JP2684561B2 - Pigmentation inhibitor - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial field of application] The present invention relates to a pigmentation inhibitor, more specifically, to the production of an enzyme itself rather than inhibiting the action or activity of the enzyme tyrosinase once produced. Thus, the present invention relates to a completely new type of pigmentation inhibitor that suppresses, that is, prevents the problematic enzyme itself from being produced in advance.

[Background Art] Melanin is present in the skin, plays an important role in protecting the body from the adverse effects of ultraviolet rays, and is an important medical and cosmetic factor. The synthesis of melanin is controlled by tyrosinase (tyrosinase,
It is said that tylosin is changed into dopa and then dopaquinone by the action of tyrosinase), which is carried out via 5,6-dioxindole and the like. Excessive amount of melanin is colored black and its uneven distribution is
It is said to be spots (melasma) and freckles (spot egg spots), both of which are cosmetic defects.

Conventionally, hydroquinone and MBEH (monobenzyl ether of hydroquinone) have been used to make the complexion beautiful skin. It has a drawback that it impairs the original physiological function of the skin and causes side effects such as irreversible vitiligo, pigment abnormality, and rash.

Therefore, paying attention to tylosinase, which is an enzyme involved in the production of melanin, vitamin C, glutathione, cysteine, etc. are used in order to reduce the amount of melanin in the skin and make the skin fair and fair. (JP-A-53-142515).

However, all of these drugs are inhibitors of the activity of the tylosinase enzyme itself, and inhibit the activity and action of the tylosinase once formed.
As described above, the conventional technical level lies in how to inhibit the activity of the enzyme once produced.

On the other hand, the present invention is intended to prevent the enzyme itself from being produced in advance, and is not intended to treat the enzyme that has been produced as in the past. In other words, it focuses on the enzyme (tylosinase), which is the root cause of the trouble, and suppresses the production of the enzyme itself, which is the most fundamental part of the melanogenesis system. Therefore, the technical idea of the present invention is basically different from the conventional technical idea.
The purpose setting itself of the invention is already different, and the starting point of the invention is novel.

Thus, suppressing the production of the enzyme itself has not been conceived in the past, technical problems, or the object of the invention itself is novel, and in the present invention, therefore, acetic acid, lactic acid, It adopts a configuration in which a specific organic acid called pyruvic acid is used. In addition, in adopting such a technical idea, there is no suggestion or even suggestion of the prior art, and it is completely novel.

That is, the present invention is novel in both the object and the configuration, and the effect is new and remarkable accordingly.

[Problems to be Solved by the Invention] As described above, the conventionally used inhibitors of tyrosinase enzyme itself not only have insufficient effect of inhibiting the activity of the enzyme itself, but all of them have Because it is a reducing agent that utilizes the reducing power of
The stability is poor, the sustained effect is poor, and the fairness effect on living cells is insufficient (JP-A-53-142515, page 1, lower right column to page 2, upper left column).

[Means for Solving the Problems] The present invention has been made to solve the above-described drawbacks all at once, and is a method of using a substance that inhibits the activity of the enzyme itself of tyrosinase that has been conventionally performed. However, no matter what type of inhibitor is used, no satisfactory substance is found in terms of both the activity inhibitory effect and the persistence of efficacy.

In other words, although various substances have been extensively tested and studied diligently from various aspects, once tyrosinase is once produced as in the past, the intended purpose was completely achieved using any drug. I came to the conclusion that I could not get it.

Therefore, the idea is completely changed, and since the cause of these dermatological troubles lies in the production of tylosinase in the first place, unless the tylosinase itself, which is the root cause of the troubles, is generated, the subsequent troubles will not occur. Recognizing that, we have set a completely new technical issue of developing a pigmentation inhibitor that does not produce tyrosinase.

In order to specifically achieve this novel purpose, tyrosinase production itself is suppressed by utilizing the autonomous melanin production regulation function of living pigment cells, regardless of the direct inhibition of the enzymatic activity of tyrosinase once produced. However, aiming at exerting a melanin production inhibitory effect, and focusing on highly stable metabolic-related organic acids, we have continued to search for a substance having a tyrosinase production inhibitory effect (hereinafter referred to as a tyrosinase production inhibitor).

As a result, we found for the first time that acetic acid, lactic acid, and pyruvic acid strongly suppress the production of tylosinase,
It was confirmed that it is suitable as a tyrosinase production suppressing substance. Furthermore, they have found that a pigmentation inhibitor containing this substance can exert a melanin production inhibitory effect and, in turn, a fair skin effect, and have completed the present invention.

The acetic acid, lactic acid and pyruvic acid referred to in the present invention are acetic acid, L-lactic acid, DL-lactic acid, D-lactic acid, pyruvic acid and alkali metal salts or alkaline earth metal salts of these carboxylic acids, or esters and acids. Refers to amide.

Although the pigmentation inhibitor of the present invention has an excellent tyrosinase production inhibitory effect in living cells, it is also characteristic in that it does not substantially inhibit the activity of the tyrosinase enzyme itself.

 Various measurement methods referred to in this specification are defined as follows.

(1) The activity inhibiting effect of tylosinase is described in "British Journal of Dermatology"
nal of Dermatology), Vol. 103, pp. 625-633 (1
980) according to the method for measuring the activity. In this way, the same reaction is carried out without changing the pH using a test sample. If the activity of the tylosinase enzyme itself is lower than that of the control, it is determined to exhibit the tylosinase activity inhibitory action.

(2) The inhibitory effect on melanin production in pigment cells is described in "Cancer Research", Vol. 42, No. 199.
It is measured according to the method described on pages 4 to 2002 (1982).

That is, 10 ml of Eagle MEM medium containing 10 v / v% fetal bovine serum
4 × 10 ⁴ mouse melanoma-derived cells B-16 are suspended in each well and cultured at 37 ° C. in the presence of 5 v / v% CO ₂ in a 25 cm ² culture vial. The main culture solution is replaced with a medium containing a tyrosinase production inhibition test sample on days 0 and 3, and the culture is cultured for 5 days. Phosphate buffer containing 0.8 w / v% sodium chloride (pH
After washing in 7.2), detach using a solution containing trypsin and EDTA, and collect cells by filtration. The cells collected on the filter paper were dried and then dried at 500n with a densitometer.
The reflected light at m is measured, and the total amount of melanin is obtained from the reflected absorbance (blackness).

According to this method, the treated group when the absorbance of the treated group is reduced to 80% or less of the absorbance of the control is determined to have the melanin production inhibitory action.

(3) The inhibitory effect of tyrosinase production is described in "Journal of the Japanese Dermatological Association", Vol. 87, No. 13, pp. 883-901 (1977).
Dopa reaction method described in
Research, "Volume 42, 1994-2.
Judgment was made according to the tylosinase confirmation method described on page 002 (1982). That is, mouse melanoma-derived cells B-16 were cultivated for 5 days in a culture medium containing the tyrosinase production-inhibiting substance of the present invention according to the method described above, whitened, and then the culture medium was discarded and 10 v / v% formalin was added. Immobilized with 0.1% w / v% L-dopa and then immersed in 0.1M phosphate buffer (pH 7.3) at 37 ° C for 3 hours, and observed under a microscope to darken the cells due to melanin production. When it can be confirmed by electrophoresis that all of the isozymes T ₁ , T ₂ , and T _{3 of} tyrosinase are reduced or eliminated, it is determined that the tyrosinase production inhibitory effect is exhibited.

Since acetic acid, lactic acid, and pyruvic acid of the present invention have a highly safe and stable tyrosinase production-inhibiting substance, it should be contained as an active ingredient, for example, injections, internal preparations, external preparations, bath preparations. It is used for pharmaceuticals such as agents, cosmetics such as emulsions, packs, creams, etc. to suppress the production of melanin and not only exert a whitening effect, but also stains,
It is highly effective in treating and preventing pigmentation diseases such as freckles and sunburn, and also imparts a moisturizing effect to the skin, and is highly effective in treating and preventing skin irritation.

The specific organic acids used in the present invention may be used alone or in combination of two or more kinds, and in the case of combined use, a synergistic effect is expected.

As is clear from the fact that these organic acids are biological components or natural products and can be directly drunk, toxicity is virtually nonexistent and there is no problem in terms of safety.

 Hereinafter, the present invention will be described in detail by experiments.

Experiment 1. Search for tyrosinase production inhibitory substances Using the metabolism-related organic acids shown in Table 1, the tyrosinase activity inhibitory action, melanin production inhibitory action and tyrosinase production inhibitory action were investigated according to the above-mentioned measurement method. Each organic acid was neutralized with sodium hydroxide to a predetermined pH in advance and used at a concentration of 10 mM.

 The results are shown in Table 1.

As is clear from the results in Table 1, acetic acid, lactic acid, and pyruvic acid, unlike other organic acids, were found to have a tyrosinase production inhibitory effect, even though they did not have a tyrosinase activity inhibitory effect. did. It was also found that acetic acid, lactic acid, and pyruvic acid produce a melanin production inhibitory action due to a tyrosinase production inhibitory action.

On the other hand, koji acid, although showing a melanin production suppressing action,
It was revealed that this was due to the activity-inhibiting effect of tylosinase, not the activity-inhibiting effect of tyrosinase.

Experiment 2. Effects of type and concentration of salt on tyrosinase production inhibitory effect According to the method of Experiment 1, acetic acid, L-lactic acid, DL-lactic acid,
Using pyruvic acid, the effect of salt type and concentration on the tyrosinase production inhibitory effect was investigated.

 The results are shown in Table 2.

As is clear from the results in Table 2, acetic acid, L-lactic acid,
DL-lactic acid and pyruvic acid show similar tyrosinase production inhibitory action as alkali metal salts such as lithium, sodium and potassium, and alkaline earth metal salts such as calcium and magnesium. It was found to have an effect.

Experiment 3. Enhancement of tyrosinase production inhibitory effect In accordance with the method of Experiment 1, the combined effect of acetic acid, L-lactic acid and pyruvic acid on the tyrosinase production inhibitory effect was investigated. Each organic acid was neutralized with sodium hydroxide to a predetermined pH in advance, used in a predetermined combination (molar ratio) and in a predetermined concentration, and then used.

 The results are shown in Table 3.

As is clear from the results in Table 3, when acetic acid, lactic acid and pyruvic acid are used alone, two or more kinds selected from them are used in combination to remarkably enhance the tyrosinase production inhibitory effect. It turned out to be possible.

Experiment 4. Clinical trial For each of the pigmentation inhibitors prepared by the methods of Examples 4, 6, 8 and 10, 60 men and women of 20 to 55 years of age (30 males) at any age who suffer from spots and freckles , 30 women, average age 34.8 years old, women 39.2 years old) used for 3 months,
We conducted a questionnaire survey on safety and efficacy.

 The results are shown in Table 4.

As is clear from the results in Table 4, the pigmentation inhibitor of the present invention can be safely used without side effects, and
It was found to be highly effective in the treatment and prevention of spots and freckles.

As is clear from the above experiments, these tyrosinase production inhibitory substances exhibit a tyrosinase production inhibitory action, exhibit a strong melanin production inhibitory effect, and can achieve a high fair-whitening effect. Furthermore, the safety of these tylosinase production-inhibiting substances is extremely high, even in view of their effective amounts.

The pigmentation inhibitor containing one or more selected from acetic acid, lactic acid, and pyruvic acid of the present invention as an active ingredient is a tyrosinase production inhibitor,
It can be used by itself, but usually has a concentration of 0.002
To 10M, preferably 0.005 to 5M,
The pH is 2.5 to 9.0, preferably 3.0 to 8.0. The daily dose for an adult is 0.01 to 1,000 g as a tyrosinase production inhibitor, preferably 0.01 to 100 g for systemic use such as intramuscular injection, and 1.0 for oral use such as an oral drug. To 200 g, external preparation, bath preparation, and further, in the case of transdermal or transmucous use of cosmetics such as emulsion and cream, it is 0.1 to 1,000 g, but this amount depends on the usage, symptoms, etc. It can be increased or decreased as appropriate.

In order to prevent or treat topical spots such as spots, freckles, and sunburn, as well as systemic pigmentation such as addisonism, tylosinase production inhibitor alone is used.
Alternatively, if necessary, a tyrosinase production inhibitor and any material, for example, a physiologically active substance, a nutrient, a penetration enhancer such as alcohol and a surfactant, a base material, a pigmentation inhibitor combined with an excipient is The shape can be freely selected according to the purpose, such as pharmaceuticals and cosmetics, or according to the usage form.

For example, an injection is a liquid or a solid injection to be dissolved in distilled water for injection at the time of use, an oral is a liquid, a powder, a granule, a capsule, a tablet, etc., and a transdermal is It can be used as a liquid preparation, an emulsion, a cream, an ointment, an external preparation such as a patch applied on one side with a cream, a bath preparation, and a cosmetic such as a lotion, emulsion, pack, cream and the like. Further, when the transdermal use method is adopted, it is also necessary to promote the permeation of the tyrosinase production suppressing substance into the skin tissue by iontophoresis in order to enhance the tyrosinase production suppressing effect and enhance the melanin production suppressing effect. For example, it is also advantageous to use a supporting electrolyte together to enhance the effect of ion introduction.

Also, if necessary, together with a tyrosinase production inhibitor, for example, vitamin C, vitamin E, glutathione, cysteine, koji acid, placenta extract, glucosamine derivative, photosensitizer No. 201 (Okayama City, manufactured by Japan Photosensitivity Research Institute, Inc.) It is also possible to appropriately use one or two or more of colloidal sulfur, hydroquinone derivative and the like in combination to suppress melanin production and further enhance the whitening effect. Furthermore, it is optional to enhance the fairness effect of the present agent by using a sunscreen agent in combination.

Next, examples of the pigmentation inhibitor of the present invention will be described.

Example 1 Injection: 8.0 parts by weight of sodium acetate, 2.0 parts by weight of L-lactic acid, L-
Sodium lactate 10 parts by weight, sodium chloride 5.3 parts by weight,
Dissolve 0.3 parts by weight of potassium chloride and 0.2 parts by weight of calcium chloride in 1,000 parts by weight of water, purify and filter according to a conventional method, fill 2 ml each of this filtrate into a sterilized glass container, and tightly stopper the injection solution. Manufactured.

This product is suitable as a pigmentation inhibitor, and can be advantageously used for the treatment and prevention of topical pigmentation diseases such as spots, freckles, and sunburns, and systemic pigmentation diseases such as adisonism.

Example 2 Oral formulation (tablet) 40 parts of L-calcium lactate pentahydrate was uniformly mixed with 45 parts by weight of maltose, 61 parts by weight of corn starch and 4 parts by weight of sucrose fatty acid ester, and tableted according to a conventional method. One tablet
A 150 mg tablet was produced.

This product is suitable as a pigmentation inhibitor, and is used in Example 1
Similar to the above case, it can be advantageously used for the treatment and prevention of pigmentation.

Example 3 Oral formulation (capsule) 10 parts by weight of calcium acetate monohydrate, 50 parts by weight of L-magnesium lactate trihydrate, 87 parts by weight of maltose and 2 parts by weight of sucrose fatty acid ester were uniformly mixed. Then, the mixture was granulated by a granule molding machine and encapsulated in gelatin capsules according to a conventional method to produce a capsule containing 150 mg of one capsule.

This product is suitable as a pigmentation inhibitor, and is used in Example 1
Similar to the above case, it can be advantageously used for the treatment and prevention of pigmentation.

Example 4 External application (ointment) 1.0 part by weight of sodium acetate trihydrate and 4.0 parts by weight of DL-calcium lactate were uniformly mixed with 10.0 parts by weight of glycerin, and the mixture was mixed with 50.0 parts by weight of petrolatum and 10.0 parts by weight of wax. Part, lanolin 10.0 parts by weight, sesame oil 14.5 parts by weight, and mint oil 0.5 parts by weight, and further homogeneously mixed to produce an ointment.

This product is suitable as a pigmentation inhibitor as in the case of Example 2, and can be advantageously used for the treatment of pigmentation disease and the like, and also for the prevention of sunburn.

Example 5 External preparation (emulsion) DL-sodium lactate 2.0 parts by weight, pyruvic acid 3.0 parts by weight, liquid paraffin 12.0 parts by weight, lanolin 4.0 parts by weight, oleic acid 3.5 parts by weight, triethanolamine 1.0 part by conventional method. Department and Octylde de Cylmyri State
3.0 parts by weight were uniformly mixed, and thereto were added an appropriate amount of preservative, perfume and 71.5 parts by weight of purified water, and the mixture was homogenized to prepare an emulsion.

This product is suitable as a pigmentation inhibitor, and is used in Example 4
Similar to the above case, it can be advantageously used for the treatment and prevention of pigmentation.

Example 6 Topical preparation (lotion) 2.0 parts by weight of sodium pyruvate, 1.0 part by weight of polyoxyethylene hydrogenated castor oil, 15.0 parts by weight of ethanol, DL
-Lactic acid 0.1 parts by weight, DL-sodium lactate 0.3 parts by weight, 1,3
-Butylene glycol 4.0 parts by weight, sodium pyrrolidonecarboxylate 0.5 parts by weight, preservative, appropriate amount of perfume and purified water 77.0 parts by weight were uniformly mixed according to a conventional method to produce a lotion.

This product is suitable as a pigmentation inhibitor, and is used in Example 5
Similar to the above, it can be advantageously used for the treatment and prevention of pigmentation diseases such as spots, freckles, and sunburn.

Example 7 Bath agent DL-sodium lactate 50 parts by weight, sodium pyruvate
15 parts by weight were mixed with 35 parts by weight of purified water, a coloring amount, and an appropriate amount of perfume to prepare a bath agent.

This product is suitable as a pigmentation inhibitor, and is suitable for the treatment or prevention of local or systemic pigmentation by diluting 100-10,000 times in hot water for bathing.
Further, this product can exert the melanin production inhibitory effect even when it is diluted with facial cleansing water, lotion and the like as in the case of hot water for bathing.

Example 8: Cosmetic (milky lotion) 0.5 part by weight of polyoxyethylene behenyl ether, 1.0 part by weight of polyoxyethylene sorbitol tetraoleate, 1.0 part by weight of lipophilic glyceryl monostearate,
0.5 parts by weight of pyruvic acid, 0.5 parts by weight of behenyl alcohol,
1.0 part by weight of avocado oil, and a suitable amount of vitamin E, a preservative, and a fragrance are dissolved by heating according to a conventional method, and 1.0 part by weight of sodium acetate, 5.0 parts by weight of 1,3-butylene glycol, and 0.1 part by weight of carboxyvinyl polymer are added thereto. And purified water 8
5.3 parts by weight was added, and the mixture was applied to a homogenizer to produce an emulsion.

This product is suitable as a pigmentation inhibitor and can be advantageously used for the treatment and prevention of pigmentation diseases such as spots, freckles, and sunburn.

Example 9 Chemical cosmetics (pack) 2.0 parts by weight of sodium acetate, 1.5 parts by weight of squalane, 0.5 parts by weight of polyoxyethylene hydrogenated castor oil, 4.0 parts by weight of glycerin Polyvinyl alcohol 15.0 according to a conventional method
By weight, the mixture was uniformly mixed with 10.0 parts by weight of ethanol and 67.0 parts by weight of purified water to produce a pack.

This product is suitable as a pigmentation inhibitor and is described in Example 8.
Similar to the above case, it can be advantageously used for the treatment and prevention of pigmentation.

Example 10 Chemical cosmetics (cream) 2 parts by weight of polyoxyethylene glycol monostearate, 5 parts by weight of self-emulsifying glyceryl monostearate, 5 parts by weight of pyruvic acid, 1 part by weight of behenyl alcohol, 1 part by weight of liquid paraffin, trioctanoic acid Glycerin (10 parts by weight), sodium hydrogen carbonate (3 parts by weight), and an appropriate amount of preservative and fragrance were dissolved by heating according to a conventional method.
-2 parts by weight of lactic acid, 5 parts by weight of 1,3-butylene glycol and 75 parts by weight of purified water were added to a homogenizer to prepare a cream.

This product is suitable as a pigmentation inhibitor and, like the case of Example 8, can be advantageously used for the treatment or prevention of pigmentation.

[Effects of the Invention] As described above, in the present invention, one or more substances selected from acetic acid, lactic acid, and pyruvic acid do not substantially inhibit the activity of the enzyme itself of tyrosinase. However, it has been newly found that it exhibits a strong inhibitory effect on tyrosinase production itself in pigment cells, and a pigmentation inhibitor containing the tyrosinase production inhibitor as an active ingredient is established.

In addition, the pigmentation inhibitor exhibits a strong melanin production inhibitory effect and can achieve a fair skin effect, so that injections, oral agents, external preparations, bath preparations and other pharmaceuticals and emulsions,
It can be advantageously used as a cosmetic such as a pack and a cream for the treatment and prevention of topical spots, freckles, sunburns and the like and systemic pigmentation diseases such as adisonism.

This pigmentation inhibitor is a natural product and can be directly drunk, and it is extremely safe from the viewpoint of its effective amount and can be used with confidence.It is also highly stable against heat and pH, heat sterilized, and stored for a long time. It is also easy and has extremely advantageous conditions for industrial production and use, and has an extremely great industrial significance in the fields of pharmaceuticals, cosmetics and the like.

Claims (1)

(57) [Claims] 1. A pigmentation inhibitor, which comprises one or more components selected from acetic acid, lactic acid and pyruvic acid as active ingredients.