Transdermal tetrapeptide and application thereof

Abstract

The invention relates to transdermal tetrapeptide and application thereof, and belongs to the technical field of active polypeptide. A novel transdermal tetrapeptide Ala-Arg-Cys-Gly is designed and synthesized, the transdermal tetrapeptide has obvious penetration enhancing capacity, the penetration enhancing effect of the transdermal tetrapeptide is similar to that of the transdermal peptide TD-1 serving as an existing penetration enhancer and is remarkably superior to that of acetyl hexapeptide-1 and menthol serving as existing penetration enhancers, and the transdermal tetrapeptide is simple in structure, small in molecular weight, high in biocompatibility, free of toxic effect on cells and suitable for being used as a transdermal drug. The method can be used for preparing penetration promoting products and has wide development prospects and application potentials. Meanwhile, transdermal tetrapeptides Arg-Arg-Cys-Gly, Ala-Lys-Cys-Gly and Ala-Arg-Met-Gly are further synthesized, and although the transdermal delivery effect of the transdermal tetrapeptides is slightly lower than that of the Ala-Arg-Cys-Gly, the transdermal tetrapeptides still have an obvious penetration promoting effect.

Description

Technical Field

[0001] The invention relates to a transdermal tetrapeptide and application thereof, belonging to the technical field of active polypeptides. Background Art

[0002] The skin is the largest organ. Whether it is transdermal drug delivery for the treatment of skin diseases or for the purpose of beauty and skin care, how to promote the transdermal absorption of active ingredients is the focus of research by scientific researchers.

[0003] Existing penetration-enhancing technologies include drug-media interactive drug delivery, ion pairing, vesicle method, microneedle, chemical penetration enhancer, physical method (laser introduction, ultrasound introduction, electroporation), etc., but these technologies have many shortcomings, such as drug-media prodrugs are difficult to synthesize, ion pairing method only works on a part of ionized drugs, the synthesis process of vesicle method is cumbersome, microneedle method will leave certain wounds, chemical penetration enhancer will destroy the normal structure of the skin, physical penetration method process is cumbersome and must rely on special instruments, etc. Biological penetration-enhancing technology uses a class of polypeptide molecules with skin penetration ability, about 10 to 30 amino acids, which can effectively promote the permeation of macromolecules and other active ingredients. Transdermal peptides are non-irritating, will not cause skin allergies and damage, have good compatibility between drugs and materials, have stable physical and chemical properties, fast onset time, and long action time.

[0004] Transdermal peptides are polypeptides with enhanced transdermal function, which can enhance and / or facilitate the penetration of active molecules through the skin, thereby increasing the dose of active molecules entering the systemic circulation or increasing the dose of active molecules reaching target organs, tissues and cells, and can be used for transdermal administration of large molecular weight drugs such as polypeptides, proteins and nucleic acid molecules.

[0005] Menthol, as a widely recognized percutaneous penetration enhancer, can destroy the lamellar structure of the stratum corneum and induce lipid cells to form a single-layer vacuolar structure, but it can cause functional damage to the stratum corneum and cause toxicity to the skin. Wan et al. used osthole as a target drug to conduct an in vitro percutaneous penetration experiment. When the dosage of menthol was as high as 0.5%, the permeation enhancement ratio was only 5.19.

[0006] Acetyl hexapeptide-1 (Ac-Nle-Ala-His-D-Phe-Arg-Trp-NH2) can induce the release of interleukins, and the release of interleukins increases the permeability of capillaries. Therefore, acetyl hexapeptide-1 has a certain permeability-promoting effect.

[0007] The transdermal peptide is transdermal peptide TD-1, with the amino acid sequence of ACSSSPSKHCG. It can overcome the skin barrier, quickly penetrate cells, retain its original structure and function, and enter cells independently of endocytosis. It can not only penetrate cells by itself, but also connect with other peptides to promote their cell penetration, thereby improving the transdermal efficiency of biomacromolecules.

[0008] Chinese patent document CN106084006A provides a polypeptide with transdermal function or transdermal enhancement function and a nucleic acid sequence encoding the polypeptide. The amino acid sequence of the polypeptide is ACSSTKKHCG, which can promote and / or enhance the transdermal penetration of other substances, including small molecule active substances, large molecule active substances such as proteins, nucleic acids, etc.

[0009] It can be seen that the prior art has proposed a technical solution that a polypeptide composed of about 10-30 amino acids can enhance the transdermal effect. However, through the prior art and the inventor's previous research, it was found that when the amino acid sequence changes, including the number and order of amino acids, the transdermal effect will also change significantly, lacking predictability. In order to enrich the product variety, different transdermal polypeptides can be further explored. Summary of the invention

[0010] In view of the shortcomings of the prior art, the present invention provides a transdermal tetrapeptide and its application. The present invention designs and synthesizes a new transdermal tetrapeptide, which has a good penetration-promoting effect, and its penetration-promoting effect is not much different from that of the transdermal peptide TD-1.

[0011] The technical solution of the present invention is as follows:

[0012] A transdermal tetrapeptide, whose amino acid sequence is: Ala-Arg-Cys-Gly, or Arg-Arg-Cys-Gly, or Ala-Lys-Cys-Gly, or Ala-Arg-Met-Gly, in order from C-terminus to N-terminus.

[0013] Preferably according to the present invention, the amino acid sequence of the transdermal tetrapeptide is: Ala-Arg-Cys-Gly.

[0014] Application of the above transdermal tetrapeptide in the preparation of transdermal products.

[0015] Preferably according to the present invention, the transdermal products include cosmetic preparations, hairdressing preparations, medicines, and foods; further preferably, the cosmetic preparations include skin care products and cosmetics.

[0016] A cosmetic preparation comprising the above transdermal tetrapeptide and at least one effective ingredient for skin care, skin function improvement or skin color regulation.

[0017] A hairdressing preparation comprises the above transdermal tetrapeptide and at least one hairdressing active ingredient, wherein the hairdressing active ingredient comprises an ingredient having the effects of promoting hair growth, preventing hair loss, and changing hair structure, composition or color.

[0018] A pharmaceutical composition comprises the transdermal tetrapeptide and at least one active pharmaceutical ingredient in an effective dose for treating a disease.

[0019] A method for assisting the transdermal penetration of active substances is to mix the above transdermal tetrapeptide with an effective dose of active substances and apply the mixture to the skin surface.

[0020] Beneficial effects:

[0021] The present invention designs and synthesizes a new transdermal tetrapeptide Ala-Arg-Cys-Gly, which has obvious penetration-promoting ability, and its penetration-promoting effect is similar to that of the existing penetration-promoting agent transdermal peptide TD-1, and is significantly better than the existing penetration-promoting agents acetyl hexapeptide-1 and menthol, and its structure is simple, molecular weight is small, biocompatibility is high, and it has no toxic effect on cells, and can be used for the preparation of penetration-promoting products, and has broad development prospects and application potential. At the same time, the present invention also synthesizes transdermal tetrapeptides Arg-Arg-Cys-Gly, Ala-Lys-Cys-Gly, and Ala-Arg-Met-Gly, and its transdermal delivery effect is slightly lower than that of Ala-Arg-Cys-Gly, but still has obvious penetration-promoting effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the synthetic route of the transdermal tetrapeptide Ala-Arg-Cys-Gly; in the figure, Aa represents amino acid.

[0023] Figure 2 This is the infrared spectrum of the transdermal tetrapeptide Ala-Arg-Cys-Gly.

[0024] Figure 3 This is the mass spectrum of the transdermal tetrapeptide Ala-Arg-Cys-Gly.

[0025] Figure 4 Schematic diagram of the Franz diffusion cell.

[0026] Figure 5 This is the cumulative permeation curve of diclofenac sodium per unit area in different treatment groups.

[0027] Figure 6 The figure is a bar graph of the cell viability of human skin fibroblasts under different concentrations of transdermal tetrapeptide.

[0028] Figure 7 This is the cumulative permeation curve of diclofenac sodium per unit area in different transdermal tetrapeptide treatment groups. DETAILED DESCRIPTION

[0029] The technical scheme of the present invention is further described below in conjunction with the examples, but the protection scope of the present invention is not limited thereto. The drugs and reagents involved in the examples, unless otherwise specified, are all common commercially available products; the experimental steps involved in the examples, unless otherwise specified, are all conventional technical operations in the art.

[0030] Example 1: Preparation of transdermal tetrapeptide

[0031] The synthetic route of transdermal tetrapeptide Ala-Arg-Cys-Gly is as follows Figure 1 As shown, the specific steps are as follows:

[0032] (1) Weigh 3 g of RINK resin (substitution degree 0.3 mmol / g) into a 150 mL reactor and soak it with 50 mL of dichloromethane (DCM) (the C-terminus is the carboxyl end);

[0033] (2) After 2 hours, wash the resin with 3 times the volume of N,N-dimethylformamide (DMF) and then drain it. Repeat this process four times and drain the resin for later use.

[0034] (3) Add 8 mL of 20% piperidine (piperidine / DMF = 1:4, volume ratio) to the reactor and shake on a decolorizing shaker for 20 min to remove the Fmoc protecting group on the resin. After removing the protecting group, wash four times with DMF 3 times the volume of the resin and then drain;

[0035] (4) Take a small amount of resin and test it using the ninhydrin (hydrated ninhydrin) method. If the resin has color, it means that the deprotection is successful.

[0036] (5) Weigh 1.5 g of amino acid Ala into a 50 mL centrifuge tube, add 5 mL of DMF to dissolve it, and then add it to the above reactor;

[0037] (6) Take 5 mL of 1-hydroxy-benzotriazole (HOBT, condensing agent) in a 50 mL centrifuge tube, add 20 mL of DMF to dissolve it, then add 3 mL of N,N-diisopropylcarbodiimide (DIC, polypeptide condensing agent), shake well for 1 min, add the solution to the above reactor after it becomes clear, and then place the reactor in a shaker at 30 °C for reaction;

[0038] (7) After 2 hours, the mixture was capped with 10 mL of acetic anhydride (acetic anhydride: DIEA (condensing agent): DCM = 1:1:2, volume ratio) for half an hour, then washed four times with 3 times the volume of the resin in DMF, and dried for later use;

[0039] (8) Add 8 mL of 20% piperidine (piperidine / DMF = 1:4, volume ratio) to the reactor and shake on a decolorizing shaker for 20 min to remove the Fmoc protecting group on the resin. After the protecting group is removed, wash four times with DMF 3 times the volume of the resin and then drain;

[0040] (9) Take a small amount of resin and test it using the ninhydrin (hydrated ninhydrin) method. If the resin has color, it means that the deprotection is successful;

[0041] (10) Weigh 1.5 g of amino acid Arg into a 50 mL centrifuge tube, add 5 mL of DMF to dissolve it, and then add it to the above reactor;

[0042] (11) Take 5 mL of 1-hydroxy-benzotriazole (HOBT, condensing agent) in a 50 mL centrifuge tube, add 20 mL of DMF to dissolve it, then add 3 mL of N,N-diisopropylcarbodiimide (DIC, polypeptide condensing agent), shake well for 1 min, add the solution to the above reactor after it becomes clear, and then place the reactor in a shaker at 30 °C for reaction;

[0043] (12) After 2 hours, the mixture was capped with 10 mL of acetic anhydride (acetic anhydride: DIEA (condensing agent): DCM = 1:1:2, volume ratio) for half an hour, then washed four times with 3 times the volume of the resin in DMF, and dried for later use;

[0044] (13) 8 mL of 20% piperidine (piperidine / DMF = 1:4, volume ratio) was added to the reactor and the resin was shaken on a decolorization shaker for 20 min to remove the Fmoc protecting group on the resin. After the protecting group was removed, the resin was washed four times with DMF 3 times the volume of the resin and then dried;

[0045] (14) Take a small amount of resin and test it using the ninhydrin (hydrated ninhydrin) method. If the resin has color, it means that the deprotection is successful.

[0046] (15) Weigh 1.5 g of amino acid Cys into a 50 mL centrifuge tube, add 5 mL of DMF to dissolve it, and then add it to the above reactor;

[0047] (16) Take 5 mL of 1-hydroxy-benzotriazole (HOBT, condensing agent) in a 50 mL centrifuge tube, add 20 mL of DMF to dissolve it, then add 3 mL of N,N-diisopropylcarbodiimide (DIC, polypeptide condensing agent), shake well for 1 min, add the clarified solution to the above reactor, and then place the reactor in a shaker at 30 °C for reaction;

[0048] (17) After 2 hours, the mixture was capped with 10 mL of acetic anhydride (acetic anhydride: DIEA (condensing agent): DCM = 1:1:2, volume ratio) for half an hour, then washed four times with 3 times the volume of the resin in DMF and dried for later use;

[0049] (18) 8 mL of 20% piperidine (piperidine / DMF = 1:4, volume ratio) was added to the reactor and the resin was shaken on a decolorization shaker for 20 min to remove the Fmoc protecting group on the resin. After the protecting group was removed, the resin was washed four times with DMF 3 times the volume of the resin and then dried;

[0050] (19) Take a small amount of resin and test it using the ninhydrin (hydrated ninhydrin) method. If the resin has color, it means that the deprotection is successful.

[0051] (20) Weigh 1.5 g of amino acid Gly into a 50 mL centrifuge tube, add 5 mL of DMF to dissolve it, and then add it to the above reactor;

[0052] (21) Take 5 mL of 1-hydroxybenzotriazole (HOBT, condensing agent) in a 50-mL centrifuge tube, add 20 mL of DMF to dissolve it, and then add 3 mL of N,N-diisopropylcarbodiimide (DIC, polypeptide condensing agent), shake well for 1 min, add the clarified solution to the above reactor, and then place the reactor in a shaker at 30 °C for reaction;

[0053] (22) After 2 hours, the mixture was capped with 10 mL of acetic anhydride (acetic anhydride: DIEA (condensing agent): DCM = 1:1:2, volume ratio) for half an hour, then washed four times with 3 times the volume of the resin in DMF and dried for later use;

[0054] (23) 8 mL of 20% piperidine (piperidine / DMF = 1:4, volume ratio) was added to the reactor and the resin was shaken on a decolorization shaker for 20 min to remove the Fmoc protecting group on the resin. After the protecting group was removed, the resin was washed four times with DMF (3 times the volume of the resin) and then dried;

[0055] (24) Take a small amount of resin and test it using the ninhydrin (hydrated ninhydrin) method. If the resin has color, it means that the deprotection is successful.

[0056] (25) The peptide was cleaved from the resin using 95% cleavage solution (trifluoroacetic acid: 1,2-ethanedithiol: triisopropylsilane: water = 95:2:2:1, volume ratio) (10 mL of cleavage solution per gram of resin) and centrifuged four times with icy ether (cleavage solution: ether = 1:9, volume ratio). Finally, it was separated and purified by HPLC and then freeze-dried to obtain the transdermal tetrapeptide Ala-Arg-Cys-Gly.

[0057] The infrared spectrum results of the transdermal tetrapeptide Ala-Arg-Cys-Gly obtained above are as follows Figure 2 As shown in the figure, the characteristic absorption peak of the transdermal tetrapeptide Ala-Arg-Cys-Gly appears at 3395cm -1 、3077cm -1 、1670cm -1 、1544cm -1 、1425cm -1 、1201cm -1 、1133cm -1 、1025cm -1 、945cm -1 They belong to the NH bond in -CO=NH- and NH 2 The stretching vibration of NH in the nucleus, the stretching vibration of the associated -NH- bond, the amide I band of -CO=NH-, the stretching vibration of -C=N, the deformation vibration absorption peak of -CH3-, and the 2 -Deformation vibration absorption peak of -, stretching vibration of -CO-, stretching vibration of -CN- bond, stretching vibration of -CS- bond, bending vibration of -OH in -COOH.

[0058] The mass spectrometry results of the transdermal tetrapeptide Ala-Arg-Cys-Gly obtained above are as follows Figure 3 As shown in the figure, the simulated mass-to-charge ratio of the transdermal tetrapeptide Ala-Arg-Cys-Gly is 405.48, and the measured mass-to-charge ratio of the mass spectrum is 405.41.

[0059] The above results proved that the transdermal tetrapeptide Ala-Arg-Cys-Gly had been successfully synthesized.

[0060] Example 2: Transdermal delivery study of transdermal tetrapeptide Ala-Arg-Cys-Gly

[0061] The diffusion cell experiment was conducted using mouse skin as the substrate. The specific steps are as follows:

[0062] 1. Preparation of ex vivo mouse skin

[0063] Take healthy mice, anesthetize them with 10% urethane solution (dosage: 8 mL / kg), first shave the coarse hair on the back of the mice, then patiently cut the hair so as not to affect the contact between the drug and the skin, then inject an overdose of urethane solution to kill the mice, cut the back skin, and finally carefully remove the subcutaneous fat and mucous tissue, clean the mouse skin with PBS buffer, spread it flat on aluminum foil, and store it in a -20℃ refrigerator for later use.

[0064] 2. Construction of in vitro percutaneous absorption experimental device

[0065] The Franz diffusion cell used in the experiment is as follows Figure 4 As shown in the figure, the speed of the star-shaped stirrer in the experiment is 500 r / min, the volume of the receiving chamber is 6.5 mL, and the effective diffusion area is 0.785 cm 2 In the experiment, PBS buffer (pH 7.4) was used as the receiving medium and the experiment was carried out in a constant temperature water bath at (32±0.5)℃.

[0066] Before the experiment, first heat the PBS buffer to be used to (32±0.5)℃, and turn on the constant temperature water bath system. Take out the prepared mouse skin, rinse it carefully with PBS buffer, wipe the liquid on the surface of the skin dry and place it on the diffusion cell, with the mouse skin stratum corneum facing up, and discard the receiving solution after balancing for 30 minutes. Add fresh PBS buffer to the receiving chamber again, place the mouse skin on the diffusion cell, drain the bubbles under the skin, and add PBS buffer dissolved with diclofenac sodium to the supply chamber. Take samples of 200μL at the 2nd, 4th, 6th, and 8th hours, and add fresh receiving solution of the same volume and temperature. After filtering the sample through a 0.45μm microporous filter membrane, dilute it to a certain multiple, and determine its concentration using a liquid chromatograph.

[0067] 3. HPLC column preparation

[0068] Chromatographic column: Amethyst C18-H (250mm×4.6mm, 5μm), mobile phase: methanol-0.4% glacial acetic acid solution (80:20), detection wavelength is 254nm, flow rate is 1.0mL / min, column temperature: 30℃, injection volume is 10μL.

[0069] 4. Drug concentration screening

[0070] Take appropriate amount of diclofenac sodium in PBS buffer to prepare 0.1, 0.2, 0.3, 0.4, 0.5, 0.6% (g / mL) diclofenac sodium test solution, and measure its 8h permeability. The results show that when the drug concentration is greater than 0.2%, its 8h permeability stops rising, so the drug concentration of diclofenac sodium solution is specified to be 0.1%.

[0071] 5. In vitro percutaneous penetration test

[0072] This experiment was divided into five groups, namely, transdermal tetrapeptide group, transdermal peptide TD-1 group, acetyl hexapeptide-1 group, menthol group, and blank control group. The above-mentioned permeation enhancers were dissolved in PBS buffer containing 0.1% diclofenac sodium, wherein the concentration of transdermal tetrapeptide was 0.08%, and the concentrations of transdermal peptide TD-1, acetyl hexapeptide-1, and menthol were 0.5%, respectively. First, the PBS buffer to be used was heated to (32±0.5)℃, and the constant temperature water bath system was turned on. Take out the prepared mouse skin, rinse it carefully with PBS buffer, wipe the liquid on the surface of the skin dry and place it on the diffusion cell, with the mouse skin stratum corneum facing up, and discard the receiving solution after balancing for 30 minutes. Add fresh PBS buffer to the receiving chamber again, place the mouse skin on the diffusion cell, drain the bubbles under the skin, and add PBS buffer dissolved with the permeation enhancer to the supply chamber. Take samples of 200μL at 2, 4, 6, and 8h, and supplement with fresh receiving solution of the same volume and temperature at the same time. The sample was filtered through a 0.45 μm microporous filter membrane and then diluted to a certain multiple, and the concentration of diclofenac sodium was determined by liquid chromatography.

[0073] Among them, the cumulative permeation amount of diclofenac sodium per unit area (Q n ) is calculated by formula (1):

[0074]

[0075] In formula (1), Q n is the cumulative permeation per unit area at the nth time point (μg / cm 2 ), C n is the drug concentration measured at the nth time point (μg / mL), V is the volume of the receptor chamber (mL), C i is the drug concentration measured at each sampling (μg / mL), V i is the volume of each sampling (mL), A is the effective diffusion area (cm 2 ).

[0076] Steady state flux J s , permeability coefficient K p , permeability enhancement ratio ER, and permeability are calculated by formulas (2), (3), (4), and (5), respectively:

[0077]

[0078]

[0079]

[0080] Permeability (%) = Q n / Initial drug loading in the supply chamber × 100% (5)

[0081] In the formula, Qp is the total amount of permeated drug (μg), A is the effective diffusion area (cm 2 ), t is the corresponding diffusion time (h), C d is the initial drug loading concentration in the supply chamber (μg / cm 3 ), K Pi is the permeability coefficient of the sample (cm / h), K P0 is the permeability coefficient of the blank control group (cm / h), Q n is the cumulative permeation per unit area at the nth time point (μg / cm 2 ).

[0082] The cumulative permeation amount of diclofenac sodium per unit area in different treatment groups is as follows Figure 5 As shown by Figure 5 It can be seen that after adding various penetration enhancers for 8 hours, the order of the cumulative permeation amount of diclofenac sodium per unit area is: Q 透皮肽TD-1 >Q 透皮四肽 >Q 乙酰基六肽-1 >Q 薄荷醇 >Q 空白对照组 The cumulative permeation per unit area of ​​the transdermal tetrapeptide was not much different from that of the transdermal peptide TD-1, and was significantly better than that of acetyl hexapeptide-1 and menthol.

[0083] The main transdermal permeation parameters of drugs in different treatment groups are shown in Table 1. The data in Table 1 show that the drug permeability of transdermal tetrapeptide is second only to transdermal peptide TD-1, and the difference between the two is not much.

[0084] Table 1. Main percutaneous permeation parameters of drugs in different treatment groups

[0085] Permeation enhancers <![CDATA[J s (μg / h cm 2 )]]> <![CDATA[K p ×10 -2 (cm / h)]]> ER Permeability Transdermal Tetrapeptide 61.52 6.15 4.42 19.3％ Transdermal Peptide TD-1 65.11 6.51 4.68 20.4％ Acetyl Hexapeptide-1 48.93 4.89 3.5 15.3％ Menthol 20.59 2.05 1.47 6.4％ Blank control group 13.90 1.39 1 4.3％

[0086] According to the research results, the transdermal tetrapeptide Ala-Arg-Cys-Gly has a good transdermal delivery rate. Compared with the existing permeation enhancers (transdermal peptide TD-1, acetyl hexapeptide-1, menthol), its transdermal delivery rate is only slightly lower than that of transdermal peptide TD-1, and significantly higher than that of acetyl hexapeptide-1 and menthol.

[0087] Example 4: Biocompatibility test

[0088] Cytotoxicity experiments are conducted in vitro to simulate the growth environment of organisms. They can screen out the cytotoxicity of batches of samples in a short period of time and are an important basis for other cell experiments. This experiment uses human skin fibroblasts as a cell model, and the concentrations of the samples to be tested are set at 60μg / mL, 80μg / mL, 100μg / mL, 120μg / mL, and 140μg / mL, and the CCK-8 method is used for determination. The specific steps are as follows:

[0089] (1) Cell Counting Kit-8 (CCK-8) reagent can be used to quickly and conveniently identify the toxicity of analytical substances. The working principle is as follows: it contains WTS-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonic acid benzene)-2H-tetrazolyl monosodium salt], which has multiple nitro groups (-NO 2 ) and aromatic sulfonic acid group (-SO 3 H) groups, which have strong light absorption, undergo photolysis to produce active substances such as free radicals or ions. These active substances further undergo redox reactions, and under the action of 1-Methoxy PMS, they are easily reduced to yellow formazan substances by dehydrogenases in cells, and the amount of the product is proportional to the cell survival rate. The yellow formazan product is characterized by high water solubility and special absorption at 450nm, and the cytotoxicity of the sample to be tested can be determined by detecting its absorbance.

[0090] Human skin fibroblasts were cultured to an appropriate density, digested with trypsin, and then prepared into a cell suspension, which was inoculated in a 96-well plate. 100 μL of the cell suspension was inoculated in each well, and the number of cells in each well was 6-8×10 3 The cells were incubated at 37°C with 5% CO 2 , incubate for 24h.

[0091] (2) This experiment set up a blank control group and an experimental group. The concentration of the test sample (i.e., transdermal tetrapeptide) in the experimental group was set to 5 concentration gradients, and at least three replicates were set for each concentration. No reagent was added to the blank control group. After the cells were firmly attached to the wall overnight, the culture medium was discarded and fresh culture medium containing different amounts of the test sample was added. After the operation was completed, the cells were incubated at 37°C and 5% CO. 2 , incubate for 24h.

[0092] (3) Take the 96-well plate out of the incubator and add 10 μL of CCK-8 detection solution to each well. Incubate the plate at 37°C and 5% CO 2 Incubate in an incubator for 3 h.

[0093] (4) After incubation, take out the sample and measure the absorbance at 450 nm.

[0094] (5) Result analysis: Using the measured absorbance value, the cell viability was calculated according to the following formula:

[0095]

[0096] In the formula, A x represents the absorbance of the experimental group at 450 nm, A 0 Represents the absorbance of the blank control group at 450 nm.

[0097] The results are as follows Figure 6 As shown, analysis shows that the cell survival rate of human skin fibroblasts of the transdermal tetrapeptide exceeds 85%, and it has basically no toxic effect on the cells.

[0098] Embodiment 4:

[0099] In addition to the above-mentioned transdermal tetrapeptide Ala-Arg-Cys-Gly, three other transdermal tetrapeptides were designed: Arg-Arg-Cys-Gly, Ala-Lys-Cys-Gly, and Ala-Arg-Met-Gly.

[0100] The transdermal tetrapeptides Arg-Arg-Cys-Gly, Ala-Lys-Cys-Gly, and Ala-Arg-Met-Gly were synthesized according to the method of Example 1, and their transdermal effects were studied according to the experimental method of Example 2.

[0101] The cumulative permeation amount of diclofenac sodium per unit area in different transdermal tetrapeptide treatment groups is as follows Figure 7 As shown by Figure 7 It can be seen that after adding different transdermal tetrapeptides for 8 hours, the drug transdermal delivery effects of transdermal tetrapeptides Arg-Arg-Cys-Gly, Ala-Lys-Cys-Gly, and Ala-Arg-Met-Gly are comparable. Although they are slightly lower than the transdermal tetrapeptide Ala-Arg-Cys-Gly, they still have obvious penetration-enhancing effects.

[0102] It is speculated that the reason why the above four tetrapeptides have a permeation-enhancing effect is that since all four tetrapeptides have ethyl, amino, thiol and methylene groups, it may be that when the tetrapeptide peptide chain contains a combination of ethyl, amino, thiol and methylene, the tetrapeptide has a certain permeation-enhancing effect.

[0103] According to the experimental method of Example 3, it was verified that the above three transdermal tetrapeptides Arg-Arg-Cys-Gly, Ala-Lys-Cys-Gly, and Ala-Arg-Met-Gly also have basically no toxic effect on cells.

Claims

1. A transdermal tetrapeptide, characterized in that: Its amino acid sequence is: Ala-Arg-Cys-Gly, or Arg-Arg-Cys-Gly, or Ala-Lys-Cys-Gly, or Ala-Arg-Met-Gly, in order from C-terminus to N-terminus.

2. The transdermal tetrapeptide according to claim 1, characterized in that Its amino acid sequence is: Ala-Arg-Cys-Gly.

3. Use of the transdermal tetrapeptide as claimed in claim 1 or 2 in the preparation of transdermal products.

4. The use according to claim 3, characterized in that The transdermal products include cosmetic preparations, hairdressing preparations, medicines, and foods.

5. The use according to claim 4, characterized in that The cosmetic preparations include skin care products and cosmetics.

6. A cosmetic preparation, characterized in that The invention comprises the transdermal tetrapeptide as claimed in claim 1 or 2, and at least one active ingredient for skin care, skin function improvement or skin color regulation.

7. A hairdressing preparation, characterized in that: The invention comprises the transdermal tetrapeptide as claimed in claim 1 or 2, and at least one hair-beautifying active ingredient, wherein the hair-beautifying active ingredient comprises an ingredient having the effects of promoting hair growth, preventing hair loss, and changing hair structure, composition or color.

8. A pharmaceutical composition, characterized in that The invention comprises the transdermal tetrapeptide as claimed in claim 1 or 2, and at least one active pharmaceutical ingredient in an effective dose for treating a disease.

9. A method for assisting the transdermal delivery of an active substance, characterized in that: The transdermal tetrapeptide according to claim 1 or 2 is mixed with an effective dose of active substance and applied to the skin surface.

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