: ① amino acids, including more than 20 kinds of amino acids including essential amino acids and non-essential amino acids for the human body; ② proteins / peptides, mainly antimicrobial peptides, pyrokinins, neuropeptides, midgut peptides, pharyngeal inhibitory neuropeptides, neurotransmitters, nematode neuropeptides, dystrophin, diuretic peptides, cardiac active peptides, visceral perikinins, short neuropeptides, American cockroach defensins, etc., with a content of up to about 57%; ③ polysaccharides, mannose, sulfated mucopolysaccharides, glucose, xylose, mucosine, fucose, galactose, arabinose, rhamnose, glucosamine hydrochloride, etc.; ④ nucleosides, mainly including uridine, guanosine, inosine, cytosine, uracil, cytidine, hypoxanthine, adenine, thymine, etc.; ⑤ Oils and fats: GC-MS analysis shows that American cockroaches contain fatty acids and fatty acid esters, alkanes, enols, olefinic acids and esters, among which the total amount of fatty acids and fatty acid esters is 26.62%. (Reference: Li Huibiao et al. Research progress on chemical composition of American cockroaches and prevention and treatment of digestive system diseases [J / OL]. Today's Pharmacy)

[0004] Although the American cockroach has not yet been included in the "Chinese Pharmacopoeia", it is included in the "Yunnan Province Traditional Chinese Medicine Standards", "Sichuan Province Traditional Chinese Medicine Standards", "Fujian Province Traditional Chinese Medicine Standards", "Hunan Province Traditional Chinese Medicine Standards" and "Hunan Traditional Chinese Medicine Preparation Specifications", indicating that it is widely used in the above-mentioned regions and has a long history of medicinal use.

[0005] Currently, the drugs developed and marketed for clinical use using Periplaneta americana as raw material after extraction mainly include "Kangfuxin Liquid", "Xinmailong Injection", "Ganlong Capsule", etc.: ① Kangfuxin Liquid is an ethanol extract of dried Periplaneta americana bodies, which can be taken orally or topically. Orally, it can be used for treating stomachache with bleeding, gastric and duodenal ulcers, and as an adjuvant treatment for yin-deficiency pulmonary tuberculosis and pulmonary tuberculosis. Topically, it can be used for the wounds of incised wounds, trauma, ulcers, fistulas, burns, scalds, and bedsore. ② Xinmailong Injection is prepared by dissolving the Xinmailong extract obtained by impregnating, decompressing and concentrating, and separating the dried Periplaneta americana. Clinically, it is mainly used as an adjuvant drug for chronic heart failure. The main active ingredients are diuretic peptides, nucleotides, inosine and compound active amino acids, which can be used to improve symptoms such as palpitation, edema, shortness of breath, dull complexion, and cyanosis of the lips caused by chronic congestive heart failure with qi-yang deficiency and blood stasis obstruction. ③ The main ingredient of Ganlong Capsule is the extraction group of Periplaneta americana, which has the effects of soothing the liver and regulating the spleen, promoting blood circulation and detoxifying. Clinically, it is mainly used for patients with chronic hepatitis B with hypochondriac pain, liver depression and spleen deficiency complicated with blood stasis syndrome. The symptoms are mainly distending pain or stabbing pain in the hypochondrium, nausea and belching, fatigue, loss of appetite, abdominal distension after eating, loose stools, pale or purple tongue color, and thready and unsmooth or string-like pulse, etc.

[0006] In the prior art, some scholars proposed to carry out drug treatment with the extract obtained. However, the extract has complex components and strong viscosity, so it is not easy to use. On the other hand, the extract contains a large amount of protein components, which are prone to corruption and deterioration and difficult to preserve. The pharmaceutical preparations prepared by refining the dried bodies of traditional Chinese medicine Periplaneta americana or its extracts have been widely used in the treatment of diseases such as ulcer repair, anti-tumor, and cardiovascular diseases due to their definite curative effects, but there are the following problems and deficiencies: ① The extract contains a large amount of peptide and protein components, which need to be stored at low temperature and transported in a cold chain. ② The components in the dried body powder or extract of Periplaneta americana are complex. Allergic reactions occasionally occur whether by injection or oral administration, causing discomfort to patients and even requiring medical treatment. ③ The quality of the medicinal materials has a great influence on the quality of the pharmaceutical preparations. Although they are all qualified according to the quality standards, the clinical observations show that the curative effects are inconsistent, which may be related to the synergistic effects of multiple components of traditional Chinese medicine.

[0007] Therefore, aiming at the bottlenecks and deficiencies of the prior art, it is necessary to develop an anti-inflammatory drug that has less impact on the viability of normal cells and lower red blood cell hemolysis activity. Summary of the Invention

[0008] The object of the present invention is to provide a truncated modified anti-inflammatory peptide derived from traditional Chinese medicine Periplaneta americana and its application. The truncated modified anti-inflammatory peptide derived from traditional Chinese medicine Periplaneta americana has the characteristics of convenient preparation process, easy large-scale industrial production, low cost, clear pharmacological activity, low toxicity, etc., and has good product development value and application prospects.

[0009] To solve the above technical problems, the present invention adopts the following technical solutions:

[0010] In a first aspect of the present invention, there is provided a truncated modified anti-inflammatory peptide derived from the traditional Chinese medicine Periplaneta americana, the amino acid sequence of the truncated modified anti-inflammatory peptide derived from the traditional Chinese medicine Periplaneta americana is shown as SEQ ID NO.1, and the basic amino acids R, H, K therein are replaced with residues in the unnatural D-conformation, and the carboxyl group of the D-histidine at the C-terminus is subjected to amidation modification.

[0011] In a second aspect of the present invention, there is provided the application of the truncated modified anti-inflammatory peptide derived from the traditional Chinese medicine Periplaneta americana in the preparation of anti-inflammatory products.

[0012] Cell-level studies have shown that when the concentration of this anti-inflammatory mimetic peptide is as high as 64 μM, it has no obvious effect on the viability of the macrophage cell line RAW264.7, and the truncated modified anti-inflammatory peptide derived from the traditional Chinese medicine Periplaneta americana has a good effect of inhibiting pro-inflammatory cytokines. Specifically, it has a significant inhibitory effect on the inflammatory mediator NO and the pro-inflammatory cytokines IL-1β, IFN-γ, IL-6 and TNF-α related to inflammation, and all of them show concentration dependence. Therefore, the truncated modified anti-inflammatory peptide derived from the traditional Chinese medicine Periplaneta americana can be used to prepare anti-inflammatory products.

[0013] Furthermore, the anti-inflammatory products include at least one of drugs, cosmetics or daily necessities.

[0014] Furthermore, the anti-inflammatory products inhibit the inflammatory mediator NO and the cytokines IL-1β, IL-6, TNF-α, IFN-γ related to inflammation.

[0015] Preferably, in the above application, the cells are macrophages.

[0016] Furthermore, the macrophages are the macrophage cell line RAW 264.7 derived from mice.

[0017] In a third aspect of the present invention, there is provided an anti-inflammatory product, including the truncated modified anti-inflammatory peptide derived from the traditional Chinese medicine Periplaneta americana.

[0018] Furthermore, the anti-inflammatory product includes antibacterial drugs or daily chemical products, and the product also includes pharmaceutically or cosmetically acceptable salts and / or acceptable excipients.

[0019] The anti-inflammatory product can be prepared into various pharmaceutical dosage forms such as injections, sterile powders for injection, powders, ointments, creams, gels, microneedles, etc. or / and daily chemical products according to conventional preparation methods.

[0020] Furthermore, the anti-inflammatory product can be introduced into muscle, endothelial, subcutaneous or mucosal tissues by injection, application, dressing, acupuncture, and other physically or chemically mediated methods, or can be introduced into the body after being mixed or / and encapsulated with other substances.

[0021] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0022] The present invention provides a truncated modified anti-inflammatory peptide derived from the traditional Chinese medicine Periplaneta americana and its application. The present invention obtains a short peptide sequence MKIFLRLYRHLVNKVLH composed of 17 amino acid residues by truncation, further replaces the basic amino acids R, H, and K with residues in the unnatural D conformation, and finally performs amidation modification on its carboxyl terminus (C-terminus) to obtain the truncated modified peptide MKIFLrLYrhLVNkVLh-NH2 designed by the present invention. The application of the truncated modified peptide in inhibiting inflammatory mediators and inhibiting pro-inflammatory related cytokines. At the same time, compared with only the truncated natural short peptide sequence, the polypeptide after unnatural amino acid modification and amidation has less impact on the viability of normal cells and has lower erythrocyte hemolysis activity.

[0023] Compared with the prior art, the truncated modified peptide derived from the traditional Chinese medicine Periplaneta americana of the present invention has significant advantages. It has a small molecular weight and its properties are similar to those of active small molecule compounds, so it is not easy to produce an immune response. On the other hand, the above-mentioned truncated modified peptide can be prepared by the method of solid-phase synthesis of polypeptides, can realize continuous cyclic production, and has a simple process and convenient operation. The crude product can be purified by semi-preparative high performance liquid chromatography (purity > 95%), which can meet the needs of product development and application. Description of the Drawings

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0025] Figure 1 It is the mass spectrometry diagram of the truncated modified anti-inflammatory peptide MKIFLrLYrhLVNkVLh-NH2 prepared by the solid-phase synthesis method;

[0026] Figure 2 It is the HPLC diagram of the truncated modified anti-inflammatory peptide derived from the traditional Chinese medicine Periplaneta americana prepared;

[0027] Figure 3 It is the spatial structure diagram of the truncated modified anti-inflammatory peptide derived from the traditional Chinese medicine Periplaneta americana simulated by a computer;

[0028] Figure 4 Effect of the natural truncated peptide modified anti-inflammatory peptide derived from the traditional Chinese medicine Periplaneta americana on cell viability;

[0029] Figure 5 Hemolytic activity results of the natural truncated peptide modified anti-inflammatory peptide derived from the traditional Chinese medicine Periplaneta americana;

[0030] Figure 6 Inhibition of the truncated modified anti-inflammatory peptide derived from the traditional Chinese medicine Periplaneta americana on the secretion of the inflammatory mediator NO by Raw264.7;

[0031] Figure 7 Inhibition of the truncated modified anti-inflammatory peptide derived from the traditional Chinese medicine Periplaneta americana on the secretion of the cytokine IL-1β by Raw264.7;

[0032] Figure 8 Inhibition of the truncated modified anti-inflammatory peptide derived from the traditional Chinese medicine Periplaneta americana on the secretion of the cytokine IL-6 by Raw264.7;

[0033] Figure 9 Inhibition of the truncated modified anti-inflammatory peptide derived from the traditional Chinese medicine Periplaneta americana on the secretion of the cytokine TNF-α by Raw264.7;

[0034] Figure 10 Inhibition of the truncated modified anti-inflammatory peptide derived from the traditional Chinese medicine Periplaneta americana on the secretion of the cytokine IFN-γ by Raw264.7.

[0035] Figure 11 Design concept of the truncated modified anti-inflammatory peptide derived from the traditional Chinese medicine Periplaneta americana of the present invention. Specific embodiments

[0036] The following will specifically elaborate on the present invention in combination with specific embodiments and examples, and the advantages and various effects of the present invention will be presented more clearly therefrom. Those skilled in the art should understand that these specific embodiments and examples are used to illustrate the present invention rather than limit the present invention.

[0037] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as having the 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 pertains. In case of contradiction, this specification shall prevail.

[0038] Unless otherwise specifically stated, various raw materials, reagents, instruments, and equipment used in the present invention can be obtained through market purchase or can be obtained by existing methods.

[0039] The general idea of the present invention is as follows:

[0040] In order to solve the problems encountered during the above-mentioned transportation, storage, and clinical use, so as to better control product quality, produce products with uniform quality, good therapeutic effects, and stable curative effects, and break through the existing technical bottlenecks and pain points:

[0041] First, the present invention discloses a truncated modified peptide derived from the traditional Chinese medicine Periplaneta americana, through the natural polypeptide of Periplaneta americana (NCBI accession number: KAJ4445100.1)

[0042] >KAJ4445100.1 hypothetical protein ANN_06899 [Periplaneta americana]

[0043] MRIRQDYEYLPPTLQEPLKQGSSCPQKEKYQEELKLATVVAKDLDHLNLSTFPRMFGGDVRGRQGNSALLRHYVGTTFADVVPMANLHITLPVPLAGRSSTWDLLGAYLQLWSLSIVREKGFYCNMKCNEGLKYRSAIFNDFKSTVSLVNKLHVAPNEKYQEVLKLATVVAKELDHFNLSTFPRIFGGDVRGREGKSARLRHNVRITFSDVVAKETLHVTLPLPLAGRSRTWDSLGDT MKIFLRLYRHLVNKVLH VAPKEKYEEELNLATVAAEELDQLNLSTLPRMFGGDFRGRQDFDWKNVIFSDQVIVSSNILCMLLSDRILIGEMSFSPTR

[0044] (References: Wang L, Xiong Q, Saelim N, Wang L, Nong W, Wan AT, Shi M, Liu X, Cao Q, Hui JHL, Sookrung N, Leung TF, Tungtrongchitr A, Tsui SKW. Genome assembly and annotation of Periplaneta americana reveal a comprehensive cockroach allergen profile. Allergy. 2023, 78(4): 1088 - 1103. doi: 10.1111 / all.15531.)

[0045] Truncation was performed to obtain a short peptide sequence consisting of 17 amino acid residues, MKIFLRLYRHLVNKVLH. Further, the basic amino acids therein were replaced with residues in the unnatural D - conformation, and finally, the carboxyl - terminal (C - terminal) was amidated to obtain the truncated modified peptide MKIFLrLYrhLVNkVLh - NH2 designed in the present invention (lowercase letters represent the corresponding D - type unnatural amino acids). The specific design idea is as Figure 11 shown.

[0046] Second, the present invention also provides a preparation method for the truncated modified peptide derived from the traditional Chinese medicine Periplaneta americana: The polypeptide MKIFLrLYrhLVNkVLh was synthesized by solid - phase peptide synthesis technology, and the corresponding raw materials were used at the positions of D - type amino acids; finally, after amidation modification of the carboxyl terminus, it was purified by semi - preparative high - performance liquid chromatography to obtain MKIFLrLYrhLVNkVLh - NH2;

[0047] Third, another object of the present invention is to provide the application of the above - mentioned truncated modified peptide derived from the traditional Chinese medicine Periplaneta americana in inhibiting inflammatory mediators and pro - inflammatory related cytokines. At the same time, compared with the native short peptide sequence alone, the polypeptide after non - natural amino acid modification and amidation has less impact on the viability of normal cells and has lower erythrocyte hemolysis activity.

[0048] From the above three points, it can be seen that compared with the prior art, the truncated modified peptide derived from the traditional Chinese medicine Periplaneta americana described in the present invention has significant advantages. It has a small molecular weight, and its properties are similar to those of active small - molecule compounds, so it is not easy to produce an immune response. On the other hand, the above - mentioned truncated modified peptide can be prepared by the method of solid - phase peptide synthesis, can realize continuous cyclic production, and has a simple process and convenient operation. The crude product can be purified by semi - preparative high - performance liquid chromatography (purity > 95%), which can meet the needs of product development and application.

[0049] Based on the above design, the truncated modified peptide derived from the traditional Chinese medicine Periplaneta americana was successfully prepared. It consists of 17 amino acid residues, with a theoretical isoelectric point pI of 11.10 and a theoretical molecular weight of 2178.73 Da.

[0050] In other embodiments, the truncated modified anti - inflammatory peptide from Periplaneta americana can also be prepared by other methods.

[0051] Based on the above design, the truncated modified peptide derived from the traditional Chinese medicine Periplaneta americana was successfully prepared. It consists of 17 amino acid residues, with a theoretical isoelectric point pI of 11.10 and a theoretical molecular weight of 2178.73 Da.

[0052] 1. The present invention provides a method for preparing a truncated modified peptide derived from the traditional Chinese medicine Periplaneta americana. Whether it is natural L - type amino acids or unnatural D - type amino acids, the raw materials are inexpensive and easily available. The solid - phase polypeptide synthesis technology and preparation process are mature and simple, fully meeting the requirements of large - scale and high - efficiency industrial preparation by modern enterprises.

[0053] 2. The present invention provides the truncated modified peptide derived from the traditional Chinese medicine Periplaneta americana:

[0054] First, the anti - inflammatory peptide of Periplaneta americana after truncation, unnatural amino acid substitution and terminal amidation modification has the characteristics and advantages of having less impact on normal cells and lower hemolytic activity.

[0055] Second, the solid - phase synthesis technology is mature, the operation is convenient, the purification is easy, and the quality is controllable, which can fully meet the requirements of subsequent transformation and application.

[0056] Third, the molecular weight of the above - mentioned truncated modified anti - inflammatory peptide is only 2179.73 Da. Its properties are similar to those of small - molecule compounds, having no immunogenicity and having the potential to be developed into an injectable drug. Therefore, compared with the prior art, the present invention has significant advancement.

[0057] Next, a truncated modified anti - inflammatory peptide of the traditional Chinese medicine Periplaneta americana and its application in the present application will be described in detail in combination with examples and experimental data. For those not specified in the examples in terms of specific technologies or conditions, the technical methods or conditions described in the technical materials such as the literature and standards in this field or according to the product instructions are followed. The methods described in this part, unless otherwise specified, are all conventional methods known in this field. The consumables and reagents used, unless otherwise specified, are all commercially available. Unless otherwise stated, the professional and scientific terms used herein have the same meanings as those well - known to those skilled in the art. In addition, any method or material similar or equivalent to the described content can also be used in the implementation of the related technologies involved in the present invention.

[0058]

Example 1

[0059] In the synthesis of this truncated modified peptide segment, 9-fluorenylmethoxycarbonyl (FMOC) was used as the protecting group at the amino terminus. 4-methyl-benzhydrylamine resin·HCl (MBHA resin) was selected as the solid-phase carrier for peptide synthesis. HOBt / DCC was used as the condensing agent for the reaction. According to the sequence of HOOC-DHis→Leu→Val→DLys→Asn→Val→Leu→DHis→DArg→Tyr→Leu→DArg→Leu→Phe→Ile→Lys→Met-NH2, the peptide chain was extended from the carboxyl terminus to the amino terminus (C-terminus → N-terminus). A mixture of 90.0% trifluoroacetic acid, 4.5% water, and 5.5% triisopropylsilane (TIA) (by mass percentage) was used to cleave the polypeptide sequence MKIFLrLYrhLVNkVLh containing 17 amino acid residues from the MBHA resin. After repeated precipitation with ether several times, its carboxyl terminus (C-terminus) was amidated and purified by preparative reverse-phase high-performance liquid chromatography RP-HPLC to obtain the target product MKIFLrLYrhLVNkVLh-NH2 truncated modified peptide. The purification process was as follows: A C18 reversed-phase preparative column (250 mm × 20 mm, 5 μm) was used; the mobile phase: 2‰ trifluoroacetic acid, 0% - 70% (by volume percentage) acetonitrile as the mobile phase, and gradient elution was carried out at a flow rate of 1.0 mL / min. The main peak eluate was collected, frozen at -80 °C for 4 hours, then placed in a freeze dryer for freeze-drying. After 12 hours of freeze-drying, a loose solid powder was obtained and stored at -20 °C in the refrigerator for later use. Identified by ESI-Q-TOF-MS, its molecular weight was consistent with the corresponding theoretical molecular weight (2179.73 Da) of the target truncated modified peptide to be prepared, m / z 1090.15 [M+2H] 2+ , m / z 727.90 [M+3H] 3+ , m / z 545.95[M+4H] 4+ ( Figure 1 ). Determined by analytical high-performance liquid chromatography, its purity > 95% ( Figure 2 ), and its spatial structure was as Figure 3 shown.

[0060]

Example 2

[0061] The MTT method was used to detect the effects of the truncated natural peptide and the truncated modified peptide on the viability of the immune cell line RAW264.7 after intervention.

[0062] Preheat the water bath to 37 °C in advance, warm the medium (DMEM + 10% FBS + 1% P / S) to 37 °C, and set the centrifuge speed to 800 - 1000 rpm. Take out the cryopreserved RAW264.7 cells from the liquid nitrogen tank. When there is no liquid nitrogen in the tube, hold the tube cap and immerse the cryopreserved part of the cells in the water bath and shake quickly. Terminate the water bath when the cells melt into ice cubes the size of rice grains (the melting process should not exceed 2 min); directly centrifuge the cryopreservation tube slowly for 90 - 120 Sec; after centrifugation, discard the supernatant, slowly add the preheated medium to the cryopreservation tube, gently resuspend the cells and then inoculate them into the culture flask, gently mix and then place them in the incubator; observe the cell attachment after 24 hours of resuscitation. After subculturing 2 - 3 times, select cells with good cell status for the MTT experiment. The specific operation is as follows:

[0063] At 37 °C, digest the cells in the logarithmic growth phase with trypsin for about 45 seconds, add complete medium to stop the digestion, gently pipette to disperse and suspend the cells, centrifuge the cell suspension at 800 rpm for 5 minutes, discard the supernatant, add an appropriate amount of medium to resuspend the precipitate to make a cell suspension, and adjust the cell count to an appropriate concentration.

[0064] After preparing the cell suspension, gently mix it. Take a 96-well plate and add 100 μL of the cell suspension (4000 cells / well) to each well. Place the culture plate in an incubator at 37 °C and 5% CO2 for 5 hours to allow the cells to attach. Carefully aspirate and discard the medium. Control group (Group A): Only add medium with a low serum content (~2% FBS) and do not add the natural truncated peptide or truncated modified peptide from Periplaneta americana. Experimental group 1 (Group B): Add 100 μL of medium with a low serum content containing the natural truncated peptide from Periplaneta americana (the final concentrations of the natural truncated peptide in Group B are 1 μM, 2 μM, 4 μM, 8 μM, 16 μM, 32 μM, 64 μM) respectively. Each group is in one column and 6 replicate wells are set. Experimental group 2 (Group C): Add 100 μL of medium with a low serum content containing the truncated modified peptide from Periplaneta americana (the final concentrations of the truncated modified peptide in Group C are 1 μM, 2 μM, 4 μM, 8 μM, 16 μM, 32 μM, 64 μM) respectively. Each group is in one column and 6 replicate wells are set.

[0065] Seal the edges of the experimental wells with sterile PBS to avoid evaporation of the culture system in the edge experimental wells due to the long experimental time. After culturing for 12 hr, add 10 μL of MTT solution (5 mg / ml, i.e., 0.5% MTT) to each well and continue culturing for 4 - 6 hr. Aspirate and discard the supernatant, add 150 μL of DMSO to each well, and incubate in a shaker at 37 °C and 80 - 120 rpm for 10 - 15 min to fully dissolve the crystals. Measure the ultraviolet absorption value of each well at a wavelength of 490 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0066] Since succinate dehydrogenase in the mitochondria of living cells can reduce exogenous MTT to water-insoluble blue-violet crystalline formazan and deposit it in the cells, the higher the cell viability, the more reduction products are produced, while dead cells do not have this function. Therefore, compared with the absorbance of the control group without peptides (cell viability is 100%), it can reflect the effects of natural truncated peptides and the truncated modified peptides described in the present invention on the cell viability of mouse-derived macrophage cell line RAW264.7 at different concentrations.

[0067] The results are as Figure 4 shown. At final concentrations of 1 μM, 2 μM, 4 μM, 8 μM, and 16 μM, natural truncated peptides and truncated modified peptides at different concentrations had no effect on the viability of RAW264.7 cells; at high concentrations of 32 μM and 64 μM, the truncated modified peptides had no effect on cell viability, while the natural truncated peptides had an inhibitory effect on cell viability, and the difference was statistically significant ( P <0.05).

[0068] The above results indicate that after non-natural amino acid substitution and carboxyl-terminal amidation modification, at high concentrations (final concentrations of 32 μM - 64 μM), the effect on cell viability can be significantly reduced. Therefore, the effect of the Periplaneta americana truncated modified peptide described in the present invention on cell viability is significantly reduced, and it has more value for transformation and development.

[0069]

Example 3

[0070] Fresh blood was obtained from male healthy volunteers (the first inventor of this application), centrifuged at 2000 rpm for 15 min at 4 °C, the supernatant was discarded, the red blood cells were gently resuspended by pipetting with injectable physiological saline, centrifuged again, and the supernatant was discarded and resuspended. This washing process was repeated three times to prepare a red blood cell suspension. Using sterile injectable physiological saline PBS buffer as the negative control and sterilized Milli-Q ultrapure water as the positive control, the hemolytic activities of Periplaneta americana natural truncated peptides and truncated modified peptides were measured respectively.

[0071] The specific steps are as follows:

[0072] ① Collect whole blood using a heparin anticoagulant tube, gently invert to allow the blood to fully contact the anticoagulant, centrifuge at 1000 rpm for 10 min at 4 °C, aspirate all the upper plasma and retain the lower red blood cells; add 4 times the volume of injectable physiological saline, gently pipette and aspirate to resuspend the red blood cells that have settled at the bottom of the centrifuge tube, and repeat the above operation 3 - 5 times until the supernatant gradually becomes colorless;

[0073] ② Prepare a red blood cell suspension with a concentration of 2% - 4% (v / v) using injectable physiological saline. Prepare solutions of the natural truncated peptide (MKIFLRLYRHLVNKVLH) and truncated modified peptide (MKIFLrLYrhLVNkVLh - NH2) derived from the traditional Chinese medicine Periplaneta americana described in this invention with concentrations of 64 μM, 32 μM, 16 μM, 8 μM, 4 μM, and 2 μM using sterilized physiological saline. Mix 100 μL of the natural truncated peptide or the truncated modified peptide of Example 1 with 100 μL of the red blood cell suspension and add it to a 96 - well plate, so that the final concentrations of the polypeptide are 32 μM, 16 μM, 8 μM, 4 μM, 2 μM, and 1 μM respectively;

[0074] ③ The negative control group is red blood cells suspended with injectable physiological saline, and the positive control uses sterilized Milli - Q ultrapure water to suspend red blood cells; Place the sample to be tested in a constant temperature shaker and incubate it at a constant temperature of (36.5 ± 0.5) °C and a rotation speed of 100 rpm for 30 min; Centrifuge the sample at 4000 rpm for 5 min at room temperature, and transfer 120 μL of the supernatant from each well to another clean 96 - well plate; Measure the absorbance at a wavelength of 490 nm using a full - wavelength microplate reader, and calculate the hemolysis percentage through the following formula. In the formula, H is the absorbance at a wavelength of 490 nm:

[0075] Hemolysis rate (%) = (H sample - H negative ) / (H positive - H negative ) × 100%.

[0076] The results of red blood cell hemolytic activity are as Figure 5 shown. The results show that when the concentration ≤ 8 μM, both the natural truncated peptide and the truncated modified peptide have no obvious hemolytic activity (hemolysis rate < 5%); When the concentrations are 16 μM and 32 μM, the hemolysis rates of the natural truncated peptide are 8.5% and 22.1% respectively, while the hemolytic activity of the truncated modified peptide described in this application is significantly reduced, only 4.8% and 9.6%. Therefore, after the replacement of non - natural D - type amino acids and the amidation modification at the carboxyl - terminal, the hemolytic toxicity of the truncated modified peptide of Periplaneta americana described in this invention is significantly reduced, and it has good application value and transformation and development prospects.

[0077]

Example 4

[0078] Inflammatory mediators refer to the chemically active substances that are released by local tissues or produced by body fluids under the action of inflammatory factors and participate in the inflammatory response. Nitric oxide (NO) is an inflammatory mediator released by immune cells. Excessive nitric oxide is associated with excessive cell death and the occurrence of inflammation. Although cell death is crucial for a healthy immune response, excessive cell death can cause the immune system to "overwork", leading to inflammatory diseases.

[0079] In the embodiments of the present application, the Griess reaction is used to determine the amount of nitric oxide (NO) contained in the culture medium of RAW 264.7 cells. Cells in the logarithmic growth phase are evenly inoculated into a 96-well plate (the number of cells inoculated per well is 1×10 4 cells / well) and cultured adherently. Subsequently, the culture medium is removed. In the blank group, DMEM high-glucose medium containing 10% fetal bovine serum is added. In the model group, the culture medium contains LPS with a final concentration of 2 μg / mL. In the experimental group, the culture medium contains truncated modified peptide MKIFLrLYrhLVNkVLh-NH2 of Periplaneta americana at different concentrations (1 μΜ, 2 μΜ, 4 μΜ, 8 μΜ, and 16 μΜ) and 2 μg / mL LPS. Cells in each group are incubated under corresponding conditions for 18 - 24 hours. After taking out the culture plate, it is centrifuged. 50 μL of the supernatant of each group is taken, and an equal volume of Griess reagent I and Griess reagent II are added. The absorbance OD 540 value is detected at a wavelength of 540 nm, and the absolute content value of NO is calculated according to the linear relationship fitted by the standard curve.

[0080] The results are as Figure 6 shown. NO is an important effector molecule and inflammatory mediator produced by macrophages and plays an important role in the occurrence and development of inflammation. After the mouse-derived macrophage cell line RAW264.7 is activated by LPS stimulation, the secretion amount of NO increases sharply. Compared with unstimulated cells, there is a highly significant difference in the NO secretion amount ( *** P <0.001), indicating that macrophages are activated and an obvious inflammatory response is generated. In the concentration range of 1 μΜ - 4 μΜ, the truncated modified peptide of Periplaneta americana does not show an inhibitory effect on the secretion of the inflammatory mediator NO. When the concentration increases to 8 μΜ, it has a certain inhibitory effect, but the difference is not statistically significant ( P >0.05), but when the concentration continues to increase to 16 μΜ, it can significantly inhibit the secretion of NO, and the difference is highly statistically significant ( *** P <0.001).

[0081]

Example 5

[0082] Cytokines IL-1β, IL-6, IFN-γ and TNF-α are important active mediators for activated macrophages to participate in host defense and regulate the inflammatory response process. ① IL-1β is an important signal molecule in the immune system, mainly produced by immune cells. It plays a key role in regulating the inflammatory response and immune response. IL-1β can activate other immune cells, promote the inflammatory process, and play a role in infection, tissue damage or autoimmune diseases. Excessive secretion and expression of IL-1β may lead to the occurrence of inflammatory diseases. ② IL-6 is a single-chain glycoprotein cytokine that can be secreted by a variety of immune cells (including macrophages and infected T cells, etc.). A large number of studies have confirmed that IL-6 plays a key role in regulating systemic inflammatory response syndrome, chronic autoimmune diseases, etc. As an important participant in the inflammatory cascade reaction, it is one of the cytokines with the most functions and the widest influence. ③ IFN-γ plays an important role in the adaptive immune response. In the inflammatory response environment, IFN-γ triggers the activation of the immune response, but over-activation can also cause tissue damage. ④ TNF-α is also an important pro-inflammatory cytokine, mainly secreted by monocytes and macrophages. At the same time, it is also a pleiotropic cytokine that plays an important role in inflammation, apoptosis and the development of the immune system.

[0083] Determining the inhibitory effects of the Periplaneta americana truncated modified peptide described in this application on pro-inflammatory cytokines IL-1β, IL-6, IFN-γ and TNF-α is crucial for evaluating its anti-inflammatory ability. Take cells in the logarithmic growth phase and evenly inoculate them into 96-well plates (the number of cells inoculated in each well is 1×10 4 cells / well), and culture them adherently. Subsequently, remove the culture medium. The blank group is added with DMEM high-glucose medium containing 10% fetal bovine serum, the model group is the culture medium containing LPS with a final concentration of 2 μg / mL, and the experimental group contains culture media with different concentrations (1 μΜ, 2 μΜ, 4 μΜ, 8 μΜ and 16 μΜ) of the Periplaneta americana truncated modified peptide MKIFLrLYrhLVNkVLh-NH2 and LPS.

[0084] The secretion and expression of the above four cytokines were detected by ELISA. The specific steps were as follows: The antibody was appropriately diluted with PBS buffer and plated at a concentration of 1 - 10 μg / ml. 0.2 ml of the diluted capture antibody was added to each well of the microtiter plate. After incubation at 37 °C for 1 hour, the coating solution was removed, and the plate was washed 3 times with washing buffer (PBS-T). The enzyme-labeled detection antibody was diluted, and 0.2 ml of the diluted detection antibody was added to each well. The plate was incubated at room temperature for 30 minutes. The detection antibody solution was removed, and the plate was washed 3 times with washing buffer (PBS-T). The corresponding substrate solution was added, and after color development in the dark for 30 minutes, the stop solution was added. The absorbance was read at an appropriate wavelength on an enzyme-linked immunosorbent assay (ELISA) reader according to the requirements of the kit instructions.

[0085] The results were as Figures 7 - 10 shown. Compared with the group treated with LPS alone, after treatment with the truncated modified peptide of Periplaneta americana at a concentration of 1 μΜ - 4 μΜ, there was no change in the secretion and expression of the above four pro-inflammatory cytokines; after treatment with the truncated modified peptide at a concentration of 8 μΜ, the expression of IL-6 was inhibited, and the difference was statistically significant ( ** P <0.01); after treatment with the truncated modified peptide at a concentration of 16 μΜ, all four cytokines detected were inhibited, and the differences were extremely significant statistically ( *** P <0.001). This indicates that the truncated modified peptide of Periplaneta americana described in the present invention has a good effect of inhibiting pro-inflammatory cytokines.

[0086] Example 6. Preparation of different dosage forms

[0087] 1. Tablets

[0088] 5 g of the truncated modified anti-inflammatory peptide derived from the traditional Chinese medicine Periplaneta americana described in the present invention, 100 g of starch, and 20 g of dextrin were mixed, and an appropriate amount of a 50% (w / v) pharmaceutical-grade polyvinylpyrrolidone (PVP) solution was added as a binder. Granulation, sieving, and tableting were carried out to obtain tablets.

[0089] 2. Cream

[0090] 5 g of the truncated modified anti-inflammatory peptide derived from the traditional Chinese medicine Periplaneta americana described in the present invention, 10 g of glyceryl monostearate, 20 g of stearic acid, 3 g of white petrolatum, 15 g of liquid paraffin, 10 g of lanolin, 1 - 2 mL of triethanolamine, and 120 mL of distilled water were mixed evenly to obtain the cream.

[0091] 3. Gel

[0092] Take 50 mL of distilled water, sprinkle an appropriate amount of carbomer (about 2 g) on the liquid surface, place it in a 50 °C water bath and stir to fully swell it. Add propylene glycol and mix evenly, then slowly add triethanolamine dropwise with stirring to make a neutral gel matrix; dissolve 2 g of the truncated modified anti-inflammatory peptide derived from the traditional Chinese medicine Periplaneta americana of the present invention in PBS buffer solution with a concentration of 0.15 M (pH = 7.2), and slowly add it to the gel matrix with continuous stirring. Finally, add the remaining distilled water to make the total volume of the preparation 100 mL to obtain the Periplaneta americana truncated modified anti-inflammatory peptide gel.

[0093] 4. Freeze-dried powder for injection

[0094] Take 1 g of the truncated modified anti-inflammatory peptide derived from the traditional Chinese medicine Periplaneta americana of the present invention and 5 g of mannitol, place them in a container, dissolve them with an appropriate amount of PBS buffer solution (0.15 M, pH 7.4), add injection water to 100 mL, shake well, add 1 - 3 g of activated carbon for injection, gently stir at room temperature for about 15 minutes, filter roughly, filter and sterilize with a 0.22 μm sterilizing filter membrane, dispense into vials, 1.0 mL per vial, adopt the method of rapid freezing, cool down by ~15 °C per minute, cool down to -50 ~ -55 °C, maintain for 1 hour, and evacuate. After freeze-drying for 12 - 18 hours, slowly raise the temperature under vacuum, with a heating rate of 5 - 10 °C per hour, stop heating when the temperature rises to room temperature (25 °C), take it out after the temperature approaches room temperature, cover and seal to obtain the freeze-dried powder injection.

[0095] Finally, it should also be noted that the term "comprising", "including" or any other variant thereof is intended to cover non-exclusive inclusion, so that a process, method, article or device comprising a series of elements not only includes those elements, but also includes other elements not expressly listed, or also includes elements inherent to such process, method, article or device.

[0096] Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications once they learn the basic creative concept. Therefore, the appended claims are intended to be construed as including the preferred embodiments and all changes and modifications falling within the scope of the present invention.

[0097] 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 equivalent technologies, the present invention is also intended to include these modifications and variations.

Claims

1. A truncated modified anti-inflammatory peptide derived from the traditional Chinese medicine American cockroach, characterized in that: The amino acid sequence of the truncated modified anti-inflammatory peptide of the traditional Chinese medicine American cockroach is shown in SEQ ID NO.1, and the basic amino acids R, H, and K therein are replaced with residues of non-natural D-type conformation, and the carboxyl group of the D-histidine at the C-terminus is amidated and modified.

2. Use of the truncated modified anti-inflammatory peptide derived from the traditional Chinese medicine American cockroach as claimed in claim 1 in the preparation of anti-inflammatory products.

3. The use according to claim 2, characterized in that: The anti-inflammatory product comprises at least one of medicine, cosmetics or daily necessities.

4. The use according to claim 2, characterized in that: The anti-inflammatory product inhibits the inflammatory mediator NO and the inflammation-related cytokines IL-1β, IL-6, TNF-α, and IFN-γ.

5. An anti-inflammatory product, characterized in that: It includes the truncated modified anti-inflammatory peptide derived from the traditional Chinese medicine American cockroach as described in claim 1.

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

7. The product according to claim 6, characterized in that The auxiliary materials include at least one of a filler, a disintegrant, a binder, an excipient, a diluent, a lubricant, a sweetener or a colorant.

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