Application of a camellia peptide in a whitening product

Abstract

The present invention discloses the application of camellia peptides in whitening products, and the amino acid sequences of the camellia peptides are at least one of Leu-Pro-Phe, Leu-Leu-Leu-Leu-Gly-His, and Ala-Pro. The camellia peptides of the present invention achieve whitening by inhibiting the activity of tyrosinase, reducing the production of melanin, and inhibiting the proliferation of melanocytes. Specifically, the camellia peptides of the present invention inhibit the activity of tyrosinase by competing with tyrosinase for the oxidation of substrates by tyrosinase, thereby reducing the production of melanin and achieving the purpose of whitening; at the same time, the camellia peptides of the present invention reduce the activity of melanocytes in the skin and inhibit the proliferation of melanocytes by interfering with the growth cycle of melanocytes, thereby reducing the production of melanin and achieving the purpose of whitening.

Description

Technical Field

[0001] The present invention relates to the technical fields of biotechnology and skin health technology, and particularly relates to the application of camellia peptides in whitening products. Background Art

[0002] Camellia japonica L. is an evergreen broad-leaved tree species, a shrub or small tree plant of the genus Camellia in the family Theaceae, and belongs to the tree species in tropical and subtropical climates. China is a major country in camellia cultivation, and the vast majority are distributed in areas south of the Yangtze River such as Zhejiang, Jiangxi, Guangxi, Yunnan, Guizhou, Hunan, and Guangdong.

[0003] Tyrosinase is a copper-containing enzyme that is widely present in animals, plants, and microorganisms and is a key enzyme in the process of melanin production. It catalyzes the oxidation of tyrosine to dopa, and then further oxidizes it to dopaquinone, and finally generates melanin. Melanin is the main determinant of the color of the skin, hair, and eyes, and its accumulation in the skin will cause problems such as age spots, freckles, and uneven skin tone. In other words, inhibiting the activity of tyrosinase is the main mechanism of whitening.

[0004] Therefore, it is of great significance to develop the application of camellia peptides that can inhibit the activity of tyrosinase, reduce melanin production, and inhibit the proliferation of melanocytes in whitening products. Summary of the Invention

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

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

[0007] In the first aspect, the present invention provides the application of camellia peptides in whitening products, and the amino acid sequences of the camellia peptides are 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 for the oxidation of the substrate, thereby reducing the production of melanin and 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 and 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, fade age spots, improve dullness, increase luster, and enhance smoothness.

[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 is not only structurally stable, but also has a small molecular weight and is easy to be absorbed through the skin; in addition, compared with animal peptides, the camellia peptide has extremely low immunogenicity because it is a plant-derived active peptide; 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, drugs, 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, cream, powder cake, body wash, soap, liquid foundation, cream foundation, essence, hand cream, body lotion, hand sanitizer, shampoo, hair conditioner, facial cleanser, facial wash, sunscreen, emollient cream, vanishing cream, nutrient solution, perfume, etc.

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

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

[0015] In the present invention, when the camellia peptide is applied to a whitening product, the camellia peptide can also be developed into a whitening product in the form of a composition together with other ingredients that also have a whitening effect; the ingredients that have a whitening effect 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 the use 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).

[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. First, extract camellia with an alkali, and then add an acid for precipitation to obtain crude camellia protein;

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

[0022] S3. Ultrafilter the protease hydrolysate, and retain the ultrafiltration fraction with a molecular weight cut-off ≤3 kDa to obtain the camellia peptide.

[0023] In the preparation process of the camellia peptide of the present invention, the ultrafiltration fraction with a molecular weight cut-off ≤3 kDa is retained because, compared with the camellia enzyme hydrolysate in the protease hydrolysate and the components with a molecular weight >3 kDa, the components with a molecular weight ≤3 kDa in the camellia peptide are 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 raw material of the camellia includes the whole camellia flower and the residue generated from the preparation of camellia for perfume, essential oil or extract.

[0025] More preferably, in step S1, the camellia is naturally air-dried or blanched and dried before extraction, and then pulverized.

[0026] Even more preferably, the blanching and drying is carried out by blanching at 80 - 100°C first and then drying at 50 - 60°C.

[0027] Even more preferably, after the pulverization treatment, it is sieved through a 40 - 60 mesh sieve.

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

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

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

[0031] More preferably, in step S1, the number of leaching times is at least 1 time.

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

[0033] More preferably, in step S1, the pH for precipitation by adding acid 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 for enzymatic hydrolysis is 6.0 - 8.0.

[0037] More preferably, in step S2, the pH regulator 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 fraction is 0 - 3 kDa.

[0039] More preferably, step S1 is specifically: adding camellia flower to the base for leaching, taking the supernatant, then adding acid for precipitation, and collecting the precipitate to obtain crude camellia protein.

[0040] More preferably, step S2 is specifically: adding crude camellia protein to water, adjusting the pH, performing enzymatic hydrolysis, then inactivating the enzyme by heating in a water bath, centrifuging, and taking the supernatant to obtain protease hydrolysate.

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

[0042] More preferably, step S3 is specifically: separating the protease hydrolysate with an ultrafiltration membrane with a molecular weight cut-off ≤ 3 kDa, retaining the ultrafiltration fraction with a molecular weight ≤ 3 kDa, and drying to obtain camellia peptides.

[0043] Compared with the prior art, the beneficial effects of the present invention are as follows:

[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 for the oxidation of the substrate, thereby reducing the production of melanin and 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 and achieving the purpose of whitening.

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

[0046] Figure 1 It is the liquid chromatography-mass spectrometry (LC-MS) base peak chromatogram (BPC) of camellia peptide.

[0047] Figure 2 It is the second-order mass spectrum of the amino acid sequence LPF of camellia peptide.

[0048] Figure 3 It is the second-order mass spectrum of the amino acid sequence LLLLGH of camellia peptide.

[0049] Figure 4 It is the second-order mass spectrum of the amino acid sequence AP of camellia peptide. DETAILED DESCRIPTION OF THE INVENTION

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

[0051] Other materials, reagents, etc. used in the examples can be obtained from commercial channels without special instructions.

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

[0053] Example 1

[0054] 1. Preparation of camellia peptide

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

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

[0057] S2. Add 300 g of crude camellia protein to 2400 mL of water, stir evenly, adjust the pH to 7.0 with 2 mol / L sodium hydroxide solution, under the condition of a water bath at 50 °C, add 6000 U / g of papain (add 6000 U of protease per g of crude camellia protein), stir at a speed of 300 r / min, and enzymatically hydrolyze for 4 h; after the enzymatic hydrolysis, 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 protease hydrolysate;

[0058] S3. Use a WTM-1812G-2 laboratory membrane separation device (Hefei Wolten Membrane Separation Equipment Co., Ltd.) to separate the protease hydrolysate with a SUEZ ultrafiltration membrane with a molecular weight of 3 kDa (material: TFM), retain the ultrafiltration components with a molecular weight cut-off ≤ 3 kDa, and then perform spray drying with an LPG-5 high-speed centrifugal spray dryer under the conditions of an inlet air temperature of 150 - 180 °C and an outlet air temperature of 80 - 100 °C to obtain camellia peptide; the molecular weights of all components in the camellia peptide are ≤ 3 kDa.

[0059] 2. Amino acid content analysis of camellia peptide

[0060] Analyze the amino acid content of the above-prepared camellia peptide according to the GB 5009.124-2016 standard "Determination Method of Amino Acids in Foods", using a S7130 automatic amino acid analyzer (SYKAM Company, Germany), with its separation column being Cation Separation Column LCA K06 / Na, 150 mm × 4.6 mm; the ammonia removal column being Ammonia Filtration Column LCA K04 / Na, 100 mm × 4.6 mm; the determination principle is that the protein in the food is hydrolyzed into free amino acids by hydrochloric acid, after being separated by the ion exchange column of the amino acid analyzer, it produces a color reaction with ninhydrin solution, and is detected at its maximum absorption peak wavelengths of 570 nm and 440 nm, and the amino acid composition and content data of 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 acid Percentage / % Serial number Amino acid 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 Glutamic acid 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. Amino acid sequence identification of camellia peptide

[0064] The amino acid sequence of the above-prepared camellia peptide was identified by HPLC / MS / MS method. The specific detection method is as follows: The X500LC-ESI-Q-TOF high-resolution liquid chromatography-mass spectrometry instrument (AB SCIEX, USA) was used to separate and detect the sample; The liquid chromatography and mass spectrometry control software used was SCIEX OS 2.0 integrated version (AB SCIEX, USA); The chromatographic column used was 1×100mm HSS T3 (1.8μm, Waters, USA).

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

[0066] Table 2 Results of amino acid sequence identification of camellia peptide

[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), "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%, and 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 is the liquid chromatography-mass spectrometry BPC chart of camellia peptide; Figure 2 is the secondary mass spectrometry chart of the amino acid sequence LPF of camellia peptide; Figure 3 is the secondary mass spectrometry chart of the amino acid sequence LLLLGH of camellia peptide; Figure 4 is the secondary mass spectrometry chart of the amino acid sequence AP of camellia peptide.

[0071] From Figures 1 - 4 and Table 1, it can be seen that 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, Ala-Pro with a mass ratio of 93.5:0.5:6.0.

[0072] Example 2

[0073] Tyrosinase is the 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 in structure and function with human tyrosinase, and is often used to screen tyrosinase inhibitors and study the mechanism of melanogenesis. In order to analyze the tyrosinase inhibitory activity of camellia peptide, the present invention uses a 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) Dissolve the camellia peptide prepared in Example 1 in water to prepare camellia peptide solutions with concentrations of 25 μg / mL, 50 μg / mL and 100 μg / mL respectively, and dissolve arbutin in water to prepare an arbutin solution with a concentration of 10 mM;

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

[0076] (3) Add 100 μL of 250 U / mL mushroom tyrosinase to each group of experiments respectively. After mixing evenly, react in a constant temperature incubator at 37 °C for 15 min, and use an enzyme-labeled instrument to detect the absorbance at 475 nm; The experiment is repeated 3 times, and the tyrosinase inhibition rate is calculated according to the formula [tyrosinase inhibition rate of the administration group (%) = (1 - absorbance of the administration group / absorbance of the blank control group) × 100%, and the administration group is 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, oxidize the substrate by tyrosinase, and then reduce the production of melanin to achieve the purpose of whitening.

[0081] Example 3

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

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

[0084] (2) Mouse skin melanoma cells B16-F10 were seeded in 6-well plates at a density of 2×10 5 cells / well and cultured in 2 mL of DMEM medium containing 10% FBS per well for 24 h. Then, the medium was discarded, and 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 DMEM medium containing 0 μg / mL of camellia peptide was used as the α-MSH group, the experiment with DMEM medium containing 25 μg / mL of camellia peptide was used as the low-dose group in the experimental group, the experiment with DMEM medium containing 50 μg / mL of camellia peptide was used as the medium-dose group in the experimental group, the experiment with DMEM medium containing 100 μg / mL of camellia peptide was used as the high-dose group in the experimental group, the experiment with DMEM medium containing 10 mM of arbutin was used as the positive control group, and the experiment with DMEM medium containing 10% FBS was used as the blank control group; then, the cells were cultured under the conditions of 37°C and 5% CO2 for 72 h, 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), digested with trypsin, and the cell pellet was collected by centrifugation at 1000 rpm for 5 min. The cells were resuspended in 2 mL of PBS and centrifuged again at 1000 rpm for 5 min to collect the cell pellet;

[0085] (3) 1 mL of 1 mol / L NaOH solution (the solvent is DMSO and water with 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. The absorbance of the lysate at 405 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader; the experiment was repeated 3 times, and the relative melanin content was calculated according to the formula [relative melanin content of the dosing group (%) = (1 - absorbance of the dosing group / average absorbance of the α-MSH group) × 100%, and the dosing 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 peptide

[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 achieving whitening.

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

[0092] Example 4

[0093] The present invention detects the effect of the camellia peptide prepared in Example 1 on the proliferation of melanocytes. The specific test method is as follows:

[0094] Mouse skin melanoma cells B16-F10 were seeded in a 96-well plate at a density of 5×10 3 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 experiment with the added camellia peptide concentration of 100 μg / mL was used as the high-dose group in the experimental group; then after culturing for 48 h under the conditions of 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 h. The solution was discarded, 150 μL of DMSO was added to each well to dissolve the crystals, and it was placed on a shaker and shaken well for 5 min. The absorbance of each well at 570 nm was detected using an enzyme-linked immunosorbent assay (ELISA) reader; 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 the proliferation of melanocytes;

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

[0096] Table 5 Cell viability of camellia peptide

[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, it 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 effects of the amino acid sequences Leu-Pro-Phe (LPF), Leu-Leu-Leu-Leu-Gly-His (LLLLGH), and Ala-Pro (AP) in the camellia peptide, the corresponding amino acid sequences were obtained by chemical synthesis and experimentally evaluated. 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. Among them, the dosage of the amino acid sequences LPF, LLLLGH, and AP was 20 μg / mL. 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 shown below:

[0102] Table 6 Tyrosinase inhibition rates of the amino acid sequences LPF, LLLLGH, and 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 contents of the amino acid sequences LPF, LLLLGH, and 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 viabilities of the amino acid sequences LPF, LLLLGH, and AP

[0108]

[0109]

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

[0111] As can be seen from Table 6-8, the amino acid sequences LPF, LLLLGH, and AP have potent inhibitory activity against tyrosinase, and can significantly reduce the production of black pigment 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 production 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 solutions of the present invention rather than to limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. An application of camellia peptide in the preparation of 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; 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. ultrafiltration of the protease hydrolysate, and retention of ultrafiltration fractions with a molecular weight cutoff of ≤3 kDa to obtain camellia peptide; In step S1, the leaching temperature is 50-60°C; In step S2, the protease is papain.

2. The use according to claim 1, characterized in that: The product is at least one of daily chemical products, medicines, 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 the preparation of a product for 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; 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. ultrafiltration of the protease hydrolysate, and retention of ultrafiltration fractions with a molecular weight cutoff of ≤3 kDa to obtain camellia peptide; In step S1, the leaching temperature is 50-60°C; In step S2, the protease is papain.

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: In step S1, the camellia raw materials include whole camellia flowers and residues produced by preparing essences, essential oils or extracts of camellia.

8. The use according to any one of claims 1 to 4, characterized in that: In step S1, the extraction is performed at least once.

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