A melanin synthesis signal antagonizing composition

Abstract

Provided is a composition comprising 20-40 parts by weight of cordyceps militaris powder, 3-25 parts by weight of theaflavins, and 1.25-4.2 parts by weight of astaxanthin, which is a whitening composition or a melanin synthesis signal antagonizing composition. The composition can be used as a main ingredient in whitening food or health food.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of whitening, in particular to a melanin synthesis signal antagonistic composition. BACKGROUND

[0002] With the development of economy, people, especially women, pay more and more attention to skin color and pursue white skin. There are a large number of reports on whitening products in the prior art.

[0003] For example, Chinese patent application CN201910064750.8 discloses a whitening composition, a whitening and skin lightening cream comprising the whitening composition and a preparation method thereof. The whitening composition comprises extracts of eight Chinese medicinal materials, i.e. ginseng, mulberry bark, angelica, licorice, tea leaves, black ganoderma, rhodiola, and ginkgo leaves, in combination with traditional whitening agents, i.e. nicotinamide and trans-amic acid, and has the synergistic whitening and reduced irritation effect.

[0004] Chinese patent application CN201310025119.X discloses the application of theaflavins in the preparation of skin care products and a skin care product containing theaflavins. The application of theaflavins in the preparation of skin care products allows the skin care products to contain theaflavins, achieving the effects of whitening and freckle removal.

[0005] Chinese patent application CN202310987021.6 discloses a microcapsule astaxanthin powder oral liquid product and a preparation method thereof. The oral liquid can fully utilize the strong and effective antioxidant performance of the microcapsule astaxanthin powder, help cells reduce oxidative stress damage, protect the health of various tissues and organs of the body, and has the effects of protecting vision, whitening and sun protection, delaying aging, preventing diabetes, preventing cardiovascular and cerebrovascular diseases, enhancing immunity, and preventing cancer.

[0006] Chinese patent application CN202211309869.5 discloses a skin external use composition containing cordycepin and having anti-aging activity and the use of the composition in the preparation of anti-aging cosmetics.

[0007] Chinese patent application CN202110077867.7 discloses a whitening and freckle-removing emulsion, which comprises angelica essential oil, cortex meliae essential oil, rose essential oil, aloe vera, grape seeds, pearl powder, saffron powder, licorice powder, methyl silicone oil, monoglyceride, an oil-in-water base, distilled water, and glycerol.

[0008] Chinese patent application CN109463730A discloses a composition of theanine and theacrine in a certain ratio, which can be used as an efficacy component of liquid beverages, candies, solid beverages and tablets, etc. to make the products have the effects of anti-microbial, anti-mutagenic, anti-metastatic, anti-angiogenic, anti-fungal, anti-diabetic, anti-inflammatory, anti-platelet aggregation, immune regulation, blood sugar reduction, anti-oxidation, anti-cancer, anti-cancer and anti-cardiovascular and cerebrovascular diseases, blood lipid regulation, weight loss and obesity prevention. However, the composition does not have the effect of whitening.

[0009] There is still a need in the art for whitening products for oral use. SUMMARY

[0010] The present application is based on the following finding of the inventors in their long-term work: Traditional whitening drugs or skin care products generally focus on the melanin production stage, reduce the production of melanin by inhibiting the tyrosinase activity in melanocytes, and thus achieve the effect of whitening. However, little attention is paid to the melanin signal initiation stage. The purpose of the present application is to seek a reasonable composition and ratio, which can reduce the production of melanin by blocking the expression of α-MSH in the α-MSH-MC1R signal pathway, and thus achieve the effect of whitening.

[0011] The inventors found that the prior art discloses the combination of theanine and theacrine to make the product have some effects in non-whitening directions. The three-component composition of the present application synergistically inhibits the expression of α-MSH and thus achieves the effect of whitening.

[0012] In one aspect, the present application provides a composition comprising a composition comprising 20-40 parts by weight of cordyceps militaris powder, 3-25 parts by weight of theacrine and 1.25-4.2 parts by weight of astaxanthin, the composition being a whitening composition or a melanin synthesis signal antagonistic composition.

[0013] In one embodiment, the composition comprises 20-35 parts by weight, 20-30 parts by weight, 23-27 parts by weight or 25 parts by weight of cordyceps militaris powder.

[0014] In one embodiment, the composition comprises 3-20 parts by weight of theacrine.

[0015] In one embodiment, the composition comprises 1.5-4 parts by weight or 2 parts by weight of astaxanthin.

[0016] In one embodiment, the composition comprises 25 parts by weight of cordyceps militaris powder, 3-25 parts of theacrine, 1.25-4.2 parts of astaxanthin.

[0017] In one embodiment, the cordyceps militaris powder has a cordycepin content of ≥0.2%. In one embodiment, the tea powder has a theacrine content of ≥40%. In one embodiment, the Haematococcus pluvialis astaxanthin oil has an astaxanthin content of ≥10%.

[0018] In one embodiment, the Cordyceps militaris powder is a dried powder of a Cordyceps militaris water extract. In one embodiment, the tea powder is a tea powder obtained by withering, rolling, fermenting, drying, and crushing tea leaves.

[0019] In another aspect, the present application provides a composition comprising 20-40 parts by weight of Cordyceps militaris powder, 60-500 parts by weight of tea powder, 62.5-210 parts by weight of astaxanthin oil or astaxanthin powder, the composition being a whitening composition or a melanin synthesis signal antagonistic composition.

[0020] In one embodiment, the composition comprises 20-35 parts by weight, 20-30 parts by weight, 23-27 parts by weight, or 25 parts by weight of Cordyceps militaris powder.

[0021] In one embodiment, the composition comprises 60-400 parts by weight of tea powder.

[0022] In one embodiment, the composition comprises 75-150 parts by weight or 100 parts by weight of astaxanthin oil.

[0023] In one embodiment, the Cordyceps militaris powder has a cordycepin content of ≥ 0.2%.

[0024] In one embodiment, the Cordyceps militaris powder is obtained by extracting Cordyceps militaris in deionized water to obtain a mixed solution, filtering, discarding the residue, concentrating, and drying.

[0025] In one embodiment, the tea powder has a theaflavin content of ≥ 40%.

[0026] In one embodiment, the tea powder is a tea powder obtained by withering, rolling, fermenting, drying, and crushing tea leaves.

[0027] In one embodiment, the astaxanthin oil has an astaxanthin content of ≥ 10%.

[0028] In one aspect, the present application provides a composition comprising 20-40 parts by weight of Cordyceps militaris powder, 7.5-62.5 parts by weight of tea powder, 12.5-42 parts by weight of astaxanthin oil or astaxanthin powder, the composition being a whitening composition or a melanin synthesis signal antagonistic composition; wherein the tea powder has a theaflavin content of ≥ 40%, and the astaxanthin oil or astaxanthin powder has an astaxanthin content of ≥ 10%.

[0029] In one embodiment, the composition comprises 20-35 parts by weight, 20-30 parts by weight, 23-27 parts by weight, or 25 parts by weight of Cordyceps militaris powder.

[0030] In one embodiment, the composition comprises 7.5-50 parts by weight of theaflavin.

[0031] In one embodiment, the composition comprises 15-40 parts by weight or 20 parts by weight astaxanthin.

[0032] In various embodiments of the present application, the composition is in liquid, gel or solid form. In various embodiments, the composition is in tablet, capsule, powder or solution form. In various embodiments, the composition is an oral composition. In various embodiments, the composition further comprises a food or nutraceutical acceptable additive, such as a carrier, diluent or excipient.

[0033] In another aspect, there is provided a method of making the composition described herein, comprising preparing cordyceps powder, theaflavins, astaxanthin into a composition, or preparing cordyceps powder, tea powder, astaxanthin oil into a composition.

[0034] In another aspect, there is provided a whitening method comprising the step of administering the composition described herein to a subject in need thereof. Preferably, the administration is oral administration.

[0035] In one embodiment, the whitening method further comprises administering an additional whitening product to the subject. Preferably, the subject is a human.

[0036] In another aspect, there is provided a method of blocking the expression of a- MSH in the a-MSH-MC1 R signaling pathway to reduce melanin production, comprising the step of contacting the composition described herein with a cell. Preferably, the cell is a melanoma cell.

[0037] In yet another aspect, there is provided a method of inhibiting tyrosinase, comprising the step of contacting the composition described herein with a cell. Preferably, the cell is a melanoma cell.

[0038] Advantages of the present application include:

[0039] 1. The present application provides a new composition for whitening, blocking the a- MSH-MC1 R signaling pathway or reducing melanin production;

[0040] 2. The composition provided by the present application can synergistically block the expression of a-MSH in the a-MSH-MC1 R signaling pathway to reduce melanin production, thereby achieving the effect of whitening.

[0041] 3. The present application has the best synergistic effect when it comprises three components and the three components are in a particular ratio (when 25 parts by weight of cordyceps powder and 2 parts by weight of astaxanthin are used, theaflavins are 3-25 parts by weight), at which time it can effectively block the a-MSH-MC1 R signaling pathway, inhibit tyrosinase activity, and achieve the effect of reducing melanin production. BRIEF DESCRIPTION OF DRAWINGS

[0042] Figure 1The α-MSH production content of each treatment group is shown. As shown in Table 2, the α-MSH production content of Example 2 is the lowest, 209.33 pg / mL, which can reduce the α-MSH content production by 29.4% compared with the control group. Significant difference is indicated: the same letter indicates that there is no significant difference between the two groups; different letters indicate that there is a significant difference between the two groups. Figure 1

[0043] The relative content of melanin of each treatment group is shown. As shown in Table 3, the relative content of melanin of Example 2 is the lowest. Significant difference is indicated: the same letter indicates that there is no significant difference between the two groups; different letters indicate that there is a significant difference between the two groups. Figure 2 Figure 2 The tyrosinase activity inhibition rate of each treatment group is shown. As shown in Table 4, the tyrosinase activity inhibition rate of Example 2 is the highest, 34.05%. Significant difference is indicated: the same letter indicates that there is no significant difference between the two groups; different letters indicate that there is a significant difference between the two groups.

[0044] Figure 3 DETAILED DESCRIPTION Figure 3 DEFINITIONS

[0045] As used herein, “cordyceps militaris powder” is cordyceps militaris added to water for extraction to obtain a mixed solution, filtration, discarding the residue, concentration, drying, and obtaining cordyceps militaris powder, which can be directly used as a solid beverage or as a food raw material. Cordyceps militaris powder can contain various components such as adenosine, cordycepin, polysaccharides, etc. “Cordycepin” is the main bioactive substance of cordyceps militaris fungus, which has attracted attention from the medical community since its discovery, and has the effects of regulating immune function, improving comprehensive disease resistance, and anti-bacterial and anti-inflammatory. In this text, cordyceps militaris powder can be characterized by its cordycepin content, and in this text, cordycepin is used as a material-based component. Those skilled in the art will understand that cordyceps militaris powder is not equivalent to cordycepin. For example, the cordycepin content of cordyceps militaris powder can be ≥0.05%. Cordyceps militaris powder can be a dried powder of cordyceps militaris water extract. In this text, cordyceps militaris powder is commercially available and can be routinely obtained by those skilled in the art. The cordyceps militaris powder in this text should meet the national food or industry standards.

[0046] As used herein, “theaflavins” are a class of benzotropolone structural compounds that can be dissolved in ethyl acetate and contain multiple hydroxyl or phenolic hydroxyl groups, which are formed after oxidation and polymerization of tea polyphenols. Although tea powder is used in the examples, those skilled in the art understand that the same amount of theaflavins or other substances containing theaflavins can be used to replace tea powder.

[0047] As used herein, “theaflavins” are a class of benzotropolone structural compounds that can be dissolved in ethyl acetate and contain multiple hydroxyl or phenolic hydroxyl groups, which are formed after oxidation and polymerization of tea polyphenols. Although tea powder is used in the examples, those skilled in the art understand that the same amount of theaflavins or other substances containing theaflavins can be used to replace tea powder.

[0048] ​​As used herein, "tea powder" is prepared by withering tea fresh leaves, then rolling, and then fermenting, and finally drying by heating to stop fermentation, and then pulverizing to obtain tea powder. The preparation method of tea powder is conventional, and can be obtained by commercial means by those skilled in the art. In this text, tea powder can be characterized by theaflavins, for example, the theaflavins content of tea powder can be ≥5%. In addition to theaflavins, tea powder can also contain other ingredients other than theaflavins. The tea powder herein shall meet the national food or industry standards. The tea powder can be black tea powder.

[0049] As used herein, "astaxanthin" is a ketocarotenoid. In this text, astaxanthin can be derived from Haematococcus pluvialis astaxanthin oil or Haematococcus pluvialis powder.

[0050] As used herein, "astaxanthin oil or powder" is astaxanthin oil or powder prepared by Haematococcus pluvialis algal bodies and spores or Haematococcus pluvialis powder as raw materials, through processes such as cell wall breaking and extraction. In this text, astaxanthin can be Haematococcus pluvialis astaxanthin oil or Haematococcus pluvialis powder. In this text, Haematococcus pluvialis astaxanthin oil can be characterized by astaxanthin, for example, the astaxanthin content of Haematococcus pluvialis astaxanthin oil can be ≥2%. The astaxanthin oil herein shall meet the national food or industry standards.

[0051] As used herein, "melanin" is a kind of amino acid-derived biological pigment synthesized by melanocytes, belonging to polyphenolic polymers, mainly divided into two types of brown / black eumelanin and red / yellow pheomelanin. The synthesis process of melanin is complex, and L-tyrosine oxidation is the starting step of melanin synthesis, and tyrosinase (TYR), tyrosinase-related protein 1 (TRP-1) and tyrosinase-related protein 2 (TRP-2) are important rate-limiting factors for melanin synthesis. The expression level of melanin synthesis rate-limiting enzyme is also regulated by a variety of proteins. α-MSH is a polypeptide produced by melanocytes and keratinocytes through autocrine and paracrine, as a starting signal, triggering G protein coupling by binding to the receptor MC1R on melanocytes, starting melanin synthesis, promoting the up-regulation of TYR, TRP-1 and TRP-2 expression, and stimulating the synthesis of black (or brown) pigment granules. The composition herein can block the expression of α-MSH in the α-MSH-MC1R signaling pathway and thus reduce the production of melanin, thereby achieving the effect of whitening.

[0052] Composition

[0053] There is no prior art report on the whitening or tyrosinase activity inhibiting effect of Cordyceps militaris powder, theaflavins and astaxanthin. In this disclosure, the inventors have combined these three components together and found that the resulting composition has whitening or tyrosinase activity inhibiting effect. Further, the inventors have found that when the three components are combined in a specific ratio, the resulting composition can achieve excellent synergistic effect in inhibiting the content of a-MSH or tyrosinase activity (i.e. the effect of inhibiting the content of a-MSH or tyrosinase activity is better than the simple addition of the individual components). Prior art has reported that when multiple ingredients with similar effect are combined to form a composition, the resulting composition can have an effect that is lower than or equal to the addition of the individual ingredients, only the addition effect or even the possibility of the individual ingredients adversely affecting each other. The above technical effect of the present disclosure is unexpected.

[0054] A composition is provided herein, which can be a whitening composition or a melanin synthesis signal antagonizing composition. The composition can comprise 20-40 parts by weight of Cordyceps militaris powder, 3-25 parts by weight of theaflavins and 1.25-4.2 parts by weight of astaxanthin. The composition can comprise 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 parts by weight of Cordyceps militaris powder. The composition can comprise 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 parts by weight of theaflavins. The composition can comprise 1.5, 2.5, 3, 3.5 or 4 parts by weight of astaxanthin. The cordycepin content of the Cordyceps militaris powder can be ≥ 0.05%, for example 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.5%, 1%, 2%, 3%, 4%, 5% or 6%. The Cordyceps militaris powder can be extracted by adding Cordyceps militaris to water to obtain a mixture, filtering, discarding the residue, concentrating and drying to obtain. The theaflavins can be added to or included in the composition in the form of tea powder. The astaxanthin can be added to or included in the composition in the form of Haematococcus pluvialis astaxanthin oil or Haematococcus pluvialis powder. The tea powder can have a theaflavins content of ≥ 5%, for example 5%, 10%, 20%, 30%, 40%, 45%, 50%, 55%, 60%, 65% or 70%. The Haematococcus pluvialis astaxanthin oil or Haematococcus pluvialis powder can have an astaxanthin content of ≥ 2%, for example 2%, 2.5%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50%.

[0055] Also provided herein is a composition comprising 20-40 parts by weight of cordyceps militaris powder, 60-500 parts by weight of tea powder, 62.5-210 parts by weight of astaxanthin oil or astaxanthin powder, the composition being a whitening composition or a melanin synthesis signal antagonistic composition. The composition can comprise 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 parts by weight of cordyceps militaris powder. The composition can comprise 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500 parts by weight of tea powder. The composition can comprise 63, 65, 70, 75, 80, 85, 90, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 parts by weight of astaxanthin oil or astaxanthin powder. The cordyceps militaris powder can have a cordycepin content of ≥ 0.05%, for example 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.5%, 1%, 2%, 3%, 4%, 5%, or 6%. The cordyceps militaris powder can be obtained by adding cordyceps militaris to water to extract, obtaining a mixed solution, filtering, discarding the residue, concentrating, and drying. The tea powder can have a theaflavins content of ≥ 5%, for example 5%, 10%, 20%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, or 70%. The astaxanthin oil or astaxanthin powder can have an astaxanthin content of ≥ 2%, for example 2%, 2.5%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%. The tea powder can be tea powder obtained by withering, rolling, fermenting, drying, and crushing tea leaves.

[0056] Also provided herein is a composition comprising 20-40 parts by weight of Cordyceps militaris powder, 7.5-62.5 parts by weight of tea powder, 12.5-42 parts by weight of astaxanthin oil or astaxanthin powder, which is a whitening composition or a melanin synthesis signal antagonistic composition. The tea powder can have a theaflavin content of > 40%. The astaxanthin oil or astaxanthin powder can have an astaxanthin content of > 10%. In this embodiment, the composition comprises 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 parts by weight of Cordyceps militaris powder. In this embodiment, the composition comprises 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, or 61 parts by weight of theaflavin. In this embodiment, the composition comprises 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 parts by weight of astaxanthin.

[0057] The composition can be in any suitable form, for example, in liquid, gel, or solid form. The composition of the present application is an oral composition, preferably in the form of a tablet, capsule, powder, solution, drink, candy, or jelly. Those skilled in the art can understand that the composition of the present application can further comprise food, health food acceptable additives, such as carriers, diluents, or excipients, etc.

[0058] The method of preparing the composition herein is not particularly limited, and can be prepared by those skilled in the art by conventional means. For example, the method can comprise preparing Cordyceps militaris powder, theaflavin, astaxanthin into a composition.

[0059] Method

[0060] The composition herein can be suitable for a subject in need thereof. A whitening method can be provided herein, which comprises the step of administering the composition herein to a subject in need thereof. In this context, the administration can be oral administration. The dose and duration of administration can be routinely determined by those skilled in the art or consumers. The whitening method can further comprise administering an additional whitening product to the subject. The subject can be any mammal in need of the composition herein, preferably a human.

[0061] The present application also provides a method for blocking the expression of α-MSH in the α-MSH-MC1R signal pathway, reducing melanin production or inhibiting tyrosinase, which comprises the step of contacting the composition of the present application with cells. Preferably, the cells can be melanoma cells. The method of the present application can be a method for non-therapeutic purposes. Of course, the method of the present application can also be used as a method for therapeutic purposes, if applicable.

[0062] The composition of the present application can be widely used in whitening food or health food.

[0063] Examples

[0064] The embodiments of the present application will be described in detail below with reference to the examples, and those skilled in the art will understand that the following examples are only used to illustrate the present application and should not be regarded as limiting the scope of the present application. If the specific conditions are not specified in the examples, the conventional conditions or the conditions recommended by the manufacturer are used. If the manufacturer of the reagent or instrument is not specified, it is a conventional product that can be obtained by purchase. If not specifically stated, the amounts listed are based on the total weight and described in parts by weight. The present application should not be interpreted as being limited to the specific examples described.

[0065] Method

[0066] 1. Detection of α-MSH protein content:

[0067] ① Inoculate B16 cell suspension (1 x 10 6 cells / well) 1 mL prepared with 10% DMEM complete medium (89% DMEM basal medium + 10% fetal bovine serum + 1% penicillin / streptomycin) in a 6-well plate, and incubate at 37°C in a 5% CO2 incubator for 24 h;

[0068] ② When the cell density reaches 60-70%, add each sample (10% DMEM complete medium as solvent) with a total concentration of 12.5 μg / mL to the sample group, and add DMEM complete medium to the control group cells, 3 replicates per group, and incubate for 48-72 h;

[0069] ③ Discard the old medium, and wash the plate once with 1 mL PBS;

[0070] ④ Discard the liquid, add 500 μL PBS to each well, and freeze in a -80°C refrigerator for 30 min (repeat freezing and thawing 3 times);

[0071] ⑤ Take out the 6-well plate, restore the room temperature, and scrape the cells in the plate with a gun head or a cell scraper and place them in an EP tube;

[0072] ⑥ Centrifuge at 5000-10000 rpm for 10 min, collect the supernatant, and use an ELISA kit (Wuhan Huamei Biotechnology Co., Ltd., CSB-E15876m) to detect the α-MSH content according to the manufacturer's instructions.

[0073] 2. Melanin content determination:

[0074] ① Inoculate 1×10⁶ B16 cell suspensions prepared with 10% DMEM complete medium (89% DMEM basal medium + 10% fetal bovine serum + 1% penicillin / streptomycin) into 24-well plates. 5 0.5 mL (cells / well), incubated at 37°C in a 5% CO2 incubator for 24 h;

[0075] ③ When the cell density reaches 60-70%, each sample group is added with a total concentration of 12.5 μg / ml (10% DMEM complete medium as solvent), and the control group cells are added with DMEM complete medium. Each group is repeated 3 times and cultured for 48-72 hours.

[0076] ③ Discard the old culture medium and add 1 ml of PBS to the well plate to wash once;

[0077] ④ Add 500 μl of trypsin to each well and incubate at 37°C for 5 min to allow for complete digestion;

[0078] ⑤ Transfer the digested cells to a 1.5 ml centrifuge tube, wash once with 500 μl of PBS, and then transfer to a centrifuge tube at a concentration of 1 × 10⁻⁶ cells / mL. 4 Centrifuge at rpm for 10 min;

[0079] ⑥ Discard the supernatant, add 200 μl of PBS solution containing 1 M NaOH (containing 10% DMSO), and incubate in an 80°C water bath for about 30 min until the melanin dissolves. Transfer 100 μl of the solution to a 96-well plate and read the plate at a wavelength of 405 nm or 475 nm.

[0080] Formula for calculating the relative content of melanin:

[0081] Relative melanin content = (absorbance value of sample treatment group - absorbance value of blank control) / (absorbance value of control group - absorbance value of blank control).

[0082] 3. Determination of tyrosinase activity inhibition rate:

[0083] ①96 well seeding with 10% DMEM complete medium (89% DMEM basal medium + 10% fetal bovine serum + 1% penicillin / streptomycin) prepared B16 cell suspension B16 cell suspension (10000 cells / well) 100 μl, 96 well plate around a circle plus 100 μl PBS solution, 37°C, 5% CO2 incubator for 24h;

[0084] ②Sample group added total concentration of 12.5 μg / ml of each sample (10% DMEM complete medium as solvent), control group cells added DMEM complete medium, 3 replicates per group, incubated for 24-48h.

[0085] ③Discard the old culture medium, add 100 μL PBS and wash twice;

[0086] ④96 well plate around a circle of 3 selected blank wells without cells as blank control, sample group, control group and blank control group added 1% concentration of octylphenol polyoxyethylene ether (1% PBST) solution 50 μl, quickly placed in -80°C freezer for 30 min, then thawed at room temperature to break the cells;

[0087] ⑤37°C warm bath for 5-10 min, then added 50 μl of L-DOPA PBS solution with a mass fraction of 10mM;

[0088] ⑥37°C incubation for 1-2h, 490nm detection of absorbance.

[0089] Using the formula to calculate the inhibition rate of tyrosinase.

[0090] Inhibition rate = (1-(sample group absorbance-blank control group absorbance) / (control group absorbance-blank control group absorbance) x 100%

[0091] Example 1-11: Preparation of the composition of example 1-11

[0092] Cordyceps militaris powder, tea powder and astaxanthin oil used in the examples were all commercially available, among which the cordycepin content of cordyceps militaris powder was 0.05%, the theaflavins content of tea powder was 5%, and the astaxanthin content of haematococcus pluvialis astaxanthin oil was 2%. According to the supplier's instructions, cordyceps militaris powder was obtained by adding cordyceps militaris to deionized water for extraction to obtain a mixed solution, filtering, discarding the residue, concentrating, and spray drying; tea powder was obtained by wilting fresh tea leaves, then rolling and fermenting, and finally stopping fermentation by heating and drying; and haematococcus pluvialis astaxanthin oil was obtained by oil solvent extraction of haematococcus pluvialis.

[0093] The components were mixed uniformly according to the weight fractions of the cordyceps militaris powder, theaflavins, and astaxanthin shown in Table 1 to obtain the compositions of Examples 1-11. Theaflavins and astaxanthin in Table 1 indicate theaflavins and astaxanthin oil of Haematococcus pluvialis in terms of the material basis composition of theaflavins and astaxanthin oil of Haematococcus pluvialis. The addition amount of theaflavins and astaxanthin oil of Haematococcus pluvialis was determined according to theaflavins and astaxanthin.

[0094] In the tables and figures herein, the compositions of Examples 1-11 are simply referred to as “Examples 1-11”. Similarly, Comparative Examples 1-8 were prepared from the materials shown in Table 1. In the tables and figures herein, the compositions of Comparative Examples 1-8 are simply referred to as “Comparative Examples 1-8”.

[0095] Table 1: Composition components of examples and comparative examples

[0096]

[0097]

[0098] Note: The use amount of theaflavins and astaxanthin oil is 20 times and 50 times the amount of theaflavins and astaxanthin, respectively.

[0099] Example 13: Efficacy determination

[0100] According to the methods of α-MSH protein content detection, melanin content determination, and tyrosinase activity inhibition rate determination described above, the compositions of Examples 1-11 and Comparative Examples 1-8 were used as treatment reagents to perform the corresponding detection. The results of α-MSH protein content detection, melanin content determination, and tyrosinase activity inhibition rate determination are shown in Table 2.

[0101] Table 2: α-MSH protein content detection, melanin content determination, and tyrosinase activity inhibition rate determination

[0102]

[0103]

[0104] Discussion

[0105] Combination Figures 1-3 The test results of Examples 1-11 and Comparative Examples 1-8 described above were analyzed, wherein: Figure 1 The α-MSH production content of each treatment group is shown, and the total concentration of each experimental group is consistent (maintaining the same concentration is to control the variable, and if the concentration is not consistent, the results obtained cannot be judged as the effect of the composition itself or the experimental results caused by the concentration difference). mean ± SD, n = 3. The difference analysis used One-way ANOVA. Figure 2The relative melanin content of each treatment group is shown, and the total concentration of each experimental group is consistent, mean ± SD, n = 3. One-way ANOVA was used for difference analysis. Figure 3 The tyrosinase activity inhibition rate of each treatment group is shown, and the total concentration of each experimental group is consistent, mean ± SD, n = 3. One-way ANOVA was used for difference analysis.

[0106] (1) The examples 1-11 and the comparative examples 3-5 were analyzed. Comparative example 3 is a two-component composition without theaflavins, comparative example 4 is a two-component composition without cordyceps militaris powder, and comparative example 5 is a composition without astaxanthin, and the content of α-MSH protein in comparative examples 3-5 is higher than that in examples 1-11, and the inhibitory rate of the composition on the generation of α-MSH protein is low, and the relative content of melanin generation in comparative examples 3-5 is relatively high, and the inhibitory rate of the composition on tyrosinase activity is low. The content of theaflavins in the composition of comparative example 1 is low, and the content of theaflavins in the composition of comparative example 6 is high. Compared with the control group, the composition of comparative example 1 and comparative example 6 only weakly inhibits the expression of α-MSH in the α-MSH-MC1R signaling pathway and reduces the generation of melanin. The content of astaxanthin in the composition of comparative example 7 is low, and the content of astaxanthin in the composition of comparative example is high, and compared with the control group, the composition of comparative examples 7 and 8 only weakly inhibits the expression of α-MSH in the α-MSH-MC1R signaling pathway and reduces the generation of melanin.

[0107] Therefore, compared with the control group, the composition of comparative examples 1-8 can improve the tyrosinase activity inhibition rate (%) and the α-MSH content generation inhibition rate, but the degree of improvement is small and is not statistically significant.

[0108] Table 3: Effect of the control group and comparative examples 1-8

[0109]

[0110]

[0111] (2) Analyze Example 1-7 and Comparative Examples 1, 6. The content of theaflavins in the composition of Comparative Example 1 is low, and the content of theaflavins in the composition of Comparative Example 6 is high. Although the compositions of Comparative Examples 1 and 6 can weakly inhibit the expression of α-MSH in the α-MSH-MC1R signal pathway and reduce the generation of melanin compared with the control group, the effect is not significant compared with the three-component compositions of Examples 1-7. In Examples 1-7, by reasonably matching the proportions of cordyceps militaris powder, theaflavins and astaxanthin, the expression of α-MSH in the α-MSH-MC1R signal pathway can be significantly inhibited, and the generation of melanin can be reduced. Especially when the cordyceps militaris powder in the composition is 25 parts, the theaflavins are 4 parts, and the astaxanthin is 2 parts (Example 2), the content of α-MSH generated is the lowest, which is 209.33 pg / mL, and the inhibition rate of α-MSH generation is 29.40% compared with the control group; at the same time, the relative content of melanin generation is the lowest, which is 0.69, and the inhibition rate of tyrosinase activity is the highest, which is 34.05%.

[0112] (3) Analyze Example 2, 8-11 and Comparative Examples 7, 8. The content of astaxanthin in the composition of Comparative Example 7 is low, and the content of astaxanthin in the composition of Comparative Example is high. Although the compositions of Comparative Examples 7 and 8 can weakly inhibit the expression of α-MSH in the α-MSH-MC1R signal pathway and reduce the generation of melanin compared with the control group, the effect is not significant compared with the three-component compositions of Examples 2, 8-11. In Examples 2, 8-11, by reasonably matching the proportions of cordyceps militaris powder, theaflavins and astaxanthin, the expression of α-MSH in the α-MSH-MC1R signal pathway can be significantly inhibited, and the generation of melanin can be reduced. Especially when the cordyceps militaris powder in the composition is 25 parts, the theaflavins are 4 parts, and the astaxanthin is 2 parts (Example 2), the content of α-MSH generated is the lowest, which is 209.33 pg / mL, and the inhibition rate of α-MSH generation is 29.40% compared with the control group; at the same time, the relative content of melanin generation is the lowest, which is 0.69, and the inhibition rate of tyrosinase activity is the highest, which is 34.05%.

[0113] (4) Analyze Example 1-11 and Comparative Example 2. Although the composition of Comparative Example 2 contains cordyceps militaris powder, theaflavins and astaxanthin, the content of each component is low, so compared with Examples 1-11, although Comparative Example 2 can inhibit the expression of α-MSH in the α-MSH-MC1R signal pathway and reduce the generation of melanin to some extent, the effect is not significant.

[0114] (5) When the composition contains 25 parts by weight of Cordyceps militaris powder and 2 parts by weight of astaxanthin, the tyrosinase activity inhibition rate is low when the theaflavins are 1 part by weight, but when the theaflavins are gradually increased to 4 parts by weight, the tyrosinase activity inhibition rate increases rapidly, and the maximum tyrosinase activity inhibition rate of 34.05% is obtained when the theaflavins are 4 parts by weight; further increasing the theaflavins will reduce the tyrosinase activity inhibition rate; when the theaflavins reach 32 parts by weight, the tyrosinase activity inhibition rate decreases to 4.18%.

[0115] Table 4

[0116]

[0117] (6) Analysis of Example 2 and Comparative Examples 3-5, Example 2 is a composition containing three components at the same time, Comparative Example 3 is a two-component composition without theaflavins, Comparative Example 4 is a two-component composition without Cordyceps militaris powder, and Comparative Example 5 is a two-component composition without astaxanthin. When the three components are combined in pairs (Comparative Examples 3-5), there seems to be no synergistic effect between the components, only with weak tyrosinase activity and α-MSH inhibitory activity, indicating that the tyrosinase activity and α-MSH inhibitory activity of each component alone is weak. However, when the three components are combined, the tyrosinase activity and α-MSH inhibitory activity of the three-component composition (e.g., Examples 1-11) far exceeds the simple superposition of the tyrosinase activity or α-MSH inhibitory activity of the three components, achieving a synergistic effect.

[0118] More surprisingly, Example 2 has better synergistic effect than Comparative Examples 3-5, at which time the composition can effectively block the α-MSH-MC1R signaling pathway, inhibit tyrosinase activity, and achieve the effect of reducing melanin production.

[0119] Table 5

[0120]

[0121] The above can show that the three-component composition containing Cordyceps militaris powder, theaflavins and astaxanthin at the same time can effectively inhibit the expression of α-MSH in the α-MSH-MC1R signaling pathway, reduce the production of melanin, and achieve the effect of whitening.

[0122] While the application has been described with reference to the illustrative embodiments, those skilled in the art will understand that various other changes, omissions, additions and / or modifications can be made without departing from the spirit and scope of the application. Further, many modifications can be made to adapt a particular situation or material to the teachings of the application without departing from its scope. Therefore, it is intended that the application not be limited to the disclosed embodiments, but will include all embodiments falling within the scope of the appended claims.

Claims

1. A composition, said composition being a skin-whitening composition or a melanin synthesis signal antagonist composition, said composition comprising a three-component composition consisting of 25 parts by weight of Cordyceps militaris powder, 3-25 parts by weight of theaflavins, and 1.25-4.2 parts by weight of astaxanthin. The cordycepin content in Cordyceps militaris powder is ≥0.05%; Theaflavins are in the form of tea powder, and the theaflavin content of the tea powder is ≥5%; Astaxanthin is in the form of astaxanthin oil or astaxanthin powder, wherein the astaxanthin content of the astaxanthin oil or astaxanthin powder is ≥2%.

2. The composition according to claim 1, wherein the composition comprises 3-20 parts by weight of theaflavins.

3. The composition according to claim 1, wherein the composition comprises 1.5-4 parts by weight of astaxanthin.

4. The composition according to claim 3, wherein the composition comprises 2 parts by weight of astaxanthin.

5. The composition according to claim 1, wherein the cordycepin content of the Cordyceps militaris powder is ≥0.2%.

6. The composition according to claim 1, wherein the astaxanthin oil is Haematococcus pluvialis astaxanthin oil and the astaxanthin powder is Haematococcus pluvialis powder.

7. The composition according to claim 1 or 2, wherein the theaflavins content of the tea powder is ≥10%.

8. The composition according to claim 7, wherein the theaflavins content of the tea powder is ≥25%.

9. The composition according to claim 8, wherein the theaflavins content of the tea powder is ≥40%.

10. The composition according to claim 1, wherein the astaxanthin content of the astaxanthin oil or astaxanthin powder is ≥5%.

11. The composition according to claim 10, wherein the astaxanthin content of the astaxanthin oil or astaxanthin powder is ≥10%.

12. A composition, said composition being a whitening composition or a melanin synthesis signal antagonist composition, said composition comprising a three-component composition consisting of 25 parts by weight of Cordyceps militaris powder, 60-500 parts by weight of tea powder, and 62.5-210 parts by weight of astaxanthin oil or astaxanthin powder, wherein the cordycepin content of the Cordyceps militaris powder is ≥0.05%; the theaflavins content of the tea powder is ≥5%; and the astaxanthin content of the astaxanthin oil or astaxanthin powder is ≥2%.

13. The composition according to claim 12, wherein the composition comprises 60-400 parts by weight of tea powder.

14. The composition according to claim 12, wherein the composition comprises 75-150 parts by weight of astaxanthin oil or astaxanthin powder.

15. The composition according to claim 14, wherein the composition comprises 100 parts by weight of astaxanthin oil or astaxanthin powder.

16. The composition according to any one of claims 12-15, wherein the cordycepin content of the Cordyceps militaris powder is ≥0.2%.

17. The composition according to any one of claims 12-15, wherein the theaflavins content of the tea powder is ≥10%.

18. The composition according to claim 17, wherein the theaflavins content of the tea powder is ≥25%.

19. The composition according to claim 18, wherein the theaflavins content of the tea powder is ≥40%.

20. The composition according to claim 12, wherein the astaxanthin content of the astaxanthin oil or astaxanthin powder is ≥5%.

21. The composition according to claim 20, wherein the astaxanthin content of the astaxanthin oil or astaxanthin powder is ≥10%.

22. The composition according to claim 1 or 12, wherein the composition is in liquid, gel or solid form.

23. The composition of claim 22, wherein the composition is in the form of tablets, capsules, powders, solutions, beverages, candies, or jellies.

24. The composition according to claim 1 or 12, wherein the composition is an oral composition.

25. The composition according to claim 1 or 12, wherein the composition further comprises food-acceptable additives.

26. The composition of claim 25, wherein the additive is a carrier, diluent, or excipient.

27. A method for preparing a composition according to any one of claims 1-26, comprising preparing a composition from Cordyceps militaris powder, theaflavins, and astaxanthin, or preparing a composition from Cordyceps militaris powder, tea powder, astaxanthin oil, or astaxanthin powder.

28. An in vitro method for blocking the α-MSH-MC1R signaling pathway, reducing melanin production, or inhibiting tyrosinase, comprising the step of contacting the composition of any one of claims 1-26 with cells.

29. The method of claim 28, wherein the cells are melanoma cells.

30. Use of the composition of any one of claims 1-26 in the preparation of food.

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