A composition containing a tripeptide and its use

Abstract

This invention discloses a composition containing a tripeptide and its applications. The composition of this invention comprises: Phe-Ala-Trp tripeptide or its salt or derivative thereof, and nicotinamide adenine dinucleotide (NAD). + This invention is the first to discover the combination of tripeptide-128 and nicotinamide adenine dinucleotide (NAD). + These two ingredients exhibit a unique synergistic effect; by combining them, their antioxidant capabilities can be significantly improved, achieving a better antioxidant effect through synergistic enhancement. In addition, tripeptide-128 and NAD... + The combination enhances CD44 gene expression, thereby improving moisturizing capabilities. This composition provides new ideas and options for the innovative application development of related products, and is of great significance for product development in areas such as maintaining skin homeostasis, preserving moisture balance, and improving skin aging.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a composition containing a tripeptide and its applications. Background Technology

[0002] Located on the surface of living organisms, the skin is the first line of physiological defense protecting tissues and organs from damage caused by mechanical, chemical, physical, and microbial invasions, acting as a barrier to protect the body. However, with age and the influence of adverse external environmental factors, the skin accumulates a large number of free radicals, causing oxidative stress and inflammatory responses, which in turn damage cellular biomolecules. Simultaneously, this also triggers the production of large amounts of matrix metalloproteinases, degrading extracellular matrix proteins, including collagen and elastin, thereby disrupting skin tissue structure, impairing skin barrier function, and accelerating skin aging. Furthermore, the production of free radicals can induce excessive melanin production, leading to skin pigmentation and the formation of dark spots, affecting skin appearance. Therefore, effective free radical scavengers not only protect the skin barrier and overall health but also effectively improve skin pigmentation and dark spot formation, maintaining an even skin tone.

[0003] Peptides possess a wide range of potential biological activities due to their diverse structures. Therefore, the search for and application of peptides with antioxidant activity plays a crucial role in protecting and restoring skin health. Prior Chinese patents CN 111777665 A, CN117143183A, and CN 118530299 A disclose a tripeptide, tripeptide salt, or derivative thereof, and their applications. These patents involve a peptide with the International Nomenclature of Cosmetic Ingredients (INCI) name of tripeptide-128 (Phe-Ala-Trp, INCI monograph ID: 39895), which exhibits antioxidant and skin-repairing effects. Although tripeptide-128 possesses antioxidant properties, its widespread use in practical applications depends not only on its effectiveness but also on the cost associated with its application concentration. Therefore, achieving better and broader efficacy and effects with lower doses of tripeptide-128 is an important research goal in this field. Currently, no compounded tripeptide-128 compositions have been developed. Summary of the Invention

[0004] The present invention aims to at least solve one of the aforementioned technical problems existing in the prior art. Therefore, the object of the present invention is to provide a composition containing a tripeptide and its application. The composition comprises: Phe-Ala-Trp tripeptide or a salt thereof or a derivative thereof, and nicotinamide adenine dinucleotide (NAD). + The composition contains Phe-Ala-Trp tripeptide and NAD+. +They exhibit significant and unique synergistic effects and cannot be replaced by other similar substances. They possess extremely strong antioxidant and moisturizing activities, thus making them effective for the development and utilization of related products.

[0005] A first aspect of the present invention provides a composition comprising: Phe-Ala-Trp tripeptide or a salt thereof or a derivative thereof, and nicotinamide adenine dinucleotide (NAD). + ).

[0006] In this invention, "comprising," "containing," "having," or "including" means that at least the specified substance, component, element, or method step is present in the product, article, or method, but does not exclude the presence of other substances, components, elements, or method steps, even if the other such substances, components, elements, or method steps have the same function as the specified ones.

[0007] In some embodiments of the present invention, the composition contains, by weight, 5-5000 parts of Phe-Ala-Trp tripeptide or its salt or derivative thereof, and 0.1-100 parts of NAD. + .

[0008] In some embodiments of the present invention, the composition contains, by weight, 10-2000 parts of Phe-Ala-Trp tripeptide or its salt or derivative thereof, and 1-100 parts of NAD. + .

[0009] In some embodiments of the present invention, the composition contains, by weight, 50-1500 parts of Phe-Ala-Trp tripeptide or its salt or derivative thereof, and 1-50 parts of NAD. + .

[0010] In some embodiments of the present invention, the composition contains, by weight, 200-1500 parts of Phe-Ala-Trp tripeptide or its salt or derivative thereof, and 5-30 parts of NAD. + .

[0011] In some embodiments of the present invention, the composition contains, by weight, 200-1200 parts of Phe-Ala-Trp tripeptide or its salt or derivative thereof, and 5-20 parts of NAD. + .

[0012] In some embodiments of the present invention, the salt is a soluble salt.

[0013] In some embodiments of the present invention, the salt is a salt formed by Phe-Ala-Trp tripeptide with metal ions, organic acids, organic bases or inorganic acids.

[0014] In some embodiments of the present invention, the metal ions include lithium, sodium, potassium, calcium, magnesium, manganese, copper, zinc and aluminum ions.

[0015] In some embodiments of the present invention, the organic base includes: ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, arginine, lysine, histidine, and piperazine.

[0016] In some embodiments of the present invention, the organic acids include: 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, pyric acid, and gluconic acid.

[0017] In some embodiments of the present invention, the inorganic acid includes hydrochloric acid, sulfuric acid, boric acid, and carbonic acid.

[0018] In some embodiments of the present invention, the derivatives include derivatives obtained by chemically modifying the Phe-Ala-Trp tripeptide.

[0019] In some embodiments of the present invention, the chemical bond modification includes at least one of N-terminal modification, C-terminal modification and CH-terminal modification.

[0020] In some embodiments of the present invention, the modifications include, but are not limited to, methylation, acetylation, hexanoylation, octanoylation, hydroxylation, carboxylation, phosphorylation, glycosylation, palmitoylation, stearylation, lauroylation, myristylation, and oleylation.

[0021] A second aspect of the invention provides the use of the compositions described in the first aspect of the invention in the preparation of pharmaceuticals or cosmetics.

[0022] In some embodiments of the present invention, the pharmaceutical or cosmetic product has at least one of the following functions:

[0023] (1) Antioxidant; and

[0024] (2) Moisturizing.

[0025] In some embodiments of the present invention, the antioxidants include, but are not limited to, anti-aging and anti-photoaging.

[0026] In some embodiments of the present invention, the pharmaceutical or cosmetic product further contains pharmaceutically or cosmetically acceptable excipients.

[0027] In this invention, the term "pharmaceutically or cosmetically acceptable carrier" refers to a conventional cosmetic ingredient or pharmaceutical carrier that can be used in the methods disclosed in this invention. See Remington's Pharmaceutical Sciences, Ewmartin, Mack Publishing Co., Easton, Pa., 15th edition (1975).

[0028] In some embodiments of the present invention, the excipients include, but are not limited to: diluents (such as starch, dextrin, sucrose, lactose, mannitol, etc.), absorbents (such as calcium sulfate, dicalcium phosphate, etc.), wetting agents (such as ethanol), binders (such as hydroxypropyl methylcellulose, povidone, etc.), disintegrants (such as sodium hydroxymethyl starch, crospovidone, 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).

[0029] In some embodiments of the present invention, the pharmaceutical or cosmetic product further contains a second active substance.

[0030] In some embodiments of the present invention, the second active substance includes a substance having at least one of the following functions:

[0031] (1) Antioxidant; and

[0032] (2) Moisturizing.

[0033] In some embodiments of the present invention, the second active substance does not have an antagonistic effect with the composition or any component thereof.

[0034] In this invention, the term "antagonistic effect" refers to the phenomenon where, when two or more substances interact in the body, one inhibits or counteracts the function or effect of the other.

[0035] The beneficial effects of this invention are:

[0036] This invention is the first to discover the combination of tripeptide-128 and nicotinamide adenine dinucleotide (NAD). + These two ingredients exhibit a unique synergistic effect; by combining them, their antioxidant capabilities can be significantly improved, achieving a better antioxidant effect through synergistic enhancement. In addition, tripeptide-128 and NAD... + The combination enhances CD44 gene expression, thereby improving moisturizing capabilities. This composition provides new ideas and options for the innovative application development of related products, and is of great significance for product development in areas such as maintaining skin homeostasis, preserving moisture balance, and improving skin aging.

[0037] Through substitution experiments, it was found that even replacing it with similar functional ingredients could not achieve a synergistic effect. Therefore, tripeptide-128 and nicotinamide adenine dinucleotide (NAD) + The effects of combining these ingredients are unexpected. Attached Figure Description

[0038] Figure 1 The results are based on the antioxidant experiment of the experimental group in Comparative Example 1.

[0039] Figure 2 The results of the antioxidant experiment are based on the experimental group in Example 1.

[0040] Figure 3 For tripeptide-128 and NAD + Results of the effect of compounding on the expression of SOD1 antioxidant gene.

[0041] Figure 4 For tripeptide-128 and NAD + Results of the effect of compounding on the expression of SOD2 antioxidant gene.

[0042] Figure 5 For tripeptide-128 and NAD + Results of the effect of compounding on the expression of moisturizing genes. Detailed Implementation

[0043] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials, reagents, or apparatus used in the embodiments and comparative examples are all available from conventional commercial sources or can be obtained by existing technical methods. Unless otherwise specified, the test or experimental methods are conventional methods in the art.

[0044] Example 1

[0045] This embodiment provides a composition, which specifically includes:

[0046] Based on parts by weight, 211 parts of tripeptide-128 and 10 parts of NAD + .

[0047] The preparation method of this composition is as follows:

[0048] Tripeptide-128 and NAD + Mix thoroughly until homogeneous, and you have the desired product.

[0049] Example 2

[0050] This embodiment provides a composition, which specifically includes:

[0051] Based on parts by weight, 250 parts of tripeptide-128 and 10 parts of NAD+ .

[0052] The preparation method of this composition is as follows:

[0053] Tripeptide-128 and NAD + Mix thoroughly until homogeneous, and you have the desired product.

[0054] Example 3

[0055] This embodiment provides a composition, which specifically includes:

[0056] Based on parts by weight, 1100 parts of tripeptide-128 and 10 parts of NAD + .

[0057] The preparation method of this composition is as follows:

[0058] Tripeptide-128 and NAD + Mix thoroughly until homogeneous, and you have the desired product.

[0059] Comparative Example 1

[0060] This embodiment provides a composition, which specifically includes:

[0061] Based on parts by weight, 211 parts of tripeptide-128 and 10 parts of GSH.

[0062] The preparation method of this composition is as follows:

[0063] The tripeptide-128 and GSH are thoroughly mixed to obtain the final product.

[0064] Test Example 1

[0065] In this test example, the antioxidant effects of the compositions in Example 1 and Comparative Example 1 were tested respectively.

[0066] Glutathione (GSH) is a recognized antioxidant and has been used as a raw material in cosmetics. Nicotinamide adenine dinucleotide (NAD) + Sirtuin 1 can assist in cellular metabolism, stress response, and gene expression regulation, playing a role in delaying aging, and is therefore a common ingredient in cosmetics. GSH and NAD + These terms are often used interchangeably in this field. In this test example, equal amounts of GSH and NAD were used. + Comparative tests were conducted to demonstrate the impact of the selection of compound components on the effectiveness of the composition.

[0067] The specific experimental method is as follows:

[0068] Human skin keratinocytes (HaCaT) were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin (P / S) under a 5% CO2, 37°C incubator environment. When the HaCaT cells reached the logarithmic growth phase, they were digested with 0.25% trypsin to obtain a cell suspension. The cell suspension was then processed at a rate of 3 × 10⁻⁶ cells / year. 5 Cells were transferred to 12-well plates at a density of 10 cells / well. The plates were then incubated for 24 hours in DMEM medium containing 10% fetal bovine serum and 1% penicillin-dextrose antibody (P / S).

[0069] After incubation, the original culture medium was removed, and the samples were divided into groups. Two experimental groups were formed based on Example 1 and Comparative Example 1. The experimental group based on Example 1 included: a blank group (CON), a model group (H2O2), and a NAD+ group. + Treatment group (H2O2+NAD) + Tripeptide treatment group (H2O2 + tripeptide-128), Example 1 group (H2O2 + tripeptide-128 + NAD) + The experimental groups based on Comparative Example 1 included: blank group (CON), model group (H2O2), GSH treatment group (H2O2+GSH), tripeptide treatment group (H2O2+tripeptide-128), and Comparative Example 1 group (H2O2+tripeptide-128+GSH).

[0070] The drug administration details for each group are shown in the table below.

[0071] Table 1. Dosing data of the experimental group based on Example 1.

[0072]

[0073] Table 1 shows the drug administration details for the experimental group based on Comparative Example 1.

[0074]

[0075] According to the tripeptide-128, GSH, and NAD in the table above +The appropriate amount of DMEM basal medium was added to prepare the corresponding culture medium. After the culture was completed, equal volumes of the corresponding DMEM basal medium were added to each group according to their grouping and incubated for another 24 hours. After incubation, the medium was removed, and equal volumes of DMEM basal medium containing 10 μM of commercially available DCFH-DA fluorescent probe were added. The cells were then incubated in the dark for 45 min. After incubation, residual probes were washed away. Then, DMEM basal medium containing 1000 μmol / L H2O2 was added to each group except the blank group and incubated for 30 min (equal volumes of DMEM basal medium without any additives were added to the blank group). After incubation, the cells were digested with 0.25% trypsin to obtain cell suspensions. The cell suspensions were then used to detect the fluorescence intensity of each group using flow cytometry, with an excitation wavelength of 488 nm and an emission wavelength of 525 nm.

[0076] For statistical analysis, the fluorescence intensity of the H2O2 group was normalized. One-way ANOVA was used to test for statistical differences between the control group (CON) and the model group, and between the model group and the single-drug and combination groups. Specifically, compared with the CON group, $$$$ P < 0.0001; compared with the model group, ****P < 0.0001; compared with Example 1 or Comparative Example 1, #### P<0.0001.

[0077] The results are as follows Figure 1 and Figure 2 As shown.

[0078] Figure 1 Based on the experimental results of Comparative Example 1, it can be found that H2O2 modeling significantly increased the free radical content. However, there was essentially no difference between the GSH-treated group and the tripeptide-treated group, indicating that GSH treatment had no effect on increasing the free radical content generated by H2O2 modeling. Tripeptide-128 had a certain inhibitory effect on the increase in free radical content generated by H2O2 modeling, but further addition of GSH, which also has antioxidant properties, did not significantly improve its free radical scavenging effect. Therefore, it can be determined that at a given concentration, tripeptide-128 and GSH do not have a synergistic effect.

[0079] Figure 2 Based on the experimental results of the experimental group in Example 1, it can be found that H2O2 modeling significantly increased the free radical content. Compared with the model group, the addition of the conventional antioxidant NAD+... + Not only did it fail to reduce or maintain free radical levels, it actually increased them further. Tripeptide-128 has a certain inhibitory effect on the increased free radical levels generated by H2O2 modeling. Furthermore, the addition of NAD... +Subsequently, this inhibitory effect was significantly enhanced, which suggests that, at a given concentration, tripeptide-128 and NAD... + It has a synergistic effect, and NAD + It can be used as a synergist for tripeptide-128.

[0080] The same experiment was performed on other embodiments, and the results were found to be similar to those in Embodiment 1.

[0081] Test Example 2

[0082] To further determine the relationship between tripeptide-128 and NAD + To determine whether the synergistic effect also manifests in other effects, the expression of relevant genes (SOD1, SOD2 and CD44) after treatment in Example 1 was tested in this test case to determine its antioxidant and moisturizing effects.

[0083] The specific experimental method is as follows:

[0084] Human skin keratinocytes (HaCaT) were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin (P / S) under a 5% CO2, 37°C incubator environment. When the HaCaT cells reached the logarithmic growth phase, they were digested with 0.25% trypsin to obtain a cell suspension. The cell suspension was then processed at a rate of 6 × 10⁻⁶ cells / year. 5 Cells were seeded at a density of 10 cells / well into 6-well culture plates. They were then incubated for 24 hours in DMEM medium containing 10% fetal bovine serum and 1% penicillin-dextrose antibody (P / S).

[0085] After incubation, the culture medium was removed, and the samples were divided into groups. The experimental groups included: a blank group (CON), a model group (H2O2), and a NAD group. + Treatment group (H2O2+NAD) + Tripeptide treatment group (H2O2 + tripeptide-128), Example 1 group (H2O2 + tripeptide-128 + NAD) + ).

[0086] Prepare the corresponding DMEM basal medium for each experimental group according to Table 1. After culture, add the corresponding DMEM basal medium in equal volumes according to the grouping situation and continue incubation for 24 hours. After incubation, wash the cells with PBS, and extract RNA using the HiPure Total RNA Mini Kit (tissue cell RNA mini-extraction kit) according to the instructions. Then, take 500 ng of the extracted RNA and use PrimeScript... TMReverse transcription was performed using the RT reagent kit with gDNAEraser (Perfect Real Time) RNA reverse transcription kit to obtain cDNA. Using the cDNA as a template, TBGreen was used... TM Premix Ex Taq TM RT-qPCR amplification was performed using Tli RNaseH Plus, and quantification was performed using a LightCycler 480 II (Roche) detection system. GAPDH was used as an internal control. 2 -△△Ct The method calculates the relative gene expression level.

[0087] For statistical analysis, mRNA levels in the CON group were normalized. One-way ANOVA was used to test the difference between the control group (CON) and NAD. + Group, Tripeptide-128 group, Tripeptide-128+NAD + The statistical differences between the groups were as follows: ***P<0.001, ****P<0.0001 compared to CON; and compared to tripeptide-128+NAD. + Compared to the group, #### P<0.0001.

[0088] The results are as follows Figure 3-5 As shown.

[0089] Superoxide dismutase (SOD) has antioxidant and anti-aging effects. Its mechanism of action is mainly to scavenge harmful superoxide anion free radicals (O2). 2- SOD has different subtypes, with SOD1 mainly found in the cytoplasm and SOD2 mainly found in the mitochondria. Both are commonly used antioxidant markers and can therefore be used to assess the antioxidant activity of raw materials.

[0090] CD44 is the main cell membrane receptor for hyaluronic acid. The binding of CD44 and hyaluronic acid activates downstream signaling pathways, regulates skin cell proliferation and differentiation, and facilitates the association of hyaluronic acid with collagen and elastin fibers to form a network structure, thereby maintaining high skin hydration and achieving a moisturizing effect. Therefore, CD44 is commonly used as a marker of moisturization and can be used to evaluate the moisturizing effect of raw materials.

[0091] Figure 3 and Figure 4 For tripeptide-128 and NAD + The effects of the combined formulation on the expression of SOD1 and SOD2 antioxidant genes were investigated. It was found that, compared to the control group, NAD... + The use of SOD-128 alone did not significantly alter the expression levels of SOD1 and SOD2, thus failing to address the oxidation problem, which confirms the validity of the above results. At a given concentration, tripeptide-128 and NAD...+ The combination of tripeptide-128 and NAD + The single application significantly enhanced the expression levels of SOD1 and SOD2, indicating that tripeptide-128 and NAD... + It has a synergistic effect in terms of antioxidant properties.

[0092] Figure 5 For tripeptide-128 and NAD + The effects of the compound on the expression of moisturizing genes were investigated. It was found that, compared to the control group, tripeptide-128 and NAD... + Using it alone actually downregulated CD44 expression, while at a given concentration, tripeptide-128 and NAD... + The combination significantly enhances CD44 expression, indicating that tripeptide-128 and NAD+ have a similar effect. + It has a synergistic effect in moisturizing.

[0093] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. Application of the composition in the preparation of pharmaceuticals or cosmetics; The composition comprises: 200-1200 parts of Phe-Ala-Trp tripeptide or its salt, and 5-20 parts of nicotinamide adenine dinucleotide (NAD+). The medicine or cosmetic has the following functions: (1) Antioxidant; and (2) Moisturizing.

2. Use according to claim 1, characterized in that, The salt is a soluble salt.

3. Use according to claim 1, characterized in that, The salt is a salt formed by Phe-Ala-Trp tripeptide with metal ions, organic acids, organic bases, or inorganic acids.

4. Use according to claim 1, characterized in that, The medicine or cosmetic also contains pharmaceutically or cosmetically acceptable excipients.

5. Use according to claim 4, characterized in that, The excipients include at least one of the following: diluent, absorbent, wetting agent, adhesive, disintegrant, lubricant, colorant, coating material, solvent, pH adjuster, antibacterial agent, isotonic adjuster, and chelating agent.

6. Use according to claim 1, characterized in that, The medicine or cosmetic also contains a second active substance.

7. Use according to claim 6, characterized in that, The second active substance includes a substance having at least one of the following functions: (1) Antioxidant; and (2) Moisturizing.

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