Anti-photoaging polypeptide and its application

Abstract

The present invention belongs to the field of biomedicine technology, and specifically discloses an anti-photoaging polypeptide, which has a ‑Tyr‑Trp‑Leu‑Ser‑Thr‑characteristic fragment; the characteristic fragment is located at the amino end, carboxyl end or any position in the middle of the polypeptide sequence; wherein the amino acid composition of the ‑Tyr‑Trp‑Leu‑Ser‑Thr‑characteristic fragment is independently selected from its corresponding D-type amino acid, or the corresponding N‑methylated amino acid. The present invention has found through cell experiments that the polypeptide can significantly inhibit the release of reactive oxygen species induced by light in HaCaT human immortalized epidermal cells, indicating that the chemically synthesized polypeptide of the present invention has potential anti-photoaging efficacy; at the same time, the polypeptide has weak cytotoxicity and antioxidant effects, and has broad application prospects in skin care products, cosmetics and cell culture matrices.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to an anti-photoaging polypeptide and an application thereof. Background Art

[0002] According to data released by the China Flavor and Fragrance Cosmetic Industry Association, the most popular services that women care about are: anti-aging care, freckle removal and whitening. Among them, the mention rate of anti-aging reached 72.2%. From the basic attributes of the anti-aging consumer group, it can be seen that the current consumer group is mainly female, accounting for as much as 72%. As the anti-aging concept gradually becomes younger, the age structure of anti-aging consumers extends to both ends, and the demand for fighting different levels of aging symptoms will continue to grow. Skin aging refers to the physiological phenomenon that the cell structure and function of skin tissue degenerate under the continuous influence of internal and external environments, and the skin texture changes (such as elasticity, tension, etc.), pigment changes, and blood vessel atrophy or hyperplasia. It is usually divided into endogenous aging (natural aging) and exogenous aging (photoaging, etc.).

[0003] The effects of sunlight on the skin are profound and are thought to account for 90% of exogenous skin aging. Periodic and continuous exposure to ultraviolet (UV) radiation is a classic and key factor in skin aging, known as photoaging. Photoaging is characterized by wrinkles, inflammation, pigmentation, sagging, and dryness. Although UVB accounts for only a small part of total UV radiation, it is most active in damaging the epidermis and dermis of the skin. Some studies have reported that UVB irradiation increases intracellular reactive oxygen species (ROS), such as superoxide anions, hydroxyl radicals, and hydrogen peroxide. ROS stimulate various signaling pathways and initiate biological processes, including cell death, cell senescence, and inflammation. At present, many peptide molecules have been found to have good antioxidant activity, but there are still few peptides with anti-photoaging properties. Summary of the invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide an anti-photoaging polypeptide that can significantly inhibit the release of reactive oxygen species induced by light in HaCaT human immortalized epidermal cells and has potential antioxidant and collagen regeneration promoting effects.

[0005] The technical solution adopted by the present invention to solve the technical problem is: an anti-photoaging polypeptide is provided, characterized in that: the anti-photoaging polypeptide has a -Tyr-Trp-Leu-Ser-Thr-characteristic fragment, and the characteristic fragment is located at the amino end, carboxyl end or any position in the middle of the polypeptide sequence; wherein the amino acid composition of the -Tyr-Trp-Leu-Ser-Thr-characteristic fragment is independently selected from its corresponding D-type amino acid, or the corresponding N-methylated amino acid.

[0006] Furthermore, the amino group at the amino terminus of the anti-photoaging polypeptide is modified with an amide bond formed by acetylation, formylation or mesylation.

[0007] Furthermore, the modification method of the carboxyl terminus of the anti-photoaging polypeptide is amidation or esterification of the carboxyl terminus of the amino acid.

[0008] Furthermore, the anti-photoaging polypeptide comprises an amino acid sequence of Tyr-Ser-Tyr-Trp-Leu-Ser-Thr-Pro-Glu-Met.

[0009] Furthermore, the anti-photoaging polypeptide comprises an amino acid sequence of Ser-Tyr-Trp-Leu-Ser-Thr-Pro-Glu.

[0010] Furthermore, the anti-photoaging polypeptide comprises an amino acid sequence of Asn-Asp-Tyr-Ser-Tyr-Trp-Leu-Ser-Thr-Pro.

[0011] Furthermore, use of an anti-photoaging polypeptide in the preparation of a drug and / or cosmetic having an anti-photoaging effect;

[0012] And / or use of an anti-photoaging polypeptide in the preparation of a drug and / or cosmetic for preventing oxidative damage diseases;

[0013] And / or use of an anti-photoaging polypeptide in the preparation of anti-ultraviolet radiation, anti-oxidation and / or anti-aging drugs and / or cosmetics.

[0014] In some specific embodiments provided by the present invention, commercially available polypeptides and Tyr-Trp-Leu-Ser-Tyr-Pro-Glu-Met-Ala-Met-NH2 were both subjected to HaCaT cell UVB radiation treatment in an anti-photoaging test. The results showed that the ROS release of the commercially available polypeptide was significantly higher than that of the untreated group, while after treatment with H-Tyr-Trp-Leu-Ser-Tyr-Pro-Glu-Met-Ala-Met-NH2, it was found that it had significant activity in inhibiting ROS release.

[0015] Furthermore, an anti-photoaging polypeptide is used in the preparation of medicines, cosmetics or cell culture matrices for cell repair and / or promotion of cell collagen production.

[0016] In some specific embodiments provided by the present invention, the polypeptide is added to the cell culture medium, and the ROS release of cells irradiated by UVB after polypeptide treatment is measured. The results show that the polypeptides all exhibit the ability to significantly inhibit ROS release, indicating that the polypeptides have potential anti-photoaging effects.

[0017] Furthermore, an anti-photoaging polypeptide is used in the preparation of a product for tissue regeneration and repair of skin photoaging damage.

[0018] In some specific embodiments provided by the present invention, polypeptides with high anti-photoaging activity are obtained by physical screening tests on polypeptide molecule libraries, and anti-photoaging polypeptides with high activity are screened out by retesting photoaging cell models, such as the sequence: H-Tyr-Trp-Leu-Ser-Tyr-Pro-Glu-Met-Ala-Met-NH2.

[0019] Specifically, the oxidation in the present invention includes but is not limited to reactive oxygen species (ROS), including superoxide (O2·-), hydroxyl (HO·), peroxide (ROO·), alkoxy (RO·) and nitric oxide (NO·).

[0020] Furthermore, the polypeptide described in the present invention refers to a class of compounds composed of amino acids connected by peptide bonds, and is a product with no limitation on the number of amino acids.

[0021] The anti-photoaging polypeptide described in the present invention is a synthetic polypeptide, which can be synthesized according to the chemical synthesis method commonly used by those skilled in the art.

[0022] Furthermore, the present invention adopts a solid phase synthesis method to prepare the above-mentioned anti-photoaging polypeptide.

[0023] Furthermore, in some specific embodiments provided by the present invention, the anti-photoaging polypeptide is synthesized using a polypeptide synthesizer according to its amino acid sequence.

[0024] The present invention discloses an anti-photoaging polypeptide and its application, which has the following beneficial effects:

[0025] (1) The present invention has found through cell experiments that the anti-photoaging polypeptides can significantly inhibit the release of reactive oxygen species induced by light in HaCaT human immortalized epidermal cells, indicating that the chemically synthesized polypeptides of the present invention have potential antioxidant and collagen regeneration promoting effects; at the same time, the polypeptides have weak cytotoxicity and anti-photoaging effects, and have broad application prospects in skin care products, cosmetics and cell culture matrices.

[0026] (2) The polypeptide provided by the present invention can inhibit the release of ROS in cells irradiated by UVB, thereby reducing cell damage. In addition, the polypeptide is a synthetic polypeptide with the advantages of good stability, weak cytotoxicity, and easy preparation. It has broad application prospects in the preparation of skin care products with anti-photoaging effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The mass spectrum and high performance liquid chromatogram of molecule 5 obtained in Example 2 of the present invention;

[0028] Figure 2 The mass spectrum and high performance liquid chromatogram of molecule 6 obtained in Example 3 of the present invention;

[0029] Figure 3 This is a diagram showing the results of the light aging resistance experiment in Example 4 of the present invention;

[0030] Figure 4 This is a diagram showing the results of a cytotoxicity experiment in Example 5 of the present invention;

[0031] Figure 5 This is a graph showing the results of the anti-photoaging apoptosis experiment in Example 6 of the present invention. DETAILED DESCRIPTION

[0032] The technical solutions in the embodiments of the present invention are clearly and completely described. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention. The technical solutions of the present invention are further described in detail below through specific embodiments.

[0033] In the present invention, unless otherwise specified, the raw materials and equipment used can be purchased from the market or are commonly used in the art. The methods in the following embodiments, unless otherwise specified, are all conventional methods in the art.

[0034] Embodiment 1:

[0035] In some specific embodiments provided by the present invention, the anti-photoaging polypeptide has an amino acid sequence as shown in any one of molecules 1 to 22:

[0036]

[0037]

[0038] Example 2: Synthesis of Molecule 5 (Tyr-Trp-Leu-Ser-Thr-Pro-Glu-Met-Ala-Met-NH2)

[0039] Step 1: Synthesis of H-Tyr(tBu)-Trp(Boc)-Leu-Ser(tBu)-Thr(tBu)-Pro-Glu(OtBu)-Met-Ala-Met-MBHA resin 5

[0040] Removal of Fmoc protecting group: Rink Amide MBHA Resin (0.68mmol / g, 1.47g) was swollen with DCM (20mL) in a peptide synthesis tube at 25℃±5℃ for 15 minutes, the solvent was drained, piperidine / DMF (volume ratio 1 / 4, 20mL) was added, and the reaction was carried out at 25℃±5℃ for 5 minutes, and then the reaction was dried. Piperidine / DMF (volume ratio 1 / 4, 20mL) was added again and the reaction was carried out at 25℃±5℃ for 10 minutes. After the reaction was completed, the reaction solution was drained, and washed with DMF (20mL) for 6 times, and then drained and waited for the next reaction.

[0041] Coupling of amino acids: HOBT (271 mg, 2 mmol), HBTU (762 mg, 2 mmol) and DIEA (516 mg, 4 mmol) were added to a DMF (20 mL) solution of Fmoc-Met-OH (740 mg, 2 mmol) respectively, and the reaction was activated in an ice bath for 10 min. After activation, the activation solution was added to the resin prepared above and reacted at room temperature for 1 hour. The reaction solution was drained and the resin was washed 6 times with DMF (20 mL) to obtain Fmoc-Met-resin 5.

[0042] Repeat the above steps to couple amino acids Fmoc-Ala-OH, Fmoc-Met-OH, and Fmoc-Glu(OtBu) one by one.

[0043] -OH, Fmoc-Pro-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Tyr( tBu)-OH to obtain Fmoc-Tyr(tBu)-Trp(Boc)-Leu-Ser(tBu)-Thr(tBu)-Pro-Glu(OtBu)-Met-Ala-Met-MBHA resin 5.

[0044] Removal of Fmoc protecting group: piperidine / DMF (volume ratio 1 / 4, 20 mL) was added to the above prepared resin, and the mixture was reacted at 25°C ± 5°C for 5 minutes, and then drained. Piperidine / DMF (volume ratio 1 / 4, 20 mL) was added again and reacted at 25°C ± 5°C for 5 minutes. After the reaction was completed, the reaction solution was drained and washed with DMF (20 mL) 6 times, then washed alternately with DCM (20 mL) and MeOH (20 mL) 2 times, and finally washed twice with MeOH (5 mL); the obtained resin was dried in a vacuum drying oven at 30°C for 3 h to obtain H-Tyr(tBu)-Trp(Boc)-Leu-Ser(tBu)-Thr(tBu)-Pro-Glu(OtBu)-Met-Ala-Met-MBHA resin 5 (2.8 g).

[0045] Step 2: Synthesis of H-Tyr-Trp-Leu-Ser-Thr-Pro-Glu-Met-Ala-Met-NH2 (molecule 5)

[0046] The H-Tyr(tBu)-Trp(Boc)-Leu-Ser(tBu)-Thr(tBu)-Pro-Glu(OtBu)-Met-Ala-Met-MBHA resin 5 (2.8 g) prepared above and a mixed solution of TFA:TIS:H2O (volume ratio 95:2.5:2.5, 28 mL) were added to a 100 mL reaction bottle, respectively, and reacted at 25°C±5°C for 3 h; after the reaction, the mixture was filtered and washed three times with 10 mL of TFA, the solution was combined and concentrated to about 5 mL; the concentrated solution was added dropwise to 50 mL of methyl tert-butyl ether, settled in an ice bath for 30 minutes, centrifuged, and washed three times with methyl tert-butyl ether (30 mL), and the solid was placed in a vacuum drying oven and dried overnight to obtain 1.1 g of a white solid, which was directly purified by Pre-HPLC to obtain a purified solution, which was concentrated and lyophilized to obtain 104.7 mg of a white solid with a purity of 98.6%.

[0047] The molecule 5 obtained in Example 1 was analyzed by high performance liquid chromatography, and its HPLC spectrum was obtained as follows: Figure 1 As shown, the chromatographic conditions are as follows:

[0048] The instrument model is Agilent 1200, and the chromatograph is Sepax HP-C18, 4.6*50mm*3um

[0049] flow: 1ml / min, buffer A: 0.1% TFA-H2O, buffer B: ACN, B%: 5% (4min) → 90% → (5min) 90% → (5.01) 5% → 7min → 5%.

[0050] The molecule 5 obtained in Example 1 was analyzed by mass spectrometry, and the MS (ESI) was obtained: m / z (M+2H + ) / 2=614.5、M+H + =1227.6, M+Na + =1249.8, (M+Na + +H + ) / 2=625.5;

[0051] Example 3: Synthesis of molecule 6 (Tyr-Trp-Leu-Ser-Thr-Pro-Glu-met-Pro-Ala-NH2)

[0052] Molecule 6 was synthesized by the method of Example 2 to obtain 104.7 mg of a white solid with a purity of 98.7%.

[0053] The molecule 6 was analyzed by mass spectrometry and obtained (M+2H + ) / 2=597.3,M+H + =1193.8, (M+Na + +H + ) / 2=608.4,(M+2Na + ) / 2=619.3.

[0054] The preparation methods of the compounds with an amide at the carbon end (such as molecules 1 to 22) are consistent with the preparation method in Example 2.

[0055] Example 4: Anti-photoaging efficacy test

[0056] Logarithmically growing HaCaT cells were inoculated into a 96-well plate with a black edge and a transparent bottom, reaching 3×10 cells per well. 4 Cells were cultured in a complete medium (4.5ml FBS (Sigma-F8318) + 45ml MEM basal medium (gibco-C11095500BT) + 0.5ml penicillin-streptomycin solution (Biosharp-BL505A)) in a carbon dioxide incubator at 37°C and 5% CO2 concentration for 24h. The old medium was discarded, 100μL of PBS was added to each well, and the plate was moved to the bottom of the UVB device for 105mJ irradiation. The control group (i.e., blank untreated) and the model group (UVB irradiation group) discarded the liquid and replaced the blank basal medium. After the irradiation was completed, the other wells were added with the basal medium containing gradient concentrations of molecules 5 and 6 (MEM basal medium (gibco-C11095500BT)) and cultured for 24h. The concentration of molecule 5 was diluted from 3.12μM to 200μM for 6 concentrations, and the concentration of molecule 6 was diluted from 0.78μM to 100μM for 8 concentrations. The positive reference drug was palmitoyl pentapeptide-4. Pre-prepare 1:1000 dilution of DCFH-DA, discard the liquid, load the probe, place the 96-well plate in an incubator, incubate for 40 minutes, wash with PBS 4 times, 3 minutes each time (low-speed oscillation on an oscillator), replace with new PBS, and use an ELISA reader to detect the signal value at 495nm. The results are shown in the figure. At the tested concentration, polypeptide molecules 5 and 6 significantly inhibited the release of ROS induced by UVB (P<0.01), indicating that the polypeptide has an anti-photoaging effect comparable to palmitoyl pentapeptide-4.

[0057] Example 5: Cytotoxicity test

[0058] Logarithmically growing HaCaT cells were inoculated into a 96-well plate with a black edge and a transparent bottom, reaching 3×10 cells per well. 4Cells were cultured in a complete medium (4.5 ml FBS (Sigma-F8318) + 45 ml MEM basal medium (gibco-C11095500BT) + 0.5 ml penicillin-streptomycin solution (Biosharp-BL505A)) in a carbon dioxide incubator at 37°C and 5% CO2 for 24 hours. The old medium was discarded, and 100 μL of PBS was added to each well. For the control group (i.e., blank and untreated), the other wells were added with basal medium (MEM basal medium (gibco-C11095500BT)) containing chemically synthesized polypeptide molecules 5 and 6. The dosage of polypeptide molecule 5 was diluted to 5 concentrations from 0.32 μM to 200 μM, and the dosage of molecule 6 was diluted to 8 concentrations from 0.012 μM to 1 mM, and cultured for 24 hours. The 96-well plate to be tested was taken out and equilibrated at room temperature for 10 minutes; 100 μL of celltiter solution was added to each well, and the plate was oscillated on an oscillator for 2 minutes. After the oscillation was completed, the plate was allowed to stand for 10 minutes; the cell activity test results were obtained by detection with an ELISA instrument. As shown in the figure, polypeptide molecules 5 and 6 had no effect on cell viability at 200 μM, proving that the ROS inhibition results in Example 3 were not caused by cytotoxicity, indicating that polypeptide molecules 5 and 6 were relatively safe.

[0059] Example 6: Photoaging apoptosis experiment

[0060] Logarithmically growing HaCaT cells were inoculated into black-edged transparent-bottom well plates to a volume of 3 × 10 cells per well. 4Cells were cultured in a complete medium (4.5 ml FBS (Sigma-F8318) + 45 ml MEM basal medium (gibco-C11095500BT) + 0.5 ml penicillin-streptomycin solution (Biosharp-BL505A)) in a carbon dioxide incubator at 37°C and 5% CO2 concentration for 24 hours. The old medium was discarded, 100 μL of PBS was added to each well, and the plate was moved to the bottom of the UVB device for 105 mJ irradiation. The control group (i.e., blank untreated) and the model group (UVB irradiation group) discarded the liquid and replaced with blank basal medium. After irradiation, the other wells were added with basal medium containing polypeptide molecules 5 and 6 (MEM basal medium (gibco-C11095500BT)). The dose of molecule 5 was diluted to 6 concentrations from 0.064 μM to 200 μM, and the dose of molecule 6 was diluted to 7 concentrations from 3 μM to 200 μM, and cultured for 24 hours. Take out the 96-well plate to be tested, balance at room temperature for 10 minutes; add 100 μL of celltiter solution to each well, oscillate on an oscillator for 2 minutes, and let stand for 10 minutes after oscillation; detect with an ELISA instrument to obtain the cell activity test results. As shown in the figure, UVB induces cell apoptosis, but polypeptide molecules 5 and 6 do not have an aggravating effect on apoptosis at the test concentration, proving that the ROS inhibition results in Example 3 are not caused by cell apoptosis, and at the same time supporting the conclusion that polypeptide molecules 5 and 6 in Example 4 are more secure.

[0061] According to the above data, polypeptide molecules 5 and 6 have antioxidant effects comparable to palmitoyl pentapeptide, and are low in toxicity and have no effect of inducing apoptosis on damaged cells. Such molecules can be used in drugs or cosmetics for cell anti-photoaging, drugs or cosmetics (skin care products) or cell culture matrices for cell repair, drugs or cosmetics (skin care products) or cell culture matrices, and products for tissue regeneration and repair of skin photoaging damage.

[0062] Those skilled in the art should understand that, although the present invention is specifically described with reference to the above embodiments, the present invention is not limited to these specific embodiments. Based on the methods and technical solutions taught by the present invention, without departing from the spirit of the present invention, those skilled in the art can make appropriate modifications or improvements, and the equivalent implementation schemes obtained therefrom are all within the protection scope of the present invention.

Claims

1. An anti-photoaging polypeptide, characterized in that: The amino acid sequence of the anti-photoaging polypeptide is H-Tyr-Trp-Leu-Ser-Thr-Pro-Glu-Met-Ala-Met-NH2; Any one of H-Tyr-Trp-Leu-Ser-Thr-Pro-Glu-D-Met-Pro-Ala-NH2.

2. Use of the anti-photoaging polypeptide according to claim 1 in the preparation of drugs and / or cosmetics with anti-photoaging effects.

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