CN116606349A - Active peptide, active peptide mutant, application of active peptide mutant and composition with melanin inhibiting effect - Google Patents

Active peptide, active peptide mutant, application of active peptide mutant and composition with melanin inhibiting effect Download PDF

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CN116606349A
CN116606349A CN202310816759.6A CN202310816759A CN116606349A CN 116606349 A CN116606349 A CN 116606349A CN 202310816759 A CN202310816759 A CN 202310816759A CN 116606349 A CN116606349 A CN 116606349A
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active peptide
melanin
modification
mutant
composition
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叶成龙
张雅琪
杨安全
胡秋艺
李哲
王思思
莫家欢
王苗苗
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Osmun Biological Co ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/02—Preparations for care of the skin for chemically bleaching or whitening the skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

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Abstract

The application provides an active peptide and an active peptide mutant with melanin inhibiting effect and application thereof, and a composition with melanin inhibiting effect, belonging to the technical field of active peptides. The amino acid sequence of the active peptide with melanin inhibiting effect is shown as SEQ ID No. 1. The active peptide provided by the application is mild, high in safety, has the effect of enhancing cell activity, has high tyrosinase activity inhibition capability, can realize melanin synthesis inhibition rate of up to 32.4% at the concentration of 100ppm, can be used for preparing medicines, cosmetics or skin care products with melanin inhibition effect, tyrosinase inhibitors and the like, and has good application prospect.

Description

Active peptide, active peptide mutant, application of active peptide mutant and composition with melanin inhibiting effect
Technical Field
The application relates to the technical field of active peptides, in particular to an active peptide and an active peptide mutant with melanin inhibiting effect, application thereof and a composition with melanin inhibiting effect.
Background
Tyrosinase is the rate-limiting enzyme for melanin production and is also an important target for inhibiting pigmentation. Many tyrosinase inhibitors have been identified at present, and kojic acid, hydroquinone, arbutin, etc. are common. Wherein, the kojic acid has certain stability and safety problems, and too high concentration of kojic acid can irritate skin to cause adverse reactions such as allergy, dermatitis and the like; in addition, kojic acid is as unstable as vitamin C and is easily oxidized. The hydroquinone also has certain safety problems, and the too high concentration of hydroquinone can cause skin rash, stinging, allergy and other symptoms; moreover, studies in rodents have shown that hydroquinone may be a carcinogen. Arbutin is a natural active substance derived from green plants and is a safer and effective whitening raw material popular at present, however, the melanin synthesis inhibition rate of arbutin even at high concentration of 1000ppm is not more than 20%, and the requirement of continuous development of industry on higher melanin synthesis inhibition rate of whitening agents cannot be met.
Therefore, it is desired to find an active ingredient which has both mildness and safety and has a high melanin synthesis-inhibiting ability.
Disclosure of Invention
The application provides an active peptide and an active peptide mutant with melanin inhibiting effect, application thereof and a composition with melanin inhibiting effect, which aims to provide an active ingredient with mildness, safety and higher melanin synthesis inhibiting capability.
In a first aspect, the present application provides an active peptide having melanin inhibiting effect, the amino acid sequence of which is shown in SEQ ID No. 1.
The active peptide with the amino acid sequence shown as SEQ ID No.1 provided by the application is mild, high in safety, has the effect of enhancing cell activity, has high tyrosinase activity inhibiting capability, can realize melanin synthesis inhibition rate of up to 32.4% (far higher than 20%) at the concentration of 100ppm, can be used for preparing whitening medicaments, cosmetics or skin care products with melanin inhibition effect, tyrosinase inhibitors and the like, and has good application prospect.
In a second aspect, the present application provides an active peptide mutant having melanin inhibiting effect, the amino acid sequence of which is a sequence having 80% or more homology with the sequence shown in SEQ ID No. 1.
Because the amino acid sequence of the active peptide mutant provided by the application has the sequence homology of more than or equal to 80% with the sequence shown in SEQ ID No.1, the active peptide mutant also has higher capability of inhibiting tyrosinase activity, can effectively inhibit melanin generation, can be used for preparing medicines, cosmetics or skin care products with melanin inhibition effect, tyrosinase inhibitors and the like, and has good application prospect.
With reference to the second aspect, in an alternative embodiment of the present application, the amino acid sequence of the active peptide mutant satisfies: at least one amino acid in the sequence shown in SEQ ID No.1 is modified.
In the above technical scheme, the amino acid sequence of the active peptide mutant differs from the sequence shown in SEQ ID No.1 only in that: the amino acid is modified, so that the active peptide mutant also has higher melanin generation inhibiting capability, can be used for preparing medicines, cosmetics or skin care products with melanin inhibiting effect, tyrosinase inhibitors and the like, and has good application prospect.
With reference to the second aspect, in an alternative embodiment of the present application, the modification comprises at least one of a glycosylation modification, a phosphorylation modification, a ubiquitination modification, an S-nitrosylation modification, an N-methylation modification, an O-methylation modification, an N-acetylation modification, an N-palmitoylation modification, an N-myristoylation modification, and a lipidation modification.
In a third aspect, the present application provides the use of an active peptide provided in the first aspect or an active peptide mutant provided in the second aspect for the preparation of a melanin inhibiting medicament.
Because the active peptide provided in the first aspect or the active peptide mutant provided in the second aspect has high melanin production inhibiting capacity, melanin inhibiting effect of the melanin inhibiting medicine prepared by the active peptide or the active peptide mutant is better.
In a fourth aspect, the present application provides the use of an active peptide provided in the first aspect or an active peptide mutant provided in the second aspect for preparing a cosmetic or skin care product having melanin inhibiting effect.
Because the active peptide provided in the first aspect or the active peptide mutant provided in the second aspect has high melanin generation inhibiting capability, the melanin inhibiting effect of cosmetics or skin care products prepared by the active peptide or the active peptide mutant is better.
In a fifth aspect, the present application provides the use of an active peptide provided in the first aspect or an active peptide mutant provided in the second aspect for the preparation of a tyrosinase inhibitor.
Because the active peptide provided in the first aspect or the active peptide mutant provided in the second aspect has higher capability of inhibiting tyrosinase activity, the tyrosinase inhibitor prepared by using the active peptide or the active peptide mutant has higher capability of inhibiting tyrosinase activity.
In a sixth aspect, the present application provides a composition having melanin inhibiting effect, which comprises the active peptide provided in the first aspect or the active peptide mutant provided in the second aspect.
Because the active peptide provided in the first aspect or the active peptide mutant provided in the second aspect has higher melanin generation inhibiting capacity, the composition containing the active peptide or the active peptide mutant has better melanin inhibiting effect.
With reference to the sixth aspect, in an alternative embodiment of the present application, the active peptide is present in the composition in an amount of 80 to 120ppm; or, the content of the active peptide mutant in the composition is 80-120ppm.
In the technical scheme, when the content of the active peptide or the active peptide mutant in the composition is 80-120ppm, the composition can show the capability of remarkably inhibiting melanin generation, and the composition is used for preparing melanin inhibiting medicines, cosmetics or skin care products, tyrosinase inhibitors and the like, and has better capability of inhibiting melanin generation.
With reference to the sixth aspect, in an alternative embodiment of the present application, the composition further includes an auxiliary material, and the auxiliary material includes a humectant.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present application and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic diagram showing molecular docking between the polypeptide LGGLTG and tyrosinase.
FIG. 2 is a HPLC chart of the polypeptide LGGLTG synthesized in example 2 of the present application.
FIG. 3 is a mass spectrum of the polypeptide LGGLTG synthesized in example 2 of the present application.
FIG. 4 is a graph showing cytotoxicity results of the polypeptide LGGLTG at different concentrations.
FIG. 5 is a graph showing the comparison of melanin content of arbutin and polypeptide LGGLTG at different concentrations on B16-F10 cells.
Detailed Description
There are a variety of tyrosinase inhibitors in the prior art, such as kojic acid, hydroquinone, arbutin, and the like. Wherein, the kojic acid has certain stability and safety problems, and too high concentration of kojic acid can irritate skin to cause adverse reactions such as allergy, dermatitis and the like; in addition, kojic acid is as unstable as vitamin C and is easily oxidized. The hydroquinone also has certain safety problems, and the too high concentration of hydroquinone can cause skin rash, stinging, allergy and other symptoms; moreover, studies in rodents have shown that hydroquinone may be a carcinogen. Arbutin is a natural active substance derived from green plants and is a safer and effective whitening raw material popular at present, however, the melanin synthesis inhibition rate of arbutin even at high concentration of 1000ppm is not more than 20%, and the requirement of continuous development of industry on higher melanin synthesis inhibition rate of whitening agents cannot be met.
Therefore, the present application provides an active peptide having a melanin inhibiting effect, which has both mildness, safety and high melanin synthesis inhibiting ability.
The application provides an active peptide with melanin inhibiting effect, the amino acid sequence of which is shown as SEQ ID No.1 (namely LGGLTG).
In the present application, LGGLTG is leucine (Leu) -glycine (Gly) -leucine (Leu) -threonine (Thr) -glycine (Gly), and the structural formula of LGGLTG is as follows:
the active peptide with the amino acid sequence shown as SEQ ID No.1 has no cytotoxicity at 1ppm-100ppm, has mildness and higher safety, has the function of enhancing cell activity, has higher capability of inhibiting tyrosinase activity, has higher affinity with tyrosinase (PDB: 2Y 9X), and has the binding energy of-6.5 kcal/mol.
Compared with the melanin synthesis inhibition rate of 18.5% of arbutin at high concentration of 1000ppm and 7.3% of arbutin at 100ppm, the melanin synthesis inhibition rate of the active peptide with the amino acid sequence shown as SEQ ID No.1 at the concentration of 100ppm provided by the application can be up to 32.4%, namely the melanin synthesis inhibition capability of the active peptide provided by the application is far higher than that of arbutin.
Therefore, the active peptide with the amino acid sequence shown as SEQ ID No.1 provided by the application can obviously inhibit melanin generation, can be used for preparing medicines, cosmetics or skin care products with melanin inhibition effect, tyrosinase inhibitors and the like, and has good application prospect.
The application also provides an active peptide mutant with melanin inhibiting effect, and the amino acid sequence of the active peptide mutant is a sequence with the sequence homology of more than or equal to 80% with the sequence shown in the SEQ ID No. 1.
Illustratively, the amino acid sequence of the active peptide mutant is 80%, 83%, 85%, 87%, 90%, 95%, 99% and 99.9% homologous to the sequence shown in SEQ ID No.1, or a range between any one point or any two.
Because the amino acid sequence of the active peptide mutant provided by the application has the sequence homology of more than or equal to 80% with the sequence shown in SEQ ID No.1, the active peptide mutant also has higher capability of inhibiting tyrosinase activity, can effectively inhibit melanin generation, and has good application prospect in the aspects of preparing medicines, cosmetics or skin care products with melanin inhibition effect, tyrosinase inhibitors and the like.
In some possible embodiments of the application, the amino acid sequence of the active peptide mutant satisfies: at least one amino acid in the sequence shown in SEQ ID No.1 is modified.
It will be appreciated that in the present application the amino acid sequence of the active peptide mutant may be modified with respect to one, two, three, four, five or all of the amino acids in the sequence shown in SEQ ID No. 1.
The amino acid sequence of the active peptide mutant meets the following conditions: when at least one amino acid in the sequence shown in SEQ ID No.1 is modified, the amino acid sequence of the active peptide mutant differs from the sequence shown in SEQ ID No.1 only in that: the amino acid is modified, so that the active peptide mutant also has higher melanin generation inhibiting capability, and the active peptide mutant has good application prospect in the aspects of preparing medicines, cosmetics or skin care products with melanin inhibiting effect, tyrosinase inhibitors and the like.
Further, the aforementioned amino acid is modified in such a manner as to include at least one of glycosylation modification, phosphorylation modification, ubiquitination modification, S-nitrosylation modification, N-methylation modification, O-methylation modification, N-acetylation modification, N-palmitoylation modification, N-myristoylation modification and lipidation modification.
The method of modifying the amino acid is not limited to the above, and for example, the amino acid modification may be a substitution of a side chain group of the amino acid with another group, and the present application is not limited to the method of modifying the amino acid.
The application provides an application of the active peptide provided by the previous description or the active peptide mutant provided by the previous description in preparing melanin inhibiting medicines.
Because the active peptide provided by the foregoing or the active peptide mutant provided by the foregoing has a high melanin production inhibiting ability, the melanin inhibiting effect of the melanin inhibiting medicine prepared by using the active peptide or the active peptide mutant is better.
In some possible embodiments of the application, the medicament has the effect of treating or preventing a melanin disorder.
The application also provides application of the active peptide provided by the previous description or the active peptide mutant provided by the previous description in preparing cosmetics or skin care products with melanin inhibiting effect.
Because the active peptide provided by the foregoing or the active peptide mutant provided by the foregoing has a high melanin production inhibiting ability, the melanin inhibiting effect of the cosmetic or skin care product prepared by using the active peptide or the active peptide mutant is better.
The application also provides application of the active peptide provided by the previous description or the active peptide mutant provided by the previous description in preparing tyrosinase inhibitors.
Because the active peptide provided by the foregoing or the active peptide mutant provided by the foregoing has a high ability to inhibit tyrosinase activity, tyrosinase inhibitors prepared by using the active peptide or the active peptide mutant have a high ability to inhibit tyrosinase activity.
In addition, the application also provides a composition with melanin inhibiting effect, which comprises the active peptide provided by the previous content or the active peptide mutant provided by the previous content.
Because the active peptide provided by the previous content or the active peptide mutant provided by the previous content has higher melanin generation inhibiting capability, the composition containing the active peptide or the active peptide mutant has better melanin inhibiting effect; the composition can be used for preparing medicines, cosmetics, skin care products, medical and aesthetic products, tyrosinase inhibitors, functional foods, etc. with melanin inhibiting effect.
Further, in some possible embodiments of the application, when the composition contains the active peptide provided in the foregoing, the content of the active peptide in the composition is 80 to 120ppm.
Illustratively, the amount of active peptide in the composition may be any one or range of values between any one of 80ppm, 85ppm, 90ppm, 95ppm, 100ppm, 110ppm and 120ppm.
The content of the active peptide in the composition is 80-120ppm, so that the composition can obviously inhibit melanin generation, and the composition can be used for preparing melanin inhibiting medicines, cosmetics or skin care products, tyrosinase inhibitors and the like, and has better melanin generation inhibiting capability.
In some possible embodiments of the application, when the composition contains the active peptide mutant provided in the foregoing, the active peptide mutant is contained in the composition in an amount of 80 to 120ppm.
Illustratively, the active peptide mutant may be present in the composition at any point or range of values between any two of 80ppm, 85ppm, 90ppm, 95ppm, 100ppm, 110ppm and 120ppm.
The content of the active peptide mutant provided by the content is 80-120ppm in the composition, so that the composition can obviously inhibit melanin generation, and the composition can be used for preparing melanin inhibiting medicines, cosmetics or skin care products, tyrosinase inhibitors and the like, and has better melanin generation inhibiting capability.
In some possible embodiments of the application, the composition further comprises an adjuvant.
As an example, the auxiliary materials may include a humectant, a preservative, a thickener, and the like, and the present application is not limited to the specific selection of the foregoing humectant, preservative, and thickener. For example, the humectant may be, but is not limited to, butylene glycol, polyglycerin-10 or sodium hyaluronate, the thickener may be, but is not limited to, xanthan gum, hydrolyzed sclerotium (SCLEROTIUM ROLFSSII) gum or acrylic acid (ester) class/C10-30 alkanol acrylate cross-linked polymer, etc., and the preservative may be, but is not limited to, phenoxyethanol or ethylhexyl glycerol.
In order to make the objects, technical solutions and advantages of the embodiments of the present application more clear, the technical solutions of the embodiments of the present application will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Example 1 molecular docking of polypeptide LGGLTG with tyrosinase
PDB format files of tyrosinase (PDB: 2Y 9X) were obtained in a PDB database (https:// www.pdbus.org /). The protonated state of the polypeptide LGGLTG was set to ph=7.4 and extended to a 3D structure using OpenBabel. Tyrosinase and the polypeptide LGGLTG were subjected to a series of pretreatment using an AutoDock tool. The docking cassette was generated by autopoly, then molecular docking was performed using autopackvina, and the interaction of the polypeptide LGGLTG with tyrosinase was analyzed by selecting the optimal binding conformation and generating an interaction map by PyMOL (i.e. fig. 1).
As can be seen from fig. 1, a plurality of groups of interaction forces are formed between the polypeptide LGGLTG and tyrosinase; wherein, hydrogen bond is formed between tyrosinase GLN-41 and polypeptide LGGLTG, hydrogen bond is formed between tyrosinase ASN-174 and polypeptide LGGLTG, hydrogen bond is formed between tyrosinase LYS-180 and polypeptide LGGLTG, and ionic interaction is formed between tyrosinase HIS-178 and polypeptide LGGLTG. Under the action of the interaction forces, the binding energy of the polypeptide LGGLTG and tyrosinase is-6.5 kcal/mol, which shows that the polypeptide LGGLTG has potential inhibition effect on the activity of tyrosinase.
EXAMPLE 2 Synthesis of polypeptide LGGLTG
The synthesis of the polypeptide LGGLTG adopts a microwave-assisted solid phase synthesis method, and the polypeptide LGGLTG is synthesized from a C end to an N end in sequence, and the specific method is as follows:
(1) Activation of the resin: 1.00g of 2-CTC resin was weighed into the reaction tube and swollen with DCM (dichloromethane) for 1h to activate the resin. The swollen resin was drained and washed twice with DCM and DMF (N, N-dimethylformamide) alternately. Adding SOCl with the volume ratio of 1:1 into the cleaned resin 2 (thionyl chloride) and DCM, stirred for 1h, filtered and the resin was washed three times alternately with DCM and DMF.
(2) Fmoc-L-glycine introduction resin: 1.25g Fmoc-L-glycine-OH (4.2 mmol) was weighed out and dissolved in 5mL DCM, mixed with the resin from step (1) and microwaved at room temperature for 1h. After the reaction was completed, the resin was alternately washed three times with DCM and DMF and dried in vacuo.
(3) And (3) end capping: the system obtained in the step (2) is dissolved in a mixed reagent of MeOH (methanol) and DIPEA (N, N-diisopropylethylamine) in a volume ratio of 9:1, reacted for 20 mm at room temperature under microwave, alternately washed twice with DCM and DMF, and finally thoroughly washed with DMF.
(4) Removing Fmoc protecting group: fmoc protecting group is removed by using 20% piperidine solution (DMF solvent) for 3min, and the reaction temperature is 75 ℃. After the reaction was completed, the reaction mixture was washed three times with DCM and DMF alternately.
(5) Coupling of the remaining amino acids: the remaining amino acids were coupled in the order Fmoc-Thr-OH (1.43 g), fmoc-Leu-OH (1.48 g), fmoc-Gly-OH (1.25 g), fmoc-Leu-OH (1.48 g) according to steps (2) to (4).
(6) Cutting resin: after all amino acids were successfully coupled, after final removal of Fmoc protecting groups, 10mL of a mixed reagent of HFIP (hexafluoroisopropanol) and DCM in a volume ratio of 1:4 was added to the deprotected system, and the reaction was carried out by microwave at room temperature for 20min to remove the resin. The solvent in the system was filtered off with suction into a collection bottle, the resin was rinsed with DCM and the filtrate was collected; and combining the liquid phase after suction filtration and the liquid phase after resin flushing to obtain a system to be enriched and purified.
(7) Enrichment and purification of polypeptides: slowly dripping diethyl ether into the system to be enriched and purified obtained in the step (6), generating light yellow solid during the period, placing the light yellow solid in a refrigerator at the temperature of minus 20 ℃ for 2 hours, centrifuging, discarding the supernatant, adding diethyl ether again, centrifuging, and discarding the supernatant. The resulting solid was dried in a fume hood, then dissolved in water and passed through a 0.22 μm PES filter, and purified by SemiPrep RP-HPLC. The collected polypeptide is characterized and confirmed by Analytical RP-HPLC and MALDI-TOF-MS, and then is freeze-dried by a freeze dryer, thus obtaining the target polypeptide LGGLTG (the amino acid sequence is shown as SEQ ID NO. 1), and the HPLC spectrogram and the mass spectrogram of the polypeptide LGGLTG are shown as figures 2 and 3 respectively.
As can be seen from FIG. 2, the area ratio of peak No.1 was as high as 99.672%, indicating that the polypeptide LGGLTG synthesized in example 2 has a higher purity (99.672%).
As can be seen from fig. 3, the molecular weight of the LGGLTG synthesized in example 2 is expected, indicating that the LGGLTG synthesized in example 2 is the target polypeptide.
EXAMPLE 3 cytotoxicity assay of polypeptide LGGLTG
The cytotoxicity test procedure was as follows:
(1) Cell plating: after B16-F10 cells in the dishes had grown, the old medium was discarded and washed once with PBS buffer (pH 7.2), after which 1mL of trypsin was added and digested for 1.5min in a 37℃cell incubator. After the digestion was completed, 3mL of DMEM complete medium was added to terminate the digestion, and the cells were collected into a 15mL centrifuge tube and centrifuged at 1000rpm for 5min. After centrifugation, the supernatant was discarded and 1mL of fresh was addedFresh medium and gently suspend the cells with a pipette. The density of the cell suspension was adjusted to 5X 10 4 Each mL was inoculated into a 96-well plate (100. Mu.L/well), and placed in a cell incubator at 37℃for 24 hours.
(2) Drug administration treatment: 10mg of polypeptide LGGLTG was weighed and dissolved in 1mL of 0.1M PBS buffer at a concentration of 10mg/mL (i.e., 10000 ppm). Diluting polypeptide LGGLTG to a working concentration by using 0.1M PBS, adding 10 mu L of diluted polypeptide LGGLTG solution into each hole of a polypeptide treatment group (6 compound holes are arranged in each group), wherein the final concentration of polypeptide LGGLTG in each hole is 100ppm, 10ppm and 1ppm respectively, and adding 10 mu L of 0.1M PBS into a control group; then placed in a cell incubator at 37℃for 24 hours.
(3) CCK8 detection: CCK8 reagent was incubated with DMEM medium at 1: 10. The old medium in the 96-well plate was discarded by a lance, 100. Mu.L of diluted CCK8 reagent was added to each well, and the mixture was placed in a cell incubator at 37℃for 1 hour. Double wavelength measurement (main wavelength 450nm, reference wavelength 630 nm) was performed using an enzyme-labeled instrument.
(4) Data analysis: the calculation formula of the cell viability is as follows: cell viability (%) = (absorbance of polypeptide treated group/average absorbance of control group) ×100%. The significant differences between the polypeptide treated and control groups were analyzed using the Student's st-test two-tailed test, and P < 0.05 was considered statistically significant, P < 0.05 being indicated by x, P < 0.01 being indicated by x, and the cytotoxicity test results being shown in fig. 4.
From FIG. 4, it can be seen that the polypeptide LGGLTG has no cytotoxicity at concentrations of 100ppm, 10ppm and 1ppm, and can significantly improve the activity of B16-F10 cells (P < 0.05); the polypeptide LGGLTG has no cytotoxicity in the concentration range of 1ppm-100ppm, and has mild and high safety.
EXAMPLE 4 melanin inhibition experiments with the polypeptide LGGLTG and arbutin
The procedure for the melanin inhibition experiments was as follows:
after the B16-F10 cells in the culture dish are grown, they are grown up in a culture dish in a form of (10-15). Times.10 4 The cells were inoculated at a density of one mL/mL into 100mm cell culture dishes (10 mL/dish) and cultured in a 37℃cell culture incubator. After 24 hours, the drug administration treatment is carried out, and the culture is carried outDishes are divided into four groups: control and sample groups (arbutin-100 ppm group, arbutin-1000 ppm group, polypeptide LGGLTG-100ppm group) and three replicates per group. After the end of the administration, the cells were cultured in a 37℃cell incubator for 24 hours, digested with trypsin and centrifuged to collect the cells, the supernatant was discarded, the cells were washed 2 times with 0.1M PBS, centrifuged and the supernatant was discarded, 300. Mu.L of 1M NaOH solution containing DMSO (dimethyl sulfoxide) at a mass ratio of 10% was added, and the cells were lysed in a 80℃water bath. After 30min, 100. Mu.L of cell lysate is taken into a 96-well plate, and the absorbance OD at 405nm is read by an enzyme-labeled instrument 405 . Finally, absorbance OD 405 The melanin content of the control group was defined as 100% in terms of melanin content, and the melanin content (%) = (OD of the sample group) of the sample group 405 OD of control group 405 ) 100% and the melanin inhibiting experimental results are shown in fig. 5.
As can be seen from FIG. 5, the melanin content of the cells at 100ppm concentration of arbutin was 92.7%, and the melanin content of the cells was reduced to 81.5% after the concentration of arbutin was increased to 1000 ppm. The melanin content of the polypeptide LGGLTG at the concentration of 100ppm can be reduced to 67.6 percent (namely, the melanin synthesis inhibition rate is as high as 32.4 percent), and the melanin synthesis inhibition capability is obviously better than that of 1000ppm of arbutin (the melanin synthesis inhibition rate is 18.5 percent); the polypeptide LGGLTG has excellent melanin inhibiting effect, and can be used as a raw material for preparing medicines, cosmetics, skin care products, medical and aesthetic products and functional foods with the melanin inhibiting effect.
In conclusion, the active peptide with the amino acid sequence shown as SEQ ID No.1 provided by the application is mild, high in safety, has the effect of enhancing cell activity, has high tyrosinase activity inhibiting capability, can realize melanin synthesis inhibition rate of up to 32.4% at the concentration of 100ppm, can be used for preparing medicines, cosmetics or skin care products with melanin inhibition effect, tyrosinase inhibitors and the like, and has good application prospect.
The embodiments described above are some, but not all embodiments of the application. The detailed description of the embodiments of the application is not intended to limit the scope of the application, as claimed, but is merely representative of selected embodiments of the application. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to be within the scope of the application.

Claims (10)

1. An active peptide with melanin inhibiting effect is characterized in that the amino acid sequence of the active peptide is shown as SEQ ID No. 1.
2. An active peptide mutant with melanin inhibiting effect, which is characterized in that the amino acid sequence of the active peptide mutant is a sequence with the sequence homology of more than or equal to 80% with the sequence shown in SEQ ID No. 1.
3. The active peptide mutant according to claim 2, wherein the amino acid sequence of the active peptide mutant satisfies: at least one amino acid in the sequence shown in SEQ ID No.1 is modified.
4. The active peptide mutant according to claim 3, wherein the modification comprises at least one of glycosylation modification, phosphorylation modification, ubiquitination modification, S-nitrosylation modification, N-methylation modification, O-methylation modification, N-acetylation modification, N-palmitoylation modification, N-myristoylation modification and lipidation modification.
5. Use of an active peptide according to claim 1 or an active peptide mutant according to any one of claims 2 to 4 for the preparation of a melanin inhibiting medicament.
6. Use of an active peptide according to claim 1 or an active peptide mutant according to any one of claims 2 to 4 for the preparation of a cosmetic or skin care product having melanin inhibiting activity.
7. Use of an active peptide according to claim 1 or an active peptide mutant according to any one of claims 2 to 4 for the preparation of a tyrosinase inhibitor.
8. A composition having melanin inhibiting effect, characterized in that the composition comprises the active peptide according to claim 1 or the active peptide mutant according to any one of claims 2 to 4.
9. The composition of claim 8, wherein the active peptide is present in the composition in an amount of 80 to 120ppm; or, the content of the active peptide mutant in the composition is 80-120ppm.
10. The composition of claim 8 or 9, further comprising an adjunct comprising a humectant.
CN202310816759.6A 2023-07-04 2023-07-04 Active peptide, active peptide mutant, application of active peptide mutant and composition with melanin inhibiting effect Pending CN116606349A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117106015A (en) * 2023-09-01 2023-11-24 海南大学 Coconut-derived active peptide and application thereof

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117106015A (en) * 2023-09-01 2023-11-24 海南大学 Coconut-derived active peptide and application thereof
CN117106015B (en) * 2023-09-01 2024-04-26 海南大学 Coconut-derived active peptide and application thereof

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