CN117717488A

CN117717488A - Composition for inhibiting melanin generation, application thereof and whitening method

Info

Publication number: CN117717488A
Application number: CN202311598603.1A
Authority: CN
Inventors: 甘礼社; 田云才; 周长新; 田勇; 文燕; 张文环; 金静维
Original assignee: Shanghai Zhizhen Zhichen Technology Co ltd; Zhejiang University ZJU
Current assignee: Shanghai Zhizhen Zhichen Technology Co ltd; Zhejiang University ZJU
Priority date: 2023-11-27
Filing date: 2023-11-27
Publication date: 2024-03-19

Abstract

The application relates to a composition for inhibiting melanin generation, an application thereof and a whitening method, wherein the composition for inhibiting melanin generation comprises an active ingredient and auxiliary materials, and the active ingredient comprises one or a combination of more of cucurbitacin B, sanggenon C and hypocrellin A. Experiments prove that the composition obtained by any monomer or any combination of cucurbitacin B, sanggenon C and hypocrellin A in the application has obvious effects of inhibiting the activity of tyrosinase in B16F10 cells and resisting melanin generation in B16F10 cells, has low cytotoxicity, and can play roles of whitening and resisting chloasma when being applied to cosmetics or pharmaceutical preparations.

Description

Composition for inhibiting melanin generation, application thereof and whitening method

Technical Field

The application relates to the technical field of cosmetics and medicines, in particular to a composition for inhibiting melanin generation, application thereof and a whitening method.

Background

Melanin is an important factor affecting the color of human skin, eyes and hair, and excessive melanin can cause skin pigmentation to cause diseases such as chloasma, etc., so that reduction of melanin synthesis is a main method for whitening and removing freckles.

The currently commonly used whitening agents such as kojic acid, arbutin, hydroquinone, L-ascorbic acid, ellagic acid, tranexamic acid and the like are widely applied to the field of whitening cosmetics, but the whitening active ingredients have the defects of obvious cytotoxicity, strong irritation, high sensitization, large adverse reaction and the like, and are difficult to meet the demands of wide consumers.

Disclosure of Invention

Aiming at the problems existing in the prior art, the embodiment of the application provides a composition for inhibiting melanin generation, application of the composition and a whitening method, and aims to effectively inhibit melanin generation, play roles of whitening and resisting chloasma, and simultaneously greatly reduce adverse reactions.

In a first aspect, embodiments of the present application provide a composition for inhibiting melanin production, the composition comprising an active ingredient comprising one or more of cucurbitacin B, sanggenon C, hypocrellin a, and an adjuvant.

According to the composition for inhibiting melanin generation, any one or any combination of cucurbitacin B, sanggenon C and hypocrellin A is used as an active substance, the cucurbitacin B, the sanggenon C and the hypocrellin A are all derived from natural raw materials, raw materials are cheap and easy to obtain, and experiments prove that any monomer of the cucurbitacin B, the sanggenon C and the hypocrellin A or the composition obtained by any combination of the cucurbitacin B, the sanggenon C and the hypocrellin A has obvious effects of inhibiting the activity of tyrosinase in B16F10 cells and resisting the melanin generation in B16F10 cells, and the composition has low cytotoxicity, and can play roles of effectively whitening and resisting chloasma when being applied to cosmetics or pharmaceutical preparations.

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

In some embodiments of the present application, the active ingredient is present in an amount of 0.001% to 90% by mass relative to the total amount of the composition.

In some embodiments of the present application, the purity of cucurbitacin B, sanggenon C, hypocrellin a is not less than 90% each.

In some embodiments of the present application, the adjunct includes at least one of a humectant, a thickener, an emulsifier, a neutralizing agent, a preservative.

In a second aspect, embodiments of the present application provide a cosmetic or pharmaceutical composition comprising a composition for inhibiting melanogenesis provided by embodiments of the first aspect of the present application and a cosmetically or pharmaceutically acceptable excipient.

In some embodiments of the present application, the cosmetic dosage form includes a cream, a milk, a patch, a mask, a tincture.

In some embodiments of the present application, the active ingredient is 0.001 to 10% by mass relative to the total amount of the cosmetic or pharmaceutical composition.

In some embodiments of the present application, the dosage form of the pharmaceutical composition includes a tablet, a capsule, a granule, a pill, an oral liquid, an emulsion, a dry suspension, a dry extract, and an injection.

In a third aspect, embodiments of the present application provide an application of a composition for inhibiting melanin generation in preparing a pharmaceutical preparation with whitening efficacy.

In a fourth aspect, embodiments of the present application provide a whitening method comprising applying an effective amount of a whitening product comprising a melanin production inhibiting composition provided by embodiments of the first aspect of the present application or a cosmetic provided by embodiments of the second aspect of the present application to the skin of a subject to be whitened.

In some embodiments of the present application, the whitening method includes applying an effective amount of cucurbitacin B from 0.001wt% to 10wt% to the skin of a whitened subject.

In some embodiments of the present application, the whitening method includes applying an effective amount of sanguisorbanone C to the skin of the whitened subject in an amount of 0.001wt% to 10 wt%.

In some embodiments of the present application, the whitening method includes applying hypocrellin to the skin of a whitened subject in an effective amount of 0.001wt% to 10 wt%.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are needed in the embodiments of the present application will be briefly described, and it is possible for a person skilled in the art to obtain other drawings according to these drawings without inventive effort.

FIG. 1 shows the inhibition of tyrosinase by active ingredients in examples of the present application;

FIG. 2 shows the IC of the inhibition of tyrosinase activity by the active ingredient in the examples of the present application ₅₀ ；

FIG. 3 shows the effect of different concentrations of active ingredient on B16F10 cell growth in the examples of the present application;

FIG. 4 shows the effect of active ingredients on B16F10 intracellular tyrosinase activity in examples of the present application;

figure 5 shows the effect of the active ingredient on melanogenesis in B16F10 cells in the examples per se.

Detailed Description

Features and exemplary embodiments of various aspects of the present application are described in detail below. In the following detailed description, numerous specific details are set forth in order to provide a thorough understanding of the present application. It will be apparent, however, to one skilled in the art that the present application may be practiced 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 showing an example of the present application. In the drawings and the following description, at least some well-known structures and techniques are not shown in order to avoid unnecessarily obscuring the present application; also, the dimensions of some of the structures may be exaggerated for clarity. Furthermore, the described features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

It is noted that relational terms such as first and second, and the like are used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. In the description of the present application, it is to be noted that the meaning of "plurality" is two or more unless otherwise indicated. Moreover, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising … …" does not exclude the presence of other like elements in a process, method, article or apparatus that comprises the element.

In the human body, the color of the skin is mainly determined by the content and distribution of pigments in the skin, which are mainly black brown melanin (including eumelanin and pheomelanin), red heme and yellow carotene. Melanin is an important factor affecting human skin, eyes and hair, and some physiological and environmental factors can affect the synthesis of melanin, and excessive melanin can cause skin pigmentation to cause diseases such as chloasma, so that melanin reduction is a main method for whitening and removing freckles.

Melanocytes in the skin use tyrosine as a substrate, melanin is produced under the oxidation action of various rate-limiting enzymes, and is transported to basal epidermal cells via melanocyte dendrites, and is carried into the stratum corneum with the metabolism of the cells, and is removed with the exfoliation of the stratum corneum. Tyrosinase is a key enzyme in the biosynthesis of melanin in the body in this process, and thus inhibition of tyrosinase transport to melanosomes and inhibition of tyrosinase activity by inhibiting tyrosinase synthesis are effective methods for reducing melanin synthesis.

Conventional tyrosinase inhibitors such as kojic acid, arbutin, hydroquinone, L-ascorbic acid, ellagic acid, tranexamic acid, etc., have significant drawbacks although they have been used as skin lightening agents. Such as kojic acid, are carcinogenic and unstable during storage, resulting in limited use in cosmetics; arbutin has poor chemical stability, can release hydroquinone during storage, and is decomposed into benzene-containing metabolites, thus having potential toxicity to bone marrow; hydroquinone can mutagenize mammalian cells, and its irritation to skin can cause burning and stinging sensations; l-ascorbic acid is sensitive to heat and is easy to degrade; ellagic acid has poor solubility and low bioavailability.

The traditional whitening active ingredients have the defects of obvious cytotoxicity, strong irritation, high sensitization, adverse reaction and the like, so that the requirements of vast consumers are hardly met, therefore, searching for safe and efficient melanin inhibitors derived from natural products and traditional Chinese medicines becomes a hot spot for research at home and abroad in recent years, and more natural products and melanin inhibitors derived from traditional Chinese medicines are applied to whitening and freckle-removing products, such as tea polyphenols in green tea, oolong tea and black tea, caffeic acid phenethyl ester in propolis, chrysin, piperlonguminine in long pepper, rhodiola rosea extract, walnut fruit extract, ginsenoside and the like.

Based on this, the inventors have made extensive studies to provide a novel composition of natural products having melanogenesis inhibitory effect, which can be applied as a whitening active ingredient to cosmetic or pharmaceutical preparations, and has low adverse reactions such as cytotoxicity, irritation, etc.

Composition for inhibiting melanogenesis

Embodiments of the first aspect of the present application provide a composition for inhibiting melanogenesis comprising an active ingredient comprising one or more combinations of cucurbitacin B, sanggenon C, hypocrellin a and an adjuvant.

Cucurbitacin B is one of the effective components of pedicellus melo, is a type of tetracyclic triterpene compound, and researches show that cucurbitacin B has various biological activities of protecting liver, diminishing inflammation, resisting tumor and the like, has obvious effect of inhibiting proliferation of tumor cells, and has obvious synergistic curative effect on malignant tumor cell treatment. Morgandone C is one of the active ingredients of the cortex mori radicis, is a natural flavonoid extract, and is found to have antioxidant and anti-inflammatory effects and a certain anticancer activity. Hypocrellin A is the main active component in hypocrellin and tabasheer fungus, is one of the main active components of tabasheer stroma, and has a certain antibacterial effect, and can be applied to medicines for preventing and treating diabetes.

Through intensive researches on cucurbitacin B, sanggenon C and hypocrellin A, the inventor discovers that the cucurbitacin B, the sanggenon C and the hypocrellin A have good performance in the field of whitening and resisting chloasma. Further pharmacological activity researches show that cucurbitacin B, sanggenone C and hypocrellin A all show strong capability of inhibiting tyrosinase activity, which indicates that the activity of intracellular tyrosinase can be inhibited to reduce the melanin generation amount of cells, so that the cucurbitacin B, the sanggenone C and the hypocrellin A have the application in the preparation of cosmetics and/or medicaments for whitening, resisting chloasma and the like. Therefore, the composition for inhibiting melanin generation and the application thereof in the whitening cosmetic or pharmaceutical preparation are all reported for the first time.

According to the embodiment of the application, the composition for inhibiting melanin generation can be used for cosmetic products such as emulsions, water aquas, creams, facial masks and the like, or can be applied to preparation of pharmaceutical preparations, and has the effects of whitening, preventing and treating chloasma.

In the embodiment of the application, cucurbitacin B, sanggenon C and hypocrellin A are prepared by the following methods:

cucurbitacin B:

extracting pedicellus melo or trichosanthes kirilowii Maxim with 10-15 times of 60-70% ethanol for 1-3 times, each time for 1-2 hours, mixing the extracting solutions, and evaporating the solvent to obtain a crude extract of cucurbitacin B;

dispersing the rough extract of cucurbitacin B with water, extracting with petroleum ether and ethyl acetate in sequence to obtain an ethyl acetate part, separating by macroporous resin, gradient eluting by ethanol, taking 60-90% of the eluting part, and crystallizing by solvent to obtain the cucurbitacin B with the purity of more than 90%.

Morgandone C:

extracting cortex Mori with 10-15 times of 70-80% ethanol for 1-3 times, each time for 1-2 hr, mixing extractive solutions, and evaporating to dry solvent to obtain crude extract of Morgandone C;

dispersing the crude extract of the sanggenon C with water, sequentially extracting with petroleum ether and ethyl acetate to obtain an ethyl acetate part, separating by macroporous resin, gradient eluting by ethanol, taking 55% -80% of the eluting part, and crystallizing by solvent to obtain the sanggenon C with the purity of more than 90%.

Hypocrellin a:

extracting hypocrellin (tabasheer and bamboo flower) with 10-15 times of 70-80% ethanol for 1-3 times (each for 1-2 hr), mixing the extractive solutions, and evaporating to dry the solvent to obtain hypocrellin A crude extract;

dispersing the crude extract of hypocrellin A with water, extracting with petroleum ether, ethyl acetate and n-butanol successively, merging the extracted parts of ethyl acetate and n-butanol, separating with macroporous resin, gradient eluting with ethanol, taking 50-80% of the eluted parts, and crystallizing with solvent to obtain hypocrellin A with purity higher than 90%.

In some embodiments, the active substance comprises cucurbitacin B, sanggenon C, and hypocrellin in a mass ratio of 2:1:2 to 1:2:1.

According to the embodiment of the application, through further pharmacological activity researches, the inventor finds that when cucurbitacin B, sanggenon C and hypocrellin A are compounded according to the proportion, the hypocrellin A has better melanin generation inhibiting effect, and shows better tyrosinase activity inhibiting characteristic compared with the equivalent monomers, namely, the cucurbitacin B, sanggenon C and hypocrellin A have synergistic effect in the proportion range.

In some embodiments, the active ingredient is 0.001% to 90% by mass relative to the total amount of the composition. Optionally, the mass percentage of the active ingredients is 0.01% -50%; further alternatively, the mass percentage of the active ingredient is 0.1% -10%.

According to the embodiment of the application, the active ingredient can effectively play a role in inhibiting melanin generation by controlling the content of the active ingredient in the composition within the range.

In some embodiments, the purity of cucurbitacin B, sanggenon C, hypocrellin a is not less than 90% each.

Through further purification treatment of cucurbitacin B, sanggenon C and hypocrellin A in the embodiment of the application, the purity of the hypocrellin B can be further improved by more than 90%, and the requirement of higher purity is further met.

According to the embodiment of the application, the impurity content in cucurbitacin B, sanggenon C and hypocrellin A is reduced as much as possible by optimizing the extraction and purification process, so that the product has higher pharmacological activity, the influence of impurity components on active components can be effectively avoided, and the risk of irritation and other adverse reactions generated by the application of the active components in cosmetics or pharmaceutical preparations is reduced.

It will be appreciated by those skilled in the art that the higher purity cucurbitacin B, sanggenon C and hypocrellin have higher pharmacological activity, but it is not intended in the examples herein that the purity of cucurbitacin B, sanggenon C and hypocrellin obtained by this definition must be above 90%, and in other examples, the purity of cucurbitacin B, sanggenon C and hypocrellin may be 60%, 50%, 40% or other purity, which all meet the corresponding pharmacological activity requirements.

In some embodiments, the adjunct includes at least one of a humectant, a thickener, an emulsifier, a neutralizing agent, a preservative.

In some embodiments, the humectant comprises glycerin, butylene glycol, 1, 3-propanediol, 1, 2-pentanediol, octanediol, and sodium hyaluronate, or a combination thereof.

In some embodiments, the thickener comprises carbomers, acrylics, and C ₁₀ -C ₃₀ An alkanol acrylate crosslinked polymer, xanthan gum, or a combination thereof.

In some embodiments, the emulsifier comprises polyglycerol-3-diisostearate, polyglycerol 2-dimerized hydroxystearate, polyglycerol-2-isostearate, polyglycerol 4-isostearate, polyglycerol 3-polyricinoleate, polyglycerol 6-polyricinoleate, glyceryl stearate, sorbitan isostearate, sorbitan oleate, and sucrose cocoate, or a combination thereof.

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

In some embodiments, the preservative comprises phenoxyethanol, p-hydroxyacetophenone, 1, 2-pentanediol, 1, 2-hexanediol, p-anisic acid, octanediol, ethylhexyl glycerol, benzoic acid, sodium benzoate, octanoyl hydroxamic acid, or a combination thereof.

Cosmetic or pharmaceutical composition

Embodiments of the second aspect of the present application provide a cosmetic or pharmaceutical composition comprising the melanogenesis-inhibiting composition of the embodiments of the first aspect of the present application and a cosmetically or pharmaceutically acceptable excipient.

The cosmetic or pharmaceutical composition of the embodiments of the present application may be added with various cosmetic or pharmaceutically acceptable excipients according to various dosage forms of the composition. For example, when the composition of the present application is prepared as a liquid agent, the excipient may be selected from water, physiological saline, and the like; when the composition of the present application is prepared as a gel, the excipient may be selected from cellulose derivatives, starches, gelatins, agar, polysaccharides, and the like; when the composition of the present application is formulated as a cream, the excipient may be selected from glycerin, vaseline, paraffin, etc.; when the composition of the present application is prepared as a foaming agent, the excipient may be selected from hydroxypropyl methylcellulose, sodium polyethylene glycol lauryl sulfate, sodium fatty alcohol polyoxyethylene ether sulfonate, and the like; when the composition of the present application is prepared as a patch, the excipient may be selected from cellulose derivatives, starches, gelatins, agar, polysaccharides, and the like.

In some embodiments, the active ingredient is 0.001 to 10% by mass relative to the total amount of the cosmetic or pharmaceutical composition.

In some embodiments, the cosmetic may be in the form of a cream, a milk, a patch, a mask, a tincture.

In some embodiments, the dosage form of the pharmaceutical composition may be a tablet, capsule, granule, pill, oral liquid, emulsion, dry suspension, dry extract, injection.

According to the embodiment of the application, the cosmetic or pharmaceutical composition is suitable for cleaning, washing and caring products such as water agent products, emulsified products, oil products and the like, and can also be used in cosmetic products such as essence, facial masks and the like, such as ointment, base makeup and the like.

Application of composition for inhibiting melanin generation in preparation of pharmaceutical preparation with whitening effect

Embodiments of the third aspect of the present application provide for the use of a composition for inhibiting melanogenesis in the preparation of a pharmaceutical formulation having a whitening effect.

Whitening method

Embodiments of the fourth aspect of the present application provide a whitening method comprising applying an effective amount of a whitening product to the skin of a subject to be whitened, the whitening product comprising a composition for inhibiting melanin formation according to embodiments of the first aspect of the present application or a cosmetic according to embodiments of the second aspect of the present application.

According to the embodiment of the present application, the whitening method provided in the embodiment of the present application may be to apply the composition for inhibiting melanin formation to the skin of the subject to be whitened, or may be to apply a whitening cosmetic such as a cream, an emulsion, a mask, etc. to which the composition for inhibiting melanin formation is added.

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

In some embodiments, the whitening method includes applying an effective amount of sanggenon C in an amount of 0.001wt% to 10wt% to the skin of the subject to be whitened.

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

Examples

The invention is illustrated by the following specific examples. It should be noted that the embodiments described below are exemplary only for explaining the present application and are not to be construed as limiting the present application. The examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the specifications of the product. Unless otherwise indicated, all parts, percentages, and ratios reported in the examples below are by weight, and all reagents used in the examples are commercially available or were obtained synthetically according to conventional methods and can be used directly without further treatment, as well as the instruments used in the examples.

Example 1: acquisition of cucurbitacin B

5kg of pedicellus melo is taken, extracted with 60L of 65% ethanol at 80 ℃ for 3 times, each time for 1h, and the solvent is evaporated to dryness, thus obtaining 389g of crude extract. Dispersing the extract with 500mL of water, sequentially extracting with petroleum ether and ethyl acetate to obtain 80g of ethyl acetate part, separating with macroporous resin, gradient eluting with ethanol, and collecting 60-90% of the eluting part to obtain 10g of sample. Then, the cucurbitacin B with the purity of more than 90% is obtained by solvent crystallization, and 3.95g of cucurbitacin B is obtained.

Example 2: acquisition of sanggenon C

Taking 5kg of cortex Mori, extracting with 60L of 80% ethanol at 50deg.C for 3 times each for 1 hr, and evaporating solvent to obtain 498g of extract. Dispersing the extract with 1000mL of water, sequentially extracting with petroleum ether and ethyl acetate to obtain 78g of ethyl acetate, separating with macroporous resin, gradient eluting with ethanol to obtain 12.5g of 55-80% eluting parts, crystallizing with solvent to obtain Morgandone C with purity greater than 90%, and obtaining 4.23g.

Example 3: acquisition of hypocrellin A

Taking 5kg of hypocrellin, extracting with 60L of 80% ethanol at 80deg.C for 3 times, each time for 0.5 hr, and evaporating solvent to obtain 435g of extract. Dispersing the extract with 1000mL of water, sequentially extracting with petroleum ether, ethyl acetate and n-butanol, combining ethyl acetate and n-butanol extraction sites to total 102g, separating with macroporous resin, gradient eluting with ethanol, collecting 50-80% elution sites to total 25.5g, and crystallizing with solvent to obtain hypocrellin A with purity higher than 90%, and obtaining 2.35g.

Example 4: evaluation of whitening efficacy of active ingredient in vitro mushroom tyrosinase model

Sample preparation

The sample preparation information is shown in Table 1 below, and the mother liquor concentrations are 10mM.

Table 1:

medicament	Numbering device	Molecular weight	Mass/mg	DMSO/μL
					Kojic acid	KA	142.11	0.3	211.10
Cucurbitacin B	CB	558.70	1.4	250.58
					Morgandone C	SC	708.71	0.8	113
Hypocrellin A	HA	546.52	1.1	201

Test method

Preparation of 50. Mu.L of each drug with an initial concentration of 40. Mu.M (final concentration of 10. Mu.M kojic acid as positive control) was mixed with 20. Mu.L of mushroom tyrosinase solution (250U/mL), the mixture was incubated at 37℃in wells of a 96-well plate for 10min, and then 130. Mu. L L-tyrosine solution (2 mM) was added to each well, and immediately the OD before incubation was measured ₄₇₅ The OD value of each well was recorded at nm wavelength as the true color subtraction of the sample, and after the reaction mixture was allowed to react at 37℃for 20min, the reaction mixture was subjected to an ELISA reader at OD ₄₇₅ Absorbance (OD) was recorded for each well at nm wavelength, 3 times per group.

The reagent loading in the test is shown in table 2 below.

Table 2:

inhibition of tyrosinase activity was calculated by the following formula:

relative absorbance a=od ₄₇₅ nm(30min)–OD ₄₇₅ nm(0min)

Inhibition (%) =1- (a) _{Pharmaceutical set} /A _{Blank group} )×100％

Experimental results

The inhibition of tyrosinase by each active ingredient is shown in fig. 1, wherein p is less than 0.05 in the comparison with model group, p is less than 0.01 in the comparison with model group, p is less than 0.001 in the comparison with model group, and p is less than 0.0001 in the comparison with model group.

Referring to fig. 1, it can be seen that, compared with the control group, three monomer active components of cucurbitacin B, sanggenon C and hypocrellin a have a stronger ability to inhibit tyrosinase activity (p < 0.01), wherein the ability of sanggenon C to inhibit tyrosinase activity is stronger than kojic acid at the same concentration (10 μm), the inhibition rate can reach 99%, and an experimental foundation is laid for subsequent development of whitening efficacy raw materials.

The examples of this application further determine the semi-Inhibitory Concentration (IC) of sanguisorbanone C and kojic acid ₅₀ ) The results are shown in FIG. 2. As can be seen with reference to FIG. 2, the IC of sanggenon C ₅₀ IC of up to 0.9702 mu M, biqu acid ₅₀ (26.09. Mu.M) is an order of magnitude higher.

The examples herein further determine the IC of a combination of sanggenon C and hypocrellin A (2:1), sanggenon C and cucurbitacin B (2:1) ₅₀ As shown in FIG. 2, it can be seen that the composition still has a potent tyrosinase inhibiting effect.

The examples of this application further examined the synergistic effect of sanggenon C with hypocrellin A and cucurbitacin B, respectively.

The evaluation was performed using a partial inhibitory concentration index (FICI), which is the sum of the values of the MIC of each drug divided by the MIC of each drug alone when the combination was administered, i.e., fici= (drug combination a)/MIC (drug combination a) +mic (drug combination B)/MIC (drug combination B).

Wherein, MIC (A drug combination) is the lowest inhibitory concentration of A drug in the drug composition when the combination is used, MIC (A drug alone) is the lowest inhibitory concentration of A drug when the combination is used, MIC (B drug combination) is the lowest inhibitory concentration of B drug in the drug composition when the combination is used, and MIC (B drug alone) is the lowest inhibitory concentration of B drug when the combination is used alone.

When FICI is less than or equal to 0.5, the two components are synergistic; adding when FICI is more than 0.5 and less than or equal to 1; the FICI is not less than 1 and not more than 2; antagonism occurs when FICI > 2.

The test results are shown in tables 3a and 3b below:

table 3a:

as can be seen from Table 3a, hypocrellin A and sanggenon C can synergistically inhibit in the range of 1:1.8-35:1 when used in combination.

Table 3b:

as shown in Table 3B, the combined administration of cucurbitacin B and sanggenon C can generate synergistic inhibition within the range of 1:1.6-40:1.

Example 5: effect of active ingredient on tyrosinase activity in skin melanoma cells of B16F10 mice

Sample preparation

The operation is carried out in a biological clean bench, the sterile environment is ensured, the sample preparation information is shown in the following table 4, and the concentration of mother liquor is 10 mu M.

Table 4:

medicament	Numbering device	Molecular weight	Mass/mg	DMSO/μL
					Glabridin	GI	324.37	0.3	154.14
Cucurbitacin B	CB	558.70	1.4	250.58
					Morgandone C	SC	708.71	0.8	113
Hypocrellin A	HA	546.52	1.1	201

RPMI1640 medium formulation (10% fbs+1% biantipenicillin/streptomycin): 50mL of fetal bovine serum inactivated by a 56℃water bath for 30min was added to 450mL of RPM 1640 basal medium, followed by 1% of diabody (penicillin+streptomycin).

Cell culture: subculturing with 10% RPMI-1640 complete medium, digesting B16F10 cells in logarithmic phase with pancreatin for 90s, adding medium to stop digestion, centrifuging at 1000rpm for 3min to remove pancreatin effect, removing supernatant, adding appropriate medium to adjust cell concentration, and preparing into cell suspension.

Experimental method

(1) Effect of active ingredients on B16F10 cell growth

B16F10 cells with good logarithmic growth state were grown at 5X 10 ³ Culturing in 96-well plate for 24 hr, taking out culture solution, washing with PBS with pH of 7.4, adding 100 μl of medicines (0.1 μM, 0.5 μM, 1.5 μM, 10 μM) with different concentrations, interfering, and culturing for 48 hr;

the supernatant was aspirated, washed 2 times with PBS at pH 7.4, and 100. Mu.L of phosphate containing 1% Triton X-100 was added to lyse the cells, followed by storage in a freezer at-80℃for 30min, followed by thawing at room temperature to complete cell disruption and lysis, adding 20. Mu.L of 2mM L-DOPA solution after pre-warming in a 37℃water bath, continuing to react in a 37℃water bath for 2h, and absorbance of the reaction mixture was measured at 405nm using a microplate reader.

Intracellular tyrosinase activity (%) = (a) _{(405 nm) pharmaceutical group} /A _{(405 nm) blank group} )×100％

Experimental results

The effect of the active ingredient on B16F10 cell growth results are shown in figure 3. Referring to FIG. 3, glabridin, cucurbitacin B, sanggenon C and hypocrellin A have maximum safe concentrations of 5. Mu.M, 1. Mu.M and 0.5. Mu.M respectively in the concentration range of 0.1-20. Mu.M, and the cell survival rate can reach more than 80%, and the toxicity is low and the safety is high.

(2) Determination of the Effect of active ingredient on the tyrosinase Activity in B16F10 cells

Good B16F10 cells in log phase were plated at 5X 10 ³ Concentration of/well was cultured in 96-well plate for 24 hours, and then taken outWashing the culture solution with PBS with pH of 7.4, adding 100 μl of the drug (glabridin as positive control), performing intervention, and culturing for 48 hr; the supernatant was aspirated, washed 2 times with PBS at pH 7.4, and 100. Mu.L of phosphate containing 1% Triton X-100 was added to lyse the cells, followed by storage in a freezer at-80℃for 30min, followed by thawing at room temperature to complete cell disruption and lysis, adding 20. Mu.L of 2mM L-DOPA solution after pre-warming in a 37℃water bath, continuing to react in a 37℃water bath for 2h, and absorbance of the reaction mixture was measured at 405nm using a microplate reader.

Experimental results

The effect of active ingredient on the tyrosinase activity in B16F10 cells is shown in fig. 4, in which fig. 4, the ratio p to model group is less than 0.05, the ratio p to model group is less than 0.01, the ratio p to model group is less than 0.001, and the ratio p to model group is less than 0.0001.

Referring to fig. 4, compared with the control group, cucurbitacin B, sanggenon C and hypocrellin a can inhibit the activity of tyrosinase in B10F10 cells at the maximum safe concentration, and hypocrellin a reduces the activity of tyrosinase in cells by about 50% at 0.5 μm, thereby affecting the formation of melanin in cells and laying a foundation for further development of cosmetic raw materials.

Example 6: effect of active ingredient on melanin content in B16F10 cells

Experimental method

B16F10 cells with good growth status in log phase were cultured in RPMI1640 medium (10% FBS+1% biantipenicillin/streptomycin) to log phase at 2.5X10 ⁵ The culture was continued by inoculating/mL to a 12-well plate, culturing 2mL per well for 24 hours, removing the supernatant, washing 2 times with PBS, adding the drug (glabridin as a positive control, 3 duplicate wells per group), and using the cells and 1640 complete medium as a blank. After 48h, the old medium was discarded, washed twice with PBS and the PBS was discarded. Digesting the cells with pancreatin, centrifuging at 1500rpm for 10min, rinsing the cells with PBS for 2 times, and discardingRemoving supernatant, adding 300 μL 1mol/L NaOH solution containing 10% DMSO into centrifuge tube, lysing cells, respectively transferring into 96-well plates at 80deg.C in water bath for 1 hr, and measuring OD with enzyme labeling instrument ₄₉₀ OD values were recorded for each well at nm wavelength.

Relative melanin content (%) =a _{(490 nm) experimental group} /A _{(490 nm) blank} ×100％

The results of the test of the effect of the active ingredient on the melanin content in B16F10 cells are shown in fig. 5. In fig. 5, the ratio p of the model group is less than 0.05, the ratio p of the model group is less than 0.01, the ratio p of the model group is less than 0.001, and the ratio p of the model group is less than 0.0001.

Referring to fig. 5, the melanin content of cucurbitacin B, sanguisorbanone C and hypocrellin a group was significantly reduced compared to the control group, indicating that these active ingredients were effective in inhibiting the production of melanin in B16F10 cells.

Example 7: preparation of sanggenon C whitening cream

The raw materials are as follows: morgandone C1%, stearic acid 8%, glyceryl monostearate 3%, olive oil 3%, acrylic acid dimethyl taurate ammonia/VP copolymer 6%, triethanolamine 12%, glycerol 18%, and pure water in balance. And determining the emulsification time to be 20 minutes, and finally obtaining the finished product of the face cream with uniform, fine and smooth appearance, luster, good consistency, coating property and physical stability.

Example 8: preparation of sanggenon C+hypocrellin A whitening mask

The mask liquid comprises the following components: CMC-Na 0.8%, glycerin 16%, butanediol 9%, aloe extract 1.09%, sanggenon C+hypocrellin A (2:1) 0.01%, phenoxyethanol 1.2%, deionized water 71.9%, pH value detection shows that the facial mask is weak acid, and the inhibition rate of tyrosinase reaches 82.1%.

And (3) a mask: the mask liquid is soaked into the non-woven fabric face mask, each tablet is soaked into 20g of the mask liquid, sensory test is carried out on the product, the product has no irritation to skin, and the quality standard of the mask is met.

Example 9: preparation of hypocrellin A tablet for treating chloasma

Mixing hypocrellin A10 g with starch 100g, adding 10% starch slurry 10g to obtain soft material, adding magnesium stearate 1g, mixing dry starch 10g, and pressing into 1000 tablets. Each tablet contains hypocrellin A10 mg.

While the present application has been described with reference to a preferred embodiment, various modifications may be made and equivalents may be substituted for elements thereof without departing from the scope of the present application. In particular, the technical features mentioned in the respective embodiments may be combined in any manner as long as there is no structural conflict. The present application is not limited to the specific embodiments disclosed herein, but encompasses all technical solutions falling within the scope of the claims.

Claims

1. A composition for inhibiting melanin formation, comprising an active ingredient and an auxiliary material, wherein the active ingredient comprises one or more of cucurbitacin B, sanggenon C and hypocrellin a.

2. The melanogenesis inhibitory composition according to claim 1, wherein the active ingredients comprise cucurbitacin B, sanggenon C and hypocrellin a in a mass ratio of 2:1:2 to 1:2:1.

3. The melanogenesis inhibitory composition according to claim 1 or 2, wherein the mass percentage of the active ingredient is 0.001% to 90% relative to the total amount of the composition; and/or

The purity of cucurbitacin B, sanggenon C and hypocrellin A is not lower than 90%.

4. The melanogenesis inhibitory composition of claim 1, wherein the adjunct comprises at least one of a humectant, a thickener, an emulsifier, a neutralizing agent, a preservative.

5. A cosmetic or pharmaceutical composition comprising the melanogenesis inhibitory composition according to any one of claims 1 to 4 and a cosmetically or pharmaceutically acceptable excipient.

6. The cosmetic or pharmaceutical composition according to claim 5, wherein the mass percentage of the active ingredient is 0.001 to 10% with respect to the total amount of the cosmetic or pharmaceutical composition.

7. The cosmetic or pharmaceutical composition according to claim 5, wherein the cosmetic dosage form comprises a cream, a milk, a patch, a mask, a tincture, and/or

The dosage forms of the pharmaceutical composition comprise tablets, capsules, granules, pills, oral liquid, emulsion, dry suspension, dry extract and injection.

8. Use of the composition for inhibiting melanin formation of any one of claims 1 to 4 in the preparation of a pharmaceutical preparation having whitening efficacy.

9. A whitening method, comprising: an effective amount of a whitening product comprising the melanogenesis inhibitory composition according to any one of claims 1 to 4 or the cosmetic according to any one of claims 5 to 7, to the skin of a subject to be whitened.

10. The method of whitening according to claim 9, comprising applying an effective amount of cucurbitacin B in an amount of 0.001 to 10wt% to the skin of a subject to be whitened, and/or

Applying an effective amount of sanguisorbanone C to the skin of a whitening subject of 0.001wt% to 10wt%, and/or

An effective amount of hypocrellin A is applied to the skin of a whitening subject in an amount of 0.001 to 10 wt%.