Amidated polypeptides and their applications

Abstract

The present invention provides amidated polypeptides and their applications. The amidated polypeptides of the present invention are obtained by amidation modification of the C-terminus of the polypeptides; wherein, the amino acid sequence of the polypeptides is as shown in SEQ ID NO:1. The amidated polypeptides of the present invention have excellent antioxidant activity, tyrosinase inhibitory activity, and melanin inhibitory activity, and are significantly superior to the products obtained by modification using other methods (such as acetylation, palmitoylation, etc.). They are not only suitable for the preparation of antioxidant products, tyrosinase inhibitors, and melanin inhibitors, but also suitable for the preparation of products with whitening effects, such as cosmetics or drugs with whitening effects.

Description

Technical Field

[0001] The present invention belongs to the technical field of polypeptide modification. More specifically, it relates to amidated polypeptides and their applications. Background Art

[0002] Tyrosinase, also known as polyphenol oxidase, catechol oxidase, casease, etc., is composed of multiple subunits. Each subunit contains two metal copper ions, and these two copper ions are covalently bound to the imino groups of three histidine residues and fixed at the active center. Additionally, there is an endogenous bridging group that links the two copper ions together to form the active center of tyrosinase. Tyrosinase plays a crucial role in the process of melanin synthesis. First, it hydroxylates monophenols to diphenols, then oxidizes ortho-diphenols to ortho-quinones, and finally ortho-quinones react to form melanin. Therefore, tyrosinase plays a key role in the process of melanin production.

[0003] Currently, whitening products on the market often reduce melanin production by inhibiting the activity of tyrosinase, thereby achieving the whitening effect. For example, the patent application with the publication number CN116731105A provides a polypeptide with an amino acid sequence as shown in SEQ ID NO: 1, which has the activity of inhibiting tyrosinase and can be used as a raw material for whitening products, but its effect is limited. Summary of the Invention

[0004] Aiming at the deficiencies of the prior art, the present invention aims to provide an amidated polypeptide. By amidating the polypeptide with an amino acid sequence as shown in SEQ ID NO: 1, the IC 50 value of its inhibition of tyrosinase is significantly reduced, thereby improving its whitening effect.

[0005] The first object of the present invention is to provide an amidated polypeptide.

[0006] The second object of the present invention is to provide the application of the above-mentioned amidated polypeptide in the preparation of whitening products.

[0007] The third object of the present invention is to provide the application of the above-mentioned amidated polypeptide in the preparation of antioxidant products.

[0008] The fourth object of the present invention is to provide the application of the above-mentioned amidated polypeptide in the preparation of tyrosinase inhibitors.

[0009] The fifth object of the present invention is to provide the application of the above-mentioned amidated polypeptide in the preparation of melanin inhibitors.

[0010] The sixth object of the present invention is to provide the related biological materials of the above-mentioned amidated polypeptide.

[0011] The seventh object of the present invention is to provide the application of the above-mentioned related biological materials in the preparation of whitening products.

[0012] The eighth object of the present invention is to provide the use of the above-related biological materials in the preparation of antioxidant products.

[0013] The ninth object of the present invention is to provide the use of the above-related biological materials in the preparation of tyrosinase inhibitors.

[0014] The tenth object of the present invention is to provide the use of the above-related biological materials in the preparation of melanin inhibitors.

[0015] The above objects of the present invention are achieved by the following technical solutions:

[0016] The present invention provides an amidated polypeptide obtained by amidation modification of the C-terminus of a polypeptide; wherein, the amino acid sequence of the polypeptide is as shown in SEQ ID NO:1. SEQ ID NO:1: AHYYD.

[0017] The functions of the above amidated polypeptide are as follows: (1) It has excellent antioxidant activity and is suitable for the preparation of antioxidant products; (2) It can effectively inhibit tyrosinase and is suitable for the preparation of tyrosinase inhibitors; (3) It can significantly inhibit the production of melanin and is suitable for the preparation of melanin inhibitors.

[0018] In summary, the above amidated polypeptide is also suitable for the preparation of whitening products. Therefore, the applications of the above amidated polypeptide in the preparation of whitening products, antioxidant products, tyrosinase inhibitors, and melanin inhibitors should all be within the protection scope of the present invention.

[0019] Based on this, the present invention also provides the above-related biological materials of the amidated polypeptide and their applications in the preparation of whitening products, antioxidant products, tyrosinase inhibitors, and melanin inhibitors. The above-related biological materials are nucleic acid molecules capable of expressing the amidated polypeptide, or recombinant DNAs, expression cassettes, transposons, vectors, or host cells containing the nucleic acid molecules.

[0020] In addition, the present invention also provides a product comprising the above amidated polypeptide and / or its related biological materials.

[0021] Preferably, the above-related biological materials are nucleic acid molecules capable of expressing the amidated polypeptide, or recombinant DNAs, expression cassettes, transposons, vectors, or host cells containing the nucleic acid molecules.

[0022] Preferably, the product is a cosmetic or a drug.

[0023] More preferably, it further contains excipients acceptable in cosmetics or drugs.

[0024] The present invention has the following beneficial effects:

[0025] The present invention performs amidation modification on a polypeptide with an amino acid sequence as shown in SEQ ID NO: 1. The obtained amidated polypeptide has excellent antioxidant activity, tyrosinase inhibitory activity, and melanin inhibitory activity, and is significantly superior to the products obtained by other modification methods (such as acetylation, palmitoylation, etc.). It is not only applicable to the preparation of antioxidant products, tyrosinase inhibitors, and melanin inhibitors, but also applicable to the preparation of products with whitening effects, such as whitening cosmetics or drugs. Description of the Drawings

[0026] Figure 1 It is a mass spectrometry identification result diagram of T1.

[0027] Figure 2 It is a mass spectrometry identification result diagram of T2.

[0028] Figure 3 It is a mass spectrometry identification result diagram of T3.

[0029] Figure 4 It is a mass spectrometry identification result diagram of T4.

[0030] Figure 5 It is a test result diagram of the antioxidant activity of T1 - T3.

[0031] Figure 6 It is a test result diagram of the safety of T1 - T2.

[0032] Figure 7 It is a test result diagram of the inhibitory activity of T1 - T2 on tyrosinase at the cellular level.

[0033] Figure 8 It is a test result diagram of the inhibitory activity of T1 - T2 on melanin. Detailed Embodiments

[0034] The following further illustrates the present invention in conjunction with the drawings of the specification and specific embodiments, but the embodiments do not impose any form of limitation on the present invention. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field.

[0035] Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0036] Example 1 Synthesis and Identification of T1 - T4

[0037] I. Synthesis of T1 - T4

[0038] In this example, Shanghai Ammonia Link Biotechnology Co., Ltd. was commissioned to assist in synthesizing a polypeptide T1 (AHYYD) with an amino acid sequence as shown in SEQ ID NO: 1, and three chemical modifications were performed on it, namely:

[0039] (1) It is amidated at its C-terminus to obtain amidated polypeptide T2 (AHYYD-NH2);

[0040] (2) It is acetylated at its N-terminus to obtain acetylated polypeptide T3 (Ac-AHYYD);

[0041] (3) It is palmitoylated at its N-terminus to obtain palmitoylated polypeptide T4 (Pal-AHYYD).

[0042] The structural formulas of T1 to T4 are specifically as follows:

[0043] T1: ;

[0044] T2: ;

[0045] T3: ;

[0046] T4: .

[0047] II. Purity Identification of T1 to T4

[0048] The purities of T1 to T4 are respectively identified by high performance liquid chromatography.

[0049] Among them, the identification conditions of high performance liquid chromatography are:

[0050] Chromatographic column: SHIMADZU Inertsil ODS-SP (4.6 * 250 mm * 5 μm);

[0051] Mobile phase A: acetonitrile (containing 0.1% ( v / v ) trifluoroacetic acid);

[0052] Mobile phase B: water (containing 0.1% ( v / v ) trifluoroacetic acid);

[0053] Elution gradient: 0 → 25 min, 99% ( v / v ) → 50% ( v / v ) A;

[0054] Flow rate: 1 mL / min;

[0055] Detection wavelength: 220 nm;

[0056] Sample loading volume: 35 μL.

[0057] The results of purity identification show that the purities of T1 to T4 are 98.51%, 98.63%, 98.63%, and 99.51% respectively.

[0058] III. Mass Spectrometry Identification of T1 to T4

[0059] The molecular weights of T1 to T4 were respectively identified by ESI-MS mass spectrometry.

[0060] Among them, the identification conditions of the ESI-MS mass spectrometry were as follows:

[0061] Mobile phase A: water (containing 1% ( v / v ) formic acid);

[0062] Mobile phase B: acetonitrile (containing 1% ( v / v ) formic acid);

[0063] Flow rate: 0.2 mL / min;

[0064] Running time: 4 min;

[0065] Running mode: positive ion mode;

[0066] Scanning range: 0 - 2000 Da.

[0067] The results of the mass spectrometry identification were as Figures 1 to 4 shown, among which, Figure 1 was the mass spectrometry identification result diagram of T1, Figure 2 was the mass spectrometry identification result diagram of T2, Figure 3 was the mass spectrometry identification result diagram of T3, Figure 4 was the mass spectrometry identification result diagram of T4. It can be seen that the molecular ion peaks m / z of T1 to T4 were 668.76, 667.35, 710.82, and 907.06 respectively, with one charge, which was consistent with the theoretical values of their molecular weights, indicating the successful synthesis of T1 to T4.

[0068] Example 2 Inhibitory Activity of T1 - T4 on Tyrosinase in Vitro

[0069] I. Solution Preparation

[0070] Sample solution: Dissolve T1 - T4 obtained in Example 1 in PBS buffer (pH = 6.8) to make their final concentrations 0.2, 0.4, 0.6, 0.8, and 1.0 mg / mL respectively.

[0071] L - tyrosine solution: Dissolve L - tyrosine in PBS buffer (pH = 6.8) to make its final concentration 0.5 mg / mL.

[0072] Tyrosinase solution: Dissolve tyrosinase in PBS buffer (pH = 6.8) to make its final concentration 500 U / mL.

[0073] II. Experimental Grouping and Treatment

[0074] Solvent background well (A1): Add 110 μL of PBS buffer (pH = 6.8) to a 96-well plate and mix well.

[0075] Solvent reaction well (A2): Add 40 μL of L-tyrosine solution and 70 μL of PBS buffer (pH = 6.8) to a 96-well plate and mix well.

[0076] Sample background well (A3): Add 40 μL of sample solution and 70 μL of PBS buffer (pH = 6.8) to a 96-well plate and mix well.

[0077] Sample reaction well (A4): Add 40 μL of L-tyrosine solution, 40 μL of sample solution and 30 μL of PBS buffer (pH = 6.8) to a 96-well plate and mix well.

[0078] After incubating each well in a constant temperature environment at 37 °C for 10 min, add 20 μL of tyrosinase solution respectively, then mix and react at 37 °C for 10 min. Measure the absorbance value at 475 nm, and calculate the tyrosinase inhibition rate according to the formula "Tyrosinase inhibition rate (%) = [1 - (A4 - A3) / (A2 - A1)] × 100%".

[0079] III. Experimental results

[0080] The results show that the IC 50 values of T1 - T3 inhibiting tyrosinase are 2.012 ± 0.088 mM, 0.979 ± 0.028 mM, 3.430 ± 0.017 mM respectively, while T4 shows no tyrosinase inhibitory activity. It can be seen that compared with unmodified T1, the tyrosinase inhibitory activity of amidated polypeptide T2 is significantly improved, the tyrosinase inhibitory activity of acetylated polypeptide T3 is significantly reduced, and even palmitoylated polypeptide T4 completely loses its tyrosinase inhibitory activity. It shows that amidation modification of T1 in the present invention can significantly improve its tyrosinase inhibitory activity, while conventional modification methods (such as acetylation modification, palmitoylation modification, etc.) cannot improve its tyrosinase inhibitory activity, and even inhibit its activity, indicating that it is precisely the specific selection of amidation modification method for T1 in the present invention that has significantly improved its tyrosinase inhibitory activity.

[0081] Antioxidant activity of T1 - T3 in Example 3

[0082] According to the instruction manual of the DPPH kit of Grees Biotechnology Co., Ltd., measure the DPPH radical scavenging ability of T1 - T3 obtained in Example 1, and the results are as Figure 5 shown.

[0083] It can be seen that, compared with unmodified T1, the DPPH free radical scavenging rate of amidated polypeptide T2 is significantly increased, while there is no significant difference in the DPPH free radical scavenging rate of acetylated polypeptide T3. It indicates that amidation modification of T1 in the present invention can significantly improve its antioxidant activity, while conventional modification methods (such as acetylation modification, etc.) cannot improve its antioxidant activity. It shows that it is precisely because the present invention specifically selects the method of amidation modification for T1 that its antioxidant activity can be significantly improved.

[0084] Example 4 Safety of T1 - T2

[0085] I. Solution preparation

[0086] Sample solution: In a laminar flow hood, dissolve the T1 - T2 obtained in Example 1 in DMEM / F - 12 medium containing 10% ( v / v ), fetal bovine serum and 1% ( v / v ), penicillin - streptomycin double antibody so that their final concentrations are 0, 15, 37.5, 75, 150, 300, 600 μM respectively, and then filter and sterilize with a 0.22 μm filter membrane to obtain.

[0087] II. B16F10 cell culture

[0088] Inoculate B16F10 cells in DMEM / F - 12 medium containing 10% ( v / v ), fetal bovine serum and 1% ( v / v ), penicillin - streptomycin double antibody, and culture in an incubator at 37 °C, saturated humidity, and containing 5% CO2 until the cells basically cover the bottom of the culture flask. Then remove the waste liquid in the culture flask, wash with PBS, digest with 1 mL of 0.25% trypsin for 1 min, add 2 mL of DMEM / F - 12 medium containing 10% ( v / v ), fetal bovine serum and 1% ( v / v ), penicillin - streptomycin double antibody to terminate the reaction, pipette and wash the bottom of the flask, transfer the liquid to a centrifuge tube, centrifuge at 1000 rpm for 5 min, remove the supernatant, add 3 mL of DMEM / F - 12 medium containing 10% ( v / v ), fetal bovine serum and 1% ( v / v ), penicillin - streptomycin double antibody to resuspend, and passage - culture at a ratio of 1:2. Conduct experiments after the cell growth state is stable.

[0089] III. Experimental grouping and treatment

[0090] Drug - adding sample wells (A 样): Melanoma cells (B16F10) in the logarithmic growth phase were seeded into 96-well plates at a density of 3000 cells / well and 100 μL per well. After being adaptively cultured in an incubator at 37 °C for 24 h, the supernatant was discarded. Then 100 μL of the sample solution was added. After culturing in an incubator at 37 °C for 48 h, the supernatant was discarded. Under light-proof conditions, 110 μL of 10% ( v / v )CCK-8 solution was added. After incubating in an incubator at 37 °C for 1 h, the absorbance value at 450 nm was measured.

[0091] Zero-adjustment well (A 调 ): 100 μL of DMEM / F-12 medium containing 10% ( v / v )fetal bovine serum and 1% ( v / v )penicillin-streptomycin double antibody was added to a 96-well plate. After being adaptively cultured in an incubator at 37 °C for 24 h, the supernatant was discarded. Then 100 μL of DMEM / F-12 medium containing 10% ( v / v )fetal bovine serum and 1% ( v / v )penicillin-streptomycin double antibody was added. After culturing in an incubator at 37 °C for 48 h, the supernatant was discarded. Under light-proof conditions, 110 μL of 10% ( v / v )CCK-8 solution was added. After incubating in an incubator at 37 °C for 1 h, the absorbance value at 450 nm was measured.

[0092] Blank well (A 空 ): Melanoma cells (B16F10) in the logarithmic growth phase were seeded into 96-well plates at a density of 3000 cells / well and 100 μL per well. After being adaptively cultured in an incubator at 37 °C for 24 h, the supernatant was discarded. Then 100 μL of DMEM / F-12 medium containing 10% ( v / v )fetal bovine serum and 1% ( v / v )penicillin-streptomycin double antibody was added. After culturing in an incubator at 37 °C for 48 h, the supernatant was discarded. Under light-proof conditions, 110 μL of 10% ( v / v )CCK-8 solution was added. After incubating in an incubator at 37 °C for 1 h, the absorbance value at 450 nm was measured.

[0093] According to the formula "Cell viability (%) = [ (A 样 - A 调 ) / (A 空 - A 调 ) ] × 100%", the cell viability after the action of the sample solution was calculated to evaluate the safety of T1 - T2.

[0094] IV. Experimental Results

[0095] The results are as Figure 6As shown, it can be seen that compared with the case of not adding T1 and T2 and only adding unmodified T1, after the action of amidated polypeptide T2 at 15 - 600 μM, there was no significant difference in the activity of B16F10 cells. This indicates that the amidated polypeptide T2 of the present invention is non-toxic to cells and has high safety.

[0096] Example 5 Inhibitory Activity of T1 - T2 on Tyrosinase at the Cellular Level

[0097] I. Solution Preparation

[0098] Sample solution: Dissolve T1 - T2 obtained in Example 1 in DMEM / F - 12 medium containing 10% ( v / v ), fetal bovine serum and 1% ( v / v ), penicillin - streptomycin double antibody, so that their final concentrations are 37.5 and 75 μM respectively, and then sterilize with a 0.22 μm needle filter.

[0099] II. Experimental Grouping and Treatment

[0100] Sample determination wells (A 测 ): Take melanoma cells (B16F10) in the logarithmic growth phase and inoculate them into a 6 - well plate at a density of 2×10 5 cells / well. After adaptively culturing in an incubator at 37 °C for 24 h, discard the supernatant and wash twice with PBS solution (pH = 7.4). Add 2 mL of sample solution and culture in an incubator at 37 °C for 48 h, then discard the supernatant. Wash twice with PBS solution (pH = 7.4) to remove non - adherent cells, and then add 400 μL of lysis solution (the volume ratio of PMSF to RIPA is 1:100), lyse at 4 °C for 30 min, and centrifuge to collect the supernatant.

[0101] Blank wells (A 空 ): Take melanoma cells (B16F10) in the logarithmic growth phase and inoculate them into a 6 - well plate at a density of 2×10 5 cells / well. After adaptively culturing in an incubator at 37 °C for 24 h, discard the supernatant and wash twice with PBS solution (pH = 7.4). Add 2 mL of DMEM / F - 12 medium containing 10% ( v / v ), fetal bovine serum and 1% ( v / v ), penicillin - streptomycin double antibody, culture in an incubator at 37 °C for 48 h, then discard the supernatant. Wash twice with PBS solution (pH = 7.4) to remove non - adherent cells, and then add 400 μL of lysis solution (the volume ratio of PMSF to RIPA is 1:100), lyse at 4 °C for 30 min, and centrifuge to collect the supernatant.

[0102] After determining and normalizing the protein concentration of the supernatant using a BCA kit, 50 μL of the protein solution was taken and mixed with 50 μL of PBS solution (pH = 7.4) and 50 μL of L-DOPA (1 mg / mL). After reacting at 37 °C for 30 min, the absorbance value was measured at 475 nm. According to the formula "Tyrosinase activity (%) = A 测 / A 空 × 100%", the tyrosinase activity after the action of the sample solution was calculated.

[0103] III. Experimental Results

[0104] The results are as Figure 7 shown. It can be seen that compared with unmodified T1, after the action of amidated polypeptide T2 at 37.5 - 75 μM, the tyrosinase activity of B16F10 cells was significantly reduced, indicating that the amidated polypeptide T2 of the present invention can significantly inhibit the activity of tyrosinase and is suitable for the preparation of whitening products.

[0105] Example 6 Inhibitory Activity of T1 - T2 on Melanin

[0106] I. Solution Preparation

[0107] Sample solution: Dissolve T1 - T2 obtained in Example 1 in DMEM / F-12 medium containing 10% ( v / v ), fetal bovine serum and 1% ( v / v ), penicillin-streptomycin double antibody, and make their final concentrations 37.5 and 75 μM respectively. Then sterilize with a 0.22 μm needle filter to obtain.

[0108] II. Experimental Grouping and Treatment

[0109] Sample determination well (A 测 ): Take melanoma cells (B16F10) in the logarithmic growth phase and inoculate them in a 6-well plate at a density of 2 × 10 5 cells / well. After adaptively culturing in an incubator at 37 °C for 24 h, discard the supernatant and wash twice with PBS solution (pH = 7.4). Add 2 mL of the sample solution and culture in an incubator at 37 °C for 48 h. Then discard the supernatant and wash twice with PBS solution (pH = 7.4) to remove non-adherent cells. Then digest the cells with trypsin, collect the digestion product and centrifuge at 1500 r / min for 10 min. Add 1 mL of NaOH solution (1 mol / L) containing 10% ( v / v ) DMSO to the centrifuged precipitate, extract in a water bath at 80 °C for 1 h, and then measure the absorbance value at 450 nm.

[0110] Blank well (A 空): Take melanoma cells (B16F10) in the logarithmic growth phase and inoculate them into a 6-well plate at a density of 2×10 5 cells / well. After adapting and culturing in an incubator at 37 °C for 24 h, discard the supernatant and wash twice with PBS solution (pH = 7.4). Add 2 mL of DMEM / F-12 medium containing 10% ( v / v fetal bovine serum and 1% ( v / v penicillin-streptomycin double antibody, and culture in an incubator at 37 °C for 48 h. Then discard the supernatant, wash twice with PBS solution (pH = 7.4) to remove non-adherent cells, then digest the cells with trypsin, collect the digestion product and centrifuge at 1500 r / min for 10 min. Add 1 mL of NaOH solution (1 mol / L) containing 10% ( v / v DMSO) to the centrifuged precipitate, extract in a water bath at 80 °C for 1 h, and then measure the absorbance value at 450 nm.

[0111] According to the formula "Melanin content (%) = A 测 / A 空 ×100%", calculate the melanin content after the action of the sample solution.

[0112] III. Experimental Results

[0113] The results are as Figure 8 shown. It can be seen that compared with unmodified T1, after the action of amidated polypeptide T2 at 37.5 - 75 μM, the melanin content of B16F10 cells decreased significantly, indicating that the amidated polypeptide T2 of the present invention can significantly inhibit melanin production and is suitable for preparing whitening products.

[0114] In addition, combining the results of Example 5 and Example 6, it can be known that the inhibitory effect of amidated polypeptide T2 on tyrosinase activity does not show a dose-dependence, while the inhibitory effect on melanin content shows a dose-dependence, indicating that the inhibitory effect of the amidated polypeptide of the present invention on melanin content is not simply dependent on the inhibition of tyrosinase activity.

[0115] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. Use of an amidated polypeptide in the preparation of a whitening product, characterized in that, The amidated polypeptide is obtained by amidation modification of the C-terminus of the polypeptide; wherein, the amino acid sequence of the polypeptide is as shown in SEQ ID NO:

1.

2. Use of an amidated polypeptide in the preparation of an antioxidant product, characterized in that, The amidated polypeptide is obtained by amidation modification of the C-terminus of the polypeptide; wherein, the amino acid sequence of the polypeptide is as shown in SEQ ID NO:1.

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