Application of camellia peptide in whitening product

Abstract

The invention discloses an application of a camellia peptide in a whitening product. The amino acid sequence of the camellia peptide is at least one of Leu-Pro-Phe, Leu-Leu-Leu-Gly-His and Ala-Pro, and the amino acid sequence of the camellia peptide is at least one of Leu-Pro-Phe, Leu-Leu-Leu-Leu-Gly-His and Ala-Pro. The camellia peptide provided by the invention realizes whitening by inhibiting the activity of tyrosinase, reducing the generation of melanin and inhibiting the proliferation of melanocytes. Specifically, the camellia peptide provided by the invention competes with tyrosinase to inhibit the activity of the tyrosinase and the oxidation of the tyrosinase on a substrate, so that the generation of melanin is reduced, and the purpose of whitening is achieved; meanwhile, the camellia peptide reduces the activity of melanocytes in the skin and inhibits the proliferation of the melanocytes by interfering the growth cycle of the melanocytes, so that the generation of melanin is reduced, and the purpose of whitening is achieved.

Description

Technical Field

[0001] The invention relates to the field of biotechnology and skin health technology, and in particular to application of camellia peptide in whitening products. Background Art

[0002] Camellia japonica L. is a shrub or small tree of the genus Camellia in the family Theaceae, belonging to tropical and subtropical climates. my country is a major camellia planting country, with most of them distributed in Zhejiang, Jiangxi, Guangxi, Yunnan, Guizhou, Hunan, Guangdong and other areas south of the Yangtze River.

[0003] Tyrosinase is a copper-containing enzyme that is widely found in animals, plants and microorganisms. It is a key enzyme in the production of melanin. It catalyzes the oxidation of tyrosine to dopa, which is further oxidized to dopaquinone, and finally produces melanin. Melanin is the main determinant of skin, hair and eye color. Its accumulation in the skin can cause spots, freckles and uneven skin color. In other words, inhibiting the activity of tyrosinase is the main mechanism of whitening.

[0004] Therefore, it is of great significance to develop a camellia peptide that can inhibit tyrosinase activity, reduce melanin production, and inhibit melanocyte proliferation for use in whitening products. Summary of the invention

[0005] The purpose of the present invention is to overcome the shortcomings of the prior art and provide an application of camellia peptide in whitening products.

[0006] To achieve the above object, the technical solution adopted by the present invention is:

[0007] In a first aspect, the present invention provides an application of a camellia peptide in a whitening product, wherein the amino acid sequence of the camellia peptide is at least one of Leu-Pro-Phe (LPF, leucine-proline-phenylalanine), Leu-Leu-Leu-Leu-Gly-His (LLLLGH, leucine-leucine-leucine-leucine-glycine-histidine), and Ala-Pro (AP, alanine-proline).

[0008] The camellia peptide of the present invention achieves whitening by inhibiting the activity of tyrosinase, reducing the production of melanin, and inhibiting the proliferation of melanocytes. Specifically, the camellia peptide of the present invention inhibits the activity of tyrosinase by competing with tyrosinase, and the oxidation of substrate by tyrosinase, thereby reducing the production of melanin, thereby achieving the purpose of whitening; at the same time, the camellia peptide of the present invention reduces the activity of melanocytes in the skin and inhibits the proliferation of melanocytes by interfering with the growth cycle of melanocytes, thereby reducing the production of melanin, thereby achieving the purpose of whitening.

[0009] In the present invention, the definition of whitening refers to the ability to brighten skin tone, even skin tone, reduce pigmentation, lighten spots, improve dullness, increase gloss, and enhance delicateness.

[0010] The camellia peptide of the present invention is an active amino acid sequence released by enzymatic hydrolysis of camellia protein, and has a whitening effect; compared with macromolecular proteins, the camellia peptide is a short-chain amino acid, which not only has a stable structure but also has a small molecular weight and is easily absorbed through the skin; in addition, compared with animal peptides, the camellia peptide has extremely low immunogenicity because it is an active peptide derived from plants; most importantly, the amino acid structure of the camellia peptide has good adaptability to skin tissue, so it is safe and non-toxic and can be used for a long time.

[0011] Preferably, the product is at least one of daily chemical products, medicines, health care products, and biological products.

[0012] In the present invention, the forms or carriers of the daily chemical products include but are not limited to toner, moisturizing lotion, facial mask, facial cream, pressed powder, shower gel, soap, liquid foundation, foundation cream, essence, hand cream, body lotion, hand soap, shampoo, conditioner, facial cleanser, facial cleanser, sunscreen, moisturizing cream, vanishing cream, nutrient solution, perfume, etc.

[0013] Preferably, the dosage form of the product is at least one of a liquid dosage form, a solid dosage form, a semisolid dosage form, and a gas dosage form.

[0014] Preferably, the mass content of camellia peptide in the whitening product is 0.001-100%.

[0015] In the present invention, when the camellia peptide is used in a whitening product, the camellia peptide can also be used together with other ingredients that also have whitening effects to develop whitening products in the form of a composition; the ingredients with whitening effects include but are not limited to: at least one of arbutin, vitamin C, kojic acid, niacinamide, licorice extract, vitamin E, glutathione, ferulic acid, ceramide, ursolic acid, ellagic acid, fruit acid, and salicylic acid.

[0016] In a second aspect, the present invention provides an application of a camellia peptide in inhibiting tyrosinase activity and / or reducing melanin production and / or inhibiting melanocyte proliferation, wherein the amino acid sequence of the camellia peptide is at least one of Leu-Pro-Phe (LPF, leucine-proline-phenylalanine), Leu-Leu-Leu-Leu-Gly-His (LLLLGH, leucine-leucine-leucine-leucine-glycine-histidine), and Ala-Pro (AP, alanine-proline).

[0017] Preferably, the camellia peptide contains Leu-Pro-Phe, Leu-Leu-Leu-Leu-Gly-His, and Ala-Pro in a mass ratio of (93.5-94):(0.4-0.5):(5.4-6).

[0018] Preferably, the molecular weight of each component in the camellia peptide is ≤3 kDa.

[0019] Preferably, the preparation method of the camellia peptide comprises the following steps:

[0020] S1. Firstly, the camellia is extracted with alkali, and then acid is added for precipitation to obtain camellia crude protein;

[0021] S2. hydrolyzing the camellia crude protein with a protease to obtain a protease hydrolysate;

[0022] S3. Ultrafilter the protein hydrolysate and retain the ultrafiltration component with a molecular weight of ≤3kDa to obtain camellia peptide.

[0023] In the preparation process of camellia peptide of the present invention, the reason why the ultrafiltration component with a molecular weight of ≤3kDa is retained is that, compared with the camellia hydrolysate in the protease hydrolysate and the component with a molecular weight >3kDa, the component with a molecular weight of ≤3kDa in the camellia peptide is more easily taken up and utilized by fibroblasts, and can better improve the functional homeostasis of dermal fibroblasts.

[0024] More preferably, in step S1, the camellia raw material includes whole camellia flowers and residues produced by preparing essences, essential oils or extracts of camellia.

[0025] More preferably, in step S1, the camellia is firstly subjected to natural air-drying or drying before soaking, and then subjected to pulverization.

[0026] More preferably, the fixing and drying is first fixing at 80-100°C and then drying at 50-60°C.

[0027] More preferably, after the pulverization process, the product is sieved through a 40-60 mesh sieve.

[0028] More preferably, in step S1, the alkali is at least one of sodium hydroxide solution, potassium hydroxide solution, calcium hydroxide solution, sodium carbonate solution and sodium bicarbonate solution.

[0029] More preferably, the concentration of the sodium hydroxide solution is 0.2-2 mol / L.

[0030] More preferably, in step S1, the solid-to-liquid ratio of the camellia and alkali is 1 g:(15-20) mL.

[0031] More preferably, in step S1, the extraction is performed at least once.

[0032] More preferably, in step S1, the extraction temperature is 50-60°C.

[0033] More preferably, in step S1, the pH of the acid added for precipitation is 3.0-4.0.

[0034] More preferably, in step S1, the acid is at least one of hydrochloric acid, sulfuric acid and nitric acid.

[0035] More preferably, in step S2, the protease is at least one of papain, trypsin, flavor protease and bromelain.

[0036] More preferably, in step S2, the pH of the enzymatic hydrolysis is 6.0-8.0.

[0037] More preferably, in step S2, the pH adjuster for enzymatic hydrolysis is at least one of sodium hydroxide, potassium hydroxide, calcium hydroxide, hydrochloric acid, sulfuric acid, acetic acid, citric acid, and phosphoric acid.

[0038] More preferably, in step S3, the molecular weight of the ultrafiltration component is 0-3 kDa.

[0039] More preferably, the step S1 specifically comprises: adding camellia to alkali for extraction, taking the supernatant, adding acid for precipitation, collecting the precipitate, and obtaining camellia crude protein.

[0040] More preferably, the step S2 specifically comprises: adding camellia crude protein to water, adjusting the pH, performing enzymatic hydrolysis, then inactivating the enzyme by heating the water bath, centrifuging, taking the supernatant, and obtaining an enzymatic hydrolyzate.

[0041] More preferably, the solid-to-liquid ratio of the camellia crude protein and water is (1-2) g: (10-20) mL.

[0042] More preferably, the step S3 specifically comprises: separating the protein hydrolysate with an ultrafiltration membrane with a molecular weight of ≤3 kDa, retaining the ultrafiltration component with a molecular weight of ≤3 kDa, and drying to obtain the camellia peptide.

[0043] Compared with the prior art, the present invention has the following beneficial effects:

[0044] The camellia peptide of the present invention achieves whitening by inhibiting the activity of tyrosinase, reducing the production of melanin, and inhibiting the proliferation of melanocytes. Specifically, the camellia peptide of the present invention inhibits the activity of tyrosinase by competing with tyrosinase, and the oxidation of substrate by tyrosinase, thereby reducing the production of melanin, thereby achieving the purpose of whitening; at the same time, the camellia peptide of the present invention reduces the activity of melanocytes in the skin and inhibits the proliferation of melanocytes by interfering with the growth cycle of melanocytes, thereby reducing the production of melanin, thereby achieving the purpose of whitening.

[0045] The camellia peptide of the present invention is an active amino acid sequence released by enzymatic hydrolysis of camellia protein, and has a whitening effect; compared with macromolecular proteins, the camellia peptide is a short-chain amino acid, which not only has a stable structure but also has a small molecular weight and is easily absorbed through the skin; in addition, compared with animal peptides, the camellia peptide has extremely low immunogenicity because it is an active peptide derived from plants; most importantly, the amino acid structure of the camellia peptide has good adaptability to skin tissue, so it is safe and non-toxic and can be used for a long time. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 This is the HPLC BPC diagram of camellia peptide.

[0047] Figure 2 This is the secondary mass spectrum of the amino acid sequence LPF of camellia peptide.

[0048] Figure 3 This is the secondary mass spectrum of the amino acid sequence LLLLGH of camellia peptide.

[0049] Figure 4 This is the secondary mass spectrum of the amino acid sequence AP of camellia peptide. DETAILED DESCRIPTION

[0050] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0051] Unless otherwise specified, other materials, reagents, etc. used in the examples can be obtained from commercial sources.

[0052] Mouse skin melanoma cells (B16-F10) were purchased from the Cell Bank of the Chinese Academy of Sciences with the catalog number TCM36.

[0053] Example 1

[0054] 1. Preparation of Camellia Peptide

[0055] This embodiment provides a method for preparing camellia peptide, comprising the following steps:

[0056] S1. Take the whole camellia flower, air dry it naturally, crush it through a 40-mesh sieve, take 100g, add 2000mL 0.3mol / L NaOH solution, extract it in a 55℃ water bath for 1 hour, extract it twice, combine the extracts, centrifuge at 10000r / min for 10min, take the supernatant, add 0.5mol / L HCl solution to adjust to pH=3.0 for precipitation, centrifuge at 10000r / min for 10min, discard the supernatant, collect the precipitate, and obtain camellia crude protein;

[0057] S2. Add 300 g of camellia crude protein to 2400 mL of water, stir evenly, adjust the pH to 7.0 with 2 mol / L sodium hydroxide solution, add 6000 U / g papain (6000 U of protease per g of camellia crude protein) in a 50°C water bath, stir at a speed of 300 r / min, and perform enzymolysis for 4 h; after the enzymolysis is completed, heat in a water bath at 90°C for 15 min to inactivate the enzyme, cool to room temperature, centrifuge at 10000 rpm for 10 min, take the supernatant, and obtain the enzymolysis solution;

[0058] S3. The protein hydrolysate was separated by using a SUEZ ultrafiltration membrane (material: TFM) with a molecular weight of 3 kDa using a WTM-1812G-2 laboratory membrane separation equipment (Hefei Woteng Membrane Separation Equipment Co., Ltd.), and the ultrafiltration component with a molecular weight of ≤3 kDa was retained. Then, the product was spray-dried using an LPG-5 high-speed centrifugal spray dryer at an inlet air temperature of 150-180°C and an outlet air temperature of 80-100°C to obtain camellia peptide. The molecular weight of each component in the camellia peptide was ≤3 kDa.

[0059] 2. Analysis of amino acid content of camellia peptide

[0060] The amino acid content of the prepared camellia peptide was analyzed according to GB 5009.124-2016 standard "Determination of Amino Acids in Foods", using an S7130 amino acid fully automatic analyzer (SYKAM, Germany), with a separation column of Cation Separation Column LCA K06 / Na, 150 mm × 4.6 mm; an ammonia removal column of Ammonia Filtration Column LCA K04 / Na, 100 mm × 4.6 mm; the determination principle is that the protein in the food is hydrolyzed by hydrochloric acid to become free amino acids, which are separated by the ion exchange column of the amino acid analyzer and then react with the ninhydrin solution to produce a color reaction, which is detected at its maximum absorption peak wavelength of 570 nm and 440 nm, and the amino acid composition and content data of the camellia peptide are obtained based on the retention time and peak area; the experimental results are shown in the following table:

[0061] Table 1 Amino acid content of camellia peptide

[0062] Serial number Amino Acids percentage / % Serial number Amino Acids percentage / % 1 Aspartic acid 10.92 9 Leucine 10.08 2 Threonine 4.25 10 Tyrosine 3.35 3 Serine 10.09 11 Phenylalanine 4.58 4 Glutamate 17.51 12 Histidine 2.34 5 Glycine 4.98 13 Lysine 4.67 6 Alanine 5.80 14 Arginine 4.90 7 Valine 5.35 15 Proline 6.59 8 Isoleucine 4.59 total 100

[0063] 3. Identification of the amino acid sequence of camellia peptide

[0064] The amino acid sequence of the prepared camellia peptide was identified by HPLC / MS / MS method. The specific detection method is as follows: X500LC-ESI-Q-TOF high-resolution liquid chromatography-mass spectrometer (AB SCIEX, USA) was used to separate and detect the sample; the liquid chromatography and mass spectrometry control software used SCIEX OS2.0 integrated version (AB SCIEX, USA); the chromatographic column used was 1×100mmHSS T3 (1.8μm, Waters, USA).

[0065] The mobile phase consisted of 0.1% (v / v) formic acid in water (A) and acetonitrile (B), and the elution program was: 0-4.00min 5.0% B, 4.00-6.00min 5.0-10.0% B, 6.00-30.00min 10.0-40.0% B, 30.00-34.00min 40.0-90.0% B, 34.00-40.00min 90% B, 40.00-42.00min 90.0-5.0%B, 42.00-52.00min5.0%B, flow rate 0.05mL / min, injection volume 1μL, column temperature 40℃; mass spectrometry detection method: scan cycle 0.642s, ESI ion source temperature 500℃, positive ion mode, spray voltage 5500V, TOF primary scan range 100-1200Da, secondary scan range 50-1200Da, working mode IDA, maximum number of candidate ions 4, dynamic exclusion turned on, and the rest of the parameters used the default values ​​of the proteomics method. Note that the dead volume of the instrument should be minimized before use. The experimental results are as follows Figure 1-4 As shown in Table 2:

[0066] Table 2 Amino acid sequence identification results of camellia peptides

[0067]

[0068]

[0069] Note: in the above table, "LPF" refers to Leu-Pro-Phe (Leucine-Proline-Phenylalanine), "LLLLGH" refers to Leu-Leu-Leu-Leu-Gly-His (Leucine-Leucine-Leucine-Leucine-Glycine-Histidine), and "AP" refers to Ala-Pro (Alanine-Proline). The relative content of LPF = peak area of ​​LPF / (peak area of ​​AP + peak area of ​​LLLLGH + peak area of ​​LPF) × 100%, the relative content of LLLLGH = peak area of ​​LLLLGH / (peak area of ​​AP + peak area of ​​LLLLGH + peak area of ​​LPF) × 100%, the relative content of AP = peak area of ​​AP / (peak area of ​​AP + peak area of ​​LLLLGH + peak area of ​​LPF) × 100%.

[0070] Figure 1 This is the HPLC BPC diagram of camellia peptide; Figure 2 This is the secondary mass spectrum of the amino acid sequence LPF of camellia peptide; Figure 3 This is the secondary mass spectrum of the amino acid sequence LLLLGH of camellia peptide; Figure 4 This is the secondary mass spectrum of the amino acid sequence AP of camellia peptide.

[0071] from Figure 1-4 As can be seen from Table 1, the amino acid sequence of the camellia peptide of the present invention includes LPF, LLLLGH and AP, and the camellia peptide contains Leu-Pro-Phe, Leu-Leu-Leu-Leu-Gly-His, and Ala-Pro in a mass ratio of 93.5:0.5:6.0.

[0072] Example 2

[0073] Tyrosinase is a key rate-limiting enzyme in the process of melanin synthesis, and the inhibition of its activity can effectively reduce the synthesis of melanin. Mushroom tyrosinase has significant similarity and homology with human tyrosinase in structure and function, and is often used to screen tyrosinase inhibitors and study the mechanism of melanin production. In order to analyze the tyrosinase inhibitory activity of camellia peptide, the present invention uses mushroom tyrosinase and L-DOPA substrate system to evaluate the inhibitory activity of camellia peptide on tyrosinase. The specific experimental process is as follows:

[0074] (1) The camellia peptide prepared in Example 1 was dissolved in water to prepare camellia peptide solutions with concentrations of 25 μg / mL, 50 μg / mL and 100 μg / mL, respectively, and arbutin was dissolved in water to prepare an arbutin solution with a concentration of 10 mM;

[0075] (2) 150 μL of 0.1M pH=6.8 phosphate buffer and 20 μL of 5mM L-DOPA solution were mixed evenly, and then 20 μL of 10mM arbutin solution, 0.1M pH=6.8 phosphate buffer, and camellia peptide solutions of different concentrations were added, respectively. Among them, the experiment with 25 μg / mL camellia peptide solution was used as the low-dose group in the experimental group, the experiment with 50 μg / mL camellia peptide solution was used as the medium-dose group in the experimental group, the experiment with 100 μg / mL camellia peptide solution was used as the high-dose group in the experimental group, the experiment with 10mM arbutin solution was used as the positive control group, and the experiment with 0.1M phosphate buffer was used as the blank control group;

[0076] (3) 100 μL of 250 U / mL mushroom tyrosinase was added to each group of experiments, mixed well, and reacted in a 37°C incubator for 15 min. The absorbance at 475 nm was detected using an ELISA reader. The experiment was repeated 3 times, and the tyrosinase inhibition rate was calculated according to the formula [tyrosinase inhibition rate of the drug-treated group (%) = (1-absorbance of the drug-treated group / absorbance of the blank control group) × 100%, where the drug-treated group was the experimental group or the positive control group];

[0077] The experimental results are shown in the following table:

[0078] Table 3 Tyrosinase inhibition rate of camellia peptide

[0079]

[0080] As can be seen from Table 3, compared with the blank control group, the experimental group containing the camellia peptide of the present invention can significantly inhibit the activity of tyrosinase. Therefore, this shows that the camellia peptide of the present invention can inhibit the activity of tyrosinase by competing with tyrosinase, tyrosinase oxidation of the substrate, and then reduce the production of melanin to achieve the purpose of whitening.

[0081] Example 3

[0082] The present invention tests the effect of the camellia peptide prepared in Example 1 on melanin production, and the specific test method is as follows:

[0083] (1) α-MSH was dissolved in a DMEM medium containing 10% FBS to prepare an induction medium containing 100 nM α-MSH; then, the camellia peptide prepared in Example 1 was dissolved in an induction medium containing 100 nM α-MSH to prepare DMEM medium containing camellia peptide at concentrations of 0 μg / mL, 25 μg / mL, 50 μg / mL and 100 μg / mL; arbutin was dissolved in an induction medium containing 100 nM α-MSH to prepare a DMEM medium containing arbutin at a concentration of 10 mM;

[0084] (2) Mouse skin melanoma cells B16-F10 were cultured at 2×10 5 The cells were inoculated in a 6-well plate at a density of cells / well, and each well was cultured with 2 mL of DMEM medium containing 10% FBS for 24 h. The medium was discarded, and then DMEM medium containing 10% FBS, arbutin DMEM medium, and DMEM medium with different concentrations of camellia peptide were added respectively. Among them, the experiment with 0 μg / mL of camellia peptide DMEM medium was used as the α-MSH group, the experiment with 25 μg / mL of camellia peptide DMEM medium was used as the low-dose group in the experimental group, and the experiment with 50 μg / mL of camellia peptide DMEM medium was used as the low-dose group in the experimental group. The experiment with the culture medium was used as the medium-dose group in the experimental group, the experiment with the addition of 100 μg / mL of camellia peptide DMEM culture medium was used as the high-dose group in the experimental group, the experiment with the addition of 10 mM arbutin DMEM culture medium was used as the positive control group, and the experiment with the addition of DMEM culture medium containing 10% FBS was used as the blank control group; then the cells were cultured at 37°C and 5% CO2 for 72 h, and the cells were washed twice with 2 mL of cold PBS (NaCl, 137 mM; KCl, 2.7 mM; Na2HPO4, 10 mM; KH2PO4, 1.8 mM; pH 7.4), the cells were digested with trypsin, and the cell pellets were collected by centrifugation at 1000 rpm for 5 min, the cells were resuspended with 2 mL of PBS, and the cells were collected by centrifugation at 1000 rpm for 5 min to obtain cell pellets;

[0085] (3) 1 mL of 1 mol / L NaOH solution (the solvent is DMSO and water in a volume ratio of 1:9) was added to the cell pellet, and the cells were lysed by heating at 90°C for 30 min to obtain a lysate, and the absorbance of the lysate at 405 nm was detected using an ELISA reader; the experiment was repeated 3 times, and the relative melanin content was calculated according to the formula [relative melanin content of the drug-treated group (%) = (1-absorbance of the drug-treated group / average absorbance of the α-MSH group) × 100%, the drug-treated group was the experimental group or the positive control group or the blank control group];

[0086] The experimental results are shown in the following table:

[0087] Table 4 Relative melanin content of camellia peptides

[0088]

[0089] Note: In the above table, compared with the blank control group, ***P<0.001; compared with the α-MSH group, # P<0.05, ### P<0.001.

[0090] As can be seen from Table 4, compared with the α-MSH group, the experimental group containing the camellia peptide of the present invention can significantly reduce the production of melanin, which is beneficial to achieve whitening.

[0091] In addition, it can be seen from Table 3 and Table 4 that the camellia peptide of the present invention can inhibit the activity of tyrosinase by competing with tyrosinase, and the oxidation of substrate by tyrosinase, thereby reducing the production of melanin and achieving the purpose of whitening.

[0092] Example 4

[0093] The present invention tests the effect of the camellia peptide prepared in Example 1 on melanocyte proliferation, and the specific test method is as follows:

[0094] Mouse skin melanoma B16-F10 cells were cultured at 5 × 10 3 The cells were inoculated in a 96-well plate at a density of 10 cells / well, and each well was cultured with 100 μL of DMEM medium containing 10% FBS for 24 h, the medium was discarded, and then 100 μL of DMEM medium containing 10% FBS with camellia peptide concentrations of 0 μg / mL, 25 μg / mL, 50 μg / mL and 100 μg / mL were added, respectively. Among them, the experiment with the added camellia peptide concentration of 0 μg / mL was used as the blank control group, the experiment with the added camellia peptide concentration of 25 μg / mL was used as the low-dose group in the experimental group, the experiment with the added camellia peptide concentration of 50 μg / mL was used as the medium-dose group in the experimental group, and the camellia peptide concentration of 100 μg / mL was used as the medium-dose group in the experimental group. The experiment with a concentration of 100 μg / mL was used as the high-dose group in the experimental group; then, after culturing for 48 hours at 37°C and 5% CO2, 20 μL of 5 mg / mL MTT solution was added to each well, and the culture was continued for 3 hours. The solution was discarded, and 150 μL of DMSO was added to each well to dissolve the crystals, and the wells were placed on an oscillator and shaken for 5 minutes. The absorbance of each well at 570 nm was detected using an enzyme marker; the experiment was repeated 6 times, and the cell viability was calculated according to the formula [cell viability of the experimental group (%) = absorbance of the experimental group / average absorbance of the blank control group × 100%]; the smaller the cell viability value, the better the inhibitory effect of camellia peptide on melanocyte proliferation;

[0095] The experimental results are shown in the following table:

[0096] Table 5 Cell viability of camellia peptides

[0097]

[0098] Note: In the above table, compared with the blank control group, **P<0.01, ***P<0.001.

[0099] As can be seen from Table 5, compared with the blank control group, the experimental group containing the camellia peptide of the present invention can significantly reduce the cell viability of melanocytes and inhibit the proliferation of melanoma cells. Therefore, this shows that the camellia peptide of the present invention reduces the activity of melanocytes in the skin and inhibits the proliferation of melanocytes by interfering with the growth cycle of melanocytes, thereby reducing the production of melanin and achieving the purpose of whitening.

[0100] Example 5

[0101] In order to clarify the whitening effect of the amino acid sequences Leu-Pro-Phe (LPF), Leu-Leu-Leu-Leu-Gly-His (LLLLGH), and Ala-Pro (AP) in camellia peptides, the inventors obtained the corresponding amino acid sequences by chemical synthesis and conducted experimental evaluation. The solid phase synthesis of the amino acid sequences LPF, LLLLGH, and AP was commissioned to a biological company. The effects of the amino acid sequences LPF, LLLLGH, and AP on tyrosinase activity, melanin production, and melanocyte proliferation were evaluated according to the experimental procedures described in Examples 2-4, respectively. The dosage of LPF, LLLLGH, and AP amino acid sequences was 20 μg / mL, and the experiment using the LPF amino acid sequence was used as the LPF group in the experimental group, the experiment using the LLLLGH amino acid sequence was used as the LLLLGH group in the experimental group, and the experiment using the AP amino acid sequence was used as the AP group in the experimental group; the experimental results are as follows:

[0102] Table 6 Tyrosinase inhibition rate of amino acid sequences LPF, LLLLGH, AP

[0103] Group Tyrosinase inhibition rate (%) Blank control group / LPF Group 75.5±2.46 LLLLGH group 66.8±2.77 AP Group 74.2±2.93

[0104] Table 7 Relative melanin content of amino acid sequences LPF, LLLLGH, AP

[0105] Group Relative melanin content (%) Blank control group 20.0±1.30 α-MSH group 100±1.06*** LPF Group <![CDATA[26.3±2.28 ### ]]> LLLLGH group <![CDATA[34.3±2.40 ### ]]> AP Group <![CDATA[27.5±2.76 ### ]]>

[0106] Note: In the above table, compared with the blank control group, ***P<0.001; compared with the α-MSH group, ### P<0.001.

[0107] Table 8 Cell viability of amino acid sequences LPF, LLLLGH, AP

[0108]

[0109]

[0110] Note: In the above table, compared with the blank control group, ***P<0.001.

[0111] From Table 6-8, we can see that the amino acid sequences LPF, LLLLGH, and AP have strong inhibitory activity on tyrosinase, and can significantly reduce the generation of melanin and inhibit the proliferation of melanocytes, thereby achieving the purpose of whitening. The above data suggest that the amino acid sequences LPF, LLLLGH, and AP in camellia peptides can inhibit the generation of melanin, help reduce the accumulation of melanin in skin cells and tissues, and ultimately achieve the purpose of whitening the skin.

[0112] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. An application of camellia peptide in whitening products, characterized in that: The amino acid sequence of the camellia peptide is at least one of Leu-Pro-Phe, Leu-Leu-Leu-Leu-Gly-His, and Ala-Pro.

2. The use according to claim 1, characterized in that: The product is at least one of daily chemical products, medicines, health care products, and biological products.

3. The use according to claim 1, characterized in that: The dosage form of the product is at least one of a liquid dosage form, a solid dosage form, a semisolid dosage form, and a gas dosage form.

4. A use of a camellia peptide in inhibiting tyrosinase activity and / or reducing melanin production and / or inhibiting melanocyte proliferation, characterized in that: The amino acid sequence of the camellia peptide is at least one of Leu-Pro-Phe, Leu-Leu-Leu-Leu-Gly-His, and Ala-Pro.

5. The use according to any one of claims 1 to 4, characterized in that: The camellia peptide contains Leu-Pro-Phe, Leu-Leu-Leu-Leu-Gly-His and Ala-Pro in a mass ratio of (93.5-94):(0.4-0.5):(5.4-6).

6. The use according to any one of claims 1 to 4, characterized in that: The molecular weight of each component in the camellia peptide is ≤3kDa.

7. The use according to any one of claims 1 to 4, characterized in that: The preparation method of the camellia peptide comprises the following steps: S1. Firstly, the camellia is extracted with alkali, and then acid is added for precipitation to obtain camellia crude protein; S2. hydrolyzing the camellia crude protein with a protease to obtain a protease hydrolysate; S3. Ultrafilter the protein hydrolysate and retain the ultrafiltration component with a molecular weight of ≤3kDa to obtain camellia peptide.

8. The use according to claim 7, characterized in that In step S1, the camellia raw materials include whole camellia flowers and residues produced by preparing essences, essential oils or extracts of camellia.

9. The use according to claim 7, characterized in that: In step S1, the extraction is performed at least once.

10. The use according to claim 7, characterized in that: In step S2, the protease is at least one of papain, trypsin, flavor protease and bromelain.

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