Preparation and application of polypeptide with whitening and anti-aging effects

Abstract

The invention belongs to the technical field of polypeptides, and particularly relates to a novel synthetic polypeptide and a synthetic method and application thereof. The skin as the largest organ of the human body can visually reflect the aging level of the whole body. Skin senescence can be divided into endogenous senescence and exogenous senescence, the main manifestation of the cellular level is the senescence of fibroblasts in a corium layer, research shows that reactive oxygen species (ROS) is an important factor for promoting cell senescence, and the cell anti-senescence effect can be achieved by reducing the generation of ROS in cells. Melanin is a heterogeneous polyphenol-like polymer which is synthesized in melanin bodies and has a complex structure, the amount and distribution of the melanin not only determine the depth of the skin color, but also cause hyperpigmentation diseases due to metabolic disorder. In recent years, research finds that the polypeptide has excellent performance in skin care due to good biocompatibility and relatively strong biological activity. The polypeptide can rapidly permeate the skin through a specific receptor and activate the regeneration and repair functions of skin cells, so that the whitening and anti-aging effects are achieved. In addition, the action mechanism of the polypeptide is relatively mild, and the polypeptide is suitable for various skin types, especially sensitive skin. At present, although multiple polypeptide products exist in the market, most of the polypeptide products integrate a single function, and comprehensive products with double effects of whitening and anti-aging are lacked. Therefore, a polypeptide product with whitening and anti-aging effects is developed, the requirements of consumers can be better met, and the polypeptide product has important market value and application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of polypeptides, and particularly relates to a novel synthetic polypeptide, its synthesis method and applications. Background Art

[0002] As the largest organ of the human body, the skin can intuitively reflect the overall aging level of the body. Skin aging can be divided into endogenous aging and exogenous aging. At the cellular level, the main manifestation is the aging of fibroblasts in the dermis. Research shows that reactive oxygen species (ROS) is an important factor contributing to cell aging. Reducing and decreasing the production of ROS in cells can achieve the effect of cell anti-aging.

[0003] Melanin is a type of polyphenol polymer with a complex structure and inhomogeneity synthesized in melanosomes. The amount and distribution of melanin not only determine the depth of skin color, but also its metabolic disorder will lead to pigmentation diseases.

[0004] In recent years, studies have found that polypeptides perform well in skin care due to their good biocompatibility and strong biological activity. Polypeptides can rapidly penetrate the skin through specific receptors, activate the regeneration and repair functions of skin cells, thereby achieving the effects of whitening and anti-aging. In addition, the mechanism of action of polypeptides is relatively mild, suitable for various skin types, especially sensitive skin.

[0005] Currently, although there are already various polypeptide products on the market, most of them focus on single functions and lack comprehensive products targeting both whitening and anti-aging effects. Therefore, developing a polypeptide product with both whitening and anti-aging effects can better meet the needs of consumers and has important market value and application prospects. Summary of the Invention

[0006] One of the purposes of the present invention is to provide a synthetic polypeptide with a brand-new structure, having dual effects of whitening and anti-aging.

[0007] Another purpose of the present invention is to provide a preparation method for a synthetic polypeptide with a brand-new structure.

[0008] The present invention provides a polypeptide compound Pro-Thr-Ser-D-Tyr with a brand-new structure, and the structural formula is as follows:

[0009]

[0010] The brand-new synthetic polypeptide has dual effects of whitening and anti-aging, improving the traditional single effect.

[0011] The polypeptide with a brand-new structure has a stable biological structure and high biological safety, solving the toxicity problem and stability problem of traditional whitening and anti-aging products.

[0012] The present invention provides the application of the polypeptide in the preparation of drugs and skin-whitening and anti-aging cosmetics.

[0013] More preferably, the polypeptide cosmetics may be facial cleanser, cream, facial mask, essence, toner, emollient lotion, body lotion or shower gel.

[0014] More preferably, the polypeptide drug may be a drug related to skin diseases. Description of the Drawings

[0015] Figure 1 Liquid-phase NMR spectrum of the polypeptide.

[0016] Figure 2 Liquid-phase mass spectrum of the polypeptide.

[0017] Figure 3 Graph showing the effect of the polypeptide on the melanin content in melanoma cells.

[0018] Figure 4 Graph of the fluorescence of reactive oxygen species in the polypeptide group and the control group of B16F10 cells.

[0019] Figure 5 Control graph of the effect of the polypeptide on reactive oxygen species (ROS) in B16F10 cells.

[0020] Figure 6 Structural formula graph of the polypeptide. Detailed Description of the Invention

[0021] The following specifically introduces the substantial content of the present invention in combination with examples, but does not limit the protection scope of the present invention thereby.

[0022] Example 1

[0023] Synthesis method of the polypeptide, the steps are as follows:

[0024] Step (1): Add 10 g of dichloro resin (loading amount 1.0 mmol / g) to a solid-phase reactor, add 10 ml each of DCM and DMF, soak for 10 min and then filter by suction to fully expand the resin.

[0025] Step (2): Weigh 33 mmol of Fmoc-D-Tyr(tBu)-OH (the second amino acid at the C-terminus), add 100 mmol of DIPEA / DMF solution to the solid-phase reactor, and react at 30 °C in an air-bath constant temperature oscillator for 120 min. After the reaction, wash the resin 5 times each with DCM and DMF.

[0026] Step (3): Add an appropriate amount of 20% piperidine / DMF solution to the solid-phase reactor, react at 30 °C in a gas bath thermostatic oscillator for 10 min. After suction filtration, add an appropriate amount of 20% piperidine / DMF solution again, and react at 30 °C in a gas bath thermostatic oscillator for 10 min. After the reaction is completed, wash the resin with DCM and DMF 5 times each.

[0027] Step (4): Take a small amount of resin and detect it by the ninhydrin method. If the resin shows a blue color in the detection, it indicates that the deprotection is successful; if the resin has no color, repeat step (3) until the resin shows a blue color after detection by the ninhydrin method.

[0028] Step (5): Weigh 33 mmol of Fmoc-Ser(tBu)-OH (the second amino acid at the C-terminus), 30 mmol of HCTU, and 100 mmol of DIPEA / DMF solution, and react at 30 °C in a gas bath thermostatic oscillator for 40 min. After the reaction is completed, wash the resin with DCM and DMF 5 times each.

[0029] Step (6): Add an appropriate amount of 20% piperidine / DMF solution to the solid-phase reactor, react at 30 °C in a gas bath thermostatic oscillator for 10 min. After suction filtration, add an appropriate amount of 20% piperidine / DMF solution again, and react at 30 °C in a gas bath thermostatic oscillator for 10 min. After the reaction is completed, wash the resin with DCM and DMF 5 times each.

[0030] Step (7): Take a small amount of resin and detect it by the ninhydrin method. If the resin shows a blue color in the detection, it indicates that the deprotection is successful; if the resin has no color, repeat step (3) until the resin shows a blue color after detection by the ninhydrin method.

[0031] Step (8): 33 mmol of Fmoc-Thr(tBu)-OH (the third amino acid at the C-terminus), 30 mmol of HCTU, and 100 mmol of DIPEA / DMF solution, and react at 30 °C in a gas bath thermostatic oscillator for 40 min. After the reaction is completed, wash the resin with DCM and DMF 5 times each.

[0032] Step (9): Add an appropriate amount of 20% piperidine / DMF solution to the solid-phase reactor, react at 30 °C in a gas bath thermostatic oscillator for 10 min. After suction filtration, add an appropriate amount of 20% piperidine / DMF solution again, and react at 30 °C in a gas bath thermostatic oscillator for 10 min. After the reaction is completed, wash the resin with DCM and DMF 5 times each.

[0033] Step (10): Take a small amount of resin and detect it by the ninhydrin method. If the resin shows a blue color in the detection, it indicates that the deprotection is successful; if the resin has no color, repeat step (3) until the resin shows a blue color after detection by the ninhydrin method.

[0034] Step (11): 33 mmol of Fmoc-Pro-OH (the fourth amino acid at the C-terminus), 30 mmol of HCTU, and 100 mmol of DIPEA / DMF solution were reacted at 30 °C for 40 min in a gas bath thermostatic oscillator. After the reaction, the resin was washed 5 times each with DCM and DMF.

[0035] Step (12): An appropriate amount of 20% piperidine / DMF solution was added to the solid-phase reactor and reacted at 30 °C for 10 min in a gas bath thermostatic oscillator. After suction filtration, an appropriate amount of 20% piperidine / DMF solution was added again and reacted at 30 °C for 10 min in a gas bath thermostatic oscillator. After the reaction, the resin was washed 5 times each with DCM and DMF.

[0036] Step (13): Take a small amount of resin and detect it by the ninhydrin method. If the resin shows a blue color, it indicates successful deprotection; if the resin has no color, repeat step (3) until the resin shows a blue color after detection by the ninhydrin method. Pro-Thr-Ser-D-Tyr was synthesized, and the structural formula is as follows:

[0037]

[0038] Example 2

[0039] 2.1 B16-F10 cell proliferation experiment:

[0040] Considering that the polypeptide is used as a cosmetic or drug, its cytotoxicity is crucial.

[0041] To test the cell safety of the polypeptide, the inventor used MTT to evaluate the cytotoxicity. B16F10 cells were inoculated in a 96-well plate, 1 ml of cell suspension per well, and cultured in an incubator at 37 °C and 5% CO2 for 24 h. The medium in the well plate was discarded, and aqueous solutions of the polypeptide with three different concentrations of 1 mg / ml, 2 mg / ml, and 3 mg / ml were added to each well, with 16 wells repeated for each concentration, 200 μL per well, and then continued to be statically cultured for 48 h; after 48 h, each well was treated with 20 μL of MTT (5 mg / mL) for 4 h, the formed purple formazan was dissolved with DMSO, and the OD value was measured at a wavelength of 490 nm using an enzyme-linked immunosorbent assay reader. Cell viability (%) = OD value of the drug administration group / OD value of the control group × 100%.

[0042] The results showed that when the polypeptide was less than 2 mg / ml, it had no toxic effect on the cells, and the cell survival rate was above 95%.

[0043] 2.2 Effect of the polypeptide on melanogenesis in B16-F10 cells:

[0044] Mouse melanoma cells B16-F10 were purchased from Wuhan Shang'en Biotechnology Co., Ltd.

[0045] B16F10 cells were seeded in 6-well plates at a seeding density of 500,000 cells per well. After adherent growth for 24 hours, a control group and three intervention groups were set up. The intervention groups were added with 1 ml of a polypeptide aqueous solution with a concentration of 1 mg / ml. DMEM medium containing 10% fetal bovine serum was used. After culturing for 48 hours, the amount of melanin synthesis was measured by the NaOH lysis method.

[0046] After the culture was completed, the supernatant was discarded and the cells were washed with PBS. The cells were digested with 0.25% trypsin and pipetted into a cell suspension. 100 μL of the cell suspension was taken for cell counting. The remaining cell suspension was centrifuged (1000 r / min, 5 min). After discarding the supernatant, 150 μL of 1 mol / L NaOH solution (containing 10% DMSO) was added to lyse the cells. The cells were lysed in a water bath at 80 °C for 30 min to 2 h to fully lyse the cells and dissolve the melanin granules. The cell lysates of each group were transferred to a 96-well plate, 100 μL was added to each well, and the absorbance value A was measured at 405 nm using an enzyme-linked immunosorbent assay (ELISA) reader. The amount of melanin synthesis was calculated.

[0047] The results are as Figure 3 shown. Compared with the control group, the polypeptide inhibited melanin production, and the melanin content in melanoma cells was significantly reduced.

[0048] Example 3

[0049] 3.1 HaCaT cell proliferation experiment:

[0050] To test the cell safety of the polypeptide, the inventors used MTT to evaluate cytotoxicity. HaCaT cells were seeded in 96-well plates, 1 ml of cell suspension was added to each well, and the plates were placed in an incubator at 37 °C and 5% CO2 for 24 h. The medium in the wells was discarded, and 200 μL of polypeptide aqueous solutions with three different concentrations of 1 mg / ml, 2 mg / ml, and 3 mg / ml were added to each well. Each concentration was repeated in 16 wells. The plates were incubated for another 48 h. After 48 h, 20 μL of MTT (5 mg / mL) was added to each well and incubated for 4 h. Dimethyl sulfoxide (DMSO) was used to dissolve the formed purple formazan, and the optical density (OD) value was measured at a wavelength of 490 nm using an ELISA reader. Cell viability (%) = OD value of the treatment group / OD value of the control group × 100%.

[0051] The results showed that when the concentration of the polypeptide was less than 2 mg / ml, it had no toxic effect on the cells, and the cell survival rate was above 95%.

[0052] 3.2 Experiment on the inhibitory effect of HaCaT cell reactive oxygen species:

[0053] Adult human immortalized keratinocyte HaCaT cells were seeded in 6-well plates at a seeding density of 500,000 cells / well. After 24 hours of cell attachment, a control group and three intervention groups were set up. In the intervention groups, 1 ml of a polypeptide aqueous solution with a concentration of 1 mg / ml was added, and in the control group, 1 ml of aqueous solution (DMEM medium with 10% fetal bovine serum) was added. The cells were cultured for 48 hours.

[0054] DCFG-DA was diluted with serum-free culture medium at a ratio of 1:1000 to a final concentration of 10 μmol / L.

[0055] The incubated cell culture medium was removed, and an appropriate volume of diluted DCFH-DA was added. The added volume should be sufficient to cover the cells, approximately 1 ml. The cells were incubated in a 37°C incubator for 20 minutes, washed three times with serum-free cell culture medium to completely remove the DCFH-DA that had not entered the cells, and the cells were collected for flow cytometry detection. FlowJo was used to analyze and process the data.

[0056] The results are as Figure 4 and Figure 5 shown. Compared with the control group, the polypeptide inhibited the production of reactive oxygen species (ROS), and the production rate of ROS in immortalized keratinocytes decreased significantly.

Claims

1. A synthetic polypeptide, characterized in that, The amino acid sequence is Pro-Thr-Ser-D-Tyr.

2. The polypeptide with whitening and anti-aging effects according to claim 1, and the structure of the polypeptide is as shown in Figure 6 of the specification.

3. Use of the polypeptide with whitening and anti-aging effects according to claim 1 or 2 in the preparation of whitening and anti-aging cosmetics.

4. Use of the polypeptide with whitening and anti-aging effects according to claim 1 or 2 in skin disease drugs.

5. A drug, characterized in that , comprising the synthetic polypeptide according to claim 1 and pharmaceutically acceptable excipients.

6. The application according to claim 3, characterized in that: The said use is the use in the preparation of whitening cosmetics for inhibiting melanin deposition.

7. The application according to claim 3, wherein: The said cosmetics are facial cleanser, cream, facial mask, essence, toner, moisturizing lotion, body lotion, body wash.

8. The synthesis method of the synthetic polypeptide as claimed in the claim, characterized in that Solid-phase synthesis is carried out one by one according to the amino acid sequence from the C-terminus to the N-terminus of the synthetic polypeptide as described in Scheme 1.

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