CN115521356B - Polypeptide with whitening effect and application thereof - Google Patents
Polypeptide with whitening effect and application thereof Download PDFInfo
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- CN115521356B CN115521356B CN202210987869.4A CN202210987869A CN115521356B CN 115521356 B CN115521356 B CN 115521356B CN 202210987869 A CN202210987869 A CN 202210987869A CN 115521356 B CN115521356 B CN 115521356B
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- polypeptide
- cells
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- melanin
- whitening
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- 229960001727 tretinoin Drugs 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- 238000009281 ultraviolet germicidal irradiation Methods 0.000 description 1
- 230000003827 upregulation Effects 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
- C07K5/0202—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing the structure -NH-X-X-C(=0)-, X being an optionally substituted carbon atom or a heteroatom, e.g. beta-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
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0055—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10)
- C12N9/0057—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10) with oxygen as acceptor (1.10.3)
- C12N9/0059—Catechol oxidase (1.10.3.1), i.e. tyrosinase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y110/00—Oxidoreductases acting on diphenols and related substances as donors (1.10)
- C12Y110/03—Oxidoreductases acting on diphenols and related substances as donors (1.10) with an oxygen as acceptor (1.10.3)
- C12Y110/03001—Catechol oxidase (1.10.3.1), i.e. tyrosinase
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/74—Biological properties of particular ingredients
- A61K2800/78—Enzyme modulators, e.g. Enzyme agonists
- A61K2800/782—Enzyme inhibitors; Enzyme antagonists
Abstract
The invention discloses a polypeptide with whitening effect, which has the amino acid sequence as follows: beta-Ala-Pro-Dab-D-Tyr-NHBzl. The research shows that the polypeptide containing D-tyrosine has the effects of inhibiting tyrosinase activity, and can negatively regulate melanin synthesis in melanoma cells and melanocytes, further inhibit melanin deposition in cells, and has the whitening effect, so that the polypeptide has the prospect of being developed into whitening cosmetics such as facial cleanser, face cream, facial mask, essence, toner, skin cream, body lotion and bath lotion. The invention also discloses application of the polypeptide in preparing whitening cosmetics.
Description
Technical Field
The invention belongs to the field of polypeptides, and particularly relates to a polypeptide with a whitening effect and application thereof.
Background
Melanin is synthesized in the melanosomes and then transferred to surrounding keratinocytes, protecting the cells from DNA damage.
Melanin is critical for protecting human skin from radiation, but abnormal melanin accumulation can lead to skin pigmentation, such as chloasma, freckles and age spots. Skin pigmentation caused by pathophysiological or environmental factors has prompted the screening of effective drugs to reduce pigmentation. To date, despite the extensive research efforts on new depigmenting agents, many depigmenting ingredients such as kojic acid, salicylic acid, nicotinamide, arbutin, hydroquinone, corticosteroids and retinoic acid have been developed, the need for new, safer and more efficient depigmenting agents has been driven by high toxicity, low stability, poor skin penetration and insufficient activity.
In order to develop new depigmenting agents, compounds of various origins, such as natural products, endogenous or engineered peptides and synthetic chemicals, are being actively explored. Some researchers have screened natural products and found that chalcone, resveratrol and coumarin have inhibitory activity against mushroom tyrosinase. Researchers have also sought to develop bioactive materials, particularly various bioactive peptides, including short-sequence oligopeptides, tripeptide compounds, dipeptides and tripeptides derived from natural products, and cysteine-containing dipeptides, all of which are used as cosmetic peptides. Peptides have strong advantages due to their stability, ease of synthesis and modification, and diverse availability. For example, the tetrapeptide PKEK inhibits UVB-induced up-regulation of genes encoding IL-1 alpha, IL-6, IL-8 and TNF-alpha and elevation of protein levels of POMC and tyrosinase, which may be suitable as skin tone modulators in cosmetics. Currently, bioactive peptides are widely used as cosmetics by cosmeceutical companies.
Disclosure of Invention
Tyrosine is a functional catalytic substrate of tyrosinase monophenolase and is a main raw material for finally forming brown melanin and eumelanin. Tyrosinase is in a close and inseparable relationship with melanin production. In the whole process of melanin formation, the most important type of speed-limiting enzyme is tyrosinase, and the quantity of melanin formation is determined by the activity of tyrosinase; tyrosine is a functional catalytic substrate of tyrosinase, and is a main raw material for finally forming brown melanin and eumelanin. The research finds that: the enantiomer D-tyrosine of L-tyrosine can inhibit the production of melanin in melanocytes induced by alpha-MSH (alpha-melanocyte stimulating hormone) treatment or uv irradiation by inhibiting the enzymatic activity of tyrosinase. Based on the above, the inventor has shown that the polypeptide containing D-tyrosine has the effect of inhibiting tyrosinase activity, can negatively regulate melanoma cells and melanin synthesis in the melanocytes, and further inhibit melanin deposition in the cells, and has the whitening effect, so that the polypeptide has the prospect of being developed into whitening cosmetics such as facial cleanser, face cream, facial mask, essence, toner, skin cream, body cream and bath lotion.
The invention aims to provide a polypeptide with whitening effect.
A polypeptide with whitening effect, the amino acid sequence of the polypeptide is as follows: beta-Ala-Pro-Dab-D-Tyr-NHBzl.
Ala represents the corresponding residue with English name of beta-Alanine and Chinese name of beta-Alanine;
pro represents the corresponding residue with the English name Proline and the Chinese name Proline;
dab represents 2, 4-diaminobutyric acid;
D-Tyr represents the corresponding residue of the English name D-Tyrosine and the Chinese name D-Tyrosine;
the chinese name NHBzl is benzyl amine.
The structure of the polypeptide is shown as formula I:
another object of the invention is to provide the use of said polypeptides in the preparation of whitening cosmetics.
Preferably, the application is the application in preparing whitening cosmetics for inhibiting melanin deposition.
More preferably, the cosmetic may be a facial cleanser, a face cream, a face mask, an essence, a toner, a skin lotion, a body lotion or a body wash.
Drawings
FIG. 1 is a graph showing the effect of polypeptides on B16F10 cell viability.
FIG. 2 shows in situ intracellular tyrosinase activity of each group measured by L-DOPA staining.
Fig. 3 shows the melanin content in each group of melanoma cells.
FIG. 4 shows the expression levels of melanin synthesis-related proteins in melanoma cells of each group; wherein, FIG. 4A is the band of the related protein, and FIG. 4B is the data statistics performed for the protein band.
Detailed Description
The following describes the essential aspects of the present invention in detail with reference to examples, but is not intended to limit the scope of the present invention.
Example 1
The synthesis method of the polypeptide comprises the following steps:
step (1), calculating the weight of each raw material (all raw materials with protecting groups) according to the weight (10 mg) of the target polypeptide: 0.24mmol Fmoc-D-Tyr (tBu) -OH, 0.72mmol Fmoc-Dab (Boc) -OH, 0.72mmol Fmoc-Pro-OH, 0.72mmol Fmoc-beta-Ala-OH, 0.72mmol NHBzl (phenylmethylamine);
putting 500mg of 2-CL resin (chloro (o-chlorophenyl) diphenylmethane) into a 150mL reactor, adding 50mL of DCM (dichloromethane) for soaking for 2 hours, pumping the DCM by a circulating water type vacuum pump, adding 50mL of DMF (N, N-dimethylformamide), putting the reactor on a decolorizing shaker for shaking for 30s, pumping the DMF by the circulating water type vacuum pump, adding 50mL of DMF again, repeating the steps four times, and pumping the resin to dryness;
step (3), 0.24mmol Fmoc-D-Tyr (tBu) -OH (C end first amino acid), 50mL DCM and 0.1mL DIEA are weighed into a reactor, and the reactor is placed in a shaking table at 30 ℃ for reaction for 2 hours; after the reaction is finished, 50mL of DCM is added, methanol and DIEA are added, unreacted functional group structures on the resin are blocked, the reaction is prevented from being participated in the next reaction, the reactor is placed in a shaking table at 30 ℃ for reaction for 30min (the molar ratio of the resin to the methanol is 1:3, the DCM is used as a solvent, the DIEA mainly provides an alkaline environment and plays a role of catalysis), after the reaction is finished, liquid in the reactor is pumped out by a circulating water type vacuum pump, 50mL of DMF is added, the reactor is placed on the decolorizing shaking table for shaking for 30s, the DMF is pumped out by the circulating water type vacuum pump, 50mL of DMF is added again, the steps are repeated four times, and the resin is pumped out;
step (4), adding 50mL of 20% piperidine solution (the volume ratio of piperidine to DMF is=1:4) into a reactor, placing the reactor on a decolorizing shaker, shaking for 20min to remove Fmoc protecting groups, after the reaction is finished, pumping off liquid in the reactor by a circulating water type vacuum pump, adding 50mL of DMF, placing the reactor on the decolorizing shaker, shaking for 30s, pumping off DMF by a circulating water type vacuum pump, adding 50mL of DMF again, repeating the steps four times, and pumping off the resin;
step (5), taking a small amount of resin, detecting by an ninhydrin method, and if the resin is blue in detection, indicating that the deprotection is successful; if the resin is not colored, repeating the step (4) until the resin is blue after being detected by an ninhydrin method;
step (6), weighing 0.72mmol Fmoc-Dab (Boc) -OH (second amino acid at C terminal), 0.72mmol HOBT (HOBT is mainly used for preventing racemization), and 0.1mL DIC (DIC main condensation reagent), adding into a reactor, adding 50mL DMF again, mixing the amino acid and resin, and placing the reactor in a constant temperature shaking table at 30 ℃ for reaction for 1 hour; after the reaction is completed, pumping out the liquid, adding 50mL of DMF, placing the reactor on a decolorizing shaker, shaking for 30s, pumping out the DMF by a circulating water type vacuum pump, repeating the steps four times, and pumping out the resin;
step (7), taking a small amount of resin, detecting by an ninhydrin method, if the resin is colorless, indicating that the reaction is complete, pumping liquid by a circulating water type vacuum pump, adding 50mL of DMF, placing the reactor on a decolorizing shaker, shaking for 30s, pumping DMF by the circulating water type vacuum pump, repeating the steps four times, and pumping the resin; if the resin has color, indicating that the condensation reaction is incomplete, continuing the reaction for 1h until colorless detection;
step (8), adding 50mL of 20% piperidine (the volume ratio of the piperidine to the DMF=1:4) into the reactor, shaking the reactor on a decolorizing shaker for 20min, pumping out liquid by using a circulating water type vacuum pump, adding 50mL of DMF again, shaking the reactor on the decolorizing shaker for 30s, pumping out DMF by using the circulating water type vacuum pump, repeating the steps four times, and pumping out resin;
step (9), detecting whether the protecting group is removed: detecting a small amount of resin by using an ninhydrin method, and if the resin is colorless and the deprotection is unsuccessful, adding piperidine again according to the step (8) to react until the resin has color; if the resin is blue, repeating the step (6);
step (10), according to the steps (6) - (9), fmoc-Dab (Boc) -OH is replaced by Fmoc-Pro-OH (third amino acid at C terminal) connecting amino acid; step (6) -step (9), replacing Fmoc-Dab (Boc) -OH with Fmoc-beta-Ala-OH (fourth amino acid at C terminal) connecting amino acid;
step (11), preparing a cutting reagent according to the volume ratio of TFE (trifluoroethanol) and DCM=1:4, adding 100mL of the cutting reagent into a reactor, placing the reactor on a decolorizing shaker for reaction for 3 hours to cleave the polypeptide from the resin while protecting a side chain from the cleavage agent, transferring the liquid to a conical flask after the reaction is finished, placing the conical flask in a constant temperature shaker at 30 ℃, dissolving 0.72mmol of NHBzl, 0.72mmol of HOBT and 0.1mL of DIC in 5mL of DMF, slowly dripping the solution into the conical flask, and reacting for 6 hours;
step (12), preparing a cutting reagent according to the volume ratio of TFA and water=19:1, spin-drying the liquid in the conical flask after the reaction is finished, adding 50mL of the cutting reagent, and placing the conical flask on a decolorizing shaking table for reaction for 2 hours at 25 ℃; after the reaction is finished, 200mL of glacial ethyl ether is added into the conical flask, the mixture is transferred into a centrifuge tube, the polypeptide is settled at the bottom of the tube by centrifugation at 3000r/min, and the supernatant is poured out to obtain a crude polypeptide product;
step (13), dissolving the crude polypeptide product with water, separating the target peptide fragment from impurities by a High Performance Liquid Chromatography (HPLC), and freeze-drying the target peptide fragment into powder; the amino acid sequence of the polypeptide is: beta-Ala-Pro-Dab-D-Tyr-NHBzl; ESI-MS m/z 539.4[ M+H ]] + The method comprises the steps of carrying out a first treatment on the surface of the The structural formula of the polypeptide is shown as formula I:
wherein, the HPLC chromatographic conditions are as follows: chromatographic column: YMC-Triart C18 (4.6X250 mm,5 μm); mobile phase: a: 0.1% aqueous trifluoroacetic acid, B:0.1% acetonitrile trifluoroacetic acid solution; flow rate: 1mL/min; detection wavelength: 214nm; sample injection volume: 25 μl; the elution procedure is as in table 1:
TABLE 1 elution procedure
Example 2
1. Material
1.1 cell lines
Mouse melanoma cells B16-F10 (cat# Procell CL-0319) were purchased from the Living technologies Co., ltd. Of Withannocel, and were self-cultured and passaged by the laboratory. DMEM containing penicillin (100 IU/mL) -streptomycin (100. Mu.g/mL) and 10% heat-inactivated FBS was used at 37℃with 5% CO 2 Is cultured in a wet incubator.
1.2 Experimental reagents
Dulbecco's Modified Eagle's Medium (DMEM), fetal Bovine Serum (FBS) and 100 Xpenicillin-streptomycin solution were purchased from Invitrogen Inc. (Grand Island, N.Y., USA). Dimethyl sulfoxide (DMSO) and 3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide (MTT) were purchased from amerco inc. (Solon, OH, USA). Alpha-melanocyte stimulating hormone (alpha-MSH), L-3, 4-dihydroxyphenylalanine (L-DOPA), sodium hydroxide and kojic acid were purchased from Sigma Chemical Co. Fetal bovine serum, gemini, inc., USA, cat# 900-108. Microphthalmia-associated transcription factor (MITF), tyrosinase (TYR) antibodies were purchased from Cell Signaling Technology; beta-actin antibodies were purchased from BD company in the united states.
1.3 laboratory apparatus
Microplate reader (model: epoch), bio-tek, usa;
inverted fluorescence microscope (model: nikon), nikon;
horizontal shaking table (model: WD-9405F), beijing Liuyi;
a small bench-top refrigerated centrifuge (model: 5418), eppendorf, germany;
palm centrifuge (model: D1008E), SCILOGEX, U.S.A.;
a low-speed automatic balancing centrifuge (model: TD 4B), shanghai Lu Xiang;
analytical balance (model: TB 214), beijing Setolis;
magnetic stirrer (model: ZNCL-BS), henna ebote technology;
ultrapure water machine (model 1810B), china Molecular;
an electric heating constant temperature water tank (model: DK-8D), shanghai Jing Hongzhi technology;
electrothermal blowing drying oven (model 101-1), beijing Zhongxing Wei industry;
full-automatic snowflake ice maker (model: IMS-50), mature snow family;
a full-temperature oscillator (model: THZ-C-1), experimental equipment factory in Taiku city;
VORTEX mixer (model: VORTEX-5), jiangsu sea door Chenbel;
ultra clean bench (model: WOL-SY 020), guangzhou Wo Lin experiment equipment;
a liquid nitrogen tank (model: YDZ-15), beijing jun Fang Keyi;
carbon dioxide constant temperature incubator (model: 15 AIC), japan SANYO.
2. Experimental methods and results
2.1 cell culture
(1) Cell resuscitation
Rapidly removing the frozen storage tube filled with the B16-F10 cells from liquid nitrogen, placing the frozen storage tube in a water bath at 37 ℃ to melt for about 60 seconds, enabling the joint of a bottle cap to be incapable of being contacted with anything, transferring the frozen storage liquid containing the cells into a 15mL centrifuge tube after complete dissolution, adding 9 mL of DMEM complete culture medium, centrifuging at 1000rpm/min for 5min, discarding supernatant, adding 1mL of DMEM complete culture medium, repeatedly blowing and uniformly mixing, transferring the cells into a cell culture bottle after complete suspension, adding 3mL of complete culture medium into the culture bottle, capping, shaking for more than 10 times by cross-shaped horizontal shaking, and placing the culture bottle into a culture box for culture.
(2) Cell passage
And when the cell adhesion area of the B16-F10 is 80-90%, starting to perform cell subculture. First, cells were gently blown, and cells that were loosely adherent and scattered were collected in a 15mL centrifuge tube. To the flask, 1mL of pancreatin containing EDTA was added, and after digestion in the incubator for 30 seconds, 1mL of complete culture broth was added to neutralize the digestion of pancreatin. Then, the cells were blown until the cells were completely detached, and the culture containing the cells was collected based on centrifugation at 1000rpm for 5min in a 15mL centrifuge tube. Finally, the culture medium in a 15mL centrifuge tube is discarded, 1mL of DMEM complete culture medium is added, the mixture is repeatedly blown and evenly mixed, after the cells are completely suspended, the mixture is transferred into a cell culture bottle, 3mL of complete culture medium is added into the culture bottle, the culture bottle is covered, and the culture bottle is evenly shaken for more than 10 times by cross-shaped horizontal shaking, and then the culture bottle is placed into an incubator for culture.
(3) Cell cryopreservation
B16-F10 cells in the logarithmic growth phase were selected and the cell cryopreservation experiments were started. Firstly, collecting cells in a centrifuge tube, adding 1mL of cell freezing solution (volume ratio of DMSO and FBS=1:9), blowing and mixing uniformly to maintain the cell density at 5×10 6 about/mL; the frozen stock solution containing the cells was transferred to a freezing tube. After marking, the materials are placed in a program cooling box, transferred to a freezing storage at the temperature of minus 80 ℃ and finally placed in liquid nitrogen for storage.
2.2 cell proliferation assay
Considering the use of polypeptides as cosmetics or pharmaceuticals, their cytotoxicity is of paramount importance.
To test the cellular safety of polypeptides, the inventors used MTT to assess cytotoxicity. B16-F10 cells were grown at 1.25X10 4 Density of individual cells/wellsInoculating to 96-well plate, placing 180 μl of cell suspension in each well, culturing in an incubator for 24h, discarding culture medium in the well plate, adding DMEM culture medium (containing 2.5% FBS) containing polypeptides (0,12.5,25,50,100 μg/mL) at different concentrations, making 5 multiple wells per concentration, 200 μl per well, and standing for 48 hr; after 48h each well was treated with 20 μl MTT (5 mg/mL) for 4 hours, the purple formazan formed was dissolved using DMSO and the OD was measured at 490nm wavelength using an enzyme-labeled instrument.
Cell activity (%) = dosing/control OD x 100%
Data were processed using GraphPad Prism 9.0 statistical software and experimental results are expressed as mean ± SD. The comparison between groups uses t-test, p <0.05 representing significant differences (same below).
The effect of the polypeptide on B16F10 cell viability is shown in FIG. 1, where the polypeptide still showed no significant cytotoxicity at a concentration of 100. Mu.g/mL relative to the control. Thus, the inventors performed further experiments using a polypeptide concentration of 100. Mu.g/mL.
2.3 measurement of cell tyrosinase Activity
Tyrosinase is the rate-limiting enzyme critical to melanin biosynthesis. To elucidate the polypeptide-mediated inhibition of tyrosinase activity, cells were incubated with L-DOPA and the intracellular tyrosinase activity was assayed in situ. The method comprises the following steps:
B16F10 cells were seeded in 96-well plates at 1.25X10 per well 4 180 μl of cell suspension per well, wall 24h, and 96-well plate were removed, and control group, model group, polypeptide group, and kojic acid group were set respectively: the control group was added with DMEM medium containing 10% fetal bovine serum, the model group (alpha-MSH group) was added with DMEM medium containing alpha-MSH (final concentration 100 nmol/L), the polypeptide group was added with DMEM medium containing alpha-MSH (final concentration 100 nmol/L) and polypeptide (final concentrations 25,50 and 100. Mu.g/mL, respectively), and the kojic acid group was added with DMEM medium containing alpha-MSH (final concentration 100 nmol/L) and kojic acid (100. Mu.g/mL); setting 5 compound holes respectively, and continuously culturing for 48 hours; the 96-well plate was removed, the medium was aspirated, the cells were fixed at room temperature for 40min by washing with 100. Mu.L of PBS, and 100. Mu.L of 1% Triton X-100 solution (pH 6.8 was added to each wellPBS buffer) for two minutes, 100. Mu.L of 10mmol/L L-DOPA (prepared from PBS buffer) was added to each well after washing the cells with PBS, the cells were stained at 37℃for 2 hours, washed with PBS, and the staining was imaged and analyzed using a microscope.
As shown in fig. 2, the intracellular tyrosinase activity of the α -MSH group (the more black dots in the figure, the higher tyrosinase activity) was significantly increased compared to the control group; compared to the α -MSH group, B16F10 cells were treated with different concentrations of the polypeptide for 48 hours and the intracellular tyrosinase activity was dose-dependently reduced. The inhibition effect of the high dose (100 mug/mL) polypeptide on tyrosinase activity is equivalent to that of positive control medicine kojic acid (P < 0.05).
2.4 determination of melanin synthesis amount
To assess the effect of the polypeptide on melanogenesis in B16F10 cells, cells were stimulated with melanogenesis stimulator α -MSH for 48 hours in the presence or absence of the polypeptide. The method comprises the following steps:
B16F10 cells were seeded in 6-well plates at 25000 cells/well, and after 24h adherence, control, model, polypeptide, kojic acid groups were set respectively: the control group was added with DMEM medium containing 10% fetal bovine serum, the model group (alpha-MSH group) was added with DMEM medium containing alpha-MSH (final concentration 100 nmol/L), the polypeptide group was added with DMEM medium containing alpha-MSH (final concentration 100 nmol) and polypeptide (final concentrations 25,50 and 100. Mu.g/mL, respectively), and the kojic acid group was added with DMEM medium containing alpha-MSH (final concentration 100 nmol/L) and kojic acid (final concentration 100. Mu.g/mL); culturing was continued 48 h. Each sample was tested in duplicate 3 times.
The synthesis amount of melanin is determined by NaOH cleavage: after the completion of the culture, the supernatant was discarded and washed with PBS, the cells were digested with 0.25% pancreatin, the cell suspension was blown off, 100. Mu.L of the cell suspension was counted, the remaining cell suspension was centrifuged (1000 r/min,5 min), after discarding the supernatant, 150. Mu.L of 1mol/L NaOH solution (containing 10% DMSO) was added to lyse the cells, the cells were sufficiently lysed and melanin particles were lysed in a water bath at 80℃for 30min to 2h, the cell lysates of each group were transferred to 96 well plates, 100. Mu.L of each well was added, and the absorbance value A was measured at 405nm with a microplate reader, and the melanin synthesis amount was calculated.
Melanin synthesis (%) =a Administration group /A Control group ×100%
As shown in fig. 3, the melanin production of the α -MSH group was significantly increased compared to the control group. Compared with the alpha-MSH group, the polypeptide can inhibit the excessive melanin generation induced by the alpha-MSH in a dose-dependent manner, and the melanin content in melanoma cells is obviously reduced. The inhibition effect of the high dose (100 mug/mL) polypeptide on melanin is equivalent to that of positive control medicine kojic acid (P < 0.05).
2.5 Effect on expression of melanin synthesis-related proteins in melanoma cells
Taking B16-F10 cells in logarithmic growth phase, digesting with 0.25% pancreatin, preparing cell suspension from complete DMEM medium containing 10% foetal calf serum and 1% diabody, inoculating into 6-well plate with 2mL each to give cell concentration of 2×10 5 /mL, at 37℃and 5% CO 2 Culturing in incubator for 12 hr, discarding culture medium, and respectively setting control group, model group, polypeptide group, and kojic acid group: the control group was added with DMEM medium containing 10% fetal bovine serum, the model group (alpha-MSH group) was added with DMEM medium containing alpha-MSH (final concentration 100 nmol/L), the polypeptide group was added with DMEM medium containing alpha-MSH (final concentration 100 nmol/L) and polypeptide (final concentrations 25,50 and 100. Mu.g/mL, respectively), and the kojic acid group was added with DMEM medium containing alpha-MSH (final concentration 100 nmol/L) and kojic acid (final concentration 100. Mu.g/mL); culturing for 48h, discarding culture solution, washing with PBS, pancreatin digestion, cleavage on ice, determining protein concentration by BCA kit, performing SDS-PAGE electrophoresis of 50 μg protein from each group, transferring to NC membrane, blocking with 5% BSA, incubating the membrane with primary antibodies (MITF, TYR and beta-actin) overnight at 4deg.C, washing the membrane with TBST, adding secondary antibody, incubating at room temperature for 2h, developing and exposing by ECL method, photographing by chemiluminescent imaging system, and analyzing by Image J.
TYR and MITF are two important proteins in melanin synthesis. The rate of melanin synthesis is directly related to the amount and activity of TYR, and MITF can regulate tyrosinase expression in melanocytes to thereby participate in regulation of melanogenesis. As shown in fig. 4, melanin synthesis-related protein TYR, MITF was expressed in large amounts in the melanoma cells of the α -MSH group compared to the control group; compared with the alpha-MSH group, the polypeptide can inhibit the expression level of melanin synthesis related proteins TYR and MITF in melanoma cells in a dose-dependent manner, the expression level of melanin synthesis related proteins TYR and MITF in the melanoma cells in each dose group of the polypeptide is obviously reduced, and particularly, the inhibition effect of the high-dose (100 mug/mL) polypeptide on melanin is equivalent to that of positive control medicine kojic acid (P < 0.05).
The results show that the polypeptide provided by the invention has the activity of inhibiting the expression of melanin synthesis related proteins, can inhibit the activity of tyrosinase, further inhibit the deposition of melanin in cells, and has the whitening effect, so that the polypeptide has the prospect of being developed into whitening cosmetics.
Claims (4)
1. A polypeptide with whitening effect, which is characterized in that: the amino acid sequence of the polypeptide is as follows: beta-Ala-Pro-Dab-D-Tyr-NHBzl; the structure of the polypeptide is shown as formula I:
2. the use of the polypeptide having a whitening effect according to claim 1 for preparing a whitening cosmetic.
3. The use according to claim 2, characterized in that: the application is the application in preparing whitening cosmetics for inhibiting melanin deposition.
4. The use according to claim 2, characterized in that: the cosmetic is facial cleanser, facial cream, facial mask, essence, toner, skin cream, body lotion and bath lotion.
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