A tripeptide, a tripeptide salt or a derivative and its application

Abstract

The present invention discloses a tripeptide, a tripeptide salt or a derivative and an application thereof, and the structure thereof is shown in Formula I. The tripeptide simultaneously has the functions of anti-oxidation, promoting cell migration, and inhibiting tyrosinase activity. Compared with the existing single-functional peptides, the tripeptide disclosed in the present invention has a wider range of application scenarios, and a smaller molecular weight (only about 500Da), and has a better transdermal potential than other macromolecular polypeptides, which is particularly beneficial for its use as a raw material for medicines or cosmetics.

Description

[0001] This application is a divisional application of the Chinese application with application number 202311086422.0, application date August 25, 2023, and invention name “A tripeptide, a tripeptide salt or derivative and its application”. Technical Field

[0002] The present invention relates to the technical field of biological peptides, and in particular to a tripeptide, a tripeptide salt or a derivative and applications thereof. Background Art

[0003] The skin is located on the surface of the body. It is the first physiological line of defense to protect the body's tissues and organs from damage by mechanical, chemical, physical and microbial invasions, and plays a barrier protection role for the body. However, with age and the influence of external adverse environmental factors, the skin will accumulate a large number of free radicals, causing oxidative stress and inflammatory reactions, which will damage cell biomolecules and destroy the skin tissue structure, resulting in impaired skin barrier function. Moreover, the free radicals produced will further induce excessive melanin production, leading to skin pigmentation and the formation of dark spots, affecting the beauty of the skin. Therefore, effective free radical scavenging can not only protect the skin barrier and health, but also effectively improve problems such as skin pigmentation and dark spot formation. In addition, promoting skin repair is also beneficial to assist in restoring the damaged skin barrier and re-establishing a healthy skin barrier protection function.

[0004] Peptides have a wide range of biological activity potentials due to their various structures. At present, a large number of peptides with single biological activities have been discovered, but peptides with multiple functions are still scarce. Therefore, if a peptide that has both antioxidant and cell migration promoting properties can be effectively developed, it will play an important role in protecting and restoring skin health. Moreover, if it also has tyrosinase inhibitory activity, it can further reduce melanin production, which is of great significance for reducing the formation of dark spots and brightening the skin. Summary of the invention

[0005] The present invention aims to solve at least one of the above-mentioned technical problems existing in the prior art. To this end, the purpose of the present invention is to provide a tripeptide, a cosmetically or pharmaceutically acceptable salt thereof or a derivative thereof and its use. The tripeptide in the present invention has the effects of anti-oxidation, promoting cell migration and inhibiting tyrosinase, and can be widely used in the fields of food, medicine and cosmetics, thus having extremely high practical value and economic value.

[0006] The first aspect of the present invention provides a tripeptide, a salt thereof or a derivative thereof, wherein the tripeptide has a structure as shown in Formula I:

[0007] X 1 -AA1-AA2-AA3-X 2

[0008] Formula I

[0009] wherein AA1 is selected from one of Trp, His, Phe, Arg or Thr;

[0010] AA2 is selected from one of Phe, Trp, Arg or Ala;

[0011] AA3 is selected from one of His, Trp, Thr or Phe;

[0012] X 1 and X 2 It is an inactive group or does not exist.

[0013] In some embodiments of the present invention, the inactive groups include protecting groups and labeling groups, such as Trt, Acm, Mob, Boc, Fmoc, Trt, Dde, Allyl, Otbu, OBzl, OMe, OAll, OFm, etc.

[0014] In some embodiments of the present invention, the X 1 and X 2 Does not exist.

[0015] In the present invention, the term "amino acid" refers to any natural or unnatural amino acid, including any L- or D-configuration amino acids. When all amino acids are in L-configuration, the peptide is called L-enantiomer; when all amino acids are in D-configuration, the peptide is called D-enantiomer. The term may also include amino acid analogs used as peptide mimetics or peptoids. The term also includes modified or unusual amino acids or synthetic derivatives of amino acids, such as diaminobutyric acid and diaminopropionic acid. In some embodiments of the present invention, tripeptides are amino acids linked together by peptide bonds, usually in a helical conformation under hydrophobic conditions. Unless otherwise indicated, the above tripeptides are usually given sequences from the amino terminus to the carboxyl terminus.

[0016] In some embodiments of the present invention, the salt comprises a cosmetically or pharmaceutically acceptable salt.

[0017] In some embodiments of the present invention, the salt is a soluble salt.

[0018] In some embodiments of the present invention, the salt is a salt formed with a metal ion, an organic acid, an organic base, or an inorganic acid.

[0019] In some embodiments of the invention, the metal ions include lithium, sodium, potassium, calcium, magnesium, manganese, copper, zinc and aluminum ions.

[0020] In some embodiments of the present invention, the organic base includes ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, arginine, lysine, histidine and piperazine.

[0021] In some embodiments of the present invention, the organic acid comprises acetic acid, citric acid, lactic acid, malonic acid, maleic acid, tartaric acid, fumaric acid, benzoic acid, aspartic acid, glutamic acid, succinic acid, oleic acid, trifluoroacetic acid, oxalic acid, pamoic acid and gluconic acid.

[0022] In some embodiments of the present invention, the inorganic acid includes hydrochloric acid, sulfuric acid, boric acid and carbonic acid.

[0023] In some embodiments of the present invention, the tripeptide derivative includes a derivative obtained by chemical bond modification of the tripeptide, and the chemical bond modification includes N-terminal modification, C-terminal modification and CH-terminal modification.

[0024] In some embodiments of the invention, the modifications include but are not limited to methylation, acetylation, hexanoylation, octanoylation, hydroxylation, carboxylation, phosphorylation, glycosylation, palmitoylation, stearoylation, lauroylation, myristoylation and oleoylation.

[0025] In some embodiments of the present invention, the tripeptide comprises an amino acid sequence as shown in SEQ ID NOs: 1-5.

[0026] In some embodiments of the present invention, the tripeptide further comprises a polypeptide sequence having amino acid residues inserted at both ends of the amino acid sequence shown in SEQ ID NOs: 1 to 5, with a sequence identity greater than or equal to 90% and retaining functional activity.

[0027] In some embodiments of the present invention, the sequence identity is greater than or equal to 92%.

[0028] In some embodiments of the present invention, the sequence identity is greater than or equal to 95%.

[0029] In some embodiments of the present invention, the sequence identity is greater than or equal to 99%.

[0030] In the present invention, the term "maintaining functional activity" means having the same or similar functional effect as the amino acid sequence shown in SEQ ID NO: 1 to 5 in at least one of anti-oxidation, promoting cell migration or inhibiting tyrosinase.

[0031] In some embodiments of the present invention, the polypeptide sequence has at least 50%, 75% or 90% of the functional effect of the amino acid sequence shown in SEQ ID NO: 1-5 in at least one of anti-oxidation, promoting cell migration or inhibiting tyrosinase.

[0032] In some embodiments of the present invention, the polypeptide sequence has the effects of anti-oxidation, promoting cell migration and inhibiting tyrosinase.

[0033] The second aspect of the present invention provides a nucleic acid molecule encoding the tripeptide, its salt or derivative thereof according to the first aspect of the present invention.

[0034] In some embodiments of the present invention, the nucleic acid molecule further comprises a nucleotide sequence having a sequence identity of ≥90%.

[0035] In some embodiments of the present invention, the nucleic acid molecule further comprises a nucleotide sequence having a sequence identity of ≥95% thereto.

[0036] In some embodiments of the present invention, the nucleic acid molecule further comprises a nucleotide sequence having a sequence identity of ≥99%.

[0037] In some embodiments of the present invention, the peptide translated from the nucleotide sequence retains at least one of the functional effects of anti-oxidation, promoting cell migration or inhibiting tyrosinase.

[0038] In some embodiments of the present invention, the peptide translated from the nucleotide sequence has the effects of anti-oxidation, promoting cell migration and inhibiting tyrosinase.

[0039] The third aspect of the present invention provides an expression vector containing the nucleic acid molecule described in the second aspect of the present invention.

[0040] In some embodiments of the present invention, the tripeptide, its salt or derivative thereof is co-encoded by one or more expression vectors.

[0041] The fourth aspect of the present invention provides a transformant containing the expression vector described in the third aspect of the present invention.

[0042] In some embodiments of the present invention, the transformants include microorganisms and cells.

[0043] In the present invention, the term "microorganism" refers broadly to bacteria, fungi, viruses and protozoa. In particular, the term is applicable to microorganisms having cells or structural components with a lipid bilayer membrane. In some embodiments of the present invention, the membrane is a cytoplasmic membrane. Bacteria may include gram-negative bacteria and gram-positive bacteria.

[0044] In some embodiments of the invention, the cell is an isolated cell.

[0045] In some embodiments of the present invention, the cell is an in situ cell, which can be a living tissue, organ or animal cell including one from a human.

[0046] In some embodiments of the invention, the cell is a eukaryotic cell.

[0047] The fifth aspect of the present invention provides a composition, which contains the tripeptide, its salt or derivative thereof as described in the first aspect of the present invention.

[0048] In some embodiments of the present invention, the composition further contains other adjuvants.

[0049] In some embodiments of the present invention, the adjuvant includes cosmetically or pharmaceutically acceptable adjuvants or carriers, food additives and adjuvants in the field of cosmetics.

[0050] In the present invention, the term "cosmetically or pharmaceutically acceptable carrier" refers to conventional cosmetic materials or pharmaceutical carriers that can be used in the methods disclosed in the present invention. See Remington's Pharmaceutical Sciences, Ewmartin, Mack Publishing Co., Easton, Pa., 15th edition (1975).

[0051] In some embodiments of the present invention, the cosmetically or pharmaceutically acceptable adjuvants include but are not limited to diluents (such as starch, dextrin, sucrose, lactose, mannitol, etc.), absorbents (such as calcium sulfate, calcium hydrogen phosphate, etc.), wetting agents (such as ethanol), binders (such as hydroxypropyl methylcellulose, povidone, etc.), disintegrants (such as sodium hydroxymethyl starch, cross-linked polyvinylpyrrolidone, etc.), lubricants (such as talc, hydrogenated vegetable oil, polyethylene glycol, etc.), colorants (such as titanium dioxide, methylene blue, etc.), coating materials, solvents, pH adjusters, antibacterial agents (such as sodium sulfite, sodium thiosulfate, etc.), isotonic adjusters (such as glucose, sodium chloride, etc.), and chelating agents (such as disodium EDTA).

[0052] In some embodiments of the present invention, the food additives include but are not limited to colorants, enzyme preparations, thickeners, leavening agents, and sweeteners.

[0053] The sixth aspect of the present invention provides the use of the tripeptide, its salt or its derivatives described in the first aspect of the present invention in the preparation of food, medicine and cosmetics.

[0054] In some embodiments of the present invention, the food, medicine and cosmetic have at least one of the following functions (1) to (3):

[0055] (1) Antioxidant;

[0056] (2) Promote cell migration;

[0057] (3) Inhibit tyrosinase.

[0058] In some embodiments of the present invention, the promotion of cell migration is specifically manifested as promoting angiogenesis, promoting wound healing, promoting skin repair, etc.

[0059] In some embodiments of the present invention, inhibiting tyrosinase can effectively inhibit melanin production, thereby whitening and brightening.

[0060] The beneficial effects of the present invention are:

[0061] The present invention discovers a tripeptide for the first time, which has the functions of anti-oxidation, promoting cell migration, and inhibiting tyrosinase activity. Compared with the existing single-functional peptides, it has a wider range of application scenarios, and has a smaller molecular weight (only about 500Da), and has better transdermal potential than other macromolecular polypeptides, which is conducive to its use as a raw material for medicines or cosmetics. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 This is the antioxidant effect of the tripeptide in the embodiment of the present invention.

[0063] Figure 2 This is the cell migration promoting effect of the tripeptide in the example of the present invention.

[0064] Figure 3 This is the tyrosinase activity inhibitory effect of the tripeptide in the example of the present invention. DETAILED DESCRIPTION

[0065] The present invention is further described in detail below by specific examples. Unless otherwise specified, the raw materials, reagents or devices used in the examples and comparative examples can be obtained from conventional commercial sources or can be obtained by prior art methods. Unless otherwise specified, the experiments or test methods are conventional methods in the art.

[0066] Design and construction of tripeptides

[0067] In the present invention, the inventors found that the tripeptide constructed based on the following formula I has the effects of anti-oxidation, promoting cell migration, and reducing melanin production.

[0068] X 1 -AA1-AA2-AA3-X 2

[0069] Formula I

[0070] wherein AA1 is selected from one of Trp, His, Phe, Arg or Thr;

[0071] AA2 is selected from one of Phe, Trp, Arg or Ala;

[0072] AA3 is selected from one of His, Trp, Thr or Phe;

[0073] X 1 and X 2 It is an inactive group or does not exist.

[0074] The optional residues of AA1 to AA3 include L and D configurations, which may be modified commonly in the art, including but not limited to methylation, acetylation, hexanoylation, octanoylation, hydroxylation, carboxylation, phosphorylation, glycosylation, palmitoylation, stearoylation, lauroylation, myristoylation and oleoylation, etc.

[0075] In the present invention, after the above tripeptide design was completed, it was handed over to a biochemical company for synthesis, and the following five tripeptides (as shown in Table 1) were used for subsequent verification experiments.

[0076] Table 1 Tripeptides with antioxidant, cell migration promoting and melanin reduction effects

[0077] Tripeptide number Tripeptide sequence Sequence number P1 Trp-Trp-His SEQ ID NO: 1 P2 His-Phe-Trp SEQ ID NO: 2 P3 Phe-Arg-Trp SEQ ID NO: 3 P4 Trp-Arg-Trp SEQ ID NO: 4 P5 Arg-Trp-Trp SEQ ID NO: 5

[0078] Verification of the Cellular Antioxidant Activity of Tripeptides

[0079] In this example, human skin fibroblasts (HSF) were used as test subjects to detect the antioxidant effect of the tripeptides.

[0080] The specific test steps are as follows:

[0081] In 5% CO 2 Human skin fibroblast (HSF) cells were cultured in a 37°C incubator using DMEM / F12 medium containing 10% fetal bovine serum. When the HSF cells were in the logarithmic growth phase, they were digested with 0.25% trypsin to obtain a cell suspension. The cell suspension was taken and 1×10 4 Cells / well were transplanted into 96-well culture plates. Incubate for 24 hours in DMEM / F12 medium containing 10% fetal bovine serum. After the incubation, the original medium was discarded, and an equal amount of DMEM / F12 medium containing 0.1 mM of the above-mentioned different tripeptides was added (an equal amount of DMEM / F12 medium containing 0.5 mM reduced glutathione (GSH) was used as a control), and the cells were incubated in the incubator for 24 hours. After the incubation, the original medium was discarded, and an equal amount of DMEM / F12 medium containing 10 μM DCFH-DA fluorescent probe was added, and the cells were incubated in the dark in the incubator for 45 minutes. After the incubation, the residual probe was washed away. Then each group was added with 1000 μM H 2 O 2 Incubate in basal medium for 30 min (without H 2 O 2The basal medium of the cells was used as a control). The fluorescence intensity of each group was detected in an ELISA instrument, with an excitation wavelength of 488 nm and an emission wavelength of 525 nm.

[0082] One-way analysis of variance (ANOVA) was used to test the 2 O 2 Treatment group (Con) and H 2 O 2 Statistical differences among the treated group (Model), the peptide-containing group (GSH, P1, etc.) and the peptide-free group (Model). Compared with the Con group, #### indicates p<0.0001; compared with the Model group, **** indicates p<0.0001. The results are shown in Figure 1 shown.

[0083] It can be found that compared with the Con group (no H 2 O 2 treatment, without peptide intervention), Model group (H 2 O 2 The fluorescence intensity of the cells treated with H- 2 O 2 After treatment, the cells produced a large number of free radicals. After treatment with polypeptides (such as GSH, P1 and other polypeptide-containing groups), the fluorescence intensity was significantly reduced compared with the Model group, and the fluorescence intensity was not significantly increased compared with the Con group, indicating that the tripeptides such as P1 in the above examples can effectively reduce the generation of cellular free radicals, and the effect is similar to that of the positive control GSH, indicating that it has an excellent antioxidant effect.

[0084] Verification of the effect of tripeptide on promoting cell migration

[0085] In this example, human immortalized keratinocytes (HaCaT) were used as test subjects to detect the promoting effect of the above tripeptides on cell migration.

[0086] The specific test steps are as follows:

[0087] In 5% CO 2 In an incubator environment of 37°C, human immortalized keratinocytes were cultured in DMEM medium containing 10% fetal bovine serum. When HaCaT cells were in the logarithmic growth phase, they were digested with 0.25% trypsin to obtain a cell suspension. The cell suspension was taken and 4×10 5Cells / well were transplanted into a 12-well culture plate. After incubation for 24 hours, a 200 μL pipette tip was used to scratch the well plate, and the cells were washed 3 times with PBS to remove the scratched cells. Then, serum-free culture medium containing 100 μM of the above-mentioned different polypeptides was added (with an equal amount of serum-free culture medium without polypeptide added as a control), and the initial scratch area was recorded by taking a photo. After incubation in the incubator for another 24 hours, the scratch area was recorded again by taking a photo. The cell migration rate was calculated by the ratio of the cell migration area after incubation to the initial scratch area.

[0088] One-way analysis of variance (ANOVA) was used to test the statistical differences between the peptide-containing group (P1, etc.) and the peptide-free group (Con), where * indicates p<0.05, ** indicates p<0.01, *** indicates P<0.001 and **** indicates p<0.0001. Figure 2 shown.

[0089] It can be found that compared with the Con group without polypeptide intervention, the cell migration rate of the group intervened with P1 to P5 in the above example increased significantly, which can indicate that the above tripeptide has a promoting effect on cell migration.

[0090] Tyrosinase inhibitory effect of tripeptides

[0091] The expression and activity of tyrosinase determine the speed and output of melanin production. Generally speaking, the higher the tyrosinase activity, the more melanin is formed in the skin. In this example, the inhibitory effect of the above tripeptide on tyrosinase is verified to demonstrate its effect of inhibiting melanin production.

[0092] The specific test steps are as follows:

[0093] Add 50 μL of the above different peptide solutions with concentrations of 1mM and 5mM respectively to a 96-well plate, then add 150 μL of 1mg / mL levodopa solution, shake and mix, and then add 50 μL of 110U / mL tyrosinase solution. Measure the absorbance value at 475nm with an enzyme reader within 10s as the initial absorbance value. Then incubate it at 37°C for 20min and measure the absorbance value again. Calculate the change in absorbance values ​​of the blank group (no peptide group) and the sample group (peptide sample group) twice. Calculate the tyrosinase inhibition rate of the sample by the following formula.

[0094]

[0095] The results are as follows Figure 3 shown.

[0096] It can be found that the above tripeptides all showed a certain tyrosinase inhibitory effect, and generally showed obvious dose dependence. The administration of the tripeptide at a high concentration greatly enhanced the tyrosinase inhibitory effect. Therefore, it can be shown that the above tripeptide has an excellent effect of inhibiting melanin production.

[0097] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. Use of a tripeptide or a salt thereof in the preparation of a drug; the drug promotes cell migration, and the promotion of cell migration is manifested in promoting wound healing and promoting skin repair; The tripeptide is Phe-Ala-Trp.

2. Use of a tripeptide or its salt in the preparation of cosmetics; the cosmetics have the function of promoting skin repair; the tripeptide is Phe-Ala-Trp.

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