CN1377640A - External preparatino for skin - Google Patents

Abstract

Provided is a highly safe and stable medicament having excellent melanogenesis inhibitory effect, namely skin whitening effect, and to provide a skin care preparation including the medicament. This skin care preparation includes as the active ingredient a 1,4-benzenediol derivative of the general formula (I) and/or a 1,4-benzoquinone derivative of the general formula (II).

Description

Preparation for external application to skin

Technical field

The preparation for external application to skin that the present invention relates to Melanin inhibitor and contain this inhibitor.In detail, the present invention relates to melanin and generate to suppress the effect excellence, and stability, safe Melanin inhibitor and contain this inhibitor as the cosmetics of effective ingredient and/or the preparation for external application to skin of health product.

Background technology

It is significantly hyperfunction resultant that pigmentation on the skin is that the melanin that caused by the activation of the pigment cellulation (melanocyte) that exists in the skin generates, and this has become a serious skin slight illness.Particularly in recent years, exploitation can improve and/or suppress this class pigmentation and the skin material useful, that promptly have a skin whitening function of staying pale has been seemed more urgent.

As suppressing the material that melanin generates, for example known ascorbic acid and its derivant, sulfur-containing amino acid such as glutathion or cysteine compounds, kojic acid and its derivant, Azelaic Acid and its derivant, tranamic acid and its derivant, hydroquinone and its derivant (special public clear 60-16906 communique, special public clear 60-56912 communique), alkyl-resorcin derivant (spy opens the 2000-38334 communique), (spy opens clear 61-289029 communique to ubiquinone derivative, Te Biaoping 9-510725 communique) etc., and then these materials can be used for emulsifiable paste as effective ingredient, ointment, emulsion etc., and smear, be attached at the position of pigmentation.But ascorbic acid and its derivant not only have problems aspect stable, and be easy to variable color or fouling in Aquo Systems.Glutathion or cysteine etc. not only foreign odor are strong, and have the problem of easy oxidation, therefore are difficult to be combined in the external preparation.And the effect onset of these chemical compounds is slow, so be difficult to obtain effect of sufficient.Azelaic Acid and its derivant, tranamic acid and its derivant are the white powder of odorless, and be also better with the compatibility of the various base materials that adopted in the preparation for external application to skin, but the inhibiting onset of both melanin is slow.

In addition, hydroquinone and its derivant, alkyl-resorcin derivant because skin irritation and/or sensitization are arranged, therefore might cause allergia contact dermatitis etc. as side effect, have some problems when using as preparation for external application to skin.

On the other hand, ubiquinone derivative, particularly ubiquinone-10 (C ₅₉H ₉₀O ₄: Mw862) be present in biological intravital cell membrane and the mitochondrion, in electron transport system, be used as coenzyme, and play an important role as the material of tyrosine kinase activity in the regulation and control melanocyte.But, extremely low through the skin absorbability because the molecular weight height, and with preparation for external application to skin in the compatibility of the various base materials that adopted poor, be difficult in coexistence stably in the base material of preparation for external application to skin.

Disclosure of an invention

The objective of the invention is to, provide melanin to generate inhibitory action, be skin-whitening effect excellence, and the high medicament of safety, stability and contain the preparation for external application to skin of this medicament as effective ingredient.

Present inventors have carried out concentrated research for solving above-mentioned problem, found that 1,4-Benzodiazepines derivant and 1, and the skin-whitening effect of 4-quinone derivatives is very excellent, and stability, safe, thereby has finished the present invention.That is, the invention provides with the quinone derivatives shown in Benzodiazepines derivant shown in the general formula (I) or the general formula (II) is the Melanin inhibitor of effective ingredient.

[in the formula, x, y are 0～6 integer, and x+y=4,5 or 6]

[in the formula, x, y are 0～5 integer, and x+y=4 or 5]

In Benzodiazepines derivant of the present invention, quinone derivatives, if the not enough stated number of the value of x+y, the toxicity of pair cell strengthens, if the value of x+y is bigger than the number of regulation, then melanin generation inhibition effect will significantly reduce, and can not get desired melanin and generate inhibitory action.In addition, the Benzodiazepines derivant is almost colourless, has the good advantage of outward appearance when being used for skin preparations for extenal use.

In this description, 1 shown in the general formula (I), 4-Benzodiazepines derivant can be represented by following simple formula.

BD-A-yH

[in the formula, BD=Benzodiazepines, A=x+y]

Therefore, shown in the formula of of the present invention (I) 1,4-Benzodiazepines derivant is BD-4-0H, BD-4-1H, BD-4-2H, BD-4-3H, BD-4-4H, BD-5-0H, BD-5-1H, BD-5-2H, BD-5-3H, BD-5-4H, BD-5-5H, BD-6-0H, BD-6-1H, BD-6-2H, BD-6-3H, BD-6-4H, BD-6-5H and BD-6-6H.From its effect and 1, the synthetic easy degree of 4-Benzodiazepines derivant and become original and consider, preferred following 1,4-Benzodiazepines derivant.

BD-4-0H (x+y=4, y=0), promptly

2,3-dimethoxy-5-methyl-6-(3,7,11,15-tetramethyl-2,6,10,14-hexadecane apos)-1,4-Benzodiazepines;

BD-4-3H (x+y=4, y=3), promptly

2,3-dimethoxy-5-methyl-6-(3,7,11,15-tetramethyl-2-hexadecane mono alkenyl)-1,4-Benzodiazepines;

BD-4-4H (x+y=4, y=4), promptly

2,3-dimethoxy-5-methyl-6-(3,7,11,15-tetramethyl-2-cetyl)-1,4-Benzodiazepines;

BD-5-0H (x+y=5, y=0), promptly

2,3-dimethoxy-5-methyl-6-(3,7,11,15,19-pentamethyl-2,6,10,14,18-eicosane pentaene base)-1,4-Benzodiazepines;

BD-5-5H (x+y=5, y=5), promptly

2,3-dimethoxy-5-methyl-6-(3,7,11,15,19-pentamethyl-eicosyl)-1,4-Benzodiazepines;

BD-6-0H (x+y=6, y=0), promptly

2,3-dimethoxy-5-methyl-6-(3,7,11,15,19,23-hexamethyl-2,6,10,14,18,22-lignocerane six thiazolinyls)-1,4-Benzodiazepines; With

BD-6-6H (x+y=6, y=6), promptly

2,3-dimethoxy-5-methyl-6-(3,7,11,15,19,23-hexamethyl-tetracosyl)-1,4-Benzodiazepines.

In addition, 1 shown in the general formula (II), the 4-quinone derivatives can be represented by following simple formula:

BQ-A-yH

[in the formula, BQ=benzoquinone, A=x+y]

Therefore, 1 shown in the formula of of the present invention (II), the 4-benzoquinone is BQ-4-0H, BQ-4-1H, BQ-4-2H, BQ-4-3H, BQ-4-4H, BQ-5-0H, BQ-5-1H, BQ-5-2H, BQ-5-3H, BQ-5-4H and BQ-5-5H.From its effect and 1, the synthetic easy degree of 4-quinone derivatives and become original and consider, preferred following 1, the 4-quinone derivatives.

BQ-4-0H (x+y=4, y=0), promptly 2,3-dimethoxy-5-methyl-6-(3,7,11,15-tetramethyl-2,6,10,14-hexadecane apos)-1,4-benzoquinone;

BQ-4-3H (x+y=4, y=3), promptly 2,3-dimethoxy-5-methyl-6-(3,7,11,15-tetramethyl-2-hexadecane mono alkenyl)-1,4-benzoquinone; With

BQ-5-0H (x+y=5, y=0), promptly 2,3-dimethoxy-5-methyl-6-(3,7,11,15,19-pentamethyl-2,6,10,14,18-eicosane pentaene base)-1,4-benzoquinone.

Above-mentioned 1,4-Benzodiazepines derivant and 1,4-quinone derivatives and melanin cellulation (B16 melanoma, HM3KO melanoma) are when directly contacting, the low concentration of 3ppm, preferred 10ppm just can show that significant melanin generates inhibitory action, and skin is had very significant whitening function.

Therefore, the invention provides the preparation for external application to skin of skin-whitening effect excellence, it is characterized in that, contain more than a kind by above-mentioned 1 4-Benzodiazepines derivant and/or 1, the Melanin inhibitor that the 4-quinone derivatives constitutes.The content of the above-mentioned Melanin inhibitor more than a kind in the preparation for external application to skin is more than the 0.0001 weight %.Above-mentioned Melanin inhibitor of the present invention, owing to just have significant effect under extremely low concentration, therefore 0.0001 weight % is above gets final product.In addition, the effect that Melanin inhibitor content of the present invention is had when above above certain concentration can not increase yet, so its content there is no need more than 5 weight %.

Therefore, in preferred version of the present invention, the preparation for external application to skin that skin-whitening is used contain 0.0001～5 weight %, preferred 0.01～1 weight % more than a kind by the present invention 1,4-Benzodiazepines derivant and/or 1, the Melanin inhibitor that the 4-quinone derivatives constitutes.

In addition, the dosage of the preparation for external application to skin that skin-whitening of the present invention is used depends on age, individual variation, people's species diversity, pigmentation situation etc., but, usually with of the present invention 1,4-Benzodiazepines derivant and/or 1, the 4-quinone derivatives is with 0.01～5mg/cm ², preferred 0.1～1mg/cm ²Amount every day directly be applied in for 1～3 time on the disease sites of skin.

In addition, in embodiments of the invention, preparation for external application to skin can also mix with other medicament, additive and/or the base that allow on the preparation.Additive can be selected from antioxidant, ultraviolet screening agent, cutin agents for defoliating, surfactant, spice, pigment, antiseptic, the pH regulator agent, chelating agen, but of the present invention 1 in order further to improve, 4-Benzodiazepines derivant and 1, the stability of 4-quinone derivatives is preferably mixed antioxidant and/or ultraviolet screening agent.In addition, preferred compositions improves the cutin agents for defoliating of the effectiveness of preparation for external application to skin, improves the natural surfactant of Percutaneously absorbable.

The antioxidant that uses among the present invention has no particular limits, so long as allow on the preparation just can, tocopherol or derivatives thereof for example.As water soluble antioxidant; sulfur-containing amino acid such as low concentration ascorbic acid or derivatives thereof, thiolaurine, glutathion for example; antioxidation such as Herba Rosmarini Officinalis extract, sage extract herb extracts; synthetized oxidation preventive agent such as butylated hydroxytoluene, Butylated hydroxyanisole can suit to cooperate the combination more than a kind or 2 kinds of these materials.

In the ultraviolet screening agent of the present invention, comprise UV absorbent and ultraviolet reflection agent.As UV absorbent; for example cinnamic acid monooctyl ester, 2-ethyl hexanoyl base-cinnamate derivates such as di-p-methoxy cinnamic acid monoglyceride; para-aminobenzoic acid monooctyl ester, N; N-dimethyl-benzoic acid ester derivants such as benzoic acid monooctyl ester; 2; 4-dihydroxy benaophenonel, 2,2 '-dihydroxy-4-methoxy benzophenone, 2-hydroxyl-4-methoxy benzophenone, 2-hydroxyl-4-methoxy benzophenone-benzophenone such as 5-sulfonate.As other UV absorbent, 3-(4 '-methyl benzal)-d for example, 1-camphane, 3-benzal-d, 1-camphane, urocanic acid, urocanic acid ethyl ester, 4-methoxyl group-the 4 '-tert-butyl group-dibenzoyl methane etc.As the ultraviolet reflection agent, for example titanium oxide, zinc oxide, mica titanium etc.

As the cutin agents for defoliating, for example Alpha-hydroxy-carboxylic acid such as citric acid, glycolic, ethylenediamine tetraacetic carboxylic acid list, two or trisodium salt, and aminoacid such as trimethyl glycine, hydroxyproline etc.

As natural surfactant, can exemplify lecithin classes such as LYSOLECITHIN SUNLECITHIN A, dioxane acyl phospholipids phatidylcholine, dioxane acylphosphatidyl ethanolamine such as single alkanoyl phosphatidylcholine especially.

In the preparation for external application to skin of the present invention, can cooperate the composition that uses in the cosmetics (for example oiliness composition, the composition of preserving moisture, thickening agent, emulsifying agent and various additive) usually.For example, can exemplify hydro carbons, various synthesizing ester, wax class, oils, higher fatty acids class, higher alcohols, polysiloxane-based etc. as the oiliness composition.

As the composition of preserving moisture, for example propylene glycol, 1,3 butylene glycol, isoamyl glycol, glycerol, Polyethylene Glycol, hyaluronate sodium, sodium chondroitin sulfate, hydrolytic collagen peptide, elastin laminin peptide, keratin peptide, sterol glucoside etc.

As thickening agent, for example water solublity synthetic macromolecular compound CVP Carbopol ETD2050, carboxymethyl cellulose or its salt etc., the high molecule chemical compound of water-soluble natural Quince smoke tree seed extract, more skin ulcer glue, xanthan gum, sodium alginate etc.

Spendable emulsifying agent; for example as polyoxyethylene addition product of the various higher alcohols of nonionic emulsifier etc.; various higher fatty acids polyethyleneglycols, diester etc.; and dehydrating glycerin Sorbitol list, diester etc.; as the various higher fatty acids soap alkali of anion emulsifier, and as alpha-oil acylglycerol α '-phosphatidylcholine (LYSOLECITHIN SUNLECITHIN A), soybean lecithin and the Ovum Gallus domesticus Flavus lecithin etc. of cationic emulsifier.In addition, as additive, for example spice, pigment, antiseptic, pH regulator agent, chelating agen etc., these materials suitable use that can combine.

In addition, as the dosage form of the preparation for external application to skin in the mode of the present invention, can be selected from w/o type lotion, O/W type lotion, W/O emulsifiable paste, O/W type emulsifiable paste, thickness micro-emulsified essence or O/W type essence etc.

Below, enumerate of the present invention 1, the synthetic example of 4-Benzodiazepines derivant.[synthesis example 1] 2,3-dimethoxy-5-methyl-6-(3,7,11,15-tetramethyl hexadecane-2,6,10,14-apos)-benzene-1,4-diphenol (BD-4-0H) synthetic

With 2,3-dimethoxy-5-methyl isophthalic acid, 4-dihydroxy benzenes 25g and anti-adjacent-geranyl linalool 25g add in the 100ml diisopropyl ether, 40 ℃ of heating for dissolving.About 40 ℃, in 1 hour, be dissolved in solution in the 25ml diisopropyl ether to wherein dripping 12.2g etherate of trifluoroboron complex.After the dropping, stir, make reaction carry out (about 1 hour) fully at uniform temp.Reactant liquor is cooled to room temperature, adds water 10ml.Whole liquid are moved into separatory funnel, remove sub-cloud.In the diisopropyl ether layer, add 200ml and contain 5% sodium hydrate aqueous solution of a small amount of sodium dithionite, leave standstill after the stirring, remove sub-cloud (be unreacted 2,3-dimethoxy-5-methyl isophthalic acid, 4-dihydroxy benzenes).Then, in the diisopropyl ether layer, add 5% sodium hydrate aqueous solution that 100ml contains a small amount of sodium dithionite, leave standstill after the stirring, remove sub-cloud.Water, saturated aqueous common salt wash the diisopropyl ether layer successively, and distilling under reduced pressure removes and desolvates.Residue obtained by the refining (carrier: silica gel, mobile phase: diisopropyl ether-hexane), obtain 12g oily BD-4-0H (yield 32%) [synthesis example 2] 2 of column chromatography, 3-dimethoxy-5-methyl-6-(3,7,11,15-tetramethyl hexadecane-2-thiazolinyl)-and benzene-1,4-diphenol (BD-4-3H) synthetic

With 2 of 25g, 3-dimethoxy-5-methyl isophthalic acid, 4-dihydroxy benzenes and 25g phytol add in the 100ml diisopropyl ether, 40 ℃ of heating for dissolving.Carry out the processing same then, obtain the buttery BD-4-3H of 20g (yield 51%) [synthesis example 3] 2,3-dimethoxy-5-methyl-6-(3,7,11,15-tetramethyl cetyl)-benzene-1,4-diphenol (BD-4-4H) synthetic with synthesis example 1

With 2 of 25g, 3-dimethoxy-5-methyl isophthalic acid, 3 of 4-dihydroxy benzenes and 25g, 4-dihydro phytol adds in the 100ml diisopropyl ether, 40 ℃ of heating for dissolving.Carry out the processing same then, obtain the buttery BD-4-4H of 8.5g (yield 22%) with synthesis example 1.[synthesis example 4] 2,3-dimethoxy-5-methyl-6-(3,7,11,15,19-pentamethyl-2,6,10,14,18-eicosane pentaene base)-benzene-1,4-diphenol (BD-5-0H) synthetic

With 2 of 5.0g, 3-dimethoxy-5-methyl isophthalic acid, 4-dihydroxy benzenes and 6.2g adjacent anti--the geranyl farnesol adds in the 50ml diisopropyl ether, 40 ℃ of heating for dissolving.Then, carry out the processing identical, obtain the buttery BD-5-0H of 12g (yield 30%) with synthesis example 1.[synthesis example 5] 2,3-dimethoxy-5-methyl-6-(3,7,11,15-tetramethyl-cetyl)-1,4-Benzodiazepines (BD-4-4H) synthetic

With 5.0g CoQ4 Coenzyme Q4 heating for dissolving in 20ml ethanol.Add the 5% palladium carbon of 1.0g, stirring at room under hydrogen stream as catalyst.After 2 hours, there are 5 normal hydrogen to be absorbed approximately.Behind reacting liquid filtering, remove catalyst, ethanol is removed in distilling under reduced pressure, obtains the buttery BD-4-4H of 4.9g (yield 98%).[synthesis example 6] is by the synthetic BD-4-0H of CoQ4 Coenzyme Q4

With 2.0g CoQ4 Coenzyme Q4 heating for dissolving in 50ml ethanol.Add the 0.2g sodium borohydride, stirring at room is until orange color dissipated.After in reactant liquor, adding the 100ml hexane, add 100ml water and 1.5ml glacial acetic acid again.Leave standstill and remove sub-cloud after the stirring.Wash hexane phase and distilling under reduced pressure then with water and remove hexane, obtain the buttery BD-4-0H of 1.8g (yield 90%).[synthesis example 7] 2,3-dimethoxy-5-methyl-6-(3,7,11,15,19,23-hexamethyl-2,6,10,14,18,22-lignocerane six thiazolinyls)-1,4-Benzodiazepines (BD-6-0H)

The Coenzyme Qq6 that 0.1g is commercially available heating for dissolving in 5ml ethanol.Add the 0.05g sodium borohydride, stirring at room is until orange color dissipated.After in reactant liquor, adding the 100ml hexane, further add 100ml water and 0.1ml glacial acetic acid.After the stirring, leave standstill and remove sub-cloud.The washing hexane mutually after, hexane is removed in distilling under reduced pressure, obtains 0.1g oily BD-6-0H (yield 100%).[synthesis example 8] 2,3-dimethoxy-5-methyl-6-(3,7,11,15,19-pentamethyl-2,6-eicosane dialkylene)-benzene-1,4-diphenol (BD-5-3H) synthetic

With 2 of 5g, 3-dimethoxy-5-methyl isophthalic acid, 3,7,11,15 of 4-dihydroxy benzenes and 5g, 19-pentamethyl-2,6-eicosane dienol adds in the 100ml diisopropyl ether, 40 ℃ of heating for dissolving.Carry out the processing identical then, obtain 2.5g oily BD-5-3H (yield 32%) with synthesis example 1.[synthesis example 9] 2,3-dimethoxy-5-methyl-6-(3,7,11,15,19-pentamethyl eicosyl)-1,4-Benzodiazepines (BD-5-5H) synthetic

BD-5-3H heating for dissolving in 20ml ethanol that 0.5g synthesis example 8 is obtained.Add the 5% palladium carbon of 0.1g, stirring at room under hydrogen stream as catalyst.After 2 hours, absorb about 2 normal hydrogen.Filtering reacting liquid is also removed catalyst, and ethanol is removed in distilling under reduced pressure, obtains 0.49g oily BD-5-5H (98%).

Below, in conjunction with the following example explanation the present invention, but they do not have any restriction to the present invention.

Embodiment

Below illustrate of the present inventionly 1, the melanin of 4-Benzodiazepines derivant generates inhibition test and toxicity test.[melanin generates inhibition test and toxicity test]

Use compd B D-4-0H[synthesis example 1 of the present invention], BD-4-3H[synthesis example 2], BD-5-0H[synthesis example 4], BQ-4-0H, BQ-4-3H and BQ-5-0H, similar compound 2 as a comparative example, 3-dimethoxy-5-methyl-6-(3,7,11-trimethyl-2,6,10-dodecane trialkenyl)-1,4-Benzodiazepines (BD-3-0H) [comparative example 1].BD-3-0H uses with the same method of synthesis example 1 and prepares from farnesol.In addition, compare with only adding alcoholic acid solution.(1) melanin generation inhibition test 1) is used to test from the B16 of mice melanoma (pernicious melanotic tumor cell) cultured cell

With B16 melanoma cultured cell 2000 cells of every hole sowing on 96 hole titer plate, in the Eagle-MEM culture medium that contains 10% hyclone and theophylline (0.09mg/ml) at 37 ℃, 5%CO ₂Exist down and cultivate.Cultivate after 1 day, the various concentrations samples compd B of 20 μ l D-3-0H, BD-4-0H, BD-4-3H, BD-5-0H, BQ-4-0H, BQ-4-3H or BQ-5-0H are added in each hole, making ultimate density is 3,10,50,100 or 200 μ g/ml.Cultivate after 72 hours, change culture medium, also add sample compound betwixt.Second day collecting cell according to the tone of cell deposition thing, judged the synthetic inhibition effect of newborn melanin.

Simultaneously for cytotoxicity according to following method, that is, and with cell with 10% formalin buffer fixing after, with the dyeing of 1% Gentian Violet, use モ ノ セ レ--mensuration with respect to the cell growth rate that contrasts, be 100% to estimate with the control cells number.

Tone according to the cell deposition thing, according to following step melanin is generated and to judge, promptly, with phosphate buffer (50mM, pH6.8) behind the washing hole, cell is peeled off with little scraper, reclaimed and centrifugalize, divide 4 grades to estimate with following standard the tone of sedimentary cell deposition thing with phosphate buffer.

-: identical with contrast

+: shoal a little

++: shallow white

+++: white

For BD-4-0H[synthesis example 1], BQ-4-0H and BD-3-0H[comparative example 1], the result who obtains is shown in the following table 1.Table 1

	Test specimen (μ g/ml)
			????3	????10
	BD-3-0H [comparative example 1]	????+++ ????70％			????+++ ????30％
BD-4-0H [comparative example 1]			????+ ????100％	????+++ ????100％
	????BQ-4-0H	????+ ????100％			????+++ ????100％

The BD-3-0H of comparative example 1, though show that at 3 μ g/ml high melanin generates the inhibition effect, simultaneously to the dependent high toxicity of B16 melanoma display density, and cell is all dead when high concentration (50 μ g/ml).Can think that thus the melanin of BD-3-0H generates the inhibition effect owing to its cytotoxicity causes.On the other hand, BD-4-0H of the present invention and BQ-4-0H can observe melanin at 3 μ g/ml and generate the inhibition effect, show significant effect at 10 μ g/ml, but the rate of growth of cell are identical with contrast, does not observe cytotoxicity.In addition, BD-4-3H, BD-5-0H, BQ-4-3H and BQ-5-0H observe melanin at 50 μ g/ml and generate the inhibition effect, show significant effect at 100 μ g/ml, but do not observe cytotoxicity.2) be used to test from people's HM3KO melanoma cultured cell

The people HM3KO melanoma cultured cell (Melanoma Res.6, p.25-30[1996]) that will obtain from Kobe University adds antibiotic such as 10% hyclone and an amount of penicillin and streptomycin, incubated at room temperature in the Eagle of Dulbecco corporate system culture medium.Will be as the BD-3-0H[comparative example 1 of test specimen], BD-4-3H[synthesis example 2], BD-5-0H[synthesis example 4], BD-6-0H[synthesis example 7] be that the amount of 3,10,30,100,300 μ g/ml joins in the above-mentioned culture medium with ultimate density, cultivate after 5 days, handle cell with trypsin and EDTA, measure cell number with Fuchs-Rosenthal Cytometer, measure cell growth rate.Afterwards, the tone of at first visual judgement cell deposition thing is then with 10 ⁶Individual cell boils dissolving in 10 minutes with the NaOH aqueous solution of 1mol/L concentration, with the absorbance of spectrophotometric determination 400nm, with the melanin amount of quantitative generation.In addition, the melanin generating suppression that will contrast (not adding sample) is as 0%, and cell growth rate carry out relative quantificationization as 100%.

The result, the BD-3-0H of comparative example 1 is 35% at the melanin generating suppression of 3 μ g/ml, at 10 μ g/ml is 60%, suppression ratio height during low concentration, but cell growth rate is 93% at 3 μ g/ml, reduce to 56% during 10 μ g/ml,, can think that melanin generates the inhibition effect and caused by cytotoxicity with coming to the same thing of B16 melanoma cultured cell.

On the other hand, adopting BD-4-0H of the present invention is that 35%, 10 μ g/ml is 58% at the melanin generating suppression of 3 μ g/ml, and cell growth rate is 90% at 3 μ g/ml, is 100% at 10 μ g/ml, does not almost observe cytotoxicity.The melanin generating suppression of BD-4-3H is that 36%, 100 μ g/ml is 65% at 30 μ g/ml.The melanin generating suppression of BD-5-0H is 42% at 30 μ g/ml, is 60% at 100 μ g/ml.

Above-mentioned result of the test shows, and is of the present invention 1,4-Benzodiazepines derivant and 1, and the 4-quinone derivatives has significant melanin and generates and suppress effect, and pair cell does not have toxicity or toxicity low significantly, can be used as Melanin inhibitor and uses.

For the toxicology characteristic, also to of the present invention 1,4-Benzodiazepines derivant is tested.(2) single administration toxicity test

With Wister is that rat and ICR-JCL are the acute toxicity of mice study when adopting various route of administration.Administration is with ultrasound wave this material to be made suspension in normal saline, be adjusted to fixed amount implement.After the administration, observe poisoning symptom continuously, obtain till the 7th day mortality rate over time, obtain LD ₅₀Value.To the example of surviving, the equal anatomic observation of dead example.Graphics calculations method by Litchfield-Wilcoxon is calculated LD ₅₀

With the male and female rat, the male and female mice is oral with 4000mg/kg, with the 4000mg/kg percutaneous, subcutaneous with 500mg/kg, with the 500mg/kg intramuscular injection, with the 1500mg/kg intraperitoneal, or with 250mg/kg intravenous injection form administration BD-4-0H, BD-4-3H, BD-5-0H observes the acute toxicity under the various situations.

As a result, male and female rat and male and female mice all do not have special the variation in the intake of body weight, feedstuff, and in anatomic observation, various internal organs are not seen unusually yet in addition.Can confirm that from this result the present invention 1,4-Benzodiazepines derivant safe.(3) sensitization test

The maximization method of testing of founding by Magnusson B.Kligman A.M carries out the sensitization test.(i) sensitization is induced

Prepare 20 Cavia porcelluss, 10 are used for sensitization and induce, and 10 remaining usefulness compare.The area unhairing of 4 * 6cm on the Cavia porcellus scapula is symmetrical in median line following a, b, c is carried out subcutaneous injection for about three groups simultaneously.As test specimen, BD-4-0H, BD-4-3H, BD-5-0H are made the aqueous suspension of 10% concentration with ultrasound wave.

A. at the right and left difference subcutaneous injection 0.05ml (adding up to 0.1ml) Freund's complete adjuvant (FCA Difco corporate system).

B. at the right and left subcutaneous injection 0.05ml test specimen (aqueous suspension of 10% concentration made from ultrasound wave) respectively.

C. at the right and left difference subcutaneous injection 0.05ml test specimen (the FCA emulsion of 10% concentration).

After 7 days, unhairing once more on scapula, and smear the vaseline that contains 10% sodium lauryl sulphate, cause slight inflammation.Strengthening sensitization by this processing induces.Subsequently, smear test specimen at this position, sealing attaches.(ii) bring out

Sensitization was induced the back the 21st day, after the Cavia porcellus flank portion 5 * 5cm place unhairing with sensitization disposal group and matched group, each test specimen was diluted with 80% ethanol water, and sealing in 24 hours attaches.Matched group also carries out same operation.(iii) estimate

It is macroscopic unusual whether observation has sensitization disposal group and matched group, and comparative evaluation sensitization disposal group has or not erythema, edema and degree thereof (slight, moderate or height).(iv) result

Of the present invention 1,4-Benzodiazepines derivant does not all cause sensitivity response to all Cavia porcelluss.Even this result shows that this compounds percutaneous long term administration also is safe.(4) pharmacological effect test

Use 5 phototoxicity pigmentation WeiserMaple Cavia porcelluss of handling through 8-methoxyl group ソ ラ one レ Application, the back after unhairing with test specimen every day of 50 μ l 1 time at 4cm ²Scope in smeared for 8 weeks, estimate the pigmentation degree of the decolorizing effect and the side effect form of expression according to following evaluation criterion.

To the sedimentary inhibition effect of pigment

Estimate some criterion

3: (decolouring) bleaches

2: bleach substantially

1: bleach a little

0: do not change

-1: blackening a little

-2: basic blackening

-3: blackening (pigmentation)

It is of the present invention 1 that test specimen uses, 4-Benzodiazepines derivant BD-4-0H, BD-4-3H and BD-5-0H, and 2 use hydroquinone as a comparative example.Each test specimen is dissolved in the dipropylene glycol with the concentration of 1 weight %, this solution is applied on the Cavia porcellus according to the method described above.In addition, as estimating point, the average ratings of 5 Weiser Maple Cavia porcelluss is put shown in the following tabulation 2.

Table 2

Test compound	Smear number of days (week)
									??1	??2	??3	??4	??5	??6	??7	??8
	Hydroquinone [comparative example 2]	??0.2	??0.4	??1.2	??0.6	??0.2	??0.0	??-1.0									??-1.2
BD-4-0H [synthesis example 1]									??0.6	??1.0	??1.8	??2.0	??2.0	??2.0	??2.2	??2.2
	BD 4-3H [synthesis example 2]	??0.4	??0.8	??1.0	??1.8	??2.0	??2.0	??2.0									??2.2
BD-5-0H [synthesis example 4]									??0.5	??1.8	??1.8	??2.0	??2.0	??2.2	??2.4	??2.4

Of the present invention 1, (BD-4-0H, BD-4-3H BD-5-0H) all can be along with the time increases decolorizing effect for 4-Benzodiazepines derivant.Relative with it, the hydroquinone of comparative example 2 decolorizing effect when the 3rd week reaches maximum, thereafter because stimulation causes pigmentation, the counter tendency of observing blackization of the 7th, 8 phases.

Below, the preparation example of the preparation for external application to skin among the present invention is shown.In addition, following combined amount all is weight %.[embodiment 1] W/O moisturizing lotion

Microwax 1.0

Cera Flava 2.0

Macadimia nut oil 2.0

Squalane 10.0

Liquid paraffin 19.0

BD-4-0H (synthesis example 1) 1.0

1,3 butylene glycol 4.0

Glycerol 3.0

NOFABLE SO-992 NOFABLE SO-902 3.0

LYSOLECITHIN SUNLECITHIN A 1.0

Polyoxyethylene (20 moles) dehydrated sorbitol mono-fatty acid ester 1.0

Alpha-tocopherol 0.02

Antiseptic is an amount of

Ion exchange water 52.98

(preparation method) adds wetting agent 1,3 butylene glycol, glycerol in ion exchange water, and 70 ℃ of heating.In addition with microwax, Cera Flava, macadimia nut oil, squalane and liquid paraffin 70 ℃ of heating for dissolving, when keeping 70 ℃, add BD-4-0H, emulsifying agent NOFABLE SO-992 NOFABLE SO-902, LYSOLECITHIN SUNLECITHIN A and polyoxyethylene (20 moles) dehydrated sorbitol mono-fatty acid ester, antioxidant alpha-tocopherol and antiseptic then.When stirring, add the water of preparing earlier in the oil phase pre-emulsifying slowly, after with homogenizer emulsified particles being homogenized, the degassing, cooling.[embodiment 2] W/O moisturizing lotion

Spermol 1.0

Cera Flava 0.5

Vaseline 2.0

Squalane 6.0

Dimethyl polysiloxane 2.0

Ethanol 5.0

BD-4-3H (synthesis example 2) 1.0

Glycerol 4.0

1,3 butylene glycol 4.0

Polyoxyethylene (10 moles) monoleate 1.0

Quince smoke tree mucus (1% aqueous solution) 20.0

Alpha-tocopherol 0.02

Antiseptic is an amount of

Spice is an amount of

Ion exchange water 52.48

(preparation method) adds glycerol, 1,3 butylene glycol He Quince smoke tree mucus in ion exchange water, and 70 ℃ of heating.In addition with spermol, Cera Flava, vaseline, squalane and dimethyl polysiloxane 70 ℃ of heating for dissolving, when keeping 70 ℃ then, add BD-4-3H, emulsifier polyoxyethylene (10 moles) monoleate, antioxidant alpha-tocopherol and antiseptic.When stirring, add the water of preparing earlier in the oil phase pre-emulsifying slowly, temperature is added ethanol after reducing to 35 ℃, after with homogenizer emulsified particles being homogenized, the degassing, cooling.[embodiment 3] W/O skin moistening emulsifiable paste

Liquid paraffin 30.0

Microwax 2.0

Vaseline 5.0

BD-5-5H (synthesis example 5) 1.0

Two oleic acid, two glyceride 5.0

Alpha-tocopherol 0.02

Antiseptic is an amount of

Spice is an amount of

Water (1)

L-sodium glutamate 1.2

L-serine 0.4

Trimethyl glycine 0.4

Ion exchange water 13.0

Water (2)

Dipropylene glycol 3.0

Ascorbic acid-α-D-pyranglucoside 0.2

Sodium glycollate 0.1

Glycolic 0.1

Ion exchange water 38.58

(preparation method) 50 ℃ of heating for dissolving, adds water (1) to when stirring in same emulsifying agent part two oleic acid two glyceride 50 ℃ of heating slowly, is mixed with emulsification composition.The oil phase (liquid paraffin, microwax and vaseline) that will contain BD-5-5H and alpha-tocopherol after 70 ℃ of heating for dissolving, the above-mentioned emulsification composition of homodisperse therein.Again with water (2) 70 ℃ of heating, under fully stirring, adding in the above-mentioned dispersion liquid, behind the homogenizer uniformly emulsify, outgas, filter and be cooled to 30 ℃.[embodiment 4] O/W skin moistening emulsifiable paste

Spermol 5.0

Stearic acid 3.0

Vaseline 5.0

Squalane 10.0

Glycerol three 2-ethylhexanoate 7.0

BD-5-0H (synthesis example 4) 0.5

Alpha-tocopherol 0.02

1,3 butylene glycol 5.0

Glycerol 5.0

Propylene glycol monostearate 3.0

Polyoxyethylene (20 moles) spermaceti alcohol ether 3.0

Triethanolamine 0.8

Antiseptic is an amount of

Spice is an amount of

Ion exchange water 52.68

(preparation method) adds triethanolamine, wetting agent 1,3 butylene glycol and glycerol in ion exchange water, 70 ℃ of heating.After will containing oil phase (spermol, stearic acid, vaseline, squalane and the glycerol three 2-ethylhexanoate) heating for dissolving of BD-5-0H and alpha-tocopherol, add emulsifying agent propylene glycol monostearate, polyoxyethylene (20 moles) spermaceti alcohol ether, antiseptic and spice, 70 ℃ of heating.Add it to pre-emulsifying of aqueous phase of preparation earlier.Then with homogenizer with the emulsified particles homogenizing after, outgas, filter and be cooled to 30 ℃.[embodiment 5] translucent microemulsified astringent

1,3 butylene glycol 6.0

Glycerol 5.0

Macrogol 4000 3.0

Macadimia nut oil 0.5

BD-4-4H (synthesis example 3) 0.3

Alpha-tocopherol 0.02

Polyoxyethylene (20 moles) sorbitan monostearate 1.5

Polyoxyethylene (5 moles) oleoyl alcohol ether 0.3

Ethanol 10.0

Spice is an amount of

Pigment is an amount of

Antiseptic is an amount of

The pH regulator agent is an amount of

Chelating agen is an amount of

Ion exchange water 73.38

(preparation method) adds wetting agent 1,3 butylene glycol, glycerol and Macrogol 4000, pigment, pH regulator agent, chelating agen in ion exchange water, and dissolves in room temperature.In addition BD-4-4H, alpha-tocopherol, macadimia nut oil, emulsifier polyoxyethylene (20 moles) sorbitan monostearate, polyoxyethylene (5 moles) oleoyl alcohol ether, antiseptic and spice are added in the ethanol, and dissolve in room temperature.Formerly Pei Zhi aqueous phase adds this alcohol phase, is mixed with microemulsifying formulations with homogenizer.[embodiment 6] heavy-gravity micro-emulsified essence

Dipropylene glycol 5.0

PEG400 5.0

Ethanol 10.0

CVP Carbopol ETD2050 0.3

Sodium alginate 0.3

Potassium hydroxide 0.15

Polyoxyethylene (20 moles) sorbitan monostearate 1.0

Dehydrated sorbitol mono-fatty acid ester 0.3

LYSOLECITHIN SUNLECITHIN A 0.2

BD-4-0H (synthesis example 1) 0.2

BD-5-5H (synthesis example 5) 0.3

Alpha-tocopherol 0.02

Alpha-tocopherol acetate 0.02

Oleyl alcohol 0.5

Spice is an amount of

Antiseptic is an amount of

Ion exchange water 73.38

(preparation method) is dissolved in CVP Carbopol ETD2050 in the ion exchange water, dissolves contract a propylene glycol and a PEG400 more successively.In addition dehydrated sorbitol mono-fatty acid ester, polyoxyethylene (20 moles) sorbitan monostearate, LYSOLECITHIN SUNLECITHIN A, BD-4-0H, BD-5-5H, oleyl alcohol, alpha-tocopherol, alpha-tocopherol acetate, spice and antiseptic are dissolved in the ethanol successively.Add it to aqueous phase of preparation earlier, use the homogenizer microemulsified.Then add the potassium hydroxide that is dissolved in a part of ion exchange water, stir, outgas and filter.

[embodiment 7] contain the O/W type essence of ultraviolet screening agent

Stearic acid 3.0

Ethanol 1.0

Cholest-12-hydroxy stearic acid ester 3.0

Liquid paraffin 5.0

Palmic acid 2-Octyl Nitrite 3.0

BD-4-3H (synthesis example 2) 0.3

BD-5-0H (synthesis example 4) 0.3

Alpha-tocopherol 0.02

1,3 butylene glycol 5.0

Polyoxyethylene (20 moles) spermaceti alcohol ether 2.0

Glyceryl monostearate 2.0

Glycerol list-2-ethyl hexanoyl base-di-p-methoxy cinnamate 4.0

4-methoxyl group-the 4 '-tert-butyl group--dibenzoyl methane 4.0

Polysiloxanes surface-treated Zinc oxide particles 2.0

Decamethyl cyclomethicone 2.0

Triethanolamine 0.8

Spice is an amount of

Antiseptic is an amount of

Ion exchange water 61.88

(preparation method) joins 1,3 butylene glycol, triethanolamine in the ion exchange water, 70 ℃ of heating for dissolving.With stearic acid, cholest-12-hydroxy stearic acid ester, liquid paraffin and Palmic acid 2-Octyl Nitrite after 70～80 ℃ of heating for dissolving; add emulsifying agent (polyoxyethylene (20 moles) spermaceti alcohol ether and glyceryl monostearate), UV absorbent (cinnamate and 4-methoxyl group-the 4 '-tert-butyl group-dibenzoyl methane), BD-4-3H, BD-5-0H, alpha-tocopherol, antiseptic and spice successively, and be heated to 70 ℃.Stir and down oil phase is added to the aqueous phase of preparation earlier, carry out pre-emulsifying.Afterwards, temperature is cooled to below 40 ℃, when adding polysiloxanes surface-treated zinc oxide fine particles and decamethyl cyclomethicone dispersion liquid, emulsified particles is homogenized, the degassing, cooling with homogenizer.[embodiment 8] w/o type emulsifiable paste

Liquid paraffin 23.0

Vaseline 6.0

Ceresin 3.0

Cera Flava 1.0

BQ-4-0H??????????????????0.5

BQ-5-5H??????????????????0.5

Alpha-tocopherol 0.02

Two dioleins 4.0

L-sodium glutamate 2.0

Pyrrolidone sodium carboxylate 3.0

1,3 butylene glycol 5.0

Glycerol 2.0

Antiseptic is an amount of

The chelating inhibitor is an amount of

Spice is an amount of

Ion exchange water 49.98

(preparation method) adds wetting agent pyrrolidone sodium carboxylate, L-sodium glutamate, 1,3 butylene glycol, glycerol and chelating inhibitor in ion exchange water, and 70 ℃ of homogeneous heating dissolvings.In addition with ceresin, Cera Flava, vaseline, liquid paraffin and two dioleins 80 ℃ of heating for dissolving, add BQ-4-0H, BQ-5-5H, alpha-tocopherol, spice and antiseptic.When stirring, the water of preparation earlier added to slowly carry out pre-emulsifying in this oil phase, after with homogenizer emulsified particles being homogenized, the degassing, slowly cooling.[test example] people's operational test 1) administration sample and test method

In order to determine the actual effect of external preparation for skin medicine of the present invention, carry out following test to application on human skin.

127 faces there is the testee of the sedimentary disease sites of pigment (pigmentation that pigmentation and other reason cause after chloasma hepaticum, freckle, senile plaque, the inflammation) as study subject.67 experimenters of half smear external preparation for skin medicine of the present invention approximately, and the back that washes one's face sooner or later other 60 every days generates hyperfunction disease sites at melanin and directly smears placebo.Use after 2 months continuously, judge its effect according to doctor's vision, and by before on-test and the photo after 2 months compare research.

As test specimen, use the w/o type moisturizing lotion of embodiment 1 preparation, placebo is to replace the w/o type moisturizing lotion of preparing behind the BD-4-0H with the equivalent ion exchange water.

In addition, when smearing the external preparation for skin medicine, according to 1, the dosage of 4-Benzodiazepines derivant is 0.05～0.5mg/cm ², smear lotion at ill position.The gained result of the test is as shown in the table.

Table 3

	The state of disease sites after 2 months
					Improve	The basic improvement	Constant	Worsen
	The lotion of embodiment 1	28 people (42%)	31 people (46%)	8 people (12%)					0 people (0%)
The lotion of placebo					3 people (5%)	10 people (17%)	43 people (72%)	4 people (6%)

2) result of the test

From the explanation of last table, when adopting external preparation for skin medicine w/o type moisturizing lotion of the present invention, the ratio of doing well,improving (improving+basic the improvement) is 88%.Relative therewith, have 78% not see improvement when adopting placebo, and observe 6% deterioration.In addition, though observe 22% improvement when adopting placebo, can think so-called placebo effect.Effect does not have bigger difference when in addition, finding the symptom difference (pigmentation that pigmentation and other reason cause after chloasma hepaticum, freckle, senile plaque, the inflammation) of pigmentation in this test.

By above result as can be known, the preparation for external application to skin among the present invention has a significant effect really to the pigmentation that improves in the application on human skin.

The effect of invention

The invention provides melanin and generate the inhibition excellence, and stable, safe Melanin inhibitor, and contain this inhibitor as the cosmetics of active ingredient and/or the preparation for external application to skin of health products. Generate inhibition owing to have significant melanin during the extremely low concentration of Melanin inhibitor of the present invention more than 0.0001 % by weight, and to skin or its cell is nontoxic or toxicity is extremely low, therefore the preparation for external application to skin that contains this Melanin inhibitor, not only have excellent skin-whitening effect, and it is safe, can use continuously for a long time.

Claims (7)

1. Melanin inhibitor, it is an effective ingredient with the quinone derivatives shown in Benzodiazepines derivant shown in the general formula (I) or the general formula (II),

In the formula, x, y are 0～6 integer, and x+y=4,5 or 6,

In the formula, x, y are 0～5 integer, and x+y=4 or 5.

2. the described Melanin inhibitor of claim 1, wherein, the Benzodiazepines derivant is 2,3-dimethoxy-5-methyl-6-(3,7,11,15-tetramethyl-2,6,10,1 4-hexadecane apos)-1, the 4-Benzodiazepines, 2,3-dimethoxy-5-methyl-6-(3,7,11,15-tetramethyl-2-hexadecane mono alkenyl)-1, the 4-Benzodiazepines, or 2,3-dimethoxy-5-methyl-6-(3,7,11,15,19-pentamethyl-2,6,10,14,18-eicosane pentaene base)-1, the 4-Benzodiazepines.

3. the described Melanin inhibitor of claim 1, wherein, quinone derivatives is 2,3-dimethoxy-5-methyl-6-(3,7,11,15-tetramethyl-2,6,10,14-hexadecane apos)-1, the 4-benzoquinone, 2,3-dimethoxy-5-methyl-6-(3,7,11,15-tetramethyl-2-hexadecane mono alkenyl)-1, the 4-benzoquinone, or 2,3-dimethoxy-5-methyl-6-(3,7,11,15,19-pentamethyl-2,6,10,14,18-eicosane pentaene base)-1, the 4-benzoquinone.

4. a preparation for external application to skin is characterized in that, contains 1～3 each described Melanin inhibitor more than a kind.

5. the described preparation for external application to skin of claim 4, wherein, the content of above-mentioned Melanin inhibitor is 0.0001～5 weight %.

6. claim 4 or 5 described preparation for external application to skin wherein, contain the additive that is selected from antioxidant, ultraviolet screening agent, cutin agents for defoliating, surfactant, spice, pigment, antiseptic, pH regulator agent, chelating agen.

7. each described preparation for external application to skin of claim 4～6, the dosage form of this preparation for external application to skin is selected from w/o type lotion, O/W type lotion, w/o type emulsifiable paste, O/W type emulsifiable paste, thickness micro-emulsified essence and O/W type essence.