Composition for inhibiting melanin production, application thereof, and whitening method

Abstract

The present application relates to a composition for inhibiting melanin production, its application, and a whitening method. The composition comprises active ingredients and excipients, wherein the active ingredients include one or more combinations of cucurbitacin B, mulberry root C, and hypocrellin A. Experimental verification demonstrates that any monomer of cucurbitacin B, mulberry root C, or hypocrellin A, or any combination thereof, exhibits significant inhibition of mushroom tyrosinase activity, inhibits tyrosinase activity in B16F10 cells, and inhibits melanin production in B16F10 cells, with low cytotoxicity. Application of the composition in cosmetic or pharmaceutical formulations can effectively whiten and combat melasma.

Description

Technical Field

[0001] The present application relates to the fields of cosmetics and pharmaceutical technology, and specifically to a composition for inhibiting melanin production, its application, and a whitening method. Background Art

[0002] Melanin is an important factor affecting the color of human skin, eyes and hair. Excessive melanin can lead to skin pigmentation, and then cause diseases such as chloasma. Therefore, reducing melanin synthesis is the main method of whitening and removing spots.

[0003] Currently commonly used whitening agents such as kojic acid, arbutin, hydroquinone, L-ascorbic acid, ellagic acid, tranexamic acid, etc., have been widely used in the field of whitening cosmetics. However, these whitening active ingredients have disadvantages such as obvious cytotoxicity, strong irritation, high sensitization and large adverse reactions, which make it difficult to meet the needs of consumers. Summary of the Invention

[0004] In response to the problems existing in the prior art, the embodiments of the present application provide a composition for inhibiting melanin production, its application, and a whitening method, aiming to effectively inhibit the production of melanin, achieve whitening and anti-chloasma effects, and at the same time significantly reduce the occurrence of adverse reactions.

[0005] In a first aspect, an embodiment of the present application provides a composition for inhibiting melanin production, the composition comprising active ingredients and excipients, wherein the active ingredients comprise one or more combinations of cucurbitacin B, sangenone C, and hypocrellin A.

[0006] The compositions for inhibiting melanin production provided in the examples of the present application use any one of cucurbitacin B, mulberry root C, and hypocrellin A, or any combination thereof, as active ingredients. Cucurbitacin B, mulberry root C, and hypocrellin A are all derived from natural raw materials that are inexpensive and readily available. Experimental verification has shown that the compositions obtained from any monomer of cucurbitacin B, mulberry root C, and hypocrellin A, or any combination thereof, have significant effects of inhibiting mushroom tyrosinase activity, inhibiting tyrosinase activity in B16F10 cells, and inhibiting melanin production in B16F10 cells, and have low cytotoxicity. Application of the compositions in cosmetics or pharmaceutical preparations can effectively whiten and combat melasma.

[0007] In some embodiments of the present application, the active ingredients include cucurbitacin B, sangenone C, and hypocrellin A in a mass ratio of 2:1:2 to 1:2:1.

[0008] In some embodiments of the present application, the mass percentage of the active ingredient relative to the total amount of the composition is 0.001% to 90%.

[0009] In some embodiments of the present application, the purity of cucurbitacin B, sangenone C, and hypocrellin A is not less than 90%.

[0010] In some embodiments of the present application, the auxiliary material includes at least one of a moisturizer, a thickener, an emulsifier, a neutralizer, and a preservative.

[0011] In a second aspect, an embodiment of the present application provides a cosmetic or pharmaceutical composition comprising the composition for inhibiting melanin production provided in an embodiment of the first aspect of the present application and a cosmetically or pharmaceutically acceptable excipient.

[0012] In some embodiments of the present application, the dosage form of the cosmetic includes ointment, cream, lotion, patch, mask, and tincture.

[0013] In some embodiments of the present application, the dosage forms of the pharmaceutical composition include tablets, capsules, granules, pills, oral liquids, emulsions, dry suspensions, dry extracts, and injections.

[0014] In a third aspect, an embodiment of the present application provides an application of a composition for inhibiting melanin production in the preparation of a pharmaceutical preparation with whitening efficacy.

[0015] In a fourth aspect, an embodiment of the present application provides a whitening method, which comprises applying an effective amount of a whitening product to the skin of a subject to be whitened, wherein the whitening product comprises the composition for inhibiting melanin production provided in the embodiment of the first aspect of the present application or the cosmetic provided in the embodiment of the second aspect of the present application.

[0016] In some embodiments of the present application, the whitening method comprises applying an effective amount of 0.001 wt% to 10 wt% of cucurbitacin B to the skin of a subject to be whitened.

[0017] In some embodiments of the present application, the whitening method comprises applying an effective amount of 0.001 wt% to 10 wt% of hypocrellin A to the skin of the subject to be whitened. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following is a brief introduction to the drawings required for use in the embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0019] Figure 1 The inhibitory effect of the active ingredients in the examples of the present application on tyrosinase is shown;

[0020] Figure 2 The effect of different concentrations of the active ingredients in the examples of this application on the growth of B16F10 cells is shown;

[0021] Figure 3 The effect of the active ingredients in the examples of the present application on tyrosinase activity in B16F10 cells is shown;

[0022] Figure 4 The effect of the active ingredients in the examples of the present application on melanin production in B16F10 cells is shown. DETAILED DESCRIPTION

[0023] The features and exemplary embodiments of various aspects of the present application will be described in detail below. In the detailed description below, many specific details are set forth in order to provide a comprehensive understanding of the present application. However, it will be apparent to those skilled in the art that the present application can be implemented without some of these specific details. The following description of the embodiments is merely intended to provide a better understanding of the present application by illustrating examples of the present application. In the accompanying drawings and the following description, at least some of the well-known structures and technologies are not shown in order to avoid unnecessary ambiguity in the present application; and, for clarity, the sizes of some structures may be exaggerated. In addition, the features, structures, or characteristics described below may be combined in any suitable manner in one or more embodiments.

[0024] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish an entity or operation from another entity or operation, and do not necessarily require or imply that there is any such actual relationship or order between these entities or operations. In the description of the present application, it should be noted that, unless otherwise stated, the meaning of "plurality" is more than two. Moreover, the term "comprises", "comprising" or any other variant thereof is intended to cover non-exclusive inclusion, so that the process, method, article or equipment comprising a series of elements not only include those elements, but also include other elements not clearly listed, or also include elements inherent to such process, method, article or equipment. In the absence of more restrictions, the elements limited by the statement "comprises..." do not exclude the presence of other identical elements in the process, method, article or equipment comprising the elements.

[0025] In the human body, skin color is primarily determined by the content and distribution of pigments within the skin. These pigments primarily include dark-brown melanin (including eumelanin and pheomelanin), red hemoglobin, and yellow carotene. Melanin is a key factor affecting the appearance of skin, eyes, and hair. Certain physiological and environmental factors can affect melanin synthesis. Excessive melanin can lead to skin pigmentation, which can in turn cause conditions like chloasma. Therefore, reducing melanin is a key approach to whitening and freckle removal.

[0026] Melanocytes in the skin use tyrosine as a substrate and produce melanin through the oxidation of multiple rate-limiting enzymes. Melanin is transported to the basal cells of the epidermis via melanocyte dendrites. With cellular metabolism, it is carried to the stratum corneum and excreted as the stratum corneum sheds. In this process, tyrosinase is a key enzyme in the biosynthesis of melanin in the body. Therefore, inhibiting tyrosinase synthesis, inhibiting tyrosinase transport to melanosomes, and inhibiting tyrosinase activity are effective methods to reduce melanin synthesis.

[0027] Although traditional tyrosinase inhibitors such as kojic acid, arbutin, hydroquinone, L-ascorbic acid, ellagic acid, and tranexamic acid have been used as skin-whitening agents, they still have significant drawbacks. For example, kojic acid is carcinogenic and unstable during storage, which limits its application in cosmetics. Arbutin has poor chemical stability and can release hydroquinone during storage and decompose into benzene-containing metabolites, which are potentially toxic to bone marrow. Hydroquinone may be mutagenic to mammalian cells and its skin irritation can cause burning and stinging sensations. L-ascorbic acid is heat-sensitive and easily degraded. Ellagic acid has poor solubility and low bioavailability.

[0028] Traditional whitening active ingredients have disadvantages such as obvious cytotoxicity, strong irritation, high sensitization and adverse reactions, which make it difficult to meet the needs of consumers. Therefore, the search for safe and effective melanin inhibitors derived from natural products and traditional Chinese medicine has become a hot topic of research at home and abroad in recent years. More and more melanin inhibitors derived from natural products and traditional Chinese medicine are being used in whitening and freckle removal products, such as tea polyphenols in green tea, oolong tea and black tea, caffeic acid phenethyl ester in propolis, chrysin, piperine in Piper longum, Rhodiola rosea extract, walnut fruit extract and ginsenosides.

[0029] Based on this, the inventors have conducted extensive research, aiming to provide a new composition of natural products with the effect of inhibiting melanin production, which can be used as a whitening active ingredient in cosmetics or pharmaceutical preparations and has low cytotoxicity, irritation and other adverse reactions.

[0030] Composition for inhibiting melanin production

[0031] An embodiment of the first aspect of the present application provides a composition for inhibiting melanin production, comprising active ingredients and excipients, wherein the active ingredients comprise one or more combinations of cucurbitacin B, sangenone C, and hypocrellin A.

[0032] Cucurbitacin B is one of the active ingredients of melon stems and is a class of tetracyclic triterpenoid compounds. Studies have shown that cucurbitacin B has multiple biological activities such as liver protection, anti-inflammatory and anti-tumor effects. It has a relatively obvious effect of inhibiting tumor cell proliferation and has significant synergistic therapeutic effects in the treatment of malignant tumor cells. Sanggenone C is one of the active ingredients of Morus alba bark and is a natural flavonoid extract. Studies have found that Sanggenone C has antioxidant and anti-inflammatory effects and has certain anti-cancer activity. Bamboored fungus A is the main active ingredient in Bamboored Fungus and Bamboo Yellow Fungus, and is one of the main active ingredients of Bamboo Yellow Fungus. Studies have shown that Bamboored fungus A has certain antibacterial effects and can be used in drugs for the prevention and treatment of diabetes.

[0033] The inventors have conducted in-depth research on cucurbitacin B, sangenone C, and hypocrellin A and found that cucurbitacin B, sangenone C, and hypocrellin A have good performance in the fields of whitening and anti-chloasma. Further pharmacological activity studies have found that cucurbitacin B, sangenone C, and hypocrellin A all show a strong ability to inhibit tyrosinase activity, indicating that they can reduce the amount of cellular melanin production by inhibiting the activity of intracellular tyrosinase. Therefore, the above-mentioned cucurbitacin B, sangenone C, and hypocrellin A have uses in the preparation of cosmetics and / or medicines for whitening, anti-chloasma, and. Therefore, the present application provides a composition for inhibiting melanin production and its use in whitening cosmetics or pharmaceutical preparations, which are both publicly reported for the first time.

[0034] According to the embodiments of the present application, the above-mentioned composition for inhibiting melanin production can be used in cosmetic products such as emulsions, lotions, creams, facial masks, or used in the preparation of pharmaceutical preparations, all of which have the effects of whitening, preventing and treating chloasma.

[0035] In the examples of the present application, cucurbitacin B, mulberry root C and hypocrellin A were prepared by the following methods:

[0036] Cucurbitacin B:

[0037] Taking melon stems or Trichosanthes kirilowii herbs, extracting them with 10-15 times the amount of 60%-70% ethanol for 1-3 times, each time for 1-2 hours, combining the extracts and evaporating the solvent to obtain a crude extract of cucurbitacin B;

[0038] The crude extract of cucurbitacin B is dispersed in water, extracted with petroleum ether and ethyl acetate in sequence to obtain the ethyl acetate fraction, which is then separated by macroporous resin and eluted with ethanol gradient. The 60% to 90% elution fraction is taken and subjected to solvent crystallization to obtain cucurbitacin B with a purity greater than 90%.

[0039] Sanggenone C:

[0040] Taking Morus alba bark medicinal material, extracting it with 10-15 times the amount of 70%-80% ethanol for 1-3 times, each time for 1-2 hours, combining the extracts and evaporating the solvent to obtain a crude extract of Sanggenone C;

[0041] The crude extract of Sanggenone C is dispersed in water, extracted with petroleum ether and ethyl acetate in sequence to obtain the ethyl acetate fraction, which is then separated by macroporous resin and eluted with ethanol gradient. The 55% to 80% elution fraction is taken and subjected to solvent crystallization to obtain Sanggenone C with a purity greater than 90%.

[0042] Hypocrellin A:

[0043] Take the medicinal material of Bamboo Red Fungus (Bamboo Yellow and Bamboo Flower), use 10 to 15 times the amount of 70% to 80% ethanol to extract 1 to 3 times, each time for 1 to 2 hours, combine the extracts and evaporate the solvent to obtain a crude extract of Bamboo Red Fungus A;

[0044] The crude extract of hypocrellin A is dispersed in water, extracted with petroleum ether, ethyl acetate and n-butanol in sequence, the ethyl acetate and n-butanol extraction fractions are combined, and then separated by macroporous resin, eluted with ethanol gradient, and the 50% to 80% elution fraction is taken and solvent crystallized to obtain hypocrellin A with a purity greater than 90%.

[0045] In some embodiments, the active substance includes cucurbitacin B, sangenone C, and hypocrellin A in a mass ratio of 2:1:2 to 1:2:1.

[0046] According to the examples of the present application, the inventors have found through further pharmacological activity studies that when cucurbitacin B, mulberry root C, and hypocrellin A are compounded in the above ratios, they have a better effect of inhibiting melanin production and exhibit better tyrosinase inhibition properties compared to equal amounts of monomers. That is, cucurbitacin B, mulberry root C, and hypocrellin A have a synergistic effect within the above ratio range.

[0047] In some embodiments, the mass percentage of the active ingredient relative to the total amount of the composition is 0.001% to 90%. Alternatively, the mass percentage of the active ingredient is 0.01% to 50%; further alternatively, the mass percentage of the active ingredient is 0.1% to 10%.

[0048] According to the embodiments of the present application, by regulating the content of the active ingredient in the composition within the above range, the active ingredient can effectively exert the effect of inhibiting melanin production.

[0049] In some embodiments, the purity of cucurbitacin B, sangenone C, and hypocrellin A is no less than 90%.

[0050] By further purifying the cucurbitacin B, sangenone C, and hypocrellin A in the examples of the present application, their purity can be further improved to more than 90%, thereby meeting higher purity requirements.

[0051] According to the embodiments of the present application, by optimizing the extraction and purification process, the content of impurities in cucurbitacin B, sangenone C, and hypocrellin A can be reduced as much as possible, so that the product has higher pharmacological activity. At the same time, the influence of impurities on the active ingredients can be effectively avoided, and the risk of adverse reactions such as irritation caused by the active ingredients in cosmetics or pharmaceutical preparations can be reduced.

[0052] Those skilled in the art will appreciate that higher purity cucurbitacin B, mulberry root C and hypocrellin A have higher pharmacological activity. However, the embodiments of the present application are not intended to limit the purity of cucurbitacin B, mulberry root C and hypocrellin A to 90% or above. In other embodiments, the purity of cucurbitacin B, mulberry root C and hypocrellin A can be 60%, 50%, 40% or other purities, which can meet the corresponding pharmacological activity requirements.

[0053] In some embodiments, the excipients include at least one of a humectant, a thickener, an emulsifier, a neutralizer, and a preservative.

[0054] In some embodiments, the humectant includes glycerin, butylene glycol, 1,3-propylene glycol, 1,2-pentanediol, caprylyl glycol, and sodium hyaluronate, or a combination thereof.

[0055] In some embodiments, the thickening agent includes carbomer, acrylic acid and C 10 -C 30 Alkyl acrylate cross-linked polymer, xanthan gum, or a combination thereof.

[0056] In some embodiments, the emulsifier includes polyglyceryl-3 diisostearate, polyglyceryl-2-dipolyhydroxystearate, polyglyceryl-2 isostearate, polyglyceryl-4 isostearate, polyglyceryl-3-polyricinoleate, polyglyceryl-6-polyricinoleate, glyceryl stearate, sorbitan isostearate, sorbitan oleate, and sucrose cocoate, or a combination thereof.

[0057] In some embodiments, the neutralizing agent comprises sodium hydroxide, potassium hydroxide, or a combination thereof.

[0058] In some embodiments, the preservative comprises phenoxyethanol, p-hydroxyacetophenone, 1,2-pentanediol, 1,2-hexanediol, p-anisic acid, caprylyl glycol, ethylhexylglycerin, benzoic acid, sodium benzoate, caprylhydroxamic acid, or a combination thereof.

[0059] Cosmetic or pharmaceutical composition

[0060] An embodiment of the second aspect of the present application provides a cosmetic or pharmaceutical composition, which comprises the composition for inhibiting melanin production according to the embodiment of the first aspect of the present application and a cosmetically or pharmaceutically acceptable excipient.

[0061] The cosmetic or pharmaceutical compositions in the embodiments of the present application can be added with various cosmetic or pharmaceutically acceptable excipients according to the various dosage forms of the composition. For example, when the composition of the present application is prepared as a liquid, the excipient can be selected from water, physiological saline, etc.; when the composition of the present application is prepared as a gel, the excipient can be selected from cellulose derivatives, starch, gelatin, agar, polysaccharides, etc.; when the composition of the present application is prepared as a cream, the excipient can be selected from glycerin, vaseline, paraffin, etc.; when the composition of the present application is prepared as a foam, the excipient can be selected from hydroxypropyl methylcellulose, sodium polyethylene glycol lauryl sulfate, sodium fatty alcohol polyoxyethylene ether sulfonate, etc.; when the composition of the present application is prepared as a patch, the excipient can be selected from cellulose derivatives, starch, gelatin, agar, polysaccharides, etc.

[0062] In some embodiments, the dosage form of the cosmetic can be ointment, cream, lotion, patch, mask,

[0063] In some embodiments, the dosage form of the pharmaceutical composition can be tablets, capsules, granules, pills, oral liquids, emulsions, dry suspensions, dry extracts, or injections.

[0064] According to the embodiments of the present application, the cosmetic or pharmaceutical composition is suitable for cleaning, washing and skin care products such as aqueous products, emulsified products, and oil products, and can also be used in beauty products such as essences and facial masks, such as ointments, foundation, etc.

[0065] Application of a composition for inhibiting melanin production in preparing a pharmaceutical preparation with whitening effect

[0066] An embodiment of the third aspect of the present application provides an application of a composition for inhibiting melanin production in the preparation of a pharmaceutical preparation with whitening efficacy.

[0067] Whitening methods

[0068] An embodiment of the fourth aspect of the present application provides a whitening method, which comprises applying an effective amount of a whitening product to the skin of a subject to be whitened, wherein the whitening product comprises a composition for inhibiting melanin production according to an embodiment of the first aspect of the present application or a cosmetic according to an embodiment of the second aspect of the present application.

[0069] According to the embodiments of the present application, the whitening method provided in the embodiments of the present application can be to apply the composition for inhibiting melanin production to the skin of the subject to be whitened, or it can be to apply whitening cosmetics to which the composition for inhibiting melanin production is added, such as creams, lotions, facial masks, etc.

[0070] In some embodiments, the whitening method comprises applying an effective amount of 0.001 wt% to 10 wt% of cucurbitacin B to the skin of a subject to be whitened.

[0071] In some embodiments, the whitening method comprises applying an effective amount of 0.001 wt% to 10 wt% of hypocrellin A to the skin of a subject to be whitened.

[0072] Example

[0073] The present invention is described below with reference to specific examples. It should be noted that the examples described below are exemplary and are only used to explain the present application, and should not be construed as limiting the present application. Where specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature in this area or in accordance with the product instructions are used. Unless otherwise stated, all parts, percentages, and ratios reported in the following examples are by weight, and all reagents used in the examples are commercially available or synthesized according to conventional methods and can be used directly without further processing, and the instruments used in the examples are commercially available.

[0074] Example 1: Acquisition of Cucurbitacin B

[0075] 5 kg of muskmelon stems were extracted three times with 60 L of 65% ethanol at 80°C for 1 hour each time. The solvent was evaporated to dryness, yielding 389 g of a crude extract. The extract was dispersed in 500 mL of water and extracted sequentially with petroleum ether and ethyl acetate to yield 80 g of the ethyl acetate fraction. The fraction was then separated on a macroporous resin and eluted with an ethanol gradient. The 60-90% fraction was sampled to yield 10 g of the sample. This was then crystallized from the solvent to obtain 3.95 g of cucurbitacin B with a purity greater than 90%.

[0076] Example 2: Acquisition of Sanggenone C

[0077] 5 kg of Morus alba bark was extracted three times with 60 L of 80% ethanol at 50°C for 1 hour each time. The solvent was evaporated to dryness to obtain 498 g of the extract. The extract was dispersed in 1000 mL of water and extracted sequentially with petroleum ether and ethyl acetate to obtain 78 g of the ethyl acetate fraction. This was then separated on a macroporous resin and eluted with an ethanol gradient. The 55-80% elution fraction (12.5 g, totaling 12.5 g) was extracted and crystallized using a solvent to obtain 4.23 g of Sanggenone C with a purity greater than 90%.

[0078] Example 3: Acquisition of Hypocrellin A

[0079] 5 kg of Hypocrellus herb was extracted three times with 60 L of 80% ethanol at 80°C for 0.5 h each time. The solvent was evaporated to dryness to obtain 435 g of extract. The extract was dispersed in 1000 mL of water and extracted sequentially with petroleum ether, ethyl acetate, and n-butanol. The ethyl acetate and n-butanol extracts were combined to yield a total of 102 g. The extract was then separated on a macroporous resin and eluted with an ethanol gradient. The 50-80% elution fraction was extracted, yielding 25.5 g. This was then crystallized from the solvent to obtain 2.35 g of Hypocrellin A with a purity greater than 90%.

[0080] Example 4: Evaluation of the whitening efficacy of active ingredients in an in vitro mushroom tyrosinase model

[0081] Sample preparation

[0082] The sample preparation information is shown in Table 1 below, and the concentration of the stock solution is 10 mM.

[0083] Table 1:

[0084] drug serial number Molecular weight Mass / mg DMSO / μL Kojic acid KA 142.11 0.3 211.10 Cucurbitacin B CB 558.70 1.4 250.58 Sanggenone C SC 708.71 0.8 113 Hypocrellin A HA 546.52 1.1 201

[0085] Test methods

[0086] Prepare 50 μL of each drug with an initial concentration of 40 μM (the final concentration was 10 μM kojic acid as a positive control), mix them with 20 μL of mushroom tyrosinase solution (250 U / mL), incubate the mixture in a 96-well plate at 37°C for 10 min, then add 130 μL of L-tyrosine solution (2 mM) to each well, and immediately measure the OD value before incubation. 475 The OD value of each well was recorded at a wavelength of nm as the sample's true color deduction. The reaction mixture was continued to react at 37°C for 20 min, and the OD value was measured using a microplate reader. 475 The absorbance (OD value) of each well was recorded at a wavelength of 0.1 nm, and each group was repeated 3 times.

[0087] The amounts of reagents added during the experiment are shown in Table 2 below.

[0088] Table 2:

[0089]

[0090] The inhibition rate of tyrosinase activity was calculated by the following formula:

[0091] Relative absorbance A = OD 475 nm(30min)–OD 475 nm(0min)

[0092] Inhibition rate (%) = 1-(A 药物组 / A 空白组 )×100%

[0093] Experimental results

[0094] The inhibitory effects of each active ingredient on tyrosinase are as follows Figure 1 As shown, Figure 1 In the table, * indicates p < 0.05 compared with the model group, ** indicates p < 0.01 compared with the model group, *** indicates p < 0.001 compared with the model group, and **** indicates p < 0.0001 compared with the model group.

[0095] Reference Figure 1 It can be seen that compared with the control group, the three monomeric active ingredients of cucurbitacin B, mulberry root ketone C and hypocrellin A all have a strong ability to inhibit tyrosinase activity (p < 0.01). Among them, the ability of mulberry root ketone C to inhibit tyrosinase activity is stronger than that of kojic acid at the same concentration (10μM), with an inhibition rate of up to 99%, laying an experimental foundation for the subsequent development of whitening efficacy raw materials.

[0096] The examples of the present application further determined the synergistic effects of Sanggenone C with Hypocrellin A and Cucurbitacin B, respectively.

[0097] The partial inhibitory concentration index (FICI) was used for evaluation. FICI was the sum of the MIC of each drug in combination divided by the MIC of each drug when used alone, that is, FICI = (drug A in combination) / MIC (drug A alone) + MIC (drug B in combination) / MIC (drug B alone).

[0098] Among them, MIC (drug A in combination) is the minimum inhibitory concentration of drug A in the pharmaceutical composition when used in combination, MIC (drug A alone) is the minimum inhibitory concentration of drug A when used alone, MIC (drug B in combination) is the minimum inhibitory concentration of drug B in the pharmaceutical composition when used in combination, and MIC (drug B alone) is the minimum inhibitory concentration of drug B when used alone).

[0099] When FICI≤0.5, it is a synergistic effect; when 0.5<FICI≤1, it is an additive effect; when 1<FICI≤2, it is an irrelevant effect; when FICI>2, it is an antagonistic effect.

[0100] The test results are shown in Table 3a and Table 3b below:

[0101] Table 3a:

[0102]

[0103] As shown in Table 3a, when used in combination, hypocrellin A and sangenone C can produce a synergistic inhibitory effect in the range of 1:1.8 to 35:1.

[0104] Table 3b:

[0105]

[0106]

[0107] As shown in Table 3b, when used in combination, cucurbitacin B and sangenone C can produce a synergistic inhibitory effect in the range of 1:1.6 to 40:1.

[0108] Example 5: Effect of active ingredients on tyrosinase activity in B16F10 mouse skin melanoma cells

[0109] Sample preparation

[0110] The operation was carried out in a biological clean bench to ensure a sterile environment. The sample preparation information is shown in Table 4 below. The concentration of the stock solution was 10 μM.

[0111] Table 4:

[0112] drug serial number Molecular weight Mass / mg DMSO / μL Glabridin GI 324.37 0.3 154.14 Cucurbitacin B CB 558.70 1.4 250.58 Sanggenone C SC 708.71 0.8 113 Hypocrellin A HA 546.52 1.1 201

[0113] Preparation of RPMI1640 culture medium (10% FBS + 1% double antibiotic penicillin / streptomycin): add 50 mL of fetal bovine serum inactivated in a 56°C water bath for 30 min to 450 mL of RPMI1640 basal culture medium, and then add 1% double antibiotic (penicillin + streptomycin).

[0114] Cell culture: Subculture in 10% RPMI-1640 complete medium. Digest B16F10 cells in the logarithmic growth phase with trypsin for 90 seconds. Add medium to terminate digestion and centrifuge at 1000 rpm for 3 minutes to remove the effects of trypsin. Remove the supernatant and add appropriate medium to adjust the cell concentration to prepare a cell suspension.

[0115] Experimental methods

[0116] (1) Effects of active ingredients on B16F10 cell growth

[0117] B16F10 cells in good logarithmic growth state were cultured at 5×10 3 The cells were cultured in a 96-well plate at a concentration of 1 μg / well for 24 h, and then the culture medium was removed, washed with PBS at pH 7.4, and 100 μL of drugs at different concentrations (0.1 μM, 0.5 μM, 1.5 μM, and 10 μM) were added for intervention, and the culture was continued for 48 h.

[0118] The supernatant was aspirated and washed twice with PBS (pH 7.4). 100 μL of phosphate-buffered saline containing 1% Triton X-100 was then added to lyse the cells. The cells were then frozen at -80°C for 30 min and then thawed at room temperature to completely rupture and decompose the cells. After pre-warming in a 37°C water bath, 20 μL of 2 mM L-DOPA solution was added. The reaction was continued in a 37°C water bath for 2 h. The absorbance of the reaction mixture was measured at 405 nm using a microplate reader.

[0119] Intracellular tyrosinase activity (%) = (A (405nm)药物组 / A (405nm)空白组 )×100%

[0120] Experimental results

[0121] The results of the effects of active ingredients on the growth of B16F10 cells are as follows Figure 2 As shown. Figure 2 In the concentration range of 0.1-20 μM, the maximum safe concentrations of glabridin, cucurbitacin B, mulberry root C, and hypocrellin A are 5 μM, 5 μM, 1 μM, and 0.5 μM, respectively. The cell survival rate can reach more than 80%, with low toxicity and high safety.

[0122] (2) Determination of the effect of active ingredients on tyrosinase activity in B16F10 cells

[0123] B16F10 cells in good condition in the logarithmic phase were cultured at a rate of 5 × 10 3 / well were cultured in a 96-well plate for 24 hours, then the culture medium was removed, washed with PBS at pH 7.4, 100 μL of drug (glabridin was used as a positive control) was added for intervention, and culture was continued for 48 hours; the supernatant was aspirated, washed twice with PBS at pH 7.4, and 100 μL of phosphate containing 1% Triton X-100 was added to lyse the cells, which were then placed in a -80°C refrigerator for 30 minutes, and then moved to room temperature to thaw to completely rupture and decompose the cells. After prewarming in a 37°C water bath, 20 μL of 2 mM L-DOPA solution was added, and the reaction was continued in a 37°C water bath for 2 hours. The absorbance of the reaction mixture was measured at 405 nm using a microplate reader.

[0124] Intracellular tyrosinase activity (%) = (A (405nm)药物组 / A (405nm)空白组 )×100%

[0125] Experimental results

[0126] The results of the effects of active ingredients on tyrosinase activity in B16F10 cells are as follows Figure 3 As shown, Figure 3In the table, * indicates p < 0.05 compared with the model group, ** indicates p < 0.01 compared with the model group, *** indicates p < 0.001 compared with the model group, and **** indicates p < 0.0001 compared with the model group.

[0127] Reference Figure 3 Compared with the control group, cucurbitacin B, mulberry root ketone C, and hypocrellin A can inhibit the activity of tyrosinase in B10F10 cells at the maximum safe concentration. Hypocrellin A reduced the activity of intracellular tyrosinase by about 50% at 0.5 μM, thereby affecting the formation of intracellular melanin, laying the foundation for further development as cosmetic raw materials.

[0128] Example 6: Effect of active ingredients on melanin content in B16F10 cells

[0129] Experimental methods

[0130] B16F10 cells with good growth status in logarithmic phase were cultured in RPMI1640 medium (10% FBS + 1% double antibiotic penicillin / streptomycin) until the logarithmic phase with a count of 2.5×10 5 / mL was inoculated into a 12-well plate, 2mL per well. After 24h of culture, the supernatant was removed, washed twice with PBS, and drugs were added (glabridin was used as a positive control, and 3 replicates were made for each group). Cells and 1640 complete medium were used as blank controls and cultured. After 48h, the old medium was discarded, and the cells were washed twice with PBS, and the PBS was discarded. The cells were digested with trypsin, centrifuged at 1500rpm for 10min, and then rinsed with PBS twice. The supernatant was discarded, and 300μL of 1mol / L NaOH solution containing 10% DMSO was added to the centrifuge tube to lyse the cells. The cells were placed in a water bath at 80℃ for 1h, and the cells were transferred to 96-well plates respectively. The OD value was measured using a microplate reader. 490 The OD value of each well was recorded at a wavelength of 1 nm.

[0131] Melanin relative content (%) = A (490nm)实验组 / A (490nm)空白组 ×100%

[0132] The test results of the effect of active ingredients on the melanin content in B16F10 cells are as follows Figure 4 shown. Figure 4 In the table, * indicates p < 0.05 compared with the model group, ** indicates p < 0.01 compared with the model group, *** indicates p < 0.001 compared with the model group, and **** indicates p < 0.0001 compared with the model group.

[0133] Reference Figure 4Compared with the control group, the melanin content of the cucurbitacin B, mulberry root C and hypocrellin A groups was significantly reduced, indicating that these active ingredients can effectively inhibit the production of melanin in B16F10 cells.

[0134] Example 7: Preparation of Hypocrellin A Anti-chloasma Tablets

[0135] Take 10g of hypocrellin A, mix it with 100g of starch, add 10g of 10% starch slurry to make a soft material, add 1g of magnesium stearate and 10g of dry starch, mix well, and press into 1000 tablets. Each tablet contains 10mg of hypocrellin A.

[0136] Although the present application has been described with reference to preferred embodiments, various modifications may be made thereto and components may be substituted with equivalents without departing from the scope of the present application. In particular, the various technical features described in the various embodiments may be combined in any manner as long as there are no structural conflicts. The present application is not limited to the specific embodiments disclosed herein, but encompasses all technical solutions within the scope of the claims.

Claims

1. A composition for inhibiting melanin production, characterized in that The invention comprises active ingredients and auxiliary materials, wherein the active ingredients comprise cucurbitacin B and sangenone C in a molar ratio of 1:1.6-40:

1.

2. The composition for inhibiting melanin production according to claim 1, wherein The mass percentage of the active ingredient relative to the total amount of the composition is 0.001% to 90%; and / or The purity of the cucurbitacin B and the mulberry root ketone C is not less than 90%.

3. The composition for inhibiting melanin production according to claim 1, wherein The auxiliary materials include at least one of a moisturizer, a thickener, an emulsifier, a neutralizer, and a preservative.

4. A cosmetic or pharmaceutical composition, characterized in that The invention comprises the composition for inhibiting melanin production according to any one of claims 1 to 3 and a cosmetically or pharmaceutically acceptable excipient.

5. The cosmetic or pharmaceutical composition according to claim 4, characterized in that The dosage forms of the cosmetics include ointments, creams, lotions, patches, facial masks, and / or The dosage forms of the pharmaceutical composition include tablets, capsules, granules, pills, oral liquids, emulsions, dry suspensions, dry extracts, and injections.

6. Use of the composition for inhibiting melanin production according to any one of claims 1 to 3 in the preparation of a pharmaceutical preparation with whitening efficacy.

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