Preparation method and application of pearl whitening anti-aging combined polypeptide and single whitening polypeptide

Abstract

The application discloses a kind of high-activity polypeptides and preparation method and application thereof. With whitening and anti-aging activity as the guide, pearl powder is subjected to enzymatic hydrolysis and ultrafiltration treatment, and parts with higher whitening and anti-aging activity are screened out through in vitro cell experiment, polypeptide identification and sequencing are carried out on the parts by high-resolution mass spectrometry, and a whitening polypeptide with a clear amino acid sequence is obtained through computer simulation and cell experiment screening. The polypeptide will be applied in the field of cosmetics or medical aesthetics, and has very high application value. The application realizes high-value utilization of freshwater products, and provides a reliable basis for deep processing of pearls.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of cosmetic, medical and beauty functional peptides, and relates to a preparation method and application of a pearl-derived high polypeptide and a composition thereof. BACKGROUND

[0002] A polypeptide is a relatively short chain of amino acids. Naturally occurring human peptides have cell communication functions, such as protein regulation, cell proliferation, cell migration, inflammation, angiogenesis and melanin production, and at the same time cause a variety of physiological processes, including defense, immunity, stress, etc. Since 2000, the use of polypeptides in pharmaceutical and cosmetic products has increased dramatically, and these polypeptide molecules have high commercial potential, especially for those with cosmetic activity (e.g., anti-aging, antioxidant, whitening). In recent years, it has been reported that deep processing products of pearls have excellent skin activity, and have the functions of whitening, anti-aging and repair. As a cosmetic raw material source, pearls contain unique pearl proteins. It is of great significance in the field of freshwater product deep processing to produce unique high-activity active peptide compositions by extracting, enzymatic hydrolysis, ultrafiltration and other means, and to screen specific functional polypeptides to improve product efficacy. SUMMARY

[0003] In a first aspect, the present application provides a polypeptide. The amino acid sequence of the polypeptide is YSLG, and the structure is shown as formula I:

[0004] .

[0005] Formula I

[0006] In some embodiments, the polypeptide YSLG provided by the present application is a whitening polypeptide.

[0007] In a second aspect, the present application provides a preparation method of the aforementioned YSLG. The preparation method of the polypeptide comprises synthesizing a target polypeptide according to the amino acid sequence YSLG using a polypeptide synthesizer; in some preferred embodiments, a polypeptide solid-phase synthesis reactor is used to synthesize the target polypeptide.

[0008] In a third aspect, the present application provides the use of the aforementioned polypeptide for preparing a cosmetic or a drug with melanin inhibitory activity. In some embodiments, the polypeptide is used for preparing a cosmetic or a drug with whitening efficacy.

[0009] In a fourth aspect, the present application provides the use of the aforementioned polypeptide for preparing a cosmetic or a drug with tyrosinase inhibitory activity. In some embodiments, YSLG with tyrosinase inhibitory activity can be used for preparing a whitening cosmetic or a drug.

[0010] In a fifth aspect, the present application provides a cosmetic composition comprising the aforementioned polypeptide YSLG.

[0011] The present application relates to a whitening anti-aging cosmetic or pharmaceutical product, including but not limited to a cosmetic or pharmaceutical product having a whitening effect, an anti-aging effect, an effect of treating or preventing a melanin hyperpigmentation disease selected from the group consisting of chloasma, freckle, senile plaque, spot, milk coffee plaque, Becker's nevus, nevus spilus, Mongolian plaque, nevus of Ota, acquired bilateral nevus of Ota-like plaque, nevus of Ito, blue nevus, junctional nevus, mixed nevus, intradermal nevus, halo nevus, congenital melanocytic nevus, Spitz nevus, dysplastic nevus, sunburn plaque.

[0012] The high-activity polypeptide YSLG provided by the present application has a whitening, anti-aging and the like activity, and is a tyrosinase inhibitor.

[0013] In a sixth aspect, the present application provides an active peptide composition, which comprises the polypeptide described above and is extracted from pearls, and the extraction method comprises the following steps:

[0014] 1) Extraction and enzymolysis of crude protein of pearl powder: the pearl powder is crushed, weak acid is added for reaction, and the precipitate is filtered to obtain crude pearl protein;

[0015] The sterilized crude pearl protein is added with pure water and protease for enzymolysis reaction at 40-60°C, the supernatant is filtered to obtain a pearl protein enzymolysis liquid;

[0016] 2) Separation and purification of the enzymolysis liquid: the supernatant obtained in step 1) is subjected to a cut-off section by using an ultrafiltration membrane experimental device, and the separation operation is performed by using an ultrafiltration membrane, and the permeate is concentrated by nanofiltration; then the cut-off liquid in the reflux tank is taken out and freeze-dried to obtain a high-activity peptide composition powder.

[0017] In some embodiments, in step 1) of the extraction method, the protease is one or more of papain, neutral protease and trypsin.

[0018] In some embodiments, in step 2) of the extraction method, the ultrafiltration membrane used is a 1K Da roll-type ultrafiltration membrane; the nanofiltration membrane used is a 200 Da roll-type nanofiltration membrane; and the operating pressure is between 15-25 bar, preferably, the operating pressure is 20 bar.

[0019] In some embodiments, the polypeptide contained has a molecular weight of between 200 Da and 1K Da.

[0020] In some embodiments, the pearl is a freshwater pearl.

[0021] In a seventh aspect, the present application provides the use of the active peptide composition described above in the preparation of a cosmetic or pharmaceutical product having a melanin inhibiting activity.

[0022] In some embodiments, the high-activity polypeptide composition obtained by the present application is applied to B16 cells in DMEM medium in a logarithmic growth phase at different concentrations. The results show that the high-activity polypeptide composition obtained by the present application has significant melanin inhibiting activity, and the high-activity polypeptide obtained by the present application is significantly higher than the widely used arbutin in melanin inhibiting activity. The high-activity polypeptide obtained by the present application has roughly equivalent inhibiting activity at a mass ratio of 100 ppm to 1000 ppm. In some specific embodiments, the high-activity polypeptide composition obtained by the present application has a melanin production inhibiting rate as high as 41.9% at a concentration of 1000 ppm.

[0023] The existing polypeptides on the market have almost no melanin inhibiting activity after being tested by the method of the present embodiment, after being subjected to B16 cell melanogenesis promotion pretreatment. In the embodiments of the present application, the provided B16 cells are in a natural state without being subjected to melanogenesis promotion test, which can better reflect the real melanin inhibiting activity of the active polypeptide composition and more truly reflect the melanin inhibiting activity of the high-activity polypeptide composition.

[0024] In some embodiments, the active polypeptide composition is used for preparing cosmetics or drugs with whitening effect.

[0025] In the eighth aspect, the active polypeptide composition described above is used for preparing cosmetics or drugs with tyrosinase inhibiting activity; in some embodiments, the tyrosinase inhibitor polypeptide composition is used for skin whitening.

[0026] In the ninth aspect, the active polypeptide composition described above is used for preparing cosmetics or drugs with collagen degradation inhibiting effect.

[0027] In the tenth aspect, the active polypeptide composition described above is used for preparing cosmetics or drugs with collagen production promoting effect.

[0028] In some embodiments, the active peptide composition is used for preparing a cosmetic or a medicine with anti-aging efficacy; in some specific embodiments, the active peptide composition obtained by the application is subjected to Col-I and MMP-1 gene expression determination, and the results show that the high-activity peptide composition provided by the application has significant anti-aging activity, and the best anti-aging activity is achieved at a mass concentration of 10 ppm, the expression amount of the matrix metalloproteinase MMP-1 gene is inhibited to 59.58% of that of the blank group, and the expression amount of the type I collagen Col-I gene is promoted to 221% of that of the blank group. It is shown that the active peptide composition provided by the application can inhibit the degradation of collagen while promoting the generation of collagen, and has a good anti-aging effect. The high-activity peptide composition is prepared by the process of enzymolysis, ultrafiltration, freeze-drying and the like of pearl powder, the high-activity peptide composition has the activities of whitening and anti-aging, and is a tyrosinase inhibitor. A whitening polypeptide is screened out, and the amino acid sequence of the polypeptide is YSLG.

[0029] Taking the whitening polypeptide and the composition thereof as the core, any corresponding adjustment or modification thereof belongs to the protection scope of the application.

[0030] Compared with the prior art, the application has the following beneficial effects:

[0031] 1. The active peptide composition with the activities of whitening and anti-aging is obtained by selecting freshwater pearls as raw materials for crude protein extraction and enzymolysis, and through the steps of enzymolysis, ultrafiltration, nanofiltration and freeze-drying, the active peptide composition has a higher melanin inhibition rate, and the active peptide composition contains only small molecule peptides.

[0032] 2. The active peptide composition with the activities of strong whitening and anti-aging is obtained by extracting pearl crude protein through the steps of enzymolysis, ultrafiltration, nanofiltration and freeze-drying and the like under the guidance of melanin inhibition activity; the active peptide composition is analyzed by ESI-LS-MS / MS, a whitening polypeptide YSLG is obtained through software simulation and cell experiment verification, and the polypeptide YSLG has great application value in the fields of cosmetics and medical beauty.

[0033] 3. The application realizes efficient preparation and screening of cosmetic functional peptides.

[0034] 4. The application realizes high-value utilization of pearl products, and provides a reliable basis for deep processing of low-value pearl products. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 A melanin production inhibition activity column chart of the high-activity peptide composition obtained in Example 1 and arbutin;

[0036] Figure 2Bar graph of melanin production inhibitory activity of whitening peptide (10 ppm). DETAILED DESCRIPTION

[0037] The following examples are further illustrations of the application and are not intended to limit the same.

[0038] Example 1

[0039] As Figure 1 described, a preparation method of a high-activity peptide from pearls and a composition thereof includes the following steps:

[0040] S1, crude protein extraction and enzymatic hydrolysis of pearl powder

[0041] Take freshwater pearls, crush the pearl powder to 40 mesh, add 25% acetic acid by volume at a material to liquid ratio of 1:5, stir at 60°C and 100 rpm for two hours, then filter with gauze, and take the precipitate as crude pearl protein. Take the sterilized crude protein as described above, add 10 times the weight of pure water to the crude protein, add papain at 50°C for enzymatic hydrolysis and stirring for 5 h, filter and take the supernatant to obtain pearl protein hydrolysate.

[0042] S2, separation and preparation of the hydrolysate

[0043] (1) Take the pearl protein hydrolysate obtained in S1 and put it into the raw material tank of the ultrafiltration membrane experimental equipment. Load 1K Da roll-type ultrafiltration membrane into the membrane shell, close the concentrated water outlet and other membrane shell valves, open the ultrafiltration passage valve, the concentrated water backflow valve and the clear liquid flow valve, start the high-pressure pump, and when the high-pressure pump is running stably, rotate the pressure control valve to adjust the pressure to 20 bar, and collect the flow-out clear liquid at the clear liquid flow valve.

[0044] (2) Load 200 Da roll-type nanofiltration membrane into the membrane shell of the ultrafiltration membrane equipment and open the corresponding valves, close the other membrane shell valves, close the concentrated water outlet valve, open the concentrated water backflow valve and the clear liquid flow valve. Put the flow-out clear liquid obtained in (1) into the equipment raw material tank, start the high-pressure pump, and when the high-pressure pump is running stably, rotate the pressure control valve to adjust the pressure to 20 bar. When the liquid in the raw material tank is about 1L, close the high-pressure pump and open the concentrated water flow-out valve, collect the concentrated liquid at the concentrated water flow-out valve, and then vacuum freeze-dry to obtain a light yellow powder, which is the pearl high-activity peptide composition. The polypeptide molecular weight contained therein is between 200 Da and 1K Da.

[0045] Example 2, melanin inhibitory activity determination

[0046] Sample preparation: 3 groups of PBS buffer were prepared, and PBS buffer was used as a blank group; arbutin was added to the PBS buffer as a control group (the mass ratio of arbutin was 1%); different amounts of the sample of Example 1 were added to a group of PBS buffer as experimental groups (the mass ratio of the active peptide composition was 1 / 10000, 1 / 1000, 1 / 100);

[0047] B16 cells in the logarithmic growth phase grown in DMEM medium were inoculated in a 100mm cell culture dish at a cell number of (10-15) x 10 4 Each well was added with 1 mL of the above sample, and due to the dilution effect, the concentration of arbutin in the culture medium was 1000 ppm; the concentration of the active peptide composition in the culture medium was 10 ppm, 100 ppm, and 1000 ppm, respectively. Three replicate wells were set for each concentration, and incubation was continued. After 24 h of culture, the cells were collected by trypsin digestion, the supernatant was discarded, and the cells were washed twice with PBS. Each tube was added with 300 μl of 1N NaOH containing 10% DMSO to lyse the cells at 80°C for 30 min. 100 μL of cell lysate was taken and added to a 96-well plate, and the absorbance was measured at 405 nm by an enzyme marker. For specific experimental results, please refer to the attached Figure 1 The results show that the high-activity peptide composition obtained in Example 1 has significant melanin inhibitory activity, and the high-activity peptide obtained in Example 1 is significantly higher than the widely used arbutin in terms of melanin inhibitory activity. At the same time, the high-activity peptide obtained in Example 1 has roughly equivalent inhibitory activity at a mass ratio of 100 ppm and 1000 ppm. At a concentration of 1000 ppm, the melanin production inhibition rate is as high as 41.9%.

[0048] In addition, the B16 cells provided in the present embodiment are in a natural state without melanocyte stimulating test, which can better reflect the true melanin inhibitory activity of the active peptide composition. The existing polypeptides on the market have almost no melanin inhibitory activity after being tested by the method of the present embodiment, which have been subjected to melanocyte stimulating pretreatment in the melanin inhibitory activity test.

[0049] Example 3 Col-I and MMP-1 gene expression determination

[0050] Sample preparation: Prepare PBS buffer as the blank group. Prepare PBS solution of the active peptide composition with a mass / volume concentration of 1 / 1000; 1 μL of PBS solution of the active peptide composition with a mass / volume concentration of 1 / 100; and 1 μL of PBS solution of the active peptide composition with a mass / volume concentration of 1 / 10. These three solutions are used as the experimental groups. VC PBS solution with a mass / volume concentration of 1 / 10 is used as the positive group.

[0051] Fibroblasts grown in FM complete medium and in the logarithmic growth phase were subjected to a 5×10⁻⁶ ion exchange rate. 4 Cells were seeded at 100 μL of culture medium per well in 6-well cell culture plates and incubated at 37°C in a 5% CO2 incubator. After 24 h, 1 μL of the above sample was added to form a blank group (PBS buffer), a positive group (VC in PBS solution, with a final concentration of 1000 ppm in the culture medium), and experimental groups (PBS solutions of different concentrations of active peptide compositions, with final concentrations of 10 ppm, 100 ppm, and 1000 ppm in the culture medium, respectively), and incubation continued. The next day, the experimental groups were irradiated with 30 mJ of UV energy and cultured for another 24 h. After culture, RNA was extracted from each group and reverse transcribed into cDNA for RT-PCR.

[0052] Please refer to Tables 2 and 3 for specific experimental results. The results show that the highly active peptide composition obtained in Example 1 has significant anti-aging activity, and the best effect is achieved at a mass concentration of 10 ppm. It can inhibit the expression level of matrix metalloproteinase MMP-1 gene to 59.58% of the blank group, while promoting the expression level of type I collagen Col-I gene to 221% of the blank group. This indicates that the active peptide composition can promote collagen production while inhibiting collagen degradation, and has a good anti-aging effect.

[0053] Example 4 Identification of Highly Active Peptides Derived from Pearl

[0054] (1) Sample reduction alkylation: Dissolve the powdered sample in ddH2O, add DTT solution to an appropriate amount of sample to make the final concentration 10 mmol / L, and reduce in a water bath at 56℃ for 1 h. Add IAA solution to make the final concentration 50 mmol / L, and react in the dark for 40 min. Desalt using a desalting column, and evaporate the solvent in a vacuum centrifuge at 45℃.

[0055] (2) LC-MS analysis: Chromatographic parameters: 300 μm i.d. × 5 mm, packed with Acclaim PepMap RPLC C18; 5 μm, 100 Å pre-column, 2) 150 μm i.d. × 150 mm, packed with Acclaim PepMap RPLC C18, 1.9 μm, 100 Å analytical column; mobile phase was 0.1% formic acid (A phase), 0.1% formic acid + 80% ACN (B phase) respectively; flow rate was 600 nL / min; total analysis time was 66 min. 4%-8% B (0-2 min), 8-28% B (2-45 min), 28%-40% B (45-55 min), 40%-95% B (55-56 min), 95% B (56-66 min). Primary mass spectrometry parameters were Resolution: 70,000, AGCtarget: 3e6, MaximumIT: 100 ms, Scanrange: 100 to 1500 m / z; secondary mass spectrometry parameters were Resolution: 17,500, AGCtarget: 5e4, MaximumIT: 50 ms, TopN: 20, NCE / steppedNCE: 28. The raw file of mass spectrometry acquisition was subjected to de novo search by software PEAKS Studio8.5 to obtain a list of peptide segments, and the molecular weight of the polypeptide contained was between 200 Da and 1 K Da.

[0056] Example 5 Molecular docking simulation of polypeptide and mushroom tyrosinase (2Y9X)

[0057] Discovery studio 2019 was used to screen the obtained low molecular peptide sequence for whitening activity. The receptor protein 2Y9X was downloaded from the PDB database, and after opening with the above software, the protein was pretreated, and the treatment process was removal of water molecules, addition of hydrogen to the protein, cleaning of the protein, and preparation of the protein. Chemdraw 20.0 was used to draw the polypeptide structure and save it as an sdf format, and the ligand was opened and prepared in Discovery studio 2019, and docking screening was performed. If the docking is successful, it is considered that the polypeptide has tyrosinase inhibitory activity;

[0058] It was found that the polypeptide with the amino acid sequence YSLG had significant tyrosinase inhibitory activity.

[0059] Example 6 Synthesis of polypeptide YSLG

[0060] In the polypeptide solid-phase synthesis reactor, 100 mg of Trityl resin was swelled by soaking in DCM for 5 min; Fmoc-L-glycine-OH was dissolved in 5 mL of DCM, and then N,N-diisopropyl ethylamine (DIEA) was added. After being fully dissolved, the mixture was added to the container containing the resin, and then nitrogen was blown to fully stir the reaction for 60 min. The resin was washed with 10 mL of N,N-dimethylformamide (DMF), and the filtrate was removed, and the resin was retained. This washing was repeated 5 times. Then, 5 mL of a 20% piperidine (Piperidine) DMF solution was added to the resin, and then nitrogen was blown to fully stir the reaction for 20 min to remove the Fmoc protecting group. Fmoc-L-isoleucine-OH, HBTU, and DIEA were added to 5 mL of DMF and fully dissolved, and then the solution was added to the resin. Nitrogen was blown to fully stir the reaction for 60 min. The resin was washed with 10 mL of N,N-dimethylformamide (DMF), and the filtrate was removed, and the resin was retained. This washing was repeated 5 times. Then, 5 mL of a 20% piperidine (Piperidine) DMF solution was added to the resin, and then nitrogen was blown to fully stir the reaction for 20 min to remove the Fmoc protecting group. Fmoc-L-serine-OH, HBTU, and DIEA were added to 5 mL of DMF and fully dissolved, and then the solution was added to the resin. Nitrogen was blown to fully stir the reaction for 60 min. During this time, ninhydrin solution was used for color reaction to detect whether the amino acid coupling reaction was complete. If the reaction was blue, the reaction was continued. If the reaction was colorless, the reaction was complete. The resin was washed with 10 mL of DMF, and the filtrate was removed, and the resin was retained. This washing was repeated 5 times. The washed resin was washed with 5 mL of DCM for 5 times, and then nitrogen was blown to dry the DCM. Then, 5 mL of a cleavage reagent was added to the resin, and then nitrogen was blown to fully stir the cleavage reaction for 50 min. The liquid was collected by filtration. The liquid was added dropwise to 40 mL of ice ethyl ether to precipitate white solids. The white solids were collected by centrifugation, and the supernatant was discarded. The white solids were dissolved in water / acetonitrile (10 / 90, with 0.1% trifluoroacetic acid added). The liquid sample was filtered through a 0.22 um filter membrane, and then injected into an HPLC for separation and purification. After the purified sample was detected by mass spectrometry and HPLC, the sample meeting the purity requirements was collected and freeze-dried to obtain a freeze-dried polypeptide powder.YSLG was synthesized by the above method.

[0061] Example 7 Melanin inhibitory activity assay (single polypeptide)

[0062] B16 cells in logarithmic growth phase, which were grown in DMEM medium, were inoculated in 100 mm cell culture dishes at a cell number of (10-15) x 10 4 Each well was added with 10 mL of culture solution, and was incubated in a carbon dioxide incubator at 37°C and a 5% CO2concentration. After 24 h, a PBS solution of YSLG was added to a concentration of 10 ppm, and the polypeptide was set in triplicate, and the incubation was continued. After 24 h of culture, the cells were collected by trypsin digestion, the supernatant was discarded, and the cells were washed twice with PBS, 300 μl of 10% DMSO in 1 N NaOH was added to each tube to lyse the cells, and the tubes were placed in a water bath at 80°C for 30 min. 100 μL of the cell lysate was taken and added to a 96-well plate, and the absorbance was measured at 405 nm by using an enzyme label meter.

[0063] The specific experimental results are shown in Table 1, and the experimental results show that YSLG has a significant melanin production inhibition rate of 18.6% at a concentration of 10 ppm.

[0064] Table 1 Melanin production inhibition rates of active peptide compositions and three whitening polypeptides

[0065]

[0066] Table 2 Expression inhibition activity of active peptide compositions on MMP-1

[0067]

[0068] Table 3 Expression promotion activity of active peptide compositions on Col-I

[0069]

[0070] The above are only preferred embodiments of the present application, and it should be noted that the above preferred embodiments should not be regarded as limiting the present application, and the protection scope of the present application should be defined by the scope defined in the claims. For ordinary skilled persons in the art, several improvements and refinements can be made without departing from the spirit and scope of the present application, and these improvements and refinements should also be regarded as the protection scope of the present application.

[0071] SEQUENCE LISTING

[0072] <110> EPODIA BIOSCIENCES CO., LTD.

[0073] <120> Preparation method and application of pearl whitening anti-aging combined polypeptide and single whitening polypeptide

[0074] <160> 1

[0075] <170> SIPOSequenceListing 1.0

[0076] <210> 1

[0077] <211> 4

[0078] <212> PRT

[0079] <213> Artificial Sequence

[0080] <400> 1

[0081] Tyr Ser Leu Gly 1 SEQUENCE LISTING <110> EPODIA BIOSCIENCES, INC. <120> Methods of making and using pearl white anti-aging combination polypeptides and single whitening polypeptides <160> 1 <170> SIPOSequenceListing 1.0 <210> 1 <211> 4 <212> PRT <213> Artificial Sequence <400> 1 Tyr Ser Leu Gly 1

Claims

1. Use of a polypeptide, characterized in that, Cosmetic for preparing a cosmetic having a melanin inhibiting activity, wherein the polypeptide has an amino acid sequence of YSLG.

2. Use according to claim 1, characterized in that, Cosmetic for preparing a cosmetic having a whitening effect.

3. Use according to claim 1, characterized in that, Cosmetic for preparing a cosmetic having a tyrosinase inhibiting activity.

4. The use according to claim 1, characterized in that, The method for preparing the polypeptide comprises synthesizing according to the amino acid sequence using a polypeptide synthesizer.

5. Use of an active peptide composition, characterized in that, Cosmetic for preparing a cosmetic having a melanin inhibiting activity, wherein the active peptide composition comprises a polypeptide having an amino acid sequence of YSLG.

6. Use according to claim 5, characterized in that, Cosmetic for preparing a cosmetic having a whitening effect.

7. Use according to claim 5, characterized in that, Cosmetic for preparing a cosmetic having a tyrosinase inhibiting activity.

8. The use according to claim 5, characterized in that, Cosmetic for preparing a cosmetic having a collagen degradation inhibiting effect.

9. The use according to claim 5, characterized in that, Cosmetic for preparing a cosmetic having a collagen production promoting effect.

10. The use according to claim 5, characterized in that, Cosmetic for preparing a cosmetic having an anti-aging effect.

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