Recombinant cell CHO@hTYR, new tyrosinase inhibitor extraction method and new tyrosinase inhibitor

Abstract

The present invention discloses a CHO cell line (CHO@hTYR) that stably expresses the human tyrosinase gene. The recombinant cell has good storage stability. And further provides a ligand fishing method for efficiently and accurately screening compounds from a complex natural product system using recombinant cells. The method has the characteristics of high sensitivity, good timeliness, and strong specificity. The method was used to screen and identify tyrosinase inhibitors in camel thorn and coffee, and new tyrosinase inhibitors 4-methoxy-5-methylcoumarin, cedarwood flavonoids, tasselflower flavonoids and 3,4-dimethoxy-5-methylcoumarin were obtained.

Description

Technical Field

[0001] Embodiments of the present disclosure relate to the field of biotechnology, and in particular to a method for extracting a tyrosinase inhibitor, a new tyrosinase inhibitor, and a skin care product containing the same. Background Art

[0002] Tyrosinase is a copper-containing polyphenol oxidase that is commonly found in animals, plants, and microorganisms. As a key rate-limiting enzyme in melanin synthesis, tyrosinase affects the pigmentation of human skin, hair, and eyes. Increased tyrosinase activity may lead to skin pigmentation and even malignant melanoma. In addition, high levels of tyrosinase may accelerate the production of neuromelanin and induce neurodegenerative diseases such as Parkinson's disease. Therefore, it is of great significance to find safe and effective tyrosinase inhibitors.

[0003] Medicinal plants are a rich and natural source of tyrosinase inhibitors, and many tyrosinase inhibitors have been isolated from natural products. However, screening active compounds from complex plant extracts has always been challenging.

[0004] The above information disclosed in this Background section is only for enhancement of understanding of the background of the inventive concept and therefore it may contain information that does not form the prior art that is already known to a person of ordinary skill in the art. Summary of the invention

[0005] The content of this disclosure is used to introduce concepts in a brief form, which will be described in detail in the detailed implementation section below. The content of this disclosure is not intended to identify the key features or essential features of the technical solution claimed for protection, nor is it intended to limit the scope of the technical solution claimed for protection.

[0006] The present invention proposes a ligand fishing method based on the display of human tyrosinase on the cell surface, which provides a more reliable and effective strategy for the screening of enzyme inhibitors.

[0007] In a first aspect, some embodiments of the present invention provide a recombinant hamster ovary cell CHO@hTYR, which is loaded with a plasmid containing a gene encoding human tyrosinase and can express human tyrosinase in the form of a membrane protein on the cell surface.

[0008] Optionally, the plasmid carrying the human tyrosinase encoding gene is a recombinant plasmid pCDNA3.1 / hTYR containing the hTYR-CD8hinge-CD8TM-CD8cyto-EGFP gene.

[0009] In a second aspect, the present invention provides a method for constructing the above-mentioned recombinant hamster ovary cell CHO@hTYR, which specifically comprises the following steps:

[0010] Step 1: CHO cell culture;

[0011] Step 2: The human tyrosinase gene was successfully inserted into the pcDNA3.1(+) vector to obtain the recombinant vector pcDNA3.1 / hTYR;

[0012] Step 3: The recombinant vector pcDNA3.1 / hTYR was stably transfected into CHO cells to obtain recombinant cells CHO@hTYR.

[0013] In the third aspect, the present invention uses recombinant cells to establish a ligand fishing method for efficiently and accurately screening compounds from a complex natural product system, and specifically provides a method for screening tyrosinase inhibitors from plant extracts, which specifically includes the following steps:

[0014] The first step is to mix and incubate the recombinant CHO@hTYR cells with the plant extract to obtain a first mixture;

[0015] Step 2: centrifuge the first mixture to remove the supernatant, and wash with buffer for multiple times to obtain a second mixture;

[0016] Step 3: Add a desorption solvent to the second mixture, vortex, and separate the recombinant cells and the desorption solution containing the enzyme inhibitor by centrifugation to obtain a tyrosinase inhibitor mixture.

[0017] In a fourth aspect, the present invention utilizes the method to screen and identify tyrosinase inhibitors in camel thorn, and separates new tyrosinase inhibitors. Specifically, a method for screening tyrosinase inhibitors from camel thorn plant extracts is provided, which specifically comprises the following steps:

[0018] The first step is to mix and incubate the recombinant CHO@hTYR cells with the camel thorn extract to obtain a first mixture;

[0019] Step 2: centrifuge the first mixture to remove the supernatant, and wash with buffer for multiple times to obtain a second mixture;

[0020] The third step is to add a desorption solvent to the second mixture, vortex, and separate the recombinant cells and the desorption solution containing the enzyme inhibitor by centrifugation to obtain a tyrosinase inhibitor mixture, wherein the tyrosinase inhibitor mixture includes 4-methoxy-5-methylcoumarin, cedarwood bioflavone, aconitum bioflavone and 3,4-dimethoxy-5-methylcoumarin.

[0021] In a fifth aspect, some embodiments of the present disclosure provide a use of 4-methoxy-5-methylcoumarin in the preparation of a tyrosinase expression inhibitor or a melanin production inhibitor.

[0022] In a sixth aspect, some embodiments of the present disclosure provide a use of cedarwood biflavone in the preparation of a tyrosinase expression inhibitor or a melanin expression inhibitor.

[0023] In a seventh aspect, some embodiments of the present disclosure provide a use of aconitum biflavone in the preparation of a tyrosinase expression inhibitor or a melanin expression inhibitor.

[0024] In an eighth aspect, some embodiments of the present disclosure provide a use of 3,4-dimethoxy-5-methylcoumarin in the preparation of a tyrosinase expression inhibitor or a melanin expression inhibitor.

[0025] In the ninth aspect, some embodiments of the present disclosure provide a skin care product, which includes a whitening ingredient, wherein the whitening ingredient is one or more of 4-methoxy-5-methylcoumarin, cedarwood bioflavone, aconitum bioflavone and 3,4-dimethoxy-5-methylcoumarin.

[0026] Optionally, the skin care product comprises one or more of: an aqueous solution, an emulsion, a cream, an ointment, a gel, and a facial mask.

[0027] Optionally, the aqueous solution may include one or more of skin care lotion, toner, and essence.

[0028] Optionally, the emulsion may include: an emulsion.

[0029] Optionally, the cream may include: a facial cream, wherein the facial cream may be a facial cream with a transparent texture or a facial cream with a non-transparent texture.

[0030] Optionally, the ointment may include: a skin care ointment.

[0031] Optionally, the facial cleanser may include: one or more of facial cleansing foam, facial cleansing water, and facial cleansing liquid.

[0032] Optionally, when the external skin care product is a facial mask, a common substrate in the art and the skin care composition of the present disclosure applied or soaked on the substrate may be used. The substrate includes, but is not limited to, silk mask paper, cotton mask paper, polyester mask paper or blended mask paper. The choice of the substrate can be selected by those skilled in the art according to actual needs. The form of the facial mask may include, but is not limited to, a peel-off mask, a jelly mask, a cream mask, an essential oil mask or a powder mask.

[0033] The above-mentioned embodiments of the present disclosure have the following beneficial effects:

[0034] The present invention establishes a ligand fishing technology based on human tyrosinase displayed on the surface of hamster ovary cells to screen potential tyrosinase inhibitors in camel thorn and coffee. This method is based on human tyrosinase and is more reliable than the existing method based on mushroom-derived tyrosinase. It provides a new idea for the rapid screening of tyrosinase inhibitors from natural products and a theoretical basis for the development of new active ingredients targeting human tyrosinase.

[0035] In this paper, a CHO cell line stably expressing human tyrosinase (CHO@hTYR) was constructed. The recombinant cell has good storage stability. A ligand fishing method for efficiently and accurately screening compounds from a complex natural product system was established using the recombinant cell. The method has the characteristics of high sensitivity, good timeliness and strong specificity. The tyrosinase inhibitors in camel thorn and coffee were screened and identified using this method. It is worth noting that the four tyrosinase inhibitors, 4-methoxy-5-methylcoumarin (1), cedarwood flavonoids (2), tachyphyllum flavonoids (3) and 3,4-dimethoxy-5-methylcoumarin (4), were isolated from camel thorn for the first time. The active ingredients of coffee are mainly linoleic acid, palmitic acid, ricinoleic acid and stearic acid. This discovery has laid a scientific foundation for the development of tyrosinase inhibitors in natural products. The results of intracellular activity test in B16 cells showed that the effective component S5 in coffee and the four compounds in camel thorn could significantly inhibit intracellular tyrosinase activity and melanin production. This work provides a new ligand fishing method and a new way to quickly screen new tyrosinase inhibitors, laying a scientific foundation for camel thorn and coffee as natural inhibitors of melanin production. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] The above and other features, advantages and aspects of the embodiments of the present disclosure will become more apparent with reference to the following detailed description in conjunction with the accompanying drawings. Throughout the accompanying drawings, the same or similar reference numerals represent the same or similar elements. It should be understood that the drawings are schematic and the elements are not necessarily drawn to scale.

[0037] Figure 1 : Agarose gel electrophoresis colony PCR

[0038] Figure 2 :Gene sequencing results of strain No. 13

[0039] Figure 3 :Fluorescence microscopy images of CHO@hTYR and control cells. (A) CHO@hTYR; (B) control cells. Left: fluorescence; right: bright field; scale bar: 100 μm.

[0040] Figure 4 :Calibration curve of mushroom tyrosinase activity

[0041] Figure 5:Storage stability of CHO@hTYR cells

[0042] Figure 6 :HPLC spectra of (a) mS0, (b) mS5, (c) mS5-blank. Peak: 1: α-arbutin; 2: quercetin; 3: dihydrotanshinone I.

[0043] Figure 7 : Reusability of CHO@hTYR: af refers to the 1st to 6th times of ligand fishing.

[0044] Figure 8 : Calibration curve for determination of limit of detection (LOD) of quercetin.

[0045] Fig. 9 : HPLC spectra of camel thorn fishing; (a) S0-A, (b) S5-A, (c) S5-A-blank.

[0046] Fig.10 : Mass spectrum of 4-methoxy-5-methylcoumarin (A).

[0047] Fig.11 : Mass spectrum of cedarwood bioflavonoids (B).

[0048] Fig.12 : Mass spectrum of aconitum biflavonoids (C).

[0049] Fig.13 : Mass spectrum of 3,4-dimethoxy-5-methylcoumarin (D).

[0050] Fig.14 : Chemical structures of 4-methoxy-5-methylcoumarin (1), cedarwood bioflavone (2), aconitum bioflavone (3), and 3,4-dimethoxy-5-methylcoumarin (4).

[0051] Fig.15 :HPLC spectra of fishing coffee; (a) S0-C, (b) S5-C, (c) S5-C-blank.

[0052] Fig.16 : GC-MS spectra of coffee active fraction (A) and standard mixture (B).

[0053] Fig.17 :Effects of active compounds from camel thorn on B16 melanoma cells-cytotoxicity # p<0.05, ## p<0.01, ### p<0.001and ####p<0.0001, compared with the blank control group; *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001, compared with the model group).

[0054] Fig.18 :Effects of active compounds from Camel thorn on B16 melanoma cells - tyrosinase inhibitory activity. α-Arbutin and kojic acid were used as positive controls. # p<0.05, ## p<0.01, ### p<0.001and #### p<0.0001, compared with the blank control group; *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001, compared with the model group).

[0055] Fig.19 :Effects of active compounds from Camel thorn on B16 melanoma cells - ability to reduce melanin content. α-Arbutin and kojic acid were used as positive controls. # p<0.05, ## p<0.01, ### p<0.001and #### p<0.0001, compared with the blank control group; *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001, compared with the model group).

[0056] Fig. 20 :Effects of coffee active component (S5) on B16 melanoma cells - cytotoxicity. α-Arbutin and kojic acid were used as positive controls (25μM). # p<0.05, ## p<0.01, 0.001 and ****p<0.0001, compared with the model group).

[0057] Fig.21 :Effects of coffee active component (S5) on B16 melanoma cells - tyrosinase inhibitory activity. α-Arbutin and kojic acid were used as positive controls (25μM). # p<0.05, ## p<0.01, and #### p<0.0001, compared with the blank control group; ***p<0.001and****p<0.0001, compared with the model group).

[0058] Fig. 22 :Effects of coffee active component (S5) on B16 melanoma cells - ability to reduce melanin content. α-Arbutin and kojic acid were used as positive controls (25μM).# p<0.05, ## p<0.01, and #### p<0.0001, compared with the blank control group; ***p<0.001and****p<0.0001, compared with the model group). DETAILED DESCRIPTION

[0059] At present, the enzymes used to screen tyrosinase inhibitors are all derived from cheap mushrooms. Since mushroom tyrosinase and human tyrosinase have significant differences in protein structure and function, the reliability of activity screening based on mushroom tyrosinase is not high. To this end, the present invention establishes a ligand fishing method based on cell surface display of human tyrosinase, which provides a more effective strategy for the screening of tyrosinase inhibitors.

[0060] In a first aspect, the present invention provides a recombinant hamster ovary cell CHO@hTYR, which is loaded with a plasmid encoding human tyrosinase and can express human tyrosinase in the form of a membrane protein on the cell surface.

[0061] Chinese hamster ovary (CHO) cells are the preferred host for the production of recombinant proteins in the biopharmaceutical industry. Cells derived from mammals can promote the proper folding and secretion of recombinant proteins, making them closer to the structure of proteins in their natural state. For example, therapeutic agents such as monoclonal antibodies and interferons produced by CHO cells are used in the treatment of cancer and various diseases. In addition, CHO recombinant cells can be used as whole-cell catalysts to improve the efficiency of catalytic reactions. The excellent proliferation and protein expression capabilities of CHO cells make them the best choice for expressing human tyrosinase.

[0062] Specifically, the plasmid loaded with the human tyrosinase encoding gene is the recombinant plasmid pCDNA3.1 / hTYR containing the hTYR-CD8hinge-CD8TM-CD8cyto-EGFP gene; wherein, hTYR is the gene of human tyrosinase; CD8TM serves as a transporter for expressing hTYR to the cell surface; CD8hinge serves as a "handle" to anchor hTYR to the cell surface; CHO-S-hTYR positive cells will express human tyrosinase in the form of membrane protein on CHO-S cells; and the EGFP tag will be used to screen the efficiency of protein expression on the membrane.

[0063] In a second aspect, the present invention provides a method for constructing the above-mentioned recombinant cell, which specifically comprises the following steps:

[0064] Step 1: CHO cell culture. In a specific embodiment, the specific operation steps are as follows: CHO-S cells are placed in CD04 culture medium and cultured in a 37° C., 5% CO 2 incubator for use.

[0065] The second step is to successfully insert the human tyrosinase gene into the pcDNA3.1(+) vector to obtain the recombinant vector pcDNA3.1 / hTYR; in a specific embodiment, the specific operation steps are: synthesize the hTYR-CD8hinge-CD8TM-CD8cyto-EGFP gene, clone it into the pCDNA3.1(+) expression vector through HindIII / NotI double enzyme digestion, and obtain the recombinant vector pcDNA3.1 / hTYR.

[0066] Step 3: Stably transfect the recombinant vector pcDNA3.1 / hTYR into CHO cells to obtain recombinant cells CHO@hTYR. In a specific embodiment, the specific operation steps are: transfer the recombinant plasmid into CHO-S cells by electroporation, and obtain positive cells by G418 pressure screening, i.e., recombinant cells CHO@hTYR, which will express human tyrosinase in the form of membrane protein on CHO-S cells.

[0067] The localization of hTYR on the surface of CHO cells was verified by cell surface enzyme activity test and fluorescence microscopy images. The recombinant cells (CHO@hTYR) were able to maintain relatively stable enzyme activity (82.59±2.70%) within 7 days and could be repeatedly fished five times. This further proves the stability and reusability of the recombinant cells (CHO@hTYR).

[0068] In the third aspect, the present invention uses recombinant cells to establish a ligand fishing method for efficiently and accurately screening compounds from a complex natural product system, and provides a method for screening tyrosinase inhibitors from plant extracts, which specifically includes the following steps:

[0069] The first step is to mix and incubate the recombinant CHO@hTYR cells with the plant extract to obtain a first mixture. In a specific embodiment, the specific operation steps are: 1×10 4 ~2×10 5 The cells were incubated with 1 mL of plant extract solution (solvent: PBS, concentration: 0.1 mg / mL) at 37°C for 30 to 120 minutes. 4 The cells were incubated with 1 mL of plant extract solution (solvent: PBS, concentration: 0.1 mg / mL) at 37°C for 30 minutes.

[0070] Specifically, the plant extract can be prepared by a common method that can be imagined in the art. In one embodiment, the plant extract is prepared by grinding the plant into fine powder, then soaking it in 80% ethanol at a solid-liquid ratio of 1:5, repeating the extraction three times, evaporating and concentrating the obtained extract, and storing it at -20°C for later use.

[0071] Specifically, the plant may be camel thorn or coffee.

[0072] The second step is to centrifuge the first mixture to remove the supernatant, and wash it with a buffer solution for multiple times to obtain a second mixture. In a specific embodiment, the buffer solution can be one of PBS, Tris-HCL, CD04, and ddH2O. Preferably, it is PBS.

[0073] The third step is to add a desorption solvent to the second mixture, and after vortexing, separate the recombinant cells and the desorption solution containing the enzyme inhibitor by centrifugation to obtain a tyrosinase inhibitor mixture. In a specific embodiment, the vortexing time is 20 to 30 minutes. In a specific embodiment, the desorption solvent can be methanol with a concentration of 10% to 50% or acetonitrile with a concentration of 10% to 50%. Preferably, the desorption solvent is methanol with a concentration of 10% to 30%. In a specific embodiment, a desorption solvent is added to the second mixture, and after vortexing for 20 to 30 minutes, the recombinant cells and the desorption solution containing the enzyme inhibitor are separated by centrifugation (4000rpm, 10min) to obtain a tyrosinase inhibitor mixture.

[0074] Through detection limit (LOD) determination experiments and selectivity verification experiments, it was determined that this method has the characteristics of high sensitivity, good timeliness and strong specificity.

[0075] In a fourth aspect, the present invention uses the method to screen and identify tyrosinase inhibitors in camel thorn, and isolates a new tyrosinase inhibitor 3,4-dimethoxy-5-methylcoumarin (4). A method for screening tyrosinase inhibitors from camel thorn plant extracts is provided, which specifically comprises the following steps:

[0076] The first step is to mix and incubate the recombinant CHO@hTYR cells with the camel thorn extract to obtain a first mixture. In a specific embodiment, the specific operation steps are: 1×10 4 ~2×10 5 The cells were incubated with 1 mL of camel thorn extract solution (solvent: PBS, concentration: 0.1 mg / mL) at 37°C for 30 to 120 minutes. 4 The cells were incubated with 1 mL of plant extract solution (solvent: PBS, concentration: 0.1 mg / mL) at 37°C for 30 minutes.

[0077] Specifically, the camel thorn extract can be prepared by a commonly used method in the art. In one embodiment, the camel thorn extract is prepared by grinding the camel thorn into fine powder, then soaking it in 80% ethanol at a solid-liquid ratio of 1:5, repeating the extraction three times, evaporating and concentrating the obtained extract, and storing it at -20°C for later use.

[0078] Step 2: centrifuge the first mixture to remove the supernatant, and wash with buffer for multiple times to obtain a second mixture;

[0079] The third step is to add a desorption solvent to the second mixture, vortex, and separate the recombinant cells and the desorption solution containing the enzyme inhibitor by centrifugation to obtain a tyrosinase inhibitor mixture, wherein the tyrosinase inhibitor mixture includes 4-methoxy-5-methylcoumarin, cedarwood bioflavone, aconitum bioflavone and 3,4-dimethoxy-5-methylcoumarin.

[0080] In a fifth aspect, some embodiments of the present disclosure provide a use of 4-methoxy-5-methylcoumarin in the preparation of a tyrosinase inhibitor or a melanin production inhibitor.

[0081] In a sixth aspect, some embodiments of the present disclosure provide a use of cedarwood biflavone in the preparation of a tyrosinase inhibitor or a melanin production inhibitor.

[0082] In a seventh aspect, some embodiments of the present disclosure provide a use of aconitum biflavone in the preparation of a tyrosinase expression inhibitor or a melanin production inhibitor.

[0083] In an eighth aspect, some embodiments of the present disclosure provide a use of 3,4-dimethoxy-5-methylcoumarin in the preparation of a tyrosinase expression inhibitor or a melanin production inhibitor.

[0084] In the ninth aspect, some embodiments of the present disclosure provide a skin care product, which includes a whitening ingredient, wherein the whitening ingredient is one or more of 4-methoxy-5-methylcoumarin, cedarwood bioflavone, edelweiss bioflavone and 3,4-dimethoxy-5-methylcoumarin.

[0085] Embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. Although certain embodiments of the present disclosure are shown in the accompanying drawings, it should be understood that the present disclosure can be implemented in various forms and should not be construed as being limited to the embodiments set forth herein. On the contrary, these embodiments are provided to provide a more thorough and complete understanding of the present disclosure. It should be understood that the drawings and embodiments of the present disclosure are only for exemplary purposes and are not intended to limit the scope of protection of the present disclosure.

[0086] It should also be noted that, for ease of description, only the parts related to the invention are shown in the drawings. In the absence of conflict, the embodiments and features in the embodiments of the present disclosure can be combined with each other.

[0087] It should be noted that the modifications of "one" and "plurality" mentioned in the present disclosure are illustrative rather than restrictive, and those skilled in the art should understand that unless otherwise clearly indicated in the context, it should be understood as "one or more".

[0088] It should be noted that the term “about” mentioned in the present disclosure, when used to define a numerical value, means ±5% of the specified numerical value.

[0089] It should be noted that the term "%" mentioned in the present disclosure means mass % unless otherwise specified.

[0090] It should be noted that the "normal temperature" mentioned in the present disclosure refers to the ambient temperature, that is, no special heating or cooling treatment has been performed.

[0091] Explanation of terms

[0092] The term "camel thorn" refers to a semi-shrubby herbaceous plant of the genus Camelus in the Fabaceae family. Camel thorn belongs to the Fabaceae family and is distributed in Central Asia and East Asia, mainly in Inner Mongolia Autonomous Region, Gansu, Qinghai and Xinjiang Uygur Autonomous Region of China. It is a representative species of arid and semi-arid areas. Because it is the main food of camels in extremely arid areas, it is called camel thorn in China. In traditional Uyghur medicine, camel thorn is used to treat fever, inflammation, headache, toothache, hypertension, hyperlipidemia, and liver diseases. It is reported that the compounds isolated from this plant have antioxidant, anti-injury, anti-inflammatory and antibacterial activities. Recently, the methanol extract of this plant was found to inhibit the activity of tyrosinase, indicating its potential for skin whitening.

[0093] The term “Coffee Arabica” belongs to the Rubiaceae family and is one of the most popular beverages in the world. Coffee has various health benefits and can prevent cardiovascular diseases, type 2 diabetes, Parkinson’s disease, and Alzheimer’s disease. The active compounds in coffee, such as gamma-tocopherol, may help prevent skin pigmentation. In addition, two key components of coffee, namely caffeic acid and ferulic acid, have been reported to work synergistically to scavenge free radicals from the skin and mitigate UV-induced oxidative damage. Although previous studies have shown that these two plants have skin-whitening effects, the active ingredients that inhibit tyrosinase have not yet been identified.

[0094] The term "quercetin" is a flavonoid monomer compound widely found in plants, which has multiple pharmacological effects such as antioxidant, anti-tumor, hypoglycemic, and hypolipidemic effects. Quercetin has certain anti-inflammatory activity, which is mainly manifested in the inhibition of inflammatory factors. There is also existing technology that proves that it can be used as a tyrosinase inhibitor.

[0095] Materials and Reagents

[0096] The B16 melanoma cell line was obtained from the Shanghai Cell Bank of the Chinese Academy of Sciences (Shanghai, China).

[0097] CD04, high-glucose medium, penicillin-streptomycin, fetal bovine serum (FBS) and other culture reagents were provided by Gibco (CA, USA).

[0098] Cell counting kit-8 (CCK-8) was purchased from ProteinTech Group (Chicago, USA). α-MSH was purchased from Beiyu Biotechnology Co., Ltd. (Shanghai, China). Chromatographic grade methanol and acetonitrile were provided by JT Technology (Beijing, China).

[0099] Camel thorn and coffee were collected from the Chengdu medicinal materials market in Sichuan Province (the former in 2021-02 and the latter in 2021-03) and preserved in the specimen room of the Chengdu Institute of Biology, Chinese Academy of Sciences.

[0100] The pcDNA3.1(+) plasmid was synthesized by GenScript.

[0101] CHO-S cells, one of the CHO cell lines, were purchased from Invitrogen.

[0102] Triton X-100 was purchased from Thermo Scientific.

[0103] instrument

[0104] A Shimadzu LC-30AD UFLC system was equipped with a SPD-20A UV detector and a BEH C18 column (2.1×100 mm, 1.7 μm, Waters, UK), and HPLC-MS analysis was performed using a QTRAP5500-MS (AB Sciex, USA).

[0105] HPLC analysis was performed using a Shimadzu LC-20AD high performance liquid chromatography system equipped with a SPD-20A UV detector and a reversed-phase Kromasil 100-5-C18 column (4.6×250 mm, 5 um, Akzonble, Sweden).

[0106] Semi-preparative HPLC was performed using the same Shimadzu LC-20AD system, but with a COSMOSIL C18 column (10×250 mm, 5 um, Nacalai Tesque Corporation, Japan).

[0107] The UV absorbance was measured using a MULTISKAN GO spectrophotometer (Thermo Scientific, USA).

[0108] Example 1 Preparation method of recombinant cell CHO@hTYR

[0109] This embodiment provides a recombinant cell CHO@hTYR, a recombinant hamster ovary cell displaying human tyrosinase on its surface.

[0110] The specific steps include:

[0111] The first step is to culture CHO cells. The specific operation is as follows: CHO-S cells are placed in CD04 culture medium and cultured in a 37° C., 5% CO 2 incubator for use.

[0112] Step 2: Successfully insert the human tyrosinase gene into the pcDNA3.1(+) vector (Supplementary Data, Part S1) to obtain the recombinant vector pcDNA3.1 / hTYR. The specific steps are as follows:

[0113] The hTYR-CD8hinge-CD8TM-CD8cyto-EGFP gene was synthesized and cloned into the pCDNA3.1(+) expression vector by HindIII / NotI double restriction digestion to obtain the recombinant vector pcDNA3.1 / hTYR.

[0114] Step 3: Stably transfect the recombinant vector pcDNA3.1 / hTYR into CHO cells to obtain recombinant cells CHO@hTYR. The specific steps are as follows:

[0115] The recombinant plasmid was transferred into CHO-S cells by electroporation, and positive cells were obtained by G418 pressure screening, namely recombinant cells CHO@hTYR, which would express human tyrosinase as membrane protein on CHO-S cells.

[0116] In the hTYR-CD8hinge-CD8TM-CD8cyto-EGFP gene, CD8TM acts as a transporter on the surface of hTYR expressing cells. CD8hinge acts as a "handle" to anchor hTYR on the cell surface. The EGFP tag will be used to screen the efficiency of protein expression on the membrane.

[0117] Verification Example 1: The recombinant vector pcDNA3.1 / hTYR was successfully constructed

[0118] hTYR-CD8hinge-CD8TM-CD8cyto-EGFP was synthesized by Shanghai Shenggong Biotechnology Co., Ltd. (Table 1 shows the primer sequence of hTYR). Figure 1 As shown in the figure, the results of colony PCR identification showed that colonies 3 and 13 were in line with expectations. The gene sequencing results of strain 13 were correct ( Figure 2 ). This proves that the recombinant vector pcDNA3.1 / hTYR was successfully constructed.

[0119] Table 1: Primer sequences of hTYRd in vectors

[0120]

[0121] Verification Example 2 Fluorescence Detection of CHO@hTYR

[0122] The transfection efficiency of CHO@hTYR was evaluated by fluorescence microscopy. Under an excitation wavelength of 488 nm, the fluorescence intensity of CHO@hTYR at 507 nm was recorded. At the same time, the pCDNA3.1(+) empty vector was transfected into CHO-S cells using the same method to obtain blank recombinant cells, which served as blank controls. Figure 3 As shown, positive cells showed obvious fluorescence ( Figure 3 A), while control cells showed no fluorescence ( Figure 3 B), confirming the successful construction of CHO@hTYR.

[0123] Verification Example 3: Enzyme activity and stability detection of recombinant cells

[0124] This experiment tests the tyrosinase activity on the surface of CHO@hTYR to evaluate the activity and stability of recombinant cells. The specific test method is:

[0125] A standard curve was drawn using mushroom tyrosinase, with the x-axis representing the concentration of mushroom tyrosinase and the y-axis representing the OD value at 475 nm, representing the amount of dopaquinone produced. The amount of substrate L-DOPA was constant at 5 μM. The standard curve (e.g. Figure 4 ). 4 The amount of product generated by the catalytic substrate of each recombinant cell was substituted into the standard curve to reflect the enzyme activity of the corresponding mushroom tyrosinase.

[0126] The test results showed that compared with mushroom tyrosinase, 1×10 4 The enzyme activity of the recombinant cells was 99.32±4.66 U. The blank recombinant cells did not show the ability to catalyze L-DOPA.

[0127] To ensure the stability of the recombinant cells, the enzyme activity was monitored continuously for 7 days. Figure 5 As shown, the enzyme activity slowly decreased and still retained more than 80% of the initial cells (ie, day 0) on the seventh day, indicating that the recombinant cells had good storage stability.

[0128] Verification Example 4: Specificity verification of the ligand fishing method based on recombinant cells

[0129] A negative ligand of TYR, dihydrotanshinone I, and two positive ligands (a-arbutin and quercetin) were mixed to prepare a standard mixture named mS0. 4CHO@hTYR cells were incubated with 1 mL of mS0 (configured at a concentration of 0.1 mg / mL) at 37°C for 30 minutes. After incubation, the mixture was centrifuged and washed three times with PBS, desorbed by adding 30% methanol, and vortexed. The recombinant cells and the desorption solution containing the enzyme inhibitor were separated by centrifugation to obtain a tyrosinase inhibitor mixture named mS5. The obtained solutions mS0 and mS5 were subjected to HPLC analysis. The blank recombinant cells repeated the above fishing experiment as a blank control, and the final eluate was called mS5-blank and subjected to HPLC analysis.

[0130] Test results such as Figure 6 As shown, CHO@hTYR can only specifically adsorb two positive ligands, indicating that the proposed method has good selectivity.

[0131] Verification Example 5 Reusability of recombinant cells

[0132] Use 5×10 4 Each CHO@hTYR cell was repeatedly exposed to 1 mL of quercetin (100 uM). 4 Each CHO@hTYR cell was incubated with 1 mL of quercetin (100uM) at 37°C for 30 minutes. After the incubation, the mixture was centrifuged and washed three times with PBS, and 30% methanol was added for desorption. After vortexing, the recombinant cells and the desorption solution containing the enzyme inhibitors were separated by centrifugation to obtain a tyrosinase inhibitor mixture, named Sa. The CHO@hTYR cells after fishing were used for subsequent repeated fishing experiments, and the above steps were repeated 5 times to obtain solutions Sb, Sc, Sd, Se, and Sf. Solutions Sa, Sb, Sc, Sd, Se, and Sf were analyzed by HPLC. Figure 7 As shown, the quercetin peak area after the sixth round of fishing was almost indistinguishable from the noise. Therefore, it can be concluded that the recombinant cells can be reused for five rounds.

[0133] Verification Example 6 Determination of the Limit of Detection (LOD) of Quercetin

[0134] The experimental process included preparing a series of quercetin solutions with concentrations of 0.1, 1, 10, 100 and 1000 μM, 4 Each CHO@hTYR cell was incubated with a series of quercetin solutions at 37°C for 30 minutes. After the incubation, the mixture was centrifuged and washed three times with PBS, and then desorbed by adding 30% methanol. After vortexing, the recombinant cells and the desorbed solution containing the enzyme inhibitor were separated by centrifugation to obtain a tyrosinase inhibitor mixture. The obtained solutions were analyzed by HPLC and a calibration curve was drawn, as shown in FIG. Figure 8As shown in , the abscissa represents the concentration of quercetin, and the ordinate represents the peak area of ​​HPLC. When the peak area value of quercetin reaches 3 times the noise value, the minimum detection limit of the method is determined to be 0.166 μM based on the corresponding quercetin concentration.

[0135] Example 2 Preparation method of camel thorn extract

[0136] This embodiment provides a camel thorn extract.

[0137] 0.82 kg of camel thorn was ground into fine powder, then soaked in 80% ethanol at a solid-liquid ratio of 1:5, and the extraction was repeated three times. The obtained extract was evaporated and concentrated, and the extraction rate was 24.85%. The obtained camel thorn extract was stored and stored at -20°C for use.

[0138] Example 3 Preparation method of coffee extract

[0139] This embodiment provides a coffee extract.

[0140] 0.5 kg of coffee was ground into fine powder, then soaked in 80% ethanol at a solid-liquid ratio of 1:5, and the extraction was repeated three times. The obtained extract was evaporated and concentrated, with an extraction rate of 29.64%. The obtained coffee extract was stored and stored at -20°C for later use.

[0141] Example 4 Screening of tyrosinase inhibitors from camel thorn extract

[0142] In this example, recombinant cells CHO@hTYR were used to screen tyrosinase inhibitors from camel thorn extracts.

[0143] The camel thorn obtained in Example 2 was prepared into a 1 mg / mL solution and named S0-A.

[0144] Use 5×10 4 CHO@hTYR cells were used to screen tyrosinase ligands; the specific steps are as follows:

[0145] 1 mL of S0-A was mixed with CHO@hTYR cells at 37°C for 30 min to promote the interaction between the target protein on the surface of the recombinant cells and the potential ligand compound. After incubation, the mixture was centrifuged at 4000 rpm for 5 min, the supernatant was discarded, and the mixture was washed three times with PBS. A desorption solvent was added, and after vortexing for 20 to 30 minutes, the recombinant cells and the desorption solution containing the enzyme inhibitor were separated by centrifugation (4000 rpm, 10 min) to obtain a tyrosinase inhibitor mixture named S5-A.

[0146] CHO cells transfected with empty vector were subjected to the same fishing procedure as blank control. The final desorption solvent was labeled as S5-A-blank.

[0147] S0-A, S5-A and S5-A-blank were analyzed by HPLC using high performance liquid chromatography under the following conditions: UV detection wavelength of 220 nm, column temperature of 35°C, injection volume of 20 μL, mobile phase of solvent A (0.1% formic acid / water) and solvent B (methanol), gradient elution of 30-100% B, elution time of 0-35 min, and flow rate of 0.8 mL / min.

[0148] Four compounds were quickly screened from camel thorn extracts, such as Fig. 9 As shown in .

[0149] Example 5 Isolation and Identification of Tyrosinase Inhibitors from Camel Thorn Extract

[0150] Guided by the active compound in S5-A, 83 g of camel thorn extract in Example 2 was taken and eluted with a silica gel column (100-200 mesh) using dichloromethane to methanol (50:1-0:1) to obtain 8 fractions. Compounds 1 (7.7 mg) and 3 (27.0 mg) were isolated from the third and seventh fractions, respectively. Compounds 2 (5.4 mg) and 4 (5.6 mg) were purified from the first and seventh fractions, respectively, using semi-preparative high performance liquid chromatography (60% MeOH).

[0151] The structures of the purified compounds 1-4 were identified by mass spectrometry and nuclear magnetic resonance.

[0152] Electrospray ionization mass spectrometry was performed in positive ion mode with a scan range of 50-1000 m / z, a spray voltage of 3.00 kV, a source temperature of 120 °C, a desolvation temperature of 450 °C, a cone gas flow rate of 50 L / h, and a desolvation gas flow rate of 800 L / h. NMR data were recorded on a Bruker DRX-600 spectrometer (Bruker, Rheinsteten, Germany).

[0153] like Figure 10-13 As shown, the m / z molecular ion fragments obtained for compounds 1-4 were 191.11 [M+H] + 、539.64[M+H] + 、539.10[M+H] + and 221.08[M+H] + .

[0154] In addition, according to 1 H and 13 C NMR data (see Table 2-3, compounds 1-4 in 1 H NMR (600 MHz) and 13C NMR (150 MHz), δ in ppm, J in Hz. These compounds were identified as 4-methoxy-5-methylcoumarin (1), cedarwood bioflavonoids (2), aconitum bioflavonoids (3) and 3,4-dimethoxy-5-methylcoumarin (4), respectively. Their structures are shown in Fig.14 .

[0155] Table 2: Compound 1-2 1 H and 13 C NMR data

[0156]

[0157]

[0158]

[0159] Table 3: Compound 3-4 1 H and 13 C NMR data

[0160]

[0161]

[0162]

[0163] Example 6 Screening of tyrosinase inhibitors from coffee extract

[0164] The coffee extract obtained in Example 3 was prepared into a 1 mg / mL solution and named S0-C. The same method as in Example 4 was used to screen tyrosinase ligands to obtain S5-C and S5-C-blank. S0-C, S5-C and S5-C-blank were analyzed by high performance liquid chromatography, and a low polarity active site was obtained from coffee, such as Fig.15 as shown in .

[0165] Example 7 Isolation and Identification of Tyrosinase Inhibitors in Coffee Extract

[0166] Taking the active compound in S5-C as a guide, 9 g of coffee extract in Example 3 was taken, fractionated with petroleum ether, and then subjected to silica gel column chromatography (100-200 mesh), eluted with petroleum ether-ethyl acetate (200:1-50:1), and the active part (3.5 g) of the ligand extraction index was obtained. The fatty acids in this component were analyzed by gas chromatography-mass spectrometry (Agilent 5977B) equipped with an HP-5ms chromatographic column (30m×0.25mm×0.25um). The chromatographic conditions were: the initial temperature was 40°C, maintained for 5 minutes, then increased to 250°C at a rate of 10°C / min, then increased to 280°C at a rate of 5°C / min, and maintained for another 5 minutes. The mass spectrometry parameters included an electron impact source with a temperature of 230°C, an interface temperature of 250°C, an electron energy of 65eV, and a mass scanning range of 30-550amu.

[0167] The compounds in S5-C in coffee extract are very low in polarity ( Fig.15 ), it is difficult to purify. Therefore, according to GB5009.168-2016 National Food Safety Standard "Determination of Fatty Acids in Foods", GC-MS internal standard method quantitative analysis was carried out, and the results were compared with 37 mixed fatty acid standards ( Fig.16 ), the main components of the active part are linoleic acid (55.18 g / 100 g), palmitic acid (26.24 g / 100 g), ricinoleic acid (6.58 g / 100 g) and stearic acid (5.31 g / 100 g).

[0168] Efficacy verification example

[0169] Experimental method 1: Cytotoxicity test experimental method

[0170] B16 melanoma cells were used for toxicity testing. Specifically, the cell survival rate of B16 melanoma cells was detected by CCK8 experiment, which specifically includes the following steps:

[0171] In the first step, B16 melanoma cells were cultured in fresh culture medium at 37° C. and 5% CO 2 .

[0172] The fresh culture medium is prepared by adding 10% fetal bovine serum and 1% penicillin-streptomycin to DMEM culture medium. In practice, cells are passaged regularly during cell culture to ensure logarithmic growth.

[0173] In the second step, when the confluence reached 80%-90%, B16 melanoma cells were detached by trypsin and cultured at 1.5×10 4 Cells were seeded at a density of 10 cells / well in 96-well plates for 24 hours.

[0174] The third step was to incubate the cells in 100 μL of culture medium containing different concentrations of the drug groups for 24 hours.

[0175] Step 4: After incubation, 10 μL of CCK-8 reagent was added to each well, and after reacting at 37°C for 1 hour, the absorbance was measured at a wavelength of 450 nm using an enzyme reader.

[0176] Among them, cell viability = (sample group OD 450 / Blank control group OD 450 )×100%

[0177] Experimental method 2: Verification experiment on the relative activity of tyrosinase in B16 melanoma cells

[0178] Specifically, the following steps are included:

[0179] In the first step, B16 melanoma cells were cultured at 2×10 5 B16 melanoma cells (2×10 cells / well in a 6-well plate) were grown in a 6-well plate for 24 h. 5 Cells / well) were exposed to 1 μM α-MSH in culture medium and treated with different concentrations of drugs for 48 h, with α-arbutin and kojic acid as positive controls.

[0180] In the second step, after treatment, B16 melanoma cells were lysed with 1% Triton X-100 and sonicated, and L-DOPA was added to each well to a final concentration of 5 mM.

[0181] The third step is to incubate at 37°C for 30 minutes, measure the absorbance at 475 nm, and calculate the relative activity of tyrosinase.

[0182] Relative activity = (OD 475 - Experimental control group OD 475 ) / (blank group OD 475 -Blank control group OD 475 )×100%.

[0183] Experimental method 3: Verification experiment on the effect on melanin content in B16 melanoma cells

[0184] The steps include:

[0185] In the first step, B16 melanoma cells were cultured at 1.25 × 10 5 The cells were incubated at a density of 1000 cells / well in a 6-well plate for 24 hours.

[0186] In the second step, after incubation, B16 melanoma cells were exposed to 1 μM α-MSH in culture medium and treated with different concentrations of drug groups and then cultured for 72 hours, with α-arbutin and kojic acid as positive controls.

[0187] In the third step, B16 melanoma cells were washed with ice-cold PBS (Phosphate Buffered Saline) and then trypsinized.

[0188] In the fourth step, the cells were lysed with 1 M NaOH and 10% DMSO at 80°C for 60 minutes to release melanin.

[0189] The fifth step is to measure the absorbance at 405 nm and express the melanin content as a percentage relative to the blank control group.

[0190] Among them, relative content = (experimental group OD 405 / Blank group OD 405 )×100%

[0191] Efficacy Verification Example 1 Effects of Compounds 1-4 on B16 Cell Viability

[0192] The effects of camel thorn active compounds 1-4 on the viability of B16 mouse melanoma cells at concentrations of 1μM, 5μM, 25μM, 50μM, 100μM, 150μM and 200μM were studied. Fig.17 It can be seen that the safe concentration range of these compounds in B16 melanoma cells is between 1-25 μM. Except for compound 4, the remaining compounds have certain toxic effects in the range of 50-100 μM and show obvious toxic effects in the range of 50-200 μM. In contrast, compound 4 and the positive control kojic acid are also safe enough at 200 μM.

[0193] Efficacy Verification Example 2 Tyrosinase Inhibition of Compounds 1-4

[0194] The inhibitory effects of different concentrations (1 μM, 5 μM and 25 μM) of compounds 1-4 on tyrosinase activity in B16 cells were evaluated. Fig.18 The α-MSH-induced tyrosinase activity in B16 cells increased to 160.91±10.00% of that in normal cells, while at concentrations of 1, 5, and 25 μM, all four compounds significantly reduced tyrosinase activity. Compound 4 (1 μM) had the most significant inhibitory effect on cells, reducing it to 122.13±2.96% of normal cells. At a concentration of 5 μM, the inhibitory effects of all compounds exceeded those of the positive controls α-arbutin and kojic acid.

[0195] Efficacy Verification Example 3 Melanin Content Reducing Ability of Compounds 1-4

[0196] The ability of different concentrations (1 μM, 5 μM and 25 μM) of compounds 1-4 to reduce the melanin content in B16 cells was evaluated. Fig.19 The compounds at three concentrations could significantly inhibit the production of melanin in the model cells, among which compounds 3 and 4 (5 μM) had the best inhibitory effect, almost restoring the melanin level to normal levels (104.35±5.65% and 98.04±7.48% of normal cells, respectively), which was significantly better than the two positive controls.

[0197] Efficacy verification example 4 Effect of active component (S5) extracted from coffee on B16 cell viability

[0198] Since the active component (S5) extracted from coffee is mainly composed of fatty acids that are difficult to separate, the inventors directly tested the inhibitory effect of the active component (S5) on α-MSH-induced B16 melanoma cell tyrosinase activity and melanin production.

[0199] The effects of coffee active component (S5) on the viability of B16 mouse melanoma cells at concentrations of 1 μM, 5 μM, 10 μM, 25 μM, 50 μM, 100 μM and 200 μM were studied. Fig. 20 As shown, S5 is safe at a concentration of 1-200ug / mL.

[0200] Efficacy Verification Example 5 Tyrosinase Inhibitory Activity of the Active Component (S5) Extracted from Coffee

[0201] The inhibitory effect of different concentrations (25ug / mL, 50ug / mL, 100ug / mL and 200ug / mL) of the active component (S5) on tyrosinase activity in B16 cells was evaluated. Fig.21 As shown in , a high concentration (100 ug / mL) of the active ingredient S5 completely restored the intracellular tyrosinase activity; when the concentration was 200 ug / mL, its inhibitory effect was even stronger.

[0202] Efficacy Verification Example 6 Melanin Content Reducing Ability of Active Component (S5) Extracted from Coffee

[0203] The inhibitory effect of different concentrations (25ug / mL, 50ug / mL, 100ug / mL and 200ug / mL) of the active component (S5) on tyrosinase activity in B16 cells was evaluated. Fig. 22 As shown in , a high concentration (100 ug / mL) of the active ingredient S5 reduced melanin production below the control level; when the concentration was 200 ug / mL, its inhibitory effect was even stronger.

[0204] Ligand fishing is a promising strategy for screening effective ingredients from complex natural products. In this paper, human tyrosinase (hTYR) was displayed on the surface of Chinese hamster ovary (CHO) cells for the first time and used as bait to develop a new ligand fishing method. The localization of hTYR on the surface of CHO cells was verified by cell surface enzyme activity test and fluorescence microscopy images. The recombinant cells (CHO@hTYR) were able to maintain relatively stable enzyme activity (82.59±2.70%) within 7 days and could be fished repeatedly five times. According to the results of ligand fishing, four tyrosinase inhibitors were isolated from camel thorn, namely 4-methoxy-5-methylcoumarin (1), cedarwood flavonoids (2), tachyphyllum flavonoids (3) and 3,4-dimethoxy-5-methylcoumarin (4). In addition, a low polarity active site was isolated from coffee. All isolated tyrosinase inhibitors significantly reduced α-MSH-induced intracellular tyrosinase activity and melanin levels in B16 cells. At the same time, the inhibitory effect of the active part in coffee (100ug / mL) was better than that of the positive control (α-arbutin and kojic acid), and it was able to restore the melanin level to normal. This study clarifies the application prospects of cell surface display technology in the field of ligand fishing and lays a chemical foundation for the skin whitening effect of camel thorn and coffee.

[0205] The above descriptions are only some preferred embodiments of the present disclosure and an explanation of the technical principles used. Those skilled in the art should understand that the scope of the invention involved in the embodiments of the present disclosure is not limited to the technical solutions formed by a specific combination of the above-mentioned technical features, but should also cover other technical solutions formed by any combination of the above-mentioned technical features or their equivalent features without departing from the above-mentioned inventive concept. For example, the above-mentioned features are replaced with the technical features with similar functions disclosed in the embodiments of the present disclosure (but not limited to) and the technical solutions formed.

Claims

1. A recombinant hamster ovary cell CHO@hTYR, loaded with a plasmid encoding human tyrosinase, can express human tyrosinase in the form of membrane protein on the cell surface.

2. The recombinant cell CHO@hTYR according to claim 1, characterized in that: The plasmid loaded with human tyrosinase encoding is a recombinant plasmid pCDNA3.1 / hTYR containing the hTYR-CD8hinge-CD8TM-CD8cyto-EGFP gene.

3. The method for constructing the recombinant cell CHO@hTYR according to any one of claims 1 to 2, comprising the following steps: Step 1: CHO cell culture; Step 2: The human tyrosinase gene was successfully inserted into the pcDNA3.1(+) vector to obtain the recombinant vector pcDNA3.1 / hTYR; Step 3: The recombinant vector pcDNA3.1 / hTYR was stably transfected into CHO cells to obtain recombinant cells CHO@hTYR.

4. A method for screening tyrosinase inhibitors from plant extracts, comprising the following steps: The first step is to mix and incubate the recombinant cell CHO@hTYR according to any one of claims 1 to 3 with a plant extract to obtain a first mixture; Step 2: centrifuge the first mixture to remove the supernatant, and wash with buffer for multiple times to obtain a second mixture; Step 3: Add a solvent to the second mixture, vortex, and separate the recombinant cells and the desorption solution containing the enzyme inhibitor by centrifugation to obtain a tyrosinase inhibitor mixture.

5. A method for screening tyrosinase inhibitors from camel thorn plant extracts, comprising the following steps: The first step is to mix and incubate the recombinant cell CHO@hTYR according to any one of claims 1 to 3 with a plant extract to obtain a first mixture; Step 2: centrifuge the first mixture to remove the supernatant, and wash with buffer for multiple times to obtain a second mixture; The third step is to add a desorption solvent to the second mixture, vortex, and separate the recombinant cells and the desorption solution containing the enzyme inhibitor by centrifugation to obtain a tyrosinase inhibitor mixture, wherein the tyrosinase inhibitor mixture includes 4-methoxy-5-methylcoumarin, cedarwood bioflavone, aconitum bioflavone and 3,4-dimethoxy-5-methylcoumarin.

6. Use of 4-methoxy-5-methylcoumarin in the preparation of a tyrosinase inhibitor or a melanin production inhibitor.

7. Use of cedarwood biflavonoids in the preparation of tyrosinase inhibitors or melanin production inhibitors.

8. Use of aconitum biflavone in the preparation of a tyrosinase expression inhibitor or a melanin production inhibitor.

9. Use of 3,4-dimethoxy-5-methylcoumarin in the preparation of a tyrosinase expression inhibitor or a melanin production inhibitor.

10. A skin care product comprising a whitening ingredient, wherein the whitening ingredient is one or more of 4-methoxy-5-methylcoumarin, cedarwood bioflavone, aconitum bioflavone and 3,4-dimethoxy-5-methylcoumarin.

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