Polypeptides and use thereof

Abstract

A group of polypeptides, which can be used as a tyrosinase inhibitor. The polypeptides have the effects of inhibiting tyrosinase activity, reducing melanin secretion, resisting oxidation, skin-whitening, spot-removing, etc. The polypeptides can be widely used in antioxidant drugs and in the preparation of skin-whitening cosmetics for resisting melanogenesis and lightening spots as a new raw material, and has good application scenarios.

Description

Peptides and their applications Technical Field

[0001] The present invention relates to the technical fields of biomedicine, medical devices, cosmetics and functional polypeptides, and in particular to polypeptides having whitening and freckle-removing effects such as inhibiting tyrosinase activity, reducing melanin secretion and resisting oxidation, and their applications. Background Art

[0002] Melanin protects the skin from ultraviolet radiation and inhibits photocarcinogenesis, but excessive melanin production can cause pigmentation, primarily manifesting as freckles, age spots, chloasma, and uneven skin tone. Melanin is a complex, multi-step conversion product of L-tyrosine. Melanin in skin cells is primarily composed of two quinone-type polymers, eumelanin and pheomelanin, and is the primary factor influencing skin color.

[0003] Tyrosinase is a copper-containing enzyme widely distributed in microorganisms, plants and animals. It regulates plant browning in plants and is responsible for skin and hair pigmentation in animals. It is also a key rate-limiting enzyme in the formation of melanin. Tyrosinase catalyzes the conversion of L-tyrosine into 3,4-dihydroxyphenylalanine (DOPA), and then catalyzes the conversion of DOPA to dopaquinone. Dopaquinone is the main factor affecting the formation of different melanins. Therefore, in theory, inhibiting tyrosinase activity can effectively inhibit the production of melanin.

[0004] Tyrosinase inhibitors currently on the market include hydroquinone, arbutin, kojic acid, and some electron-rich phenols, which have been shown to effectively inhibit tyrosinase activity. Hydroquinone is unstable and easily oxidized. It was later discovered to be highly irritating to the skin, causing irritation or contact dermatitis. Furthermore, if used inappropriately, it can cause excessive discoloration of the skin, leading to localized whitening. Consequently, hydroquinone has been banned from cosmetics. Arbutin is a derivative of hydroquinone with a similar mechanism of action. Because arbutin is a sugar ligand of hydroquinone, its structural toxicity to melanocytes is significantly reduced. However, at high concentrations, it can also exhibit adverse reactions similar to those of hydroquinone. Furthermore, numerous experiments have shown that substances with strong tyrosinase inhibition effects in vitro do not necessarily inhibit melanin production within cells. Therefore, some tyrosinase inhibitors do not have a strong ability to inhibit melanin production during use.

[0005] Studies in recent years have found that polypeptide sequences exhibit a good inhibitory effect on tyrosinase. This polypeptide competitively binds to the tyrosinase target and exhibits a higher tyrosinase inhibitory effect. At the same time, short peptide sequences can exhibit better transdermal efficacy and higher safety.

[0006] In summary, given that currently common tyrosinase inhibitors can whiten the skin but also have a certain degree of skin damage, there is a need to continuously search for natural, better transdermal efficacy, higher safety, good biocompatibility, stable and effective tyrosinase inhibitors, melanin production inhibitors, and antioxidants for widespread application in the pharmaceutical and cosmetic fields. Summary of the Invention

[0007] The present invention provides a group of polypeptides that exhibit strong tyrosinase inhibition, the ability to inhibit melanin production in B16 cells, antioxidant activity, and low cytotoxicity. These polypeptides are suitable for use in the preparation of cosmetics, tissue fillers, pharmaceuticals, foods, or agricultural products. Furthermore, the polypeptides provided by the present invention have a low molecular weight and excellent transdermal properties, making them particularly suitable for use in the cosmetics field.

[0008] In a first aspect of the present invention, a polypeptide is provided, wherein the amino acid sequence of the polypeptide is CPD (SEQ ID NO: 2), CRVI (SEQ ID NO: 4) and / or CFFY (SEQ ID NO: 5).

[0009] In a second aspect, the present invention provides a tyrosinase inhibitor, comprising a polypeptide having an amino acid sequence of CPD (SEQ ID NO: 2), CRVI (SEQ ID NO: 4) and / or CFFY (SEQ ID NO: 5).

[0010] In a third aspect, the present invention provides a melanin inhibitor, which comprises a polypeptide having an amino acid sequence of CPD (SEQ ID NO: 2), CRVI (SEQ ID NO: 4) and / or CFFY (SEQ ID NO: 5).

[0011] In a fourth aspect, the present invention provides an antioxidant, comprising a polypeptide having an amino acid sequence of CPD (SEQ ID NO: 2), CRVI (SEQ ID NO: 4) and / or CFFY (SEQ ID NO: 5).

[0012] In a fifth aspect, the present invention provides a whitening polypeptide, wherein the amino acid sequence of the whitening polypeptide is CPD (SEQ ID NO: 2), CRVI (SEQ ID NO: 4) and / or CFFY (SEQ ID NO: 5).

[0013] In a sixth aspect of the present invention, a polypeptide is provided for use in inhibiting tyrosinase, inhibiting melanin, anti-oxidation, whitening or removing spots, wherein the amino acid sequence of the polypeptide is CPD (SEQ ID NO: 2), CRVI (SEQ ID NO: 4) and / or CFFY (SEQ ID NO: 5).

[0014] The seventh aspect of the present invention provides a method for preparing the above-mentioned polypeptide, which is prepared by solid phase synthesis or recombinant expression and purified.

[0015] Preferably, the polypeptide is synthesized through artificial solid phase synthesis and purified by desalting on a reverse phase HPLC column or filtering and purifying the crude polypeptide solution using a filter membrane.

[0016] The purity of the prepared polypeptide is greater than 95%.

[0017] The preparation method further comprises obtaining the finished powder by freeze-drying technology.

[0018] Specifically, the preparation method comprises the following steps:

[0019] 1) Weighing a resin into a reactor, soaking it with dichloromethane, then washing it with nitrogen and nitrogen-dimethylformamide (DMF) and draining it; the resin is 2-chloroacetyl-L-lysine resin (2CL).

[0020] 2) Weigh appropriate amounts of Fmoc-X1-OH, N,N-diisopropylethylamine (DIEA), and DMF and add them to a reactor for reaction; wherein X1 is the first amino acid of the above-mentioned polypeptide.

[0021] 3) adding a decapping solution into the reactor to remove the Fmoc protecting group on the resin; the decapping solution is a DMF solution containing piperidine.

[0022] 4) Weigh appropriate amounts of Fmoc-X2-OH, hydroxybenzotriazole (HOBT), diisopropylcarbodiimide (DIC), and DMF into a reactor for reaction; wherein X2 is the second amino acid of the above polypeptide.

[0023] 5) adding a decapping solution into the reactor to remove the Fmoc protecting group on the resin; the decapping solution is a DMF solution containing piperidine.

[0024] 6) If the peptide has only two amino acids, proceed to the next step. If it has more than two amino acids, repeat steps 4) and 5) until the last amino acid is added.

[0025] 7) Using a cutting solution to cut the polypeptide from the resin to obtain a crude polypeptide; the cutting solution is a DCM solution containing trifluoroacetic acid TFA.

[0026] 8) Purify by desalting with HPLC reverse phase column chromatography or filter and purify the crude polypeptide solution using a filter membrane.

[0027] 9) The purified polypeptide product is freeze-dried to obtain a finished product powder.

[0028] In an eighth aspect, the present invention provides the use of a polypeptide in the preparation of cosmetics, foods, agricultural products, medicines, and tissue fillers, wherein the amino acid sequence of the polypeptide is CPD (SEQ ID NO: 2), CRVI (SEQ ID NO: 4), and / or CFFY (SEQ ID NO: 5).

[0029] The ninth aspect of the present invention provides a composition comprising a polypeptide and an auxiliary material; wherein the amino acid sequence of the polypeptide is CPD (SEQ ID NO: 2), CRVI (SEQ ID NO: 4) and / or CFFY (SEQ ID NO: 5).

[0030] Preferably, the auxiliary material is selected from one or more of excipients, diluents, humectants, emulsifiers, pH regulators, thickeners, disintegrants, lubricants, surfactants, suspending agents, gelling agents, preservatives, stabilizers, colorants or fragrances.

[0031] Preferably, the composition is a cosmetic, a food, an agricultural product, a medicine or a tissue filler.

[0032] The composition can treat, prevent or repair skin pigmentation problems, inhibit melanin production, whiten, remove spots, and have anti-oxidation effects.

[0033] The composition is used in preparing products for treating, preventing or repairing skin pigmentation problems, inhibiting melanin production, whitening, removing spots and anti-oxidation.

[0034] The composition may contain 0.01-99.5% by weight (e.g., 0.01%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.5%) of the polypeptide.

[0035] The composition can be administered by any suitable route, such as enteral (e.g. oral) or parenteral (e.g. intravenous, intramuscular, subcutaneous, intradermal, intraorgan, intranasal, intraocular, instillation, intracerebral, intrathecal, transdermal, intrarectal, etc.) routes.

[0036] The composition can be in any suitable dosage form, including but not limited to tablets, pills, powders, granules, capsules, lozenges, syrups, liquids, emulsions, microemulsions, suspensions, sprays, aerosols, powder sprays, lotions, ointments, plasters, pastes, patches, eye drops, nasal drops, sublingual tablets, suppositories, aerosols, effervescent tablets, pills, gels, injectable preparations, and the like.

[0037] The tenth aspect of the present invention provides a method for preparing a composition, which comprises mixing the above-mentioned polypeptide with auxiliary materials.

[0038] In the eleventh aspect of the present invention, a method for reducing the melanin content in the skin or inhibiting melanin production is provided, the method comprising administering a polypeptide, the above-mentioned tyrosinase inhibitor, melanin inhibitor, antioxidant, whitening polypeptide or the above-mentioned composition, wherein the amino acid sequence of the polypeptide is CPD (SEQ ID NO: 2), CRVI (SEQ ID NO: 4) and / or CFFY (SEQ ID NO: 5).

[0039] In the twelfth aspect of the present invention, a method for whitening skin or lightening spots is provided, the method comprising administering a polypeptide, the above-mentioned tyrosinase inhibitor, melanin inhibitor, antioxidant, whitening polypeptide or the above-mentioned composition, wherein the amino acid sequence of the polypeptide is CPD (SEQ ID NO: 2), CRVI (SEQ ID NO: 4) and / or CFFY (SEQ ID NO: 5).

[0040] In the thirteenth aspect of the present invention, a method for treating or preventing pigmentation is provided, which comprises administering a polypeptide, the above-mentioned tyrosinase inhibitor, melanin inhibitor, antioxidant, whitening polypeptide or the above-mentioned composition, wherein the amino acid sequence of the polypeptide is CPD (SEQ ID NO: 2), CRVI (SEQ ID NO: 4) and / or CFFY (SEQ ID NO: 5).

[0041] In the fourteenth aspect of the present invention, a method for skin antioxidant is provided, which comprises administering a polypeptide, the above-mentioned antioxidant or the above-mentioned composition, wherein the amino acid sequence of the polypeptide is CPD (SEQ ID NO: 2), CRVI (SEQ ID NO: 4) and / or CFFY (SEQ ID NO: 5).

[0042] The administration methods include but are not limited to oral administration, injection, coating, topical application, etc.

[0043] One or more amino acids in the amino acid sequence of the polypeptide of the present invention are modified by acetylation, amidation, formylation, hydroxylation, fatty acid chain modification, methylation or phosphorylation.

[0044] Preferably, the fatty acid chain is a fatty acid with a carbon chain length of 10-30, such as 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30.

[0045] Preferably, the fatty acid chain is a saturated fatty acid or an unsaturated fatty acid.

[0046] In one embodiment of the present invention, the fatty acid chain is C 25 H 42 N2O7(Pal-Glu(OSu)-OH).

[0047] Molecular weight and isoelectric point of the polypeptide of the present invention:

[0048] The molecular weight of CPD (SEQ ID NO: 2, Cys-Pro-Asp) is 333 Da and the isoelectric point is 3.5.

[0049] CRVI (SEQ ID NO: 4, Cys-Arg-Val-Ile) has a molecular weight of 489 Da and an isoelectric point of 8.2.

[0050] CFFY (SEQ ID NO: 5, Cys-Phe-Phe-Tyr) has a molecular weight of 578 Da and an isoelectric point of 5.6.

[0051] The cosmetics described herein may include lotions, cosmetic milks, facial cleansers, makeup removers, makeup remover oils, makeup remover milks, facial masks, facial creams, essence waters, essence milks, essence oils, essence creams, eye creams, foundations, bath lotions, hair conditioners, hair waters, body perfumes, and the like. Cosmetics refer to industrial chemicals or fine chemical products that are applied to any part of the human body, such as the skin, hair, nails, lips, and teeth, by smearing, spraying, or other similar methods for the purpose of cleansing, maintaining, beautifying, modifying, or altering the appearance of the body, or correcting body odor, to maintain a healthy appearance.

[0052] The medicines described in the present invention include but are not limited to: freckle-removing medicines, pigmentation-treating medicines or light-blocking agents (for internal or external use).

[0053] The tissue filler of the present invention can be used for repairing and filling soft tissues of the face, ears, chest, hands, joints, etc.

[0054] The food of the present invention includes but is not limited to: health care products, oral liquids, and is used for improving pigmentation, resisting oxidative free radicals, whitening, etc.

[0055] Beneficial effects of the present invention:

[0056] 1. The polypeptide provided by the present invention has extremely strong antioxidant activity, with an oxygen free radical scavenging rate of between 70% and 90%; it has an extremely strong tyrosinase activity inhibitory effect, with a half-inhibitory concentration (IC50) between 7.35 and 10.2 μmol / L, which is much lower than the half-inhibitory concentration of 902 μmol / L of the commonly used tyrosinase inhibitor arbutin; at the same time, the polypeptide has an extremely strong ability to inhibit the production of melanin in B16 cells. After co-culture with B16 cells, the relative content of melanin is between 52% and 67%; this is better than or equivalent to arbutin, indicating that the polypeptide of the present invention has an excellent ability to inhibit melanin production, can block the formation of melanin in cells from the source, and can effectively reduce melanin and achieve whitening or freckle removal effects.

[0057] 2. The polypeptide provided by the present invention has low cytotoxicity. Under the conditions of 0.5-2 mg / mL, it does not inhibit the growth of L929 cells and is non-cytotoxic. Even at a high concentration dose (2 mg / mL), the growth of L929 cells is still not inhibited. The polypeptide is highly safe and stable and has no cytotoxicity, which can solve the problem that high concentrations of arbutin are prone to produce adverse reactions.

[0058] 3. The polypeptides provided by the present invention have a small molecular weight and excellent transdermal performance. After modification with fatty acid side chains, the transdermal effects of the polypeptides CPD (SEQ ID NO: 2), CRVI (SEQ ID NO: 4), or CFFY (SEQ ID NO: 5) are increased by 3.0, 4.9, and 3.2 times, respectively, making them particularly suitable for use in whitening and freckle-removing cosmetics.

[0059] 4. After screening a large number of amino acid sequence fragments, the present invention can efficiently bind to the tyrosinase target, showing excellent tyrosinase activity inhibition effect. At the same time, it has a strong ability to inhibit melanin production, and can also take into account free radical scavenging and antioxidant effects. The preparation process is simple and the cost is low. It can be widely used in the fields of whitening and freckle removal cosmetics, agricultural products, foods, tissue fillers, pharmaceuticals, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings, in which:

[0061] Figure 1: Peptide cytotoxicity results (2 mg / mL).

[0062] Figure 2: Cytotoxicity results of different concentrations of PEP-2, PEP-4, PEP-5 and arbutin.

[0063] Figure 3: The half-maximal effective concentration of PEP-2 for inhibiting melanin production in B16 cells.

[0064] Figure 4: The half-maximal effective concentration of PEP-4 for inhibiting melanin production in B16 cells.

[0065] Figure 5: The half-maximal effective concentration of PEP-5 for inhibiting melanin production in B16 cells.

[0066] Figure 6: Chromatogram of fatty acid side chain modification of PEP-2, where the left figure is the detection diagram of fatty acid side chain modification PEP2C, and the right figure is the detection diagram of PEP2 (left peak) and modified PEP2C (right peak).

[0067] Figure 7: Chromatogram of PEP-4 fatty acid side chain modification (PEP4C).

[0068] Figure 8: Chromatogram of PEP-5 fatty acid side chain modification (PEP5C).

[0069] Figure 9: Comparison of transdermal effects of PEP-2 before and after fatty acid side chain modification.

[0070] Figure 10: Comparison of transdermal effects of PEP-4 before and after fatty acid side chain modification.

[0071] Figure 11: Comparison of transdermal effects of PEP-5 before and after fatty acid side chain modification. DETAILED DESCRIPTION

[0072] Below in conjunction with the accompanying drawings in the embodiment of the present invention, the technical scheme in the embodiment of the present invention is clearly and completely described. Obviously, the embodiment described is only a partial embodiment of the present invention, rather than all. Based on the embodiment in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work premise belong to the scope of protection of the present invention. The reagents adopted in the present invention are all commercially available products.

[0073] Example 1: Chemical synthesis of polypeptides

[0074] The sequences of the polypeptides designed by the present invention and the control polypeptides are shown in Table 1.

[0075] Table 1: Peptide sequence (PEP sequence)

[0076] The steps for peptide synthesis are as follows:

[0077] 1) Weigh 2CL (2-chloroacetyl-L-lysine resin) resin into a reactor, soak it with dichloromethane, then wash it with nitrogen and nitrogen-dimethylformamide (DMF) and drain it dry.

[0078] 2) Weigh appropriate amounts of Fmoc-X1-OH, N,N-diisopropylethylamine (DIEA), and DMF into a reactor for reaction, where X1 is the first amino acid of the above-mentioned polypeptide.

[0079] 3) Add a decapping solution (20% by volume piperidine in DMF solution) to the reactor to remove the Fmoc protecting group on the resin.

[0080] 4) Weigh appropriate amounts of Fmoc-X2-OH, hydroxybenzotriazole (HOBT), diisopropylcarbodiimide (DIC), and DMF into a reactor for reaction.

[0081] Wherein X2 is the second amino acid of the above polypeptide.

[0082] 5) Add decapping solution (20% by volume piperidine in DMF solution) to the reactor to remove the Fmoc protecting group on the resin.

[0083] 6) If the peptide has only two amino acids, proceed to the next step. If it has more than two amino acids, repeat steps 4) and 5) until the last amino acid is added.

[0084] 7) The polypeptide was cleaved from the resin using a cleavage solution (1% by volume of trifluoroacetic acid (TFA) in DCM) to obtain a crude polypeptide.

[0085] 8) Dissolve the crude polypeptide in pure water, and then desalt and purify it by HPLC reverse phase column chromatography or filter and purify the crude polypeptide solution using a filter membrane.

[0086] 9) The purified polypeptide finished product qualified liquid is freeze-dried using a vacuum freeze dryer to obtain a finished powder.

[0087] Example 2: Fatty acid side chain modification of polypeptides

[0088] 1) Side chain modification

[0089] According to the backbone polypeptide (PEP series) and fatty acid activated product (Pal-Glu (OSu) -OH, C 25 H 42 N2O7) in a molar ratio of 1:1.5, the reaction temperature was 25°C, the reaction pH was 4.5, the reaction time was 2h, and the reaction was terminated at pH 11.5.

[0090] 2) Purify by desalting with HPLC reverse phase column chromatography or filter and purify the crude polypeptide solution using a filter membrane.

[0091] 3) The purified polypeptide product liquid is freeze-dried using a vacuum freeze dryer to obtain a finished product powder.

[0092] Test Example 1: Cytotoxicity Test

[0093] The experiment was conducted in accordance with GB / T 16886.5-2017 Biological evaluation of medical devices Part 5: In vitro cytotoxicity test method.

[0094] 1) Experimental Materials

[0095] L929 cells (from ATCC), MEM medium, PBS, Hank's balanced salt solution (HBSS), penicillin-streptomycin and fetal bovine serum (FBS), 0.25% trypsin (containing EDTA), DMSO, and MTT detection kit.

[0096] The experimental groups were selected from arbutin and PEP series peptides (PEP-1 to PEP-15), each of which was added at 2 mg / mL, and a blank control was used.

[0097] 2) Experimental steps

[0098] The cultured cells were removed from the culture flask using enzymatic digestion (trypsin / EDTA) and the cell suspension was centrifuged at 200 g for 3 min. The cell suspension was resuspended in culture medium to adjust the density to 1×10 5 Use a multichannel pipette to add 100 μL of culture medium to the outer wells of a 96-well tissue culture microtiter plate, and add 100 μL of a 1×10 5 cells / mL of cell suspension (=1×10 4 The cells were incubated for 24 h (5% CO 2 , 37° C., >90% humidity).

[0099] After 24 hours of incubation, aspirate the culture medium. Add 100 μL of treatment medium containing the appropriate concentration of sample extract, negative control, positive control, or solvent (blank) to each well. Test groups were treated with 100% (2 mg / mL), 75% (1.5 mg / mL), 50% (1 mg / mL), and 25% (0.5 mg / mL) sample extract. Negative controls used culture medium as a blank. Incubate cells for 24 hours (5% CO2, 37°C, >90% humidity).

[0100] After 24 hours of treatment, examine each plate under a phase-contrast microscope to determine systematic errors in cell seeding and the growth characteristics of control and experimental cells. After inspection, carefully remove the culture medium and add 50 μL of MTT solution to each test well. Incubate the plates in a 37°C incubator for an additional 2 hours. Then, discard the MTT solution and add 100 μL of isopropanol solution to each well. Shake the plates and measure absorbance at 570 nm (reference wavelength 650 nm) using a microplate reader.

[0101] 3) Data Analysis

[0102] Where:

[0103] OD 570e : Average optical density of 100% extract of test sample;

[0104] OD570b : average optical density of blank;

[0105] If the cell viability drops to <70% of the blank, it is potentially cytotoxic.

[0106] 4) Test results are shown in Table 2 and Figure 1.

[0107] Table 2: Cytotoxicity data of PEP series peptides at 2 mg / mL

[0108] As can be seen from Table 2 and Figure 1, when the concentration of the PEP series polypeptides of the present invention is 2 mg / mL, the cell survival rate is high (greater than 70%), no obvious cytotoxicity is shown, and the safety to the human body is high.

[0109] The cell viability of polypeptides PEP-2, PEP-4, and PEP-5 at different concentrations (0.5 mg / mL to 2 mg / mL) was further tested, as shown in Table 3 and Figure 2.

[0110] Table 3: Cytotoxicity data of PEP series peptides

[0111] As can be seen from Table 3 and Figure 2, within the concentration range of 0.5-2 mg / mL, peptides PEP-2, PEP-4, and PEP-5 did not inhibit the growth of L929 cells, indicating that the above peptides were non-cytotoxic. However, arbutin in the control group significantly inhibited the growth of L929 cells at a low concentration of 0.5 mg / mL, with a cell survival rate of only 43%, indicating a certain degree of cytotoxicity. When the concentration was increased to 2 mg / mL, the cell survival rate was less than 10%, indicating strong cytotoxicity. This also verifies that arbutin can cause adverse reactions such as irritation and skin whitening at high concentrations, limiting its application in whitening products such as cosmetics and foods.

[0112] Test Example 2: Tyrosinase activity inhibition test

[0113] Since the processes of cellular melanin production and conversion are relatively complex, inhibiting tyrosinase activity in vitro can be used as a preliminary test method for peptide screening.

[0114] 1) Test method

[0115] Set up solvent background wells (T a ), solvent reaction hole (T b ), sample background hole (T c ), sample reaction well (T d ). Among them, the Ta group is the solvent background group, without adding substrate L-tyrosine solution and sample solution; Tb Group 1 is the solvent reaction group, in which substrate L-tyrosine solution is added but no sample solution is added; T c Group 1 is the sample background group, without adding substrate L-tyrosine solution but with adding sample solution; T d The first group is the sample reaction group, which adds both the substrate L-tyrosine solution and the sample solution. Each group is set up with three replicates; a blank group and a control group are also set up.

[0116] The sample is PEP-1 to PEP-11 polypeptides, and the solvent is a PBS solution with a pH of about 7.

[0117] The sample solution is a polypeptide solution formed by dissolving the polypeptide in a solvent.

[0118] According to the settings of each group, a certain volume of L-tyrosine solution, sample solution / solvent, and PBS buffer were added to each well of the corresponding group in turn, mixed thoroughly, and incubated at a constant temperature of 37°C for 10 minutes. Tyrosinase solution was then added to each well in turn. After mixing and reacting at 37°C for 5 minutes ± 5 seconds, the wells were immediately placed in a microplate reader for measurement at a wavelength of 475 nm.

[0119] 2) Calculation of tyrosinase activity inhibition rate:

[0120] Where: Y—tyrosinase activity inhibition rate; A d —Absorbance of sample reaction well; A c —Absorbance of sample background well; A b —Average absorbance of solvent reaction wells; A a —Average absorbance of solvent background wells.

[0121] 3) Half maximal inhibitory concentration (IC 50 value)

[0122] The concentration of the tested polypeptide sample was used as the horizontal axis and the corresponding tyrosinase inhibition rate was used as the vertical axis to draw a curve and obtain the regression equation (R 2 ≥0.9). According to the regression equation, the sample concentration corresponding to 50% tyrosinase inhibition rate was calculated, which is the IC 50 value.

[0123] 4) The test results are shown in Table 4.

[0124] Table 4: Inhibition of tyrosinase activity

[0125] The results showed that the half-inhibitory concentration of PEP polypeptide in vitro tyrosinase IC 50 Both are lower than the commonly used in vitro tyrosinase inhibitor kojic acid IC 50 concentration (60 μmol / L) and arbutin IC50 The concentration (902 μmol / L) indicates that all PEP polypeptides have tyrosinase inhibitory ability. Furthermore, the amino acid sequence of the polypeptide can be screened in combination with the results of intracellular melanin.

[0126] Test Example 3: B16 cell melanin production inhibition test

[0127] 1) Experimental Materials

[0128] Mouse melanoma cells (B16), DMEM (high glucose) medium, PBS, Hank's balanced salt solution (HBSS), penicillin-streptomycin and fetal bovine serum (FBS), 0.25% trypsin (containing EDTA), dimethyl sulfoxide (DMSO), and thiazolyl blue (MTT) detection kit.

[0129] The PEP series polypeptides were all prepared by the method of Example 1.

[0130] 2) Experimental steps

[0131] Well-grown B16 cells were passaged onto 6 mm dishes and plated at a density of 250,000 cells / well in a 4 mL volume per well. After plating, the cells were incubated in a CO2 incubator for 24 hours, after which the culture medium in the cell culture wells was discarded. In the experimental group, the peptide samples were prepared using DMEM complete medium and cultured at 37°C in 5% CO2. The blank group was cultured using DMEM complete medium at 37°C in 5% CO2.

[0132] 4 mL of experimental group (concentration of 0.5 mg / mL), blank group culture medium and commonly used tyrosinase inhibitor arbutin as control group (concentration of 0.5 mg / mL) were added respectively, and the 6-well plate was placed in a 37 ° C incubator for continued cultivation. The culture was terminated at the 48th hour of cultivation, the supernatant was collected, and the cells were centrifuged at 4000 r / min for 10 min. 500 μL of trypsin was added to the cells. After standing for 2 minutes, the cells were collected with 1 mL of PBS, centrifuged at 4000 r / min for 10 minutes, and 500 μL of NaOH solution (1 mol / L) containing 10% DMSO was added to blow the precipitate fully. After that, it was placed in an 80 ° C water bath for 1 hour to completely dissolve the melanin particles. 150 μL was aspirated into a 96-well plate, and the OD was detected by microplate reader. 390 The absorbance value at .

[0133] 3) Calculation of relative melanin content:

[0134] Relative content of melanin = OD of experimental group 390 Value / Blank Group OD 390 value × 100%

[0135] 4) Experimental results

[0136] ①The results of melanin detection experiment are shown in Table 5.

[0137] Table 5: Melanin detection test results

[0138] By detecting the relative content of melanin, the ability and effect of inhibiting melanin production in cells can be more directly characterized. The results show that the relative content of melanin of PEP-2, PEP-4 and PEP-5 is 52%, 59% and 67% respectively. The melanin inhibition ability is the strongest among the polypeptides, while the melanin content of the commonly used arbutin is 65%; that is, at the same concentration, the melanin content of polypeptides PEP-2 and PEP-4 is significantly lower than that of arbutin, which also shows that the polypeptides PEP-2 and PEP-4 of the present invention have excellent ability to inhibit melanin production, can block the formation of melanin in cells from the source, and have significant effects of reducing melanin and whitening and removing spots. The effect of polypeptide PEP-5 is basically equivalent to that of arbutin, but based on arbutin, it will produce cytotoxicity at a concentration of 0.5 mg / mL (as shown in Test Example 1), while the polypeptides of the present invention are non-cytotoxic, and even at high concentrations (2 mg / mL), they still do not produce obvious cytotoxicity. Therefore, the polypeptide PEP-5 has the advantages of safety, stability, and good biocompatibility, and can replace arbutin and be used in whitening food, medicine or cosmetics.

[0139] At the same time, although the relative melanin content of polypeptides PEP-1, PEP-3, PEP-7 and PEP-9 is also lower than that of the blank control group, their ability to inhibit melanin production is slightly lower than that of arbutin; while the melanin content of polypeptides PEP-6, PEP-8, PEP-10, PEP-12 to PEP-15 samples is significantly higher than that of the blank control group, and therefore they do not have the ability to inhibit melanin production.

[0140] In addition, the results of PEP-6, PEP-8, and PEP-11 show that not all peptides containing cysteine ​​have a melanin-inhibiting effect. Some peptides even have a tendency to enhance the formation of melanin. Although PEP-6 and PEP-8 only insert one amino acid on the basis of PEP-1, they completely lose the ability to inhibit melanin. PEP-2 has one more amino acid than PEP-7, and PEP-8 and PEP-9 only differ in the order of amino acids, but the melanin-inhibiting ability of the peptides is also significantly different. This also shows that the composition of the peptide amino acid sequence has a certain influence on the final melanin-inhibiting ability.

[0141] ② Determination of the median effective concentration of PEP-2, PEP-4, and PEP-5

[0142] The results are shown in Figures 3-5. By calculation, the half-inhibitory concentrations of PEP-2, PEP-4, and PEP-5 were 2.4 mmol / L, 1.9 mmol / L, and 1.7 mmol / L, respectively.

[0143] Test Example 4: Antioxidant Performance Determination

[0144] 1) Experimental Materials

[0145] PBS, antioxidant Trolox, total antioxidant capacity detection kit (ABTS method) - (Biyuntian, S0119). PEP series polypeptides were prepared according to the method of Example 1.

[0146] 2) Experimental steps

[0147] ① Preparation of samples to be tested:

[0148] The polypeptide sample to be tested was prepared using PBS so that the concentration of the sample to be tested was 1 mmol / L.

[0149] ② Preparation for standard curve determination:

[0150] Select PBS to dilute the standard and dilute the 10mM Trolox standard solution to 0.15, 0.3, 0.6, 0.9, 1.2 and 1.5mM.

[0151] ③Determination of total antioxidant capacity:

[0152] a. Add 200 μl of ABTS working solution to each well of a 96-well plate.

[0153] b. Add 10 μl of PBS solution to the blank control wells; add 10 μl of Trolox standard solution of various concentrations to the standard curve detection wells; add 10 μl of various samples to the sample detection wells and mix gently.

[0154] c. After incubation at room temperature for 5 minutes, measure A734 (absorbance at 734 nm).

[0155] d. Calculate the total antioxidant capacity of the sample based on the standard curve. If the sample's absorbance is outside the standard curve range, dilute or concentrate the sample appropriately before measuring.

[0156] 3) Free radical scavenging ability (Trolox relative value, %)

[0157] Free radical scavenging ability = sample measured value / 1mmol / L Trolox measured value × 100%

[0158] Table 6: Antioxidant performance determination

[0159] The results are shown in Table 6. The free radical scavenging ability of PEP-2, PEP-4, PEP-5, PEP-7, and PEP-9 is about 85% or more. In particular, PEP-2, PEP-4, and PEP-5 have extremely strong antioxidant properties and also have a strong ability to inhibit melanin production (Test Example 3).

[0160] Test Example 5: Transdermal Test

[0161] 1) Experimental steps

[0162] ① Sample preparation

[0163] a. Preparation of the oil phase: Weigh 2 parts of glyceryl monostearate, 12 parts of stearic acid, and 6 parts of liquid paraffin, stir and heat until completely dissolved;

[0164] b. Preparation of aqueous phase: Weigh 1 part of triethanolamine and 76 parts of purified water, stir and heat until completely dissolved;

[0165] c. Use a homogenizer to homogenize and slowly add the water phase to the oil phase to form a creamy sample after cooling;

[0166] d. PEP-2, PEP-4, PEP-5 (prepared in Example 1) and the corresponding fatty acid side chain modified proteins PEP-2C, PEP-4C, PEP-5C (prepared in Example 2) were taken separately and added to the above creamy sample to prepare a cream containing 100 mg / mL sample.

[0167] ② The chromatogram of the fatty acid-modified peptide product PEP-2C is shown in Figure 6. From the chromatograms before and after modification in the left and right panels of Figure 6, it can be seen that the fatty acid chains have been successfully modified onto the peptide. The chromatograms of the fatty acid-modified peptide products PEP-4C and PEP-5C are shown in Figures 7-8.

[0168] ③ Press 50mg / cm 2 Dosage (peptide addition amount is about 5mg / cm 2 ), the samples were added to pig skin, and the transdermal effect was detected using a transdermal diffusion test system.

[0169] 2) Experimental results

[0170] The results are shown in Figures 9-11. It can be seen that after about 15 hours, the concentrations of the polypeptide samples before and after modification reached a peak, and then tended to stabilize. After 24 hours, the concentrations of the fatty acid chain modified polypeptides PEP-2C, PEP-4C, and PEP-5C were 0.464, 0.624, and 0.505 μg / mL, respectively, while the concentrations of PEP-2, PEP-4, and PEP-5 without modified fatty acid chains were all lower than 0.2 μg / mL. The transdermal effects of the fatty chain modified polypeptides PEP-2C, PEP-4C, and PEP-5C (Example 2) were 3.0, 4.9, and 3.2 times that of the unmodified fatty acid chain PEP-2, PEP-4, and PEP-5 (Example 1), respectively. This shows that the polypeptide has a good transdermal effect and is particularly suitable for use in the field of whitening cosmetics.

[0171] Test Example 6: Effect of (PEP-2) Peptide Sequence Order

[0172] Based on the above test examples, the peptides PEP-2, PEP-4, and PEP-5 were screened for their ability to stably and effectively inhibit tyrosinase, inhibit melanin production, and scavenge free radicals. Further, the effects of variations in the amino acid sequence within the peptides on their performance were investigated. The peptide sample preparation method was the same as in Example 1; the methods for evaluating tyrosinase inhibition, melanin production, and free radical scavenging effects are described in Test Examples 2-4. The performance test results for the different peptide samples are shown in Table 7.

[0173] Table 7: Performance test results of different sequences of amino acids constituting PEP-2

[0174] As can be seen from Table 7, although the types of polypeptide amino acids are the same, changing the order of amino acids will cause certain changes in the technical effects of the polypeptide. For example, adjusting the amino acid sequence to CDP will slightly improve the in vitro tyrosine inhibition effect and free radical scavenging ability, but it no longer has the effect of inhibiting B16 cell melanin production, and even promotes melanin production, making it unable to effectively whiten and remove spots on the skin.

[0175] Adjusting the amino acid sequence of the peptides PCD and DCP significantly reduced the in vitro tyrosine inhibition effect and completely lost the inhibitory effect on B16 cell melanin production. This indicates that the order of the peptide amino acid sequence has a significant impact on peptide screening and peptide performance (among which, when the amino acid sequence is adjusted to PCD, the melanin content increases by more than 2 times, while the antioxidant capacity decreases slightly; when the amino acid sequence is adjusted to DCP, its free radical scavenging ability decreases significantly, to only 56.7% of PEP-2).

[0176] Test Example 7: Effect of (PEP-4) Peptide Sequence Order

[0177] Similarly, the influence of the amino acid sequence order of the PEP-4 and PEP-5 polypeptides was investigated, and Test Examples 7 and 8 were conducted. The peptide sample preparation method was the same as in Example 1; the methods for evaluating tyrosinase inhibition, melanin production, and free radical scavenging effects are described in Test Examples 2-4. The performance test results of the different peptide samples are shown in Table 8.

[0178] Table 8: Performance test results of different sequences of amino acids constituting PEP-4

[0179] As shown in Table 8, adjusting the order of the peptide amino acid sequence can significantly affect its tyrosine inhibition IC 50 The inhibitory effect on B16 cell melanin production was reduced with the increase of the concentration, and the free radical scavenging ability was also reduced to varying degrees. This also shows that the amino acid sequence and order have a certain influence on the effect of the peptide.

[0180] Test Example 8: Effect of (PEP-5) Peptide Sequence Order

[0181] The peptide preparation and testing methods were as described above. The amino acid sequence of the peptide PEP-5 was adjusted. The performance test results of different peptide samples are shown in Table 9.

[0182] Table 9: Performance test results of different amino acid sequences constituting PEP-5

[0183] As can be seen from Table 9, adjusting the order of amino acids in polypeptide PEP-5 has a significant effect on the inhibitory effect of polypeptide samples on B16 cell melanin production: the in vitro tyrosine inhibition ability of polypeptide 5-1 sample (CFYF) has been improved, but the antioxidant capacity has been reduced, and melanin production has been promoted instead; the in vitro tyrosine inhibition ability of polypeptide 5-2 (CYFF) has been reduced, and the melanin content has been greatly increased, indicating that the polypeptide has almost no ability to inhibit melanin production, and the free radical scavenging ability has also been significantly reduced; the free radical scavenging ability of polypeptide 5-3 (FFYC) has been improved, but it no longer has the effect of inhibiting B16 cell melanin production.

[0184] The peptide PEP-5 also has strong tyrosinase inhibition, melanin production inhibition, and free radical scavenging capabilities, taking into account both whitening and antioxidant effects. This also shows that the order of the peptide amino acid sequence has a significant impact on the performance of the peptide.

[0185] In summary, changes in the amino acid sequence can affect the performance of the polypeptide. The polypeptides PEP-2, PEP-4, and PEP-5 of the present invention can simultaneously meet the performance requirements of whitening and antioxidant properties, have excellent melanin inhibition and free radical scavenging capabilities, and can meet the use requirements of whitening and antioxidant products.

[0186] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection of the present invention.

[0187] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any appropriate manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A tyrosinase inhibitor, characterized in that The tyrosinase inhibitor comprises a polypeptide, and the amino acid sequence of the polypeptide is CPD (SEQ ID NO: 2), CRVI (SEQ ID NO: 4) and / or CFFY (SEQ ID NO: 5).

2. A melanin inhibitor, characterized in that The melanin inhibitor comprises a polypeptide, and the amino acid sequence of the polypeptide is CPD (SEQ ID NO: 2), CRVI (SEQ ID NO: 4) and / or CFFY (SEQ ID NO: 5).

3. An antioxidant, characterized in that The antioxidant comprises a polypeptide, and the amino acid sequence of the polypeptide is CPD (SEQ ID NO: 2), CRVI (SEQ ID NO: 4) and / or CFFY (SEQ ID NO: 5).

4. The tyrosinase inhibitor according to claim 1, the melanin inhibitor according to claim 2, or the antioxidant according to claim 3, characterized in that: One or more amino acids in the amino acid sequence of the polypeptide are modified by acetylation, amidation, formylation, hydroxylation, fatty acid chain modification, methylation or phosphorylation; preferably, fatty acid chain modification is adopted, and the fatty acid chain is a saturated or unsaturated fatty acid with a carbon chain length of 10-30.

5. The tyrosinase inhibitor according to claim 1, the melanin inhibitor according to claim 2, or the antioxidant according to claim 3, characterized in that: The polypeptide is obtained by solid phase synthesis or recombinant expression and purification.

6. The use of a polypeptide for inhibiting tyrosinase, inhibiting melanin, anti-oxidation, whitening or removing spots, characterized in that: The amino acid sequence of the polypeptide is CPD (SEQ ID NO: 2), CRVI (SEQ ID NO: 4) and / or CFFY (SEQ ID NO: 5); Preferably, one or more amino acids in the amino acid sequence of the polypeptide are modified by acetylation, amidation, formylation, hydroxylation, fatty acid chain modification, methylation or phosphorylation.

7. A composition for inhibiting melanin production, whitening, removing spots or anti-oxidation, characterized in that: The composition comprises a polypeptide and excipients; wherein the amino acid sequence of the polypeptide is CPD (SEQ ID NO: 2), CRVI (SEQ ID NO: 4) and / or CFFY (SEQ ID NO: 5).

8. The composition according to claim 7, characterized in that The auxiliary materials are selected from one or more of excipients, diluents, humectants, emulsifiers, pH regulators, thickeners, disintegrants, lubricants, surfactants, suspending agents, gelling agents, preservatives, stabilizers, colorants or fragrances.

9. The composition according to claim 7, characterized in that The composition is a cosmetic, food, agricultural product, medicine or tissue filler.

10. A method for reducing the melanin content in the skin or inhibiting melanin production, characterized in that: The method comprises administering a polypeptide, the tyrosinase inhibitor according to claim 1, the melanin inhibitor according to claim 2, the antioxidant according to claim 3, or the composition according to any one of claims 7 to 9, wherein the amino acid sequence of the polypeptide is CPD (SEQ ID NO: 2), CRVI (SEQ ID NO: 4), and / or CFFY (SEQ ID NO: 5).

11. A method for whitening skin or lightening spots, characterized in that: The method comprises administering a polypeptide, the tyrosinase inhibitor according to claim 1, the melanin inhibitor according to claim 2, the antioxidant according to claim 3, or the composition according to any one of claims 7 to 9, wherein the amino acid sequence of the polypeptide is CPD (SEQ ID NO: 2), CRVI (SEQ ID NO: 4), and / or CFFY (SEQ ID NO: 5).

12. A method for skin anti-oxidation, characterized in that: The method comprises administering a polypeptide, the antioxidant of claim 3, or the composition of any one of claims 7-9, wherein the amino acid sequence of the polypeptide is CPD (SEQ ID NO: 2), CRVI (SEQ ID NO: 4), and / or CFFY (SEQ ID NO: 5).

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