A hyaluronidase-binding cyclic peptide

Abstract

The present application relates to the field of biotechnology, in particular to a kind of hyaluronidase binding cyclic peptide, the cyclic peptide is obtained by screening phage display technology, its structure includes X 1(n) CX3X4X5X6X7X8X9X 10 CX 12(m) It has the efficacy of inhibiting hyaluronidase activity, higher transdermal absorption capacity and / or anti-inflammatory, can be used as hyaluronidase (especially PH20) inhibitor, applied in the fields of medicine, food and cosmetics.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a cyclic peptide that binds to hyaluronidase, and the application of said cyclic peptide in the fields of medicine, food, and cosmetics. Background Technology

[0002] Hyaluronidases (HYALs) include HYAL-1, HYAL-2, HYAL-3, HYAL-4, PH20, and HYAL-P1. Hyaluronidases are closely related to human health. For example, non-patent literature (Zhang Yuesheng. Investigation on recombinant expression and fermentation process of human hyaluronidase in Pichia pastoris [D]. Jiangnan University, 2023. DOI:10.27169 / d.cnki.gwqgu.2023.002353.) discloses that hyaluronidase can mediate inflammatory responses in humans (e.g., activate related signaling pathways, stimulate inflammatory responses, upregulate the expression of inflammation-related genes), contribute to tumor progression (including invasion, proliferation, and migration), and is also associated with skin aging and wrinkle formation. To inhibit hyaluronidase, various hyaluronidase inhibitors have been disclosed in the prior art. For example, patent document (CN113398182A) discloses a traditional Chinese medicine composition for inhibiting hyaluronidase activity and a hyaluronidase inhibitor. Patent document (CN113150069B) also discloses the application of adenosine heptapeptide in the preparation of a hyaluronidase inhibitor. The amino acid sequence of the adenosine heptapeptide is MATGNAD.

[0003] PH20, also known as sperm adhesion molecule 1 (SPAM1), is a GPI-anchored single-chain membrane protein present in almost all mammals. High expression of PH20 is closely related to disease development. For example, non-patent literature (Fu, Y., Zhang, X., Liu, X. et al. The DNMT1-PAS1-PH20 axis drives breast cancer growth and metastasis. Sig Transduct Target Ther 7, 81 (2022). https: / / doi.org / 10.1038 / s41392-022-00896-1) discloses that PH20 is a member of the human hyaluronidase family and can degrade hyaluronic acid in the extracellular matrix, promoting breast cancer invasion and metastasis. However, no relevant content on inhibiting PH20 has been found in the existing technology.

[0004] The concept of phage display technology was first proposed by Smith in 1985. It utilizes gene recombination technology to fuse and recombine exogenous gene fragments into phage plasmids, displaying the proteins or peptides encoded by the exogenous gene on their surface. Phage display technology is a powerful tool for constructing libraries for high-throughput screening of protein-protein interaction ligands. By using random phage peptide libraries, phages containing peptides with certain functional characteristics can be screened, mimicking the evolution of DNA molecular sequences to optimize the screening process. However, no existing technology for screening peptides that bind to and / or hyaluronidase using phage display technology was found; therefore, this invention is proposed. Summary of the Invention

[0005] In a first aspect, the present invention provides a cyclic peptide, the amino acid sequence of which includes X 1(n) CX3X4X5X6X7X8X9X 10 CX 12(m) .

[0006] Wherein, n or m is independently selected from integers from 0 to 10, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Preferably, n or m is independently selected from integers from 0 to 3, such as 0, 1, 2, or 3.

[0007] Preferably, X1, X3, X4, X5, X6, X7, X8, X9, X 10 or X 12 It is not cysteine. It can be any of the following: alanine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

[0008] Preferably, X3 is selected from any one of F, W, H, S, or Y. More preferably, X3 is any one of F, W, or H. More preferably, X3 is selected from any one of F or W.

[0009] Preferably, X4 is selected from any one of M, R, S, E, T, H, or W. More preferably, X4 is selected from any one of M, R, E, S, T, or H. More preferably, X4 is selected from any one of M, R, E, or S.

[0010] Preferably, X5 is selected from any one of R, E, T, Q, or V. More preferably, X5 is selected from any one of R, T, or E.

[0011] Preferably, X6 is selected from any one of M, L, H, Q, or V. More preferably, X6 is selected from any one of M or Q. More preferably, X6 is M.

[0012] Preferably, X7 is selected from any one of S, G, or E. More preferably, X7 is selected from any one of S or G.

[0013] Preferably, X8 is selected from any one of E, M, V, T, or A. More preferably, X8 is selected from any one of E, A, T, or M. Even more preferably, X8 is selected from any one of E, A, or M.

[0014] Preferably, X9 is selected from any one of L, M, or V. More preferably, X9 is selected from any one of L or M.

[0015] Preferred, X 10 Selected from S, E, F, H, or V. Further preferred, X 10 Choose from any one of S, E, H or V.

[0016] Further preferred, X3 is selected from any one of F, W, H, S or Y; X4 is selected from any one of M, R, S, E, T, H or W; X8 is selected from any one of E, M, V, T or A.

[0017] More preferably, X3 is selected from F, W, or H; X4 is selected from M, R, E, S, T, or H; X5 is selected from R, T, or E; X6 is selected from M or Q; X7 is selected from S, E, or G; X8 is selected from E, A, T, or M; X9 is selected from L or M; X 10 Choose from any one of S, E, H, F or V.

[0018] More preferably, X3 is selected from F or W; X4 is selected from M, E, R or S; X5 is selected from R, T or E; X6 is M; X7 is selected from S or G; X8 is selected from E, A or M; X9 is selected from L or M; X 10 Choose from any one of S, E, H or V.

[0019] The cyclic peptide is formed by cyclization through a covalent bond between two cysteine ​​residues.

[0020] In one specific embodiment of the present invention, the cyclic peptide comprises any one of the following:

[0021] 1) X3 is F, X4 is M, X5 is R, X6 is M, X7 is S, X8 is E, X9 is L, X 10 Preferably, the amino acid sequence of the cyclic peptide comprises SEQ ID NO: 1, or comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 1.

[0022] 2) X3 is W, X4 is S, X5 is E, X6 is M, X7 is G, X8 is M, X9 is L, X10 Preferably, the amino acid sequence of the cyclic peptide comprises SEQ ID NO: 2, or comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 2.

[0023] 3) X3 is F, X4 is E, X5 is R, X6 is M, X7 is S, X8 is E, X9 is L, X 10 V, preferably, the amino acid sequence of the cyclic peptide contains SEQ ID NO: 3, or contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 3.

[0024] 4) X3 is F, X4 is M, X5 is R, X6 is M, X7 is S, X8 is E, X9 is L, X 10 Preferably, the amino acid sequence of the cyclic peptide comprises SEQ ID NO: 4, or comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 4.

[0025] 5) X3 is F, X4 is T, X5 is R, X6 is M, X7 is S, X8 is T, X9 is L, X 10 For example, preferably, the amino acid sequence of the cyclic peptide contains SEQ ID NO: 5, or contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 5.

[0026] 6) X3 is H, X4 is T, X5 is R, X6 is H, X7 is S, X8 is E, X9 is L, X 10 For F, preferably, the amino acid sequence of the cyclic peptide contains SEQ ID NO: 6, or contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 6.

[0027] 7) X3 is F, X4 is R, X5 is T, X6 is M, X7 is G, X8 is A, X9 is M, X 10 For example, preferably, the amino acid sequence of the cyclic peptide contains SEQ ID NO: 7, or contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 7.

[0028] 8) X3 is F, X4 is W, X5 is R, X6 is L, X7 is S, X8 is V, X9 is L, X 10 For example, preferably, the amino acid sequence of the cyclic peptide contains SEQ ID NO: 8, or contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 8.

[0029] 9) X3 is H, X4 is H, X5 is R, X6 is Q, X7 is E, X8 is T, X9 is L, X 10For F, preferably, the amino acid sequence of the cyclic peptide contains SEQ ID NO: 9, or contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 9.

[0030] 10) X3 is S, X4 is W, X5 is Q, X6 is V, X7 is S, X8 is E, X9 is L, X 10 V, preferably, the amino acid sequence of the cyclic peptide contains SEQ ID NO: 10, or contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 10.

[0031] 11) X3 is Y, X4 is H, X5 is V, X6 is M, X7 is S, X8 is E, X9 is V, X 10 For F, preferably, the amino acid sequence of the cyclic peptide contains SEQ ID NO: 11, or contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 11.

[0032] Preferably, the amino acid sequence of the cyclic peptide comprises one or more of SEQ ID NO: 1-5, 7 or 9, or comprises an amino acid sequence that is at least 90% identical to one or more of SEQ ID NO: 1-5, 7 or 9. More preferably, the amino acid sequence of the cyclic peptide comprises one or more of SEQ ID NO: 1-4 or 7, or comprises an amino acid sequence that is at least 90% identical to one or more of SEQ ID NO: 1-4 or 7.

[0033] Preferably, the amino acid sequence of the cyclic peptide comprises one or more of SEQ ID NO: 1-3, 5, 7 or 8, or comprises an amino acid sequence that is at least 90% identical to one or more of SEQ ID NO: 1-3, 5, 7 or 8. More preferably, the amino acid sequence of the cyclic peptide comprises one or more of SEQ ID NO: 1-2, 7 or 8, or comprises an amino acid sequence that is at least 90% identical to one or more of SEQ ID NO: 1-2, 7 or 8.

[0034] Preferably, the cyclic peptide binds to hyaluronidase, and more preferably inhibits hyaluronidase activity.

[0035] Preferably, the hyaluronidase includes one or more of HYAL-1 (hyaluronidase 1), HYAL-2 (hyaluronidase 2), HYAL-3 (hyaluronidase 3), HYAL-4 (hyaluronidase 4), or PH20 (posterior head 20).

[0036] In one specific embodiment of the present invention, the hyaluronidase is PH20.

[0037] Preferably, the cyclic peptide can be prepared by chemical synthesis or prokaryotic expression.

[0038] In one specific embodiment of the present invention, the cyclic peptide is synthesized by chemical synthesis, such as solid-phase synthesis.

[0039] In a second aspect, the present invention provides a method for screening the above-mentioned cyclic peptides, the method comprising using phage display technology.

[0040] Preferably, the screening method includes:

[0041] 1) Construct a phage display library, wherein the phage display library contains an octapeptide-tetradecapeptide cyclic peptide backbone, and the cyclic peptide backbone contains cysteine.

[0042] Preferably, after activating and culturing the cyclic peptide strain, serially diluted phages are added to a randomly amplified bacterial library to obtain a recombinant phage library;

[0043] In one embodiment of the present invention, a random TG1 bacterial library is inoculated into 2YT medium and cultured until OD = 0.3-0.6. Ten times the amount of helper phage is added, mixed, and incubated. After centrifugation to obtain bacterial cells, the culture is re-inoculated into 2YT resistant medium and cultured overnight. The overnight culture is centrifuged at high speed, the supernatant is collected, PEG / NaCl solution is added, mixed, and allowed to stand overnight. The precipitate is collected after high-speed centrifugation of the Shajingshu solution, and resuspended in sterile PBS solution to obtain the amplified phage culture.

[0044] 2) Use the phage display library obtained in 1) to screen for hyaluronidase and obtain target phages that bind to hyaluronidase. Preferably, the screening is performed 1-10 times, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 times.

[0045] 3) Amplify and sequence the target phage that binds to hyaluronidase.

[0046] In one embodiment of the present invention, hyaluronidase is diluted with PBS buffer and modified with biotin dissolved in PBS. After purification to remove excess biotin, hyaluronidase is bound to streptokinase affinity beads and blocked with BSA solution. Simultaneously, the prepared phage library is blocked with BSA solution. The blocked phage library is added to streptokinase-bound magnetic beads for incubation. The phage solution is discarded, and the magnetic beads are washed three times with elution buffer. The bound phages are eluted with elution buffer, and the elution buffer is quickly added to Tris-HCl for neutralization. The phage titer in the elution buffer is measured, and TG1 infection amplification is performed for the next round of screening. The above steps are repeated twice to complete three rounds of screening. Positive phage clones are sequenced, and the sequencing results are translated into amino acid sequences.

[0047] Preferably, the hyaluronidase includes HYAL-1 (hyaluronidase 1) and HYAL-2 (hyaluronidase 2).

[0048] 2) One or more of HYAL-3 (hyaluronidase 3), HYAL-4 (hyaluronidase 4), or PH20 (posteriorhead 20).

[0049] In one specific embodiment of the present invention, the hyaluronidase is PH20.

[0050] Preferably, the amino acid sequence of the octapeptide-tetradecapeptide cyclic peptide backbone includes: X (0-10) CX (6-12) CX (0-10) .

[0051] Among them, X (0-10) Represents 0-10 arbitrary amino acids. X (6-12) It represents 6-12 arbitrary amino acids. C represents cysteine.

[0052] In one specific embodiment of the present invention, the amino acid sequence of the octapeptide-tetradecapeptide cyclic peptide backbone includes CX. (8-12) C, X (0-2) CX (8) CX (0-2) X (0-3) CX (5-6) CX (0-3) .

[0053] In a third aspect, the present invention provides a method for preparing the above-mentioned cyclic peptide, wherein the preparation method includes chemical synthesis or prokaryotic expression.

[0054] In one specific embodiment of the present invention, the cyclic peptide is synthesized by chemical synthesis, such as solid-phase synthesis.

[0055] Preferably, the preparation method includes chemically synthesizing a linear polypeptide and then oxidizing it into a cyclic peptide to obtain a cyclic peptide.

[0056] In a fourth aspect, the present invention provides a hyaluronidase inhibitor comprising the above-described cyclic peptide, the cyclic peptide obtained by the above-described screening method, or the cyclic peptide obtained by the above-described preparation method.

[0057] In a fifth aspect, the present invention provides the use of the above-described cyclic peptide, the cyclic peptide obtained by the above-described screening method, or the cyclic peptide obtained by the above-described preparation method as a hyaluronidase inhibitor.

[0058] In a sixth aspect, the present invention provides a drug, food, or cosmetic comprising the above-described cyclic peptide, the cyclic peptide obtained by the above-described screening method, the cyclic peptide obtained by the above-described preparation method, or the above-described hyaluronidase inhibitor.

[0059] Preferably, the drug further comprises pharmaceutically acceptable excipients.

[0060] Preferably, the food product further comprises food-grade ingredients that are acceptable in the food industry.

[0061] Preferably, the cosmetic product further includes excipients acceptable in the cosmetics field.

[0062] Preferably, the pharmaceutically acceptable, food-acceptable, or cosmetic-acceptable excipients may include excipients conventional in the art, such as one or more of diluents, excipients, fillers, absorption enhancers, surfactants, suspending agents, pigments, flavoring agents, humectants, binders, disintegrants, lubricants, pH adjusters, antioxidants, metal ion chelators, antibacterial agents, or isotonic adjusters.

[0063] Preferably, the drug can be any suitable dosage form, including but not limited to tablets, pills, powders, granules, capsules, lozenges, syrups, emulsions, suspensions, injections, sprays, aerosols, powder inhalers, lotions, ointments, pastes, iron supplements, eye drops, nasal drops, suppositories, effervescent tablets, drop pills, or gels, etc.

[0064] Preferably, the various dosage forms of the drug can be prepared according to conventional production methods in the chemical field.

[0065] Preferably, the food product can be any known food product, such as dairy products, biscuits, pastries, beverages, etc. Preferably, the food product is selected from at least one form of solid, dairy, solution, powder, or suspension products.

[0066] Preferably, the cosmetic product can be one or more of the following: gel, lotion, serum, eye cream, toner, facial cleanser, foundation, mask, massage cream, or spray.

[0067] Preferably, the cosmetic is used to inhibit hyaluronidase activity, increase the skin's transdermal absorption capacity, and to have anti-inflammatory, anti-aging, repairing, moisturizing, soothing, whitening, spot-removing, anti-wrinkle, or anti-aging effects on the skin.

[0068] Preferably, the cosmetic has the effects of inhibiting hyaluronidase activity, increasing the skin's transdermal absorption capacity, and having anti-inflammatory, anti-aging, repairing, moisturizing, soothing, whitening, spot-removing, anti-wrinkle, or anti-aging effects on the skin.

[0069] Preferably, the drug can be administered via any suitable route of administration, such as gastrointestinal administration (e.g., oral administration) or non-gastrointestinal administration (e.g., intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection, nasal drops, eye drops, intrathecal administration, or transdermal administration, etc.).

[0070] Preferably, the drug is for human or veterinary use.

[0071] Preferably, the drug, food, or cosmetic may contain 0.01-99.5% by weight or volume (e.g., 0.01%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%).

[0072] 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 99.5% of an active substance (e.g., the cyclic peptide described in this invention).

[0073] A seventh aspect of the present invention provides a method for treating and / or preventing a disease, the method comprising administering to a subject an effective amount of the above-described cyclic peptide, the cyclic peptide obtained by the above-described screening method, the cyclic peptide obtained by the above-described preparation method, or the above-described drug.

[0074] Preferably, the diseases include those for which inhibiting hyaluronidase activity is beneficial for treatment, such as tumors (including but not limited to breast cancer).

[0075] An eighth aspect of the present invention provides a cosmetic method comprising administering to a subject an effective amount of the aforementioned cyclic peptide, the cyclic peptide obtained by the aforementioned screening method, the cyclic peptide obtained by the aforementioned preparation method, or the aforementioned cosmetic product.

[0076] A ninth aspect of the present invention provides an application of the above-described cyclic peptide, the cyclic peptide obtained by the above-described screening method, and the cyclic peptide obtained by the above-described preparation method, said application including the application in inhibiting hyaluronidase, particularly the application in the fields of pharmaceuticals, food, or cosmetics.

[0077] In a tenth aspect, the present invention provides the application of the above-described cyclic peptide, the cyclic peptide obtained by the above-described screening method, and the cyclic peptide obtained by the above-described preparation method in the fields of medicine, food, or cosmetics.

[0078] Preferably, the application includes:

[0079] I) Use in the preparation of drugs that inhibit hyaluronidase activity, have anti-inflammatory effects, or treat or prevent diseases;

[0080] II) Application in the preparation of cosmetics or food products that inhibit hyaluronidase activity, increase skin transdermal absorption, and have anti-inflammatory, anti-aging, repairing, moisturizing, soothing, whitening, spot-removing, anti-wrinkle, or anti-aging effects.

[0081] Preferably, the diseases include those for which inhibiting hyaluronidase activity is beneficial for treatment, such as tumors (including but not limited to breast cancer).

[0082] Preferably, the anti-inflammatory effect includes inhibiting the expression of inflammatory factors, wherein the inflammatory factors preferably include IL-1β.

[0083] In an eleventh aspect, the present invention provides an anti-inflammatory method, the method comprising using the above-described cyclic peptide, the cyclic peptide obtained by the above-described screening method, the cyclic peptide obtained by the above-described preparation method, or the above-described drug, food, or cosmetic. Preferably, the anti-inflammatory effect comprises inhibiting the expression of inflammatory factors, wherein the inflammatory factors preferably include IL-1β.

[0084] A twelfth aspect of the present invention provides a method for inhibiting hyaluronidase activity, the method comprising using the above-described cyclic peptide, the cyclic peptide obtained by the above-described screening method, the cyclic peptide obtained by the above-described preparation method, or the above-described pharmaceutical, food, or cosmetic product. Preferably, the hyaluronidase comprises one or more of HYAL-1, HYAL-2, HYAL-3, HYAL-4, or PH20. In one specific embodiment of the present invention, the hyaluronidase is PH20.

[0085] The term "prevention" as used in this invention refers to a method implemented to prevent or delay the occurrence of a disease, condition, or symptom in the body.

[0086] The term "treatment" as used in this invention refers to slowing down, interrupting, preventing, controlling, stopping, reducing, or reversing the progression or severity of a sign, symptom, disorder, condition, or disease after the disease has begun to develop, but does not necessarily involve the complete elimination of all disease-related signs, symptoms, conditions, or disorders.

[0087] The term "effective amount" as used in this invention refers to the amount or dose of the product of this invention that provides the desired treatment after being administered to a subject in one or more doses.

[0088] The "subject" described in this invention can be a human or a non-human animal, or a cell, tissue, or organ of a human or non-human animal. The non-human animal can be a wild animal, a zoo animal, an economically important animal, a pet, or a laboratory animal, etc. Preferably, the non-human animal includes, but is not limited to, pigs, cattle, sheep, donkeys, foxes, minks, jackals, camels, dogs, cats, rabbits, mice (e.g., rats, mice, guinea pigs, hamsters, gerbils, chinchillas, or squirrels, etc.) or monkeys, etc.

[0089] The "method" or "application" described in this invention may be for therapeutic purposes or for non-therapeutic purposes.

[0090] The term "comprising" in this invention is an open-ended description, including the specified ingredients or steps described, as well as other specified ingredients or steps that do not substantially affect the description.

[0091] The term "and / or" as used in this invention includes all combinations of items connected by the term, and should be regarded as each combination having been individually listed in this invention. For example, "A and / or B" includes "A", "B", and "A and B"; and "A, B and / or C" includes "A", "B", "C", "A and B", "A and C", "B and C", and "A and B and C".

[0092] The terms "pharmaceutically acceptable," "food acceptable," or "cosmetic acceptable" as used in this invention refer to the biological activity and properties of the active substances in the applied product that neither significantly irritate the organism nor inhibit its activity.

[0093] The amino acid residue "X" mentioned in this invention can be a natural amino acid, a non-natural amino acid, or an analogue of a natural amino acid. The abbreviations and full names of twenty natural amino acids are listed in Table 1 below:

[0094] Table 1

[0095]

[0096]

[0097] The present invention has the following beneficial effects:

[0098] 1) This invention utilizes phage display technology to screen various polypeptide backbones, identifying specific backbones capable of binding hyaluronidase, i.e., X. 1(n) CX3X4X5X6X7X8X9X 10 CX 12(m)Compared to existing methods for screening peptide drugs (such as biochemical separation techniques, bioinformatics methods based on sequence homology, and modification and synthesis of existing natural peptides), the screening method of this invention is simple to operate and can efficiently and quickly obtain peptides with high affinity.

[0099] 2) The polypeptide of the present invention combines the advantages of small molecule drugs and protein drugs, such as small relative molecular mass, good stability, strong biological penetration, strong affinity for target, high specificity, few side effects, low immunogenicity, and low production cost.

[0100] 3) The polypeptides of the present invention are chemically synthesized and then oxidized into rings to form cyclic peptides. Compared with linear polypeptides, cyclic peptides have a unique and stable protein-like 3D structure, stronger target affinity and specificity, and resistance to protein hydrolysis, which can greatly improve stability in use. Compared with linear polypeptides, the cyclic peptides of the present invention have better transdermal effects.

[0101] 4) The cyclic peptide of the present invention can significantly inhibit the activity of hyaluronidase (especially PH20) and has excellent transdermal effect and anti-inflammatory effect, and has broad application prospects in the development of pharmaceuticals, food or cosmetics and raw material addition. Attached Figure Description

[0102] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings, wherein:

[0103] Figure 1 : Schematic diagram of the phage display library screening process;

[0104] Figure 2 : Phage enrichment results after three rounds of screening;

[0105] Figure 3 : Linear polypeptide (P1-L) (before oxidation) mass spectrometry;

[0106] Figure 4 Mass spectrometry of cyclic peptide (P1) (after oxidation);

[0107] Figure 5 Liquid chromatography pattern of purified cyclic peptide (P1);

[0108] Figure 6 : Linear peptide (P2-L) (before oxidation) mass spectrometry;

[0109] Figure 7 Mass spectrometry of cyclic peptide (P2) (after oxidation);

[0110] Figure 8 Liquid chromatography pattern of purified cyclic peptide (P2);

[0111] Figure 9: Graph showing the inhibition of hyaluronidase activity by cyclic peptides (P1-P12);

[0112] Figure 10 Transdermal properties of linear peptides (P1-L) and cyclic peptides (P1);

[0113] Figure 11 Transdermal properties of cyclic peptides (P2-P12);

[0114] Figure 12 Anti-inflammatory effects of cyclic peptides (P1-P12). Detailed Implementation

[0115] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0116] Example 1: Library Construction

[0117] The library construction steps are as follows Figure 1 As shown, it specifically includes:

[0118] (1) Design the backbone of an octapeptide-tetradecapeptide random cyclic peptide library, the backbone structure of which includes CX (8-12) C, X (0-2) CX (8) CX (0-2) X (0-3) CX (5-6) CX (0-3) Primers were designed using Snapgene software. After mixing equal volumes of primers with the same backbone, the mixture was boiled at 100°C for 10 minutes and allowed to cool naturally to form complementary DNA double strands. After mixing different backbone DNAs, the backbone DNA and pcantab plasmid were digested with HindIII and NotI enzymes and then ligated using T4 ligase.

[0119] (2) The TG1 strain was spread on 2YT antibiotic-free solid plates and cultured overnight at 37°C. Single colonies were picked and inoculated into 2YT liquid medium and cultured overnight at 37°C and 220 r / min. 5 mL of the bacterial culture was then inoculated into 500 mL of 2YT liquid medium and cultured until OD500. 600 =0.8~1, ice bath for 30 min, 2000 r / min, collect the precipitate for 20 min, discard the supernatant, add 300 mL of pre-cooled purified water and wash twice, 2000 r / min, collect the precipitate for 20 min, add 300 mL of 10% pre-cooled glycerol, 2000 r / min, collect the precipitate for 20 min and wash twice, to obtain a total of 5 mL of TG1 competent cells.

[0120] (3) After the plasmid was recovered and desalted using the Cycle-Pure recovery kit, 10 μg was added to TG1 competent cells and incubated on ice for 30 min. The plasmid was then dispensed into electroporation cups in 200 μL tubes and electroporated at 2.5 kV. The electroporated cells were collected using 1 mL of SOC liquid medium. All the transformed cells were collected and cultured at 37℃ and 220 r / min for 1 h to activate them.

[0121] (4) Collect the precipitate at 6000 r / min, spread it on a 2YT solid plate, and incubate overnight at 37℃. Take another 10 μL of the transformed bacterial culture, dilute it to 10^8 with 2YT medium, and add it to 2YT solid medium for detection. This constructs a 2×10^6 bacterial culture. 9 Random TG1 glycerol bacteria.

[0122] (5) Collect single clones on large plates using 2YT liquid medium to obtain random TG1 glycerol bacterial libraries. Add an equal volume of 30% glycerol and store at -80℃.

[0123] (6) Inoculate the random TG1 glycerol bacterial bank into 2YT liquid medium, add glucose and 0.1% ampicillin to a final concentration of 2%-5%, until OD is reached. 600 =0.1, incubate at 37℃ for 2 hours until OD 600 Reaching 0.3-0.6;

[0124] (7) Calculate the number of colonies in the culture, the calculation method being OD. 600 ×10 9 × the volume of the culture, add 10 times the number of bacteria of helper phage M13K07, incubate at 37°C for 2 hours, then incubate at 37°C and 220 rpm for 2 hours with shaking.

[0125] (8) Centrifuge the above culture at 4℃ for 6000 rpm for 20 min and discard the supernatant.

[0126] (9) Add the collected precipitate to 2YT liquid culture medium, and add 0.1% ampicillin and 0.1% kanamycin. Incubate overnight at 37°C and 220 rpm.

[0127] (10) Centrifuge the above culture at 4℃ for 6000 rpm for 20 min, collect the supernatant, add 1 / 5 of the supernatant volume of PEG / NaCl solution, mix by inverting several times, and let stand overnight at 4℃.

[0128] (11) After centrifuging the above solution at 1000 rpm for 30 min, collect the precipitate, add 1 mL of sterile PBS solution and resuspend it to obtain the amplified phage solution.

[0129] (12) Take 10 μL of the amplified phage solution to determine the titer. The titer determination method is as follows: add the phage solution to 90 μL of 2YT liquid culture medium, and dilute it 10 times in successive 10-fold dilutions for a total of 10 dilutions. Take 10 μL of each diluted phage solution and add it to 90 μL of OD medium. 600nm Take 10 μL of a TG1 solution with a concentration of 0.9 and add it dropwise into a 2YT solid plate containing ampicillin resistance. Incubate overnight at 37°C with the plate inverted position. Store the remaining solution at 4°C for later use.

[0130] Example 2: Selection of Cyclic Peptide Phage Display Library

[0131] (1) Dilute hyaluronidase with PBS buffer, add biotin solution and bind at 37°C for 2 hours. Purify the modified hyaluronidase solution with a desalting column, balance the system with PBS and remove excess unbound biotin.

[0132] (2) Add 2-10 μg of hyaluronidase solution to 100 μL of streptavidin magnetic beads as the experimental group; add 2-10 μg of hyaluronidase solution to 100 μL of non-bound magnetic beads as the control group. Incubate at 37℃ for 1 h. After full binding, discard the unbound enzyme solution, wash the magnetic beads three times with 1 mL of TBS solution, add 0.5%-2% BSA solution to block the magnetic beads, and add 3 times the volume of TBS solution and mix thoroughly.

[0133] (3) Take a titer > 10 12 The phage solution prepared in Example 1 was incubated with 0.5%-2% BSA and 3 times the volume of TBS solution.

[0134] (4) The phage solution treated in step (3) was added equally to the experimental group and the control group, and incubated at 37°C for 4 hours with the inverted incubation.

[0135] (5) Use a magnetic rack to pick up the magnetic beads, discard the phage solution, wash three times with TBST, then wash twice with TBS, add 200 μL of 1M glycine (pH=3.0) and let stand, use a magnetic rack to pick up the magnetic beads, remove the eluent, and quickly neutralize to neutral pH with 50 μL of Tris-HCl.

[0136] (6) Determination of phage titer in elution buffer: The titer of recombinant phage in elution buffer was determined according to the procedure in Example 1.

[0137] (7) Add the remaining elution buffer to the TG1 bacterial culture and let it stand for infection. After that, centrifuge at 6000 rpm for 10 min to collect the bacterial cells. Spread the cells on 0.1% ampicillin-resistant solid 2YT plates to obtain the screening library. Incubate overnight at 37°C for amplification.

[0138] (8) The obtained screening library was amplified according to the method in Example 1 for the next round of screening.

[0139] (9) Repeat steps (1)-(8) twice to complete the second and third rounds of selection.

[0140] Ensure that the amount of phage introduced for screening is always maintained at 5×10 12 The results of the three rounds of selection are shown in Table 2 and Figure 2 As shown, the phage enrichment rates in the first, second, and third rounds were 0.9, 12.5, and 32.9, respectively. The phage enrichment rate gradually increased, which is due to the elution of a large number of non-specific and relatively weakly specific phages during the initial screening. As the screening progressed, the phage enrichment rate (enrichment rate = number of experimental groups in the output group / number of control groups) gradually increased. The enrichment rate of the experimental group was 10 times higher than that of the control group, indicating that the phage peptide library specifically bound to hyaluronidase and was effectively enriched.

[0141] Table 2 Results of three rounds of screening for hyaluronidase in the cyclic peptide phage library.

[0142]

[0143] (10) Positive phage clones with a positive to negative ratio greater than 20 were sent to Novogene for high-throughput sequencing. The primers for library construction were SEQ ID NO: 13 and SEQ ID NO: 14.

[0144] SEQ ID NO: 13: 5'---ATCTCCAACTGGCTACTTACGTTCAT---3';

[0145] SEQ ID NO: 14: 5'---CCGTGCATTTGGGCAATCAGCTTTTAC---3'.

[0146] (11) The high-throughput results were analyzed in Matlab. The gene sequence and corresponding amino acid sequence between the two primers were the affinity peptide sequences displayed by the phage (Table 3). The top 12 peptides with enrichment were obtained, and it was determined that the top 11 sequences with enrichment all conformed to X. 1(n) CX3X4X5X6X7X8X9X 10 CX 12(m) The backbone demonstrates that sequences conforming to this backbone have a strong binding affinity to hyaluronidase.

[0147] Table 3. Enriched peptide sequences obtained from high-throughput sequencing.

[0148]

[0149]

[0150] Example 3: Synthesis of polypeptides using solid-phase synthesis

[0151] (1) Weigh 2-Cl-CTC (2-chlorotriphenylmethyl chloride) resin into a reactor, soak it in dichloromethane first, then wash the resin with N,N-dimethylformamide (DMF) and dry it.

[0152] (2) Weigh an appropriate amount of Fmoc-X (上述多肽的第一个氨基酸) -OH, N,N-diisopropylethylamine (DIEA), and DMF are added to the reactor for reaction.

[0153] (3) Add a decapping solution (DMF solution of 20% piperidine by volume) to the reactor to remove the Fmoc protecting groups on the resin.

[0154] (4) Weigh an appropriate amount of Fmoc-X (上述多肽的第二个氨基酸) -OH, hydroxybenzotriazole (HOBt), diisopropylcarbodiimide (DIC), and DMF are added to the reactor for reaction.

[0155] (5) Add a decapping solution (20% DMF solution of piperidine by volume) to the reactor to remove the Fmoc protecting groups on the resin.

[0156] (6) If the polypeptide has only 2 amino acids, proceed to the next step. If it has more than 2 amino acids, repeat steps (4) and (5) until the last amino acid is added.

[0157] (7) The polypeptide was cut off from the resin using a cutting solution (ratio of trifluoroacetic acid: water: triisopropylsilane = 95%: 2.5%: 2.5%) to obtain crude polypeptide.

[0158] (8) Dissolve the crude polypeptide in pure water, and then purify it by HPLC reversed-phase column chromatography or filter and purify the crude polypeptide solution using a filter membrane. Freeze-dry the polypeptide using a vacuum freeze dryer to obtain the finished linear polypeptide powder with a purity of over 95%, which is the linear polypeptide corresponding to Table 3 in Example 2.

[0159] (9) The linear peptide was dissolved in PBS (100mM) buffer at pH 7.4 and quantified using a UV-Vis spectrophotometer.

[0160] (10) Add 10 times the amount of GSSG and 20% DMSO to the linear peptide, and finally dilute with PBS solution to a final peptide concentration of 50 μM and a total volume of 500 μL.

[0161] (11) After mixing evenly, the reaction system is placed in a constant temperature shaker at 37°C and reacted for 12 hours to oxidize the linear peptide into a ring. Finally, the cyclic peptide is separated by high performance liquid chromatography.

[0162] (12) The purified cyclic peptide product qualified liquid was freeze-dried in a vacuum freeze dryer to obtain the finished product powder, and cyclic peptide P1-P12 was obtained.

[0163] Sequencing and mass spectrometry verification showed that this embodiment obtained the cyclic peptides P1-P12 of each sequence in Table 3 of Example 2. Exemplary results are shown below. Figure 3-8 .

[0164] Taking the P1-P2 cyclic peptide as an example, high-throughput sequencing yielded the synthesis spectrum, and mass spectrometry detected the structure of the linear peptides (P1-L, P2-L) before oxidation and the cyclic peptides (P1, P2) formed after oxidation. Figure 3 , 4 and Figure 6 , 7 The final cyclic peptide powder was obtained by desalting and purification using liquid chromatography-reversed-phase column chromatography (HPLC). Figure 5 and Figure 8 This demonstrates that the linear polypeptide was successfully synthesized and cyclically oxidized.

[0165] The steps for mass spectrometry verification and molecular weight results are shown in Table 4, and the steps for HPLC reversed-phase column chromatography desalting and purification are shown in Table 5. The HPLC purification results are shown in Table 6.

[0166] Table 4

[0167]

[0168] Table 5

[0169]

[0170]

[0171] Table 6

[0172]

[0173]

[0174] Example 4 uses a colorimetric method to test the inhibitory effect of cyclic peptides on hyaluronidase activity.

[0175] (1) Hyaluronidase catalytic activation:

[0176] Prepare a hyaluronidase pH20 solution with an enzyme activity between 100-1000U using purified water, add 0.1mL of 0.25mM calcium chloride solution, and incubate in a 37℃ water bath for 1 hour.

[0177] (2) Cyclic peptide binding activity assay:

[0178] Preparation of sodium acetate solution: Take 577 μL of glacial acetic acid, dilute to 50 mL with deionized water, mix well to obtain solution A. Weigh 6.80 g of sodium acetate, dilute to 250 mL with deionized water, mix well to obtain solution B. Take 4.80 mL of solution A and 45.20 mL of solution B, dilute to 100 mL with deionized water, mix well.

[0179] Experimental group: Cyclic peptides P1 to P12 were prepared with purified water to a concentration of 0.1 mg / mL. 0.5 mL of each group's cyclic peptide solution was added to the hyaluronidase solution and incubated in a water bath at 37°C for 1 h.

[0180] Positive control group (PC): Weigh 15.5 mg of dipotassium glycyrrhizate, add it to 10 mL of acetate buffer and dissolve it completely (to prepare 0.15% dipotassium glycyrrhizate), take 0.5 mL and add it to hyaluronidase solution, and incubate at 37°C for 1 h.

[0181] Negative control group (NC): Untreated hyaluronidase solution.

[0182] (3) Substrate reaction: Sodium hyaluronate substrate solution: Sodium hyaluronate solid was prepared into a 0.5 mg / mL solution using sodium acetate solution. 0.5 mL of sodium hyaluronate solution was added to the above positive control group, negative control group, or experimental group respectively.

[0183] The mixture was obtained by bathing in a water bath at 37°C for 50 minutes.

[0184] (4) Termination of reaction: Add 0.1 mL of 0.4 mol / L sodium hydroxide solution to the mixed solution to terminate the reaction.

[0185] (5) Chromogen activation: Take 0.5 mL of 0.1 mol / L sodium borate solution and add it to the mixed solution to terminate the reaction. Incubate at 100℃ for 30 min, then immediately in an ice-water bath for 30 min.

[0186] (6) Colorimetric reaction: Add 1 mL of p-DMAB colorimetric reagent to the chromogen-activated mixed solution and incubate at 30°C for 15 min. Measure OD. 584nm The competitive inhibition rate of each cyclic peptide against hyaluronidase was confirmed.

[0187] The results are as follows Figure 9 As shown, the P1 to P11 cyclic peptides all exhibited the ability to significantly downregulate the activity of hyaluronidase PH20, indicating that the interaction between the cyclic peptides screened and prepared in this invention and hyaluronidase affects the structure of hyaluronidase to a certain extent, leading to changes in activity.

[0188] In addition, although the enrichment of P12 cyclic peptide was also high, its inhibition rate on hyaluronidase activity was only 5.06%, while the inhibition rates of P1-P11 cyclic peptides on hyaluronidase activity were all greater than 20%. Among them, the inhibition rates of P1-P5, P7, and P9 cyclic peptides on hyaluronidase activity were greater than 30%, especially the inhibition rates of P1, P2, P3, P4, and P7 cyclic peptides, which could reach more than 40%, showing significantly better effects than the positive control group (PC).

[0189] The P1-P11 cyclic peptide sequence follows the backbone X. 1(n) CX3X4X5X6X7X8X9X 10 CX 12(m) Where n or m is independently selected from integers from 0 to 3; X1, X3, X4, X5, X6, X7, X8, X9, X 10 or X 12 X3 is any amino acid except cysteine; X4 is any one of F, W, H, S, or Y; X5 is any one of R, E, T, H, or W; X6 is any one of M, L, H, Q, or V; X7 is any one of S, G, or E; X8 is any one of E, M, V, T, or A; X9 is any one of L, M, or V; X 10 Selected from any one of S, E, F, H or V, it is demonstrated that the cyclic peptide backbone has a high affinity for hyaluronidase and can exhibit a significant inhibitory effect.

[0190] Example 5: Comparison of transdermal effects of linear peptides and cyclic peptides

[0191] This embodiment verifies the transdermal efficiency of linear peptides and cyclic peptides, specifically:

[0192] (1) Preparation of oil phase: Take glyceryl monostearate: stearic acid: liquid paraffin = 1:6:3, stir and heat until completely melted, mix thoroughly to dissolve completely;

[0193] (2) Aqueous phase preparation: Weigh 1 part of triethanolamine and 76 parts of purified water, stir and heat until completely dissolved;

[0194] (3) Use a homogenizer to fully disperse the aqueous phase, slowly add the aqueous phase to the oil phase, mix well, cool down and let the sample solidify to prepare a cream-like sample;

[0195] (4) The linear peptides and cyclic peptides synthesized in liquid phase in Example 3 were added to the cream sample and thoroughly mixed to prepare a 100 mg / mL sample.

[0196] (5) At 50mg / cm 2 The dosage (approximately 5 mg / cm³ for linear or cyclic peptides) is as follows. 2Each sample was added to pigskin, and the transdermal diffusion test system was used to detect the transdermal effect.

[0197] Exemplary comparison results are as follows Figure 10 As shown, after approximately 13 hours, the concentrations of both linear and cyclic peptides reached their peak and then stabilized. After 24 hours, the concentration of the cyclic peptide (P1) was 0.753 μg / mL, while the concentration of the linear peptide (P1-L) remained below 0.1 μg / mL throughout the experimental period. The transdermal permeability of the cyclic peptide oxidized to a ring was 8 times that of the linear peptide. Additionally, as shown in Table 7 and... Figure 11 As shown, the concentrations of P2-P12 cyclic peptides were all greater than 0.3 μg / mL, significantly higher than the corresponding linear peptide concentrations. Comparing the concentrations of linear and cyclic peptides after 24 hours, it was found that the transdermal permeability of oxidized cyclic peptides was significantly improved compared to linear peptides. This indicates that, compared to linear peptides, cyclic peptides oxidized to rings can significantly improve the transdermal permeability of peptides, making them suitable for use in cosmetic raw materials.

[0198] Table 7

[0199]

[0200]

[0201] Example 6: Comparison of the anti-inflammatory effects of cyclic peptides

[0202] (1) Inoculation: RAW cells were seeded into 24-well plates and incubated at 37°C in a 5% CO2 incubator for 18-24 hours.

[0203] (2) Sample feeding: After the cells in the 24-well plates have grown for 18-24 hours, samples are fed, with 3 replicates for each sample. Cell culture medium is added to both the blank control group (BC group) and the negative control group (NC group). 50 ppm of cyclic peptide P1-P12 is added to each sample group, and 0.001% dexamethasone is added to the positive control group (PC group). The cells are incubated at 37°C in a 5% CO2 incubator for 18-24 hours.

[0204] (3) LPS induction: After culturing for 18-24h, the culture medium in the plate was aspirated and washed once with PBS. Cell culture medium was added to the BC group, and cell culture medium containing LPS was added to the NC group, sample group and PC group. The cells were cultured at 37℃ and in a 5% CO2 incubator for 18-24h.

[0205] (4) Detection of inflammatory factors: Take the cell supernatant from each well and detect the inflammatory factors in the cells according to the ELISA kit instructions.

[0206] The results are as follows Figure 12As shown, the experimental results of the BC and NC groups demonstrate that the addition of LPS can significantly stimulate the occurrence of inflammation. The results of the PC and NC groups, compared with those of the PC and NC groups, demonstrate that dexamethasone can inhibit IL-1β expression, proving the validity of this experimental model. By comparing the results of the NC group with those of each sample group, it was found that, except for the P12 cyclic peptide, the P1-P11 cyclic peptides could all inhibit IL-1β expression, proving that the cyclic peptides screened in this invention have significant anti-inflammatory effects. Specifically, after treatment with P1-P3, P5, P7, and P8 cyclic peptides, the concentration of the inflammatory factor IL-1β decreased by more than 27%; particularly, after treatment with P1, P2, P7, and P8 cyclic peptides, the concentration of IL-1β decreased by more than 36%, exhibiting even better anti-inflammatory effects.

[0207] Although specific embodiments of the invention have been described, those skilled in the art will recognize that various changes and modifications can be made to the invention without departing from its scope or spirit. Therefore, the invention is intended to cover all such changes and modifications falling within the scope of the appended claims and their equivalents.

[0208] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0209] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

Claims

1. A cyclic peptide, characterized in that, The amino acid sequence of the cyclic peptide includes X 1(n) CX3X4X5X6X7X8X9X 10 CX 12(m) ; in, n or m are integers independently selected from 0 to 10; X1, X3, X4, X8 or X 12 It is not cysteine; X5 is selected from any one of R, E, T, Q, or V; X6 is selected from any one of M, L, H, Q or V; X7 is selected from any one of S, G, or E; X9 is selected from L, M, or V; X 10 Choose from any one of S, E, F, H or V.

2. The cyclic peptide according to claim 1, characterized in that, X3 is selected from any one of F, W, H, S or Y; X4 is selected from any one of M, R, S, E, T, H or W; X8 is selected from any one of E, M, V, T or A.

3. The cyclic peptide according to claim 1 or 2, characterized in that, X3 is selected from F, W, or H; X4 is selected from M, R, E, S, T, or H; X5 is selected from R, T, or E; X6 is selected from M or Q; X7 is selected from S, E, or G; X8 is selected from E, A, T, or M; X9 is selected from L or M; X 10 Selected from any one of S, E, H or V; Preferably, X3 is selected from F or W; X4 is selected from M, E, R or S; X6 is M; X7 is selected from S or G; and X8 is selected from E, A or M.

4. The cyclic peptide according to claim 1, characterized in that, The amino acid sequence of the cyclic peptide includes SEQ ID NO: 1-11.

5. A method for preparing the cyclic peptide according to any one of claims 1-4, characterized in that, The preparation methods include chemical synthesis or prokaryotic expression.

6. The preparation method according to claim 5, characterized in that, The preparation method includes chemically synthesizing a linear polypeptide and then oxidizing it into a ring to obtain a cyclic peptide.

7. A method for screening cyclic peptides according to any one of claims 1-4, the screening method comprising using phage display technology; Preferably, the screening method includes: 1) Construct a phage display library, wherein the phage display library contains an octapeptide-tetradecapeptide cyclic peptide backbone, wherein the cyclic peptide backbone contains cysteine; 2) Use the phage display library obtained in 1) to screen for hyaluronidase and obtain target phages that bind to hyaluronidase; 3) Amplify and sequence the target phage that binds to hyaluronidase.

8. The cyclic peptide according to any one of claims 1-4, the cyclic peptide obtained by the preparation method according to any one of claims 5-6, or the cyclic peptide obtained by the screening method according to claim 7, for use as a hyaluronidase inhibitor.

9. The application of the cyclic peptide according to any one of claims 1-4, the cyclic peptide obtained by the preparation method according to any one of claims 5-6, or the cyclic peptide obtained by the screening method according to claim 7 in the fields of medicine, food, or cosmetics.

10. The application according to claim 9, characterized in that, The applications include: I) Use in the preparation of drugs that inhibit hyaluronidase activity, have anti-inflammatory effects, or treat or prevent diseases; II) Application in the preparation of cosmetics or food products that inhibit hyaluronidase activity, increase skin transdermal absorption, and have anti-inflammatory, anti-aging, repairing, moisturizing, soothing, whitening, spot-removing, anti-wrinkle, or anti-aging effects.

11. A drug, food, or cosmetic, characterized in that, The aforementioned drugs, food, or cosmetics include the cyclic peptides according to any one of claims 1-4, the cyclic peptides obtained by the preparation methods according to any one of claims 5-6, or the cyclic peptides obtained by the screening methods according to claim 7.

12. A method for inhibiting hyaluronidase activity, characterized in that, The method includes the cyclic peptides of any one of claims 1-4, the cyclic peptides obtained by the preparation methods of any one of claims 5-6, the cyclic peptides obtained by the screening method of claim 7, or the pharmaceutical, food, or cosmetic product of claim 11.

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