Composition with whitening effect and preparation method and application thereof

Abstract

The application discloses a composition with whitening effect and a preparation method and application thereof, and comprises stevia rebaudiana B. extract, N-acetylneuraminic acid and tricholoma gobgyi alcohol extract with a mass ratio of (0.5-5):10:(3-22). The application can prepare an oral preparation with whitening effect by compounding stevia rebaudiana B. extract, N-acetylneuraminic acid and tricholoma gobgyi alcohol extract with a specific ratio, which can down-regulate the expression amount of tyrosinase gene, inhibit melanin secretion and reduce the activity of tyrosinase.

Description

Technical Field

[0001] This invention belongs to the field of composition preparation technology, specifically relating to compositions with whitening effects, their preparation methods, and applications. Background Technology

[0002] Skin tone primarily depends on the amount and distribution of melanin in the basal layer of the epidermis. Melanin synthesis is regulated by multiple factors, with tyrosinase being the key rate-limiting enzyme in the melanin synthesis pathway. Kojic acid and its derivatives, arbutin, and hydroquinone have tyrosinase inhibitory effects, but due to issues such as cytotoxicity, carcinogenicity, and genotoxicity, they are difficult to use in oral skin whitening products. Glycyrrhizin in licorice extract can directly inhibit tyrosinase activity, but it is easily degraded in the stomach and intestinal environment, affecting its whitening effect. Summary of the Invention

[0003] The purpose of this invention is to provide a composition with whitening effect, a method for preparing the same, and its application, so as to solve at least one aspect of the problems and defects mentioned in the background art.

[0004] To achieve the above objectives, the present invention provides the following technical solution: The composition with whitening effect includes stevia extract, N-acetylneuraminic acid and pleurotus ostreatus extract in a mass ratio of (0.5~5):10:(3~22).

[0005] As a further aspect of the present invention, the mass ratio of stevia extract, N-acetylneuraminic acid and physalis extract in the whitening composition is (0.5~5):10:(18~22).

[0006] The present invention also provides a method for preparing the composition having a whitening effect, comprising the following steps: Stevia extract, N-acetylneuraminic acid, and Pleurotus ostreatus extract were mixed to obtain a composition with whitening effect.

[0007] As a further aspect of the present invention, the stevia extract is obtained by first extracting stevia leaves with an aqueous ethanol solution of 30% to 60% by volume and then purifying them.

[0008] As a further aspect of the present invention, the material-to-liquid ratio of the first extraction is 1g:10~15mL.

[0009] As a further aspect of the present invention, the temperature of the first extraction is 30~40℃.

[0010] As a further aspect of the present invention, the first extraction time is 30~150 min.

[0011] As a further embodiment of the present invention, the first purification is performed by chromatography using polyamide resin and / or macroporous adsorption resin.

[0012] As a further aspect of the present invention, the pH of the extract during the first purification is 2.0 to 4.5.

[0013] As a further aspect of the present invention, an aqueous ethanol solution is used as the eluent during the first purification.

[0014] As a further embodiment of the present invention, the ethanol extract of Pleurotus ostreatus is obtained by second extraction of Pleurotus ostreatus with an aqueous ethanol solution of 50%~70% by volume, followed by second purification.

[0015] As a further aspect of the present invention, the material-to-liquid ratio of the second extraction is 1g:40~50mL.

[0016] As a further aspect of the present invention, the temperature of the second extraction is 40~50℃.

[0017] As a further aspect of the present invention, the second extraction time is 30~150 min.

[0018] As a further aspect of the present invention, the second purification uses an ultrafiltration membrane.

[0019] The present invention also provides the use of the whitening composition or the whitening composition obtained by the preparation method in the preparation of oral formulations for enhancing whitening effects.

[0020] As a further aspect of the present invention, the concentration of the whitening composition in the oral formulation is 150~1500µg / mL, preferably 250~1000µg / mL.

[0021] As a further aspect of the present invention, the oral preparation is one of a solution, granules, capsules, tablets, and ointment.

[0022] As a further aspect of the present invention, the enhanced whitening effect includes downregulating the expression level of the tyrosinase gene.

[0023] As a further aspect of the present invention, the enhanced whitening effect includes inhibiting melanin secretion.

[0024] As a further aspect of the present invention, the enhanced whitening effect includes reducing tyrosinase activity.

[0025] The present invention has at least the following technical effects: by using a specific ratio of stevia extract, N-acetylneuraminic acid and Pleurotus ostreatus extract to form an oral preparation with whitening effect, which can downregulate the expression of tyrosinase gene, inhibit melanin secretion and reduce tyrosinase activity. Attached Figure Description

[0026] To facilitate understanding by those skilled in the art, the present invention will be further described below with reference to the accompanying drawings.

[0027] Figure 1 This is a graph showing the inhibitory effects of stevia extract, N-acetylneuraminic acid, and Pleurotus ostreatus extract on tyrosinase activity in Experimental Example 2 of this invention. Figure 2 This is a graph showing the inhibitory effect of compositions a~e on tyrosinase activity in Experimental Example 2 of the present invention. This indicates that compared with the arbutin group, p<0.01. This indicates that compared with the arbutin group, p < 0.001; Figure 3 This is a typical image of the melanin signal intensity in the head of a zebrafish after sample treatment in Experiment Example 3 of this invention; Figure 4 This is an analysis diagram of the melanin signal intensity in the head of a zebrafish after sample treatment in Experimental Example 3 of this invention. This indicates that p < 0.05 compared to the model control group. This indicates that p < 0.001 compared to the model control group; Figure 5 This is a graph showing the OD475nm values ​​of zebrafish tyrosinase activity after sample treatment in Experimental Example 3 of this invention. This indicates that p < 0.05 compared to the model control group. This indicates that p < 0.01 compared to the model control group. This indicates that p < 0.001 compared to the model control group; Figure 6 This is a graph showing the relative expression level of the zebrafish tyr gene after sample treatment in Experiment Example 3 of this invention. This indicates that p < 0.001 compared to the model control group. Detailed Implementation

[0028] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are only for explaining the invention and are not intended to limit the invention; that is, the described embodiments are merely some embodiments of the invention, and not all embodiments.

[0029] Therefore, the following detailed description of embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0030] In some embodiments of the present invention, the mass ratio of stevia extract, N-acetylneuraminic acid and pleurotus ostreatus extract in the whitening composition is (0.5~5):10:(3~22).

[0031] In some embodiments of the present invention, the mass ratio of stevia extract, N-acetylneuraminic acid and pleurotus ostreatus extract in the whitening composition is (0.5~5):10:(18~22).

[0032] In some embodiments of the present invention, the mass ratio of stevia extract, N-acetylneuraminic acid and pleurotus ostreatus extract in the whitening composition is (2~5):10:(18~22).

[0033] In some embodiments of the present invention, the mass ratio of stevia extract, N-acetylneuraminic acid and pleurotus ostreatus extract in the whitening composition is (2.5~3.5):10:(19.5~20.5).

[0034] In some embodiments of the present invention, the method for preparing the stevia extract includes the following steps: Stevia leaves were extracted with an ethanol aqueous solution with a volume fraction of 40%~60% at a material-to-liquid ratio of 1g:10~15mL. The extraction was performed by ultrasonic extraction at 30~40℃ for 1~3 times, with each extraction time being 30~70min. The crude extract was obtained by filtration. The pH of the crude extract was adjusted to 2.0-4.5, and the mixture was subjected to chromatography with polyamide resin and macroporous adsorption resin in sequence. The eluent was eluted with 60%-80% ethanol aqueous solution, and the eluent was dried to obtain stevia extract.

[0035] In some embodiments of the present invention, the polyamide resin has a size of 30-60 mesh.

[0036] In some embodiments of the present invention, the macroporous adsorption resin is AB-8 type macroporous adsorption resin.

[0037] In some embodiments of the present invention, the preparation method of the Pleurotus ostreatus extract includes the following steps: Extract the golden top pine mushroom with 50%~60% ethanol at a material-to-liquid ratio of 1g:40~50mL, and ultrasonically extract at 40~50℃ for 1~3 times, with each extraction time being 20~50min. Filter to obtain crude extract. The crude extract was filtered through a 5-10 kDa ultrafiltration membrane and dried to obtain the physalis extract.

[0038] In the following examples, N-acetylneuraminic acid with a purity of ≥98% was provided by Yikelei Biotechnology (Group) Co., Ltd.

[0039] Example 1 This embodiment provides a method for preparing a composition with whitening effect, including the following steps: S1. Preparation of Stevia extract Take dried stevia leaves, crush them through a 60-mesh sieve, and extract them with 60% ethanol aqueous solution at a material-to-liquid ratio of 1g:15mL. Extract twice with ultrasound at 40℃, each extraction lasting 60min. After extraction, plate and frame filtration and centrifugation were performed, and the supernatant was collected to obtain the crude extract; Adjust the pH of the crude extract to 3.5-4.0, perform chromatography on a polyamide resin (30-60 mesh) column, elute with 80% ethanol aqueous solution, and collect the first eluent. After the first eluent is de-alcoholized, it is loaded onto an AB-8 macroporous adsorption resin column and eluted with 80% ethanol aqueous solution. The second eluent is then collected. The second eluent was concentrated under vacuum at 60°C, freeze-dried, pulverized, and passed through an 80-mesh sieve to obtain stevia extract.

[0040] S2. Preparation of Pleurotus ostreatus extract. Take fresh golden-topped lateral ear fruiting bodies, wash them, dry them at 50℃, and crush them through a 60-mesh sieve. Extract with 60% ethanol aqueous solution at a material-to-liquid ratio of 1g:50mL. Ultrasonic extraction was performed twice at 45℃ for 40min each time. After extraction, the mixture was filtered and centrifuged to remove mycelial residue and obtain crude extract. The crude extract was filtered using a 5kDa ultrafiltration membrane to remove macromolecular polysaccharides and proteins. The permeate was collected, decolorized and purified by activated carbon to obtain the purified solution. The purified solution was concentrated under reduced pressure and then freeze-dried to obtain the alcohol extract of Pleurotus ostreatus.

[0041] S3. Stevia extract was prepared into a 0.1 mg / mL solution using deionized water. N-acetylneuraminic acid was prepared into a 1 mg / mL solution using deionized water. The alcoholic extract of Pleurotus ostreatus was prepared into a 1 mg / mL solution using deionized water. Stevia extract solution, N-acetylneuraminic acid solution and Pleurotus ostreatus extract solution were mixed according to the volume amounts shown in Table 1 to obtain compositions a~e with whitening effects.

[0042] Table 1. Raw material composition of compositions a~e

[0043] Example 2 The difference from Example 1 is as follows: Step S3 is adjusted as follows: Take 3 parts by weight of stevia extract, 10 parts by weight of N-acetylneuraminic acid and 20 parts by weight of pine nut extract to obtain a composition f with whitening effect.

[0044] Cellular and animal experiments were conducted on stevia extract, pleurotus ostreatus extract, N-acetylneuraminic acid, and the combination a~f.

[0045] Experimental Example 1: Cytotoxicity Evaluation 1. Testing materials 1.1 Sample solution preparation Stevia extract, N-acetylneuraminic acid, and Pleurotus ostreatus extract were dissolved separately in phosphate-buffered saline (PBS) to prepare stock solutions with a concentration of 100 mg / mL. The stock solutions were filtered through a 0.45 μm filter to remove impurities, and then filtered through a 0.22 μm filter for sterilization before being aliquoted into 2 mL centrifuge tubes to obtain sample solution stock solutions, which were then stored at -20°C.

[0046] 1.2 Experimental Cells Mouse melanoma B16 cells were provided by Xiangya Hospital of Central South University. The resuscitated B16 cells were transferred into cell culture flasks and grown in 1640 medium at 37°C with 5% CO2. The medium was changed every other day. When the cells reached 80%–90% confluence, they were passaged and cultured for 5 generations before use.

[0047] 1.3 Instrument Consumables CO2 cell culture incubator (Heracell 240i, Thermo); biosafety cabinet (BSC-1500IIA2-X, Biobase); inverted microscope (AE31, Motic); low-speed centrifuge (L550, Hunan Xiangyi); micropipette (Researchplus, Eppendorf); 60mm cell culture dish (705001, Nest); 6-well cell culture plate (703002, Nest); flow cytometer (C6, BD); microplate reader (YongChuang SM600, Shanghai, China); fluorescence microscope (Nikon TE2000, Tokyo, Japan); spectrophotometer (NanoDrop 2000, Thermo Fisher Scientific, Waltham, MA, USA).

[0048] 1640 medium (C11875500BT, Gibco); fetal bovine serum (AUS-01S-02, Cell-Box); tyrosinase activity assay kit (BC4050, Solarbio); CCK-8 assay kit (500T, Solarbio); trypsin digestion solution (T1300, Solarbio); PBS (G4202, Cellbio).

[0049] 2. Experimental Procedure A cell-free control group, a blank control group (complete culture medium), and an experimental group (each group used culture medium supplemented with sample solutions at concentrations of 0.0001 mg / mL, 0.001 mg / mL, 0.01 mg / mL, 0.1 mg / mL, 1 mg / mL, and 10 mg / mL, respectively) were set up.

[0050] Take one 96-well plate, add 100 μL of cell suspension to each well, and seed the plate to make the number of cells to be tested 2 × 10⁶. 3 Cell / well (edge ​​wells were filled with sterile PBS to eliminate edge effects), 5 replicates per group, and cultured adherently in a 37°C, 5% CO2 environment for 24 h.

[0051] The blank control group cells were treated with culture medium without sample solution; the experimental group cells were exposed to culture medium containing different concentrations of sample solution. After the indicated incubation time of 24 hours, 10 µL of Cell Counting Kit-8 (CCK-8) reagent was added to the corresponding wells, and the cells were incubated at 37°C for 1 hour. Finally, the absorbance at 450 nm was recorded, and the cell viability was calculated.

[0052] 3. Experimental Results When preparing N-acetylneuraminic acid sample solutions, it was found that the culture medium could be rapidly decolorized at sample solution concentrations of 1 mg / mL and 10 mg / mL, while this phenomenon was not observed in other sample solutions.

[0053] The results showed that N-acetylneuraminic acid exhibited strong cytotoxicity at 10 mg / mL and high cell activity within the concentration range of 0.0001–1 mg / mL. The extract of *Pleurotus ostreatus* showed no significant effect on cell activity at any concentration. The stevia extract had varying effects on B16 cell activity at different concentrations, with strong cytotoxicity at 10 mg / mL and good cell activity at 0.01 mg / mL.

[0054] Experimental Example 2: Evaluation of Tyrosinase Activity Inhibition 1. Experimental Materials 1.1 Solution Preparation Sample solution: Using PBS solution as a solvent, sample solutions of stevia extract with a concentration of 0.1 mg / mL, N-acetylneuraminic acid with a concentration of 1 mg / mL, and Pleurotus ostreatus extract with a concentration of 1 mg / mL were prepared; compositions a~e prepared in Example 1.

[0055] Tyrosinase solution: Add PBS (pH=6.8) to tyrosinase to prepare an enzyme solution of 5 KU / mL. Aliquot the solution into 1.5 mL EP tubes and store in liquid nitrogen. Dilute with PBS before use to prepare a tyrosinase solution of 500 U / mL. Prepare and use immediately.

[0056] L-DOPA solution (substrate): L-DOPA-(phenyl-d3) was dissolved in a small amount of DMSO and then diluted to volume with PBS to prepare a substrate solution with a concentration of 5 mmol / L. It was stored at 4°C protected from light.

[0057] Resveratrol solution (positive control): Resveratrol was provided by Macklin, catalog number R817263-25g. The solution was prepared using an alcohol solution to a concentration of 10 mg / mL and stored at 4°C protected from light.

[0058] Arbutin solution (positive control): Arbutin was provided by Solarbio, catalog number A7380-5G. The solution was prepared using PBS at a concentration of 4 mg / mL and stored at 4°C protected from light.

[0059] 1.3 Instrument Consumables Micropipette (Top Pette, Dalong); Microplate reader (Tecan Infinite M200, Tecan, Switzerland); 96-well cell culture plate (701002, NEST).

[0060] Tyrosinase 25KU (T8830-25KU, Solarbio); L-DOPA-(phenyl-d3) (L464642-10mg, Aladdin); Kojic acid (K424322-1ml, Aladdin); PBS solution (G4202, Severin); DMSO (dimethyl sulfoxide) (500ml, Solarbio).

[0061] 2. Experimental Procedure 2.2 Determination of the inhibition rate of single component on tyrosinase activity Referring to the reagent addition amounts in Table 2, L-DOPA solution, sample solution, and PBS buffer were added to each well in sequence, mixed thoroughly, and incubated at 37 ℃ for 10 min. Then, 20 L of tyrosinase solution was added to each well in sequence, and the mixture was stirred at 37 ℃ for 5 min ± 5 s. The mixture was then immediately placed in the microplate reader for measurement at a wavelength of 475 nm.

[0062] The time from adding tyrosinase solution to measuring absorbance was kept consistent for each well (5 min ± 5 s), and the experiment was repeated 5 times in parallel. The inhibition rate was calculated based on the absorbance values, and the formula for calculating the inhibition rate is shown below: Inhibition rate (%) = ×100%.

[0063] Wherein, A (solvent reaction well group) is the absorbance when there is a substrate but no analyte; B (solvent background well group) is the absorbance when there is neither a substrate nor an analyte; C (sample reaction well group) is the absorbance when there is both an analyte and a substrate; and D (sample background well group) is the absorbance when there is an analyte but no substrate.

[0064] Table 2 Sample loading table for tyrosinase activity inhibition test

[0065] 3. Experimental Results The results are as follows Figure 1 , Figure 2 As shown in the figure. The results showed that the inhibitory effects of stevia extract, N-acetylneuraminic acid, and pleurotus ostreatus extract on tyrosinase activity were all worse than resveratrol, but better than arbutin. Compositions a-e all had some inhibitory effect on tyrosinase, among which compositions a and c had a more significant inhibitory effect on tyrosinase than arbutin, while compositions b and d had no significant difference in inhibitory effect on tyrosinase compared with arbutin. This indicates that different tyrosinase regulatory effects can be achieved by adjusting the ratio of stevia extract, N-acetylneuraminic acid, and pleurotus ostreatus extract in the compositions, with composition c showing the best inhibitory effect on tyrosinase.

[0066] Experiment Example 3: Evaluation of Oral Whitening Efficacy 1. Testing materials 1.1. Sample solution preparation The composition f prepared in Example 2 was used as the formulation, and standard dilution water was used as the solvent.

[0067] Positive control: POLA Whitening Pills (hereinafter referred to as POLA Whitening Pills), grayish-white tablets, batch number B862, POLA Co., Ltd., solvent is ultrapure water.

[0068] 1.2. Laboratory Animals Zebrafish were raised in aquarium water at 28℃ (water quality: 200 mg of readily soluble sea salt added per 1 L of reverse osmosis water; conductivity 450~550 μS / cm; pH 6.5~8.5; hardness 50~100 mg / L CaCO3), provided by the aquarium breeding center of Hangzhou Huante Biotechnology Co., Ltd., with laboratory animal use license number: SYXK (Zhejiang) 2022-0004. The breeding and management met the requirements of international AAALAC certification (certification number: 001458), and the IACUC ethics review number: IACUC-2026-202604100010-01.

[0069] 1.3. Instruments, Consumables and Reagents Dissecting microscope (SZX7, OLYMPUS, Japan); CCD camera (VertA1, Shanghai Tusen Vision Technology Co., Ltd., China); Precision electronic balance (CP214, OHAUS, USA); 6-well plate (Zhejiang Belamber Biotechnology Co., Ltd., China); Microinjector (IM300, Narishige, Japan); Needle puller (PC-10, Narishige, Japan); Electronic turbidimeter (DensiCHEK™ Plus, bioMérieux, USA); Fully automated rapid sample grinder (JXFSTPRP-24L, Shanghai Jingxin Laboratory Equipment Technology Department, China); High-speed refrigerated centrifuge (Heraeus Fresco17, ThermoFisher, Germany); Digital display constant temperature water bath (HH-4, Changzhou Guohua Electric Co., Ltd., China); Multifunctional microplate reader (SPARK, TECAN, Austria); 96-well plate (Nest) Biotech, China); Standard PCR amplification instrument (T100, BIO-RAD, Singapore); Real-time PCR instrument (CFX Connect, BIO-RAD, Singapore); UV-Vis spectrophotometer (Nanodrop2000, Thermo, USA); Microplate mini centrifuge (BE-6100, Haimen Qilinbei Instrument Manufacturing Co., Ltd., China); Low-skirted 96-well plate (transparent) (HSP9601, Bio-rad, USA); Optical adhesive sealing film B (MSB1001, Bio-rad, USA); Automated nucleic acid extractor (Auto-Pure32A, Hangzhou Aosheng Instrument Co., Ltd., China).

[0070] Melanocyte-stimulating hormone (batch number J13HS188447, Shanghai Yuanye Biotechnology Co., Ltd., China); ChamQ Universal SYBR qPCR Master Mix (batch number 027E2201CD, Vazyme, China); FastKing cDNA First-Strand Synthesis Kit (Genomic De-generated) (batch number X1213, Tiangen Biotech (Beijing) Co., Ltd., China); Universal RNA Extraction TL Kit C (Catalog No. TL2204001643C, Foshan Aowei Biotechnology Co., Ltd., China); Sodium hydroxide (Batch No. H1609020, Shanghai Aladdin Biochemical Technology Co., Ltd., China); Levodopa (Batch No. I170522, Shanghai Aladdin Biochemical Technology Co., Ltd., China); Melanin (Batch No. BCCH4437, SIGMA, USA); RIPA lysis buffer (Batch No. 052124240710, Shanghai Beyotime Biotechnology Co., Ltd., China); BCA protein concentration assay kit (Batch No. 21B05A89, Wuhan Boster Biological Engineering Co., Ltd., China); PMSF (Batch No. 013124240321, Shanghai Beyotime Biotechnology Co., Ltd., China); Methylcellulose (Batch No. C2004046, Shanghai Aladdin Biochemical Technology Co., Ltd., China).

[0071] 2. Detection Method 2.1. MTC Measurement Wild-type AB strain zebrafish, 3 days post-fertilization (3 dpf), were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well (experimental group). Water-soluble samples (concentrations shown in Table 3) were administered, along with a normal control group and a model control group, with a volume of 3 mL per well. Except for the normal control group, all other experimental groups underwent yolk sac injection of melanocyte-stimulating hormone (MSH) to establish a zebrafish melanin-increasing model. After treatment at 28℃ for 1 day, the MTC of the samples in the model zebrafish was measured.

[0072] 2.2. Evaluation of oral skin whitening efficacy (phenotype) Wild-type AB strain zebrafish (3 dpf) were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well. Water-soluble samples (concentrations shown in Table 4) were administered, along with a positive control of POLA whitening pills at a concentration of 25.0 μg / mL. A normal control group and a model control group were also included, with a volume of 3 mL per well. Except for the normal control group, all experimental groups received melanocyte-stimulating hormone injection into the yolk sac to establish a zebrafish melanin-increasing model. After treatment at 28℃ for one day, 10 zebrafish from each experimental group were randomly selected and photographed under a dissecting microscope. Data were analyzed and collected using ImageJ advanced image processing software to analyze the melanin signal intensity in the zebrafish head. The statistical analysis results of this index were used to evaluate the oral whitening efficacy of the samples. Statistical results are expressed as mean ± SE. Statistical analysis was performed using SPSS software, with p < 0.05 indicating statistical significance.

[0073] 2.3. Evaluation of oral skin whitening efficacy (tyrosinase activity) Wild-type AB strain zebrafish (3 dpf) were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well. Water-soluble samples (concentrations shown in Table 5) were administered, along with a positive control of POLA whitening pills at a concentration of 25.0 μg / mL. A normal control group and a model control group were also included, with a volume of 3 mL per well. Except for the normal control group, all experimental groups received melanocyte-stimulating hormone injection into the yolk sac to establish a zebrafish melanin-increasing model. The experiment was conducted in triplicate. After treatment at 28℃ for one day, zebrafish samples were collected. Data were collected using a multi-functional microplate reader according to the tyrosinase activity assay instructions. Tyrosinase activity in the zebrafish was analyzed, and the oral whitening efficacy of the samples was evaluated based on the statistical analysis results of the above indicators. Statistical results are expressed as mean ± SE. Statistical analysis was performed using SPSS software; p < 0.05 indicated statistical significance.

[0074] 2.4. Evaluation of oral skin whitening efficacy (relative expression level of tyr gene) Wild-type AB strain zebrafish (3 dpf) were randomly selected and placed in 6-well plates, with 30 zebrafish treated in each well. Water-soluble samples (concentrations shown in Table 8) were administered, along with a positive control of POLA whitening pills at a concentration of 25.0 μg / mL. A normal control group and a model control group were also included, with a volume of 3 mL per well. Except for the normal control group, all experimental groups received melanocyte-stimulating hormone injection into the yolk sac to establish a zebrafish melanin-increasing model. The experiment was conducted in triplicate. After treatment at 28℃ for 1 day, total RNA was extracted from each group of zebrafish using an automated nucleic acid extractor. The concentration and purity of total RNA were determined using a UV-Vis spectrophotometer. 2.00 μg of total RNA from the zebrafish sample was used to synthesize 20.0 μL of cDNA according to the cDNA first-strand synthesis kit instructions. The expression of β-actin and tyr genes was detected by q-PCR. β-actin was used as an internal control for gene expression, and the relative RNA expression level of the tyr gene was calculated. Statistical results are expressed as mean ± SE. Statistical analysis using SPSS software showed that p < 0.05 was statistically significant.

[0075] 3. Test Results 3.1. MTC Measurement Under the conditions of this experiment, the maximum detectable concentration (MTC) for the oral whitening effect of the combined formula was 1000 μg / mL. See Table 3 for details.

[0076] Table 3 Results of the experiment on the concentration of oral whitening efficacy of the samples (n = 30)

[0077] 3.2. Evaluation of oral skin whitening efficacy (phenotype) Under the conditions of this experiment, the combined formula exhibited oral whitening effects, specifically by reducing the intensity of melanin signaling in the head of zebrafish. See Table 4 for details. Figure 3 and Figure 4 The yellow dashed box indicates the area being analyzed.

[0078] Table 4. Results of the oral whitening efficacy (phenotype) evaluation of the samples (n = 10)

[0079] 3.3. Evaluation of oral skin whitening efficacy (tyrosinase activity) Under the conditions of this experiment, the combined formula exhibited oral whitening effects, specifically by reducing the activity of tyrosinase in zebrafish. See Table 5 for details. Figure 5 .

[0080] Table 5. Evaluation results of oral whitening efficacy of samples (tyrosinase activity) (n = 3)

[0081] 3.4. RNA extraction results and gene primer sequence information At the experimental endpoint, total RNA was extracted from zebrafish, and the RNA concentration and A260 / A280 ratio were measured using a UV-Vis spectrophotometer (Table 6). The A260 / A280 ratios were all between 1.8 and 2.2, indicating that the extracted total RNA from zebrafish was of good quality and could be used for subsequent q-PCR experiments. Primer sequences are shown in Table 7.

[0082] Table 6. Total RNA concentration and A260 / A280 ratio (n = 3)

[0083] Table 7 Primer sequence information

[0084] 3.5. Evaluation of oral skin whitening efficacy (relative expression level of tyr gene) The tyr gene (tyrosinase gene) is closely related to melanin synthesis. It encodes tyrosinase, a copper-containing oxidoreductase that is the rate-limiting enzyme in melanin synthesis and directly affects melanin production. The expression and activity of the tyr gene determine the rate and quantity of melanin production; the higher the tyrosinase activity, the more melanin is formed in the skin.

[0085] Under the conditions of this experiment, the combined formula exhibited oral whitening effects, specifically by downregulating the relative expression level of the tyr gene in zebrafish. See Table 8 for details. Figure 6 .

[0086] Table 8. Evaluation results of oral whitening efficacy of samples (relative expression level of tyr gene) (n = 3)

[0087] The results showed that composition f had the effects of downregulating the relative expression level of the tyr gene, inhibiting melanin secretion, and reducing tyrosinase activity. The effect was better when the concentration of composition f was 500 µg / mL.

[0088] The above description is merely an example and illustration of the structure of the present invention. Those skilled in the art can make various modifications or additions to the specific embodiments described, or use similar methods to replace them, as long as they do not deviate from the structure of the invention or exceed the scope defined in the claims, all of which should fall within the protection scope of the present invention.

Claims

1. A composition having a whitening effect, characterized in that, It includes stevia extract, N-acetylneuraminic acid and pleurotus ostreatus extract, in a mass ratio of (0.5~5):10:(3~22).

2. The method for preparing the composition with whitening effect as described in claim 1, characterized in that, Includes the following steps: Stevia extract, N-acetylneuraminic acid, and Pleurotus ostreatus extract were mixed to obtain a composition with whitening effect.

3. The preparation method according to claim 2, characterized in that, The stevia extract is obtained by first extracting stevia leaves with an aqueous ethanol solution of 30% to 60% by volume, followed by a first purification.

4. The preparation method according to claim 3, characterized in that, The material-to-liquid ratio of the first extraction is 1g:10~15mL; And / or, the temperature of the first extraction is 30~40℃; And / or, the first extraction time is 30~150 min.

5. The preparation method according to claim 3, characterized in that, The first purification uses polyamide resin and / or macroporous adsorption resin for chromatography; And / or, the pH of the extract during the first purification is 2.0 to 4.5; And / or, the first purification uses an aqueous ethanol solution as the eluent.

6. The preparation method according to claim 2, characterized in that, The *Pleurotus ostreatus* ethanol extract is obtained by second extraction of *Pleurotus ostreatus* with an aqueous ethanol solution of 50%~70% by volume, followed by second purification.

7. The preparation method according to claim 6, characterized in that, The material-to-liquid ratio for the second extraction is 1g:40~50mL; And / or, the temperature of the second extraction is 40~50℃; And / or, the second extraction time is 30~150 min; And / or, the second purification uses an ultrafiltration membrane.

8. The use of the whitening composition as described in claim 1 or the whitening composition obtained by any one of the preparation methods described in claims 2 to 7 in the preparation of an oral formulation for enhancing the whitening effect.

9. The application according to claim 8, characterized in that, The concentration of the whitening composition in the oral formulation is 150~1500µg / mL; And / or, the oral formulation is one of a solution, granules, capsules, tablets, and ointment.

10. The application according to claim 8, characterized in that, The enhanced whitening effect includes downregulating the expression level of the tyrosinase gene; And / or, the whitening effect includes inhibiting melanin secretion; And / or, the enhanced whitening effect includes reducing tyrosinase activity.

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