JP2011195519A - Melanin production inhibitor, glutathione production promoter, hyaluronic acid production promoter and involucrin production promoter, and skin cosmetic - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a melanin production inhibitor, a glutathione production promotor, a hyaluronic acid production promotor and an involucrin production promotor, and a skin cosmetic having an excellent activities, and excellent in safety, environmental property and productivity.SOLUTION: This melanin production inhibitor is provided by including at least any of rutinose and an alkyl rutinose; the glutathione production promotor is provided by containing at least any of the rutinose and alkyl rutinose; the hyaluronic acid production promotor is provided by containing methyl rutinoside; the involucrin production promotor is provided by containing the alkyl rutinose; and the skin cosmetic is provided by containing at least any of the rutinose and alkyl rutinose.

Description

  The present invention relates to a melanin production inhibitor, a glutathione production promoter, a hyaluronic acid production promoter, an involucrin production promoter, and a skin cosmetic.

  Melanin also plays a role in protecting the body from ultraviolet rays in the skin, but overproduction and uneven accumulation cause skin darkening and spots. In general, melanin is changed from tyrosine to dopa and from dopa to dopaquinone by the action of an enzyme tyrosinase biosynthesized in pigment cells, and then formed through an intermediate such as 5,6-dihydroxyindophenol. Therefore, in order to prevent and improve skin darkness (skin pigmentation), that is, for whitening, it is considered effective to suppress the production of melanin or to lightly bleach already produced melanin. It is done.

Glutathione is a tripeptide composed of three amino acids, glutamic acid, cysteine and glycine, and is a compound having a major intracellular cysteine residue. The role of glutathione in cells is radical scavenging, regulation of cell functions by redox, SH donors of various enzymes, and is also known as an antioxidant component. It is thought that the action expression of glutathione is derived from the cysteine residue. However, it has been reported that the amount of glutathione in the skin decreases with aging, which reduces the ability of the skin to oxidize and damages cellular components such as DNA and proteins. It is considered to be.
In other words, the promotion of glutathione production in the skin increases the cysteine residue, which is the main body of action, leading to increased protection against oxidative stress that declines with aging, and suppressing damage to oxidative stress due to ultraviolet rays, It is expected that skin aging can be prevented, treated, or improved against pigmentation such as spots.
So far, for example, bilberry extract or walnut extract (for example, see Patent Document 1), gardenia plant extract (for example, see Patent Document 2), etc. have been disclosed as having glutathione production promoting action. Yes.

In addition, when cellular functions decline due to aging or pathological conditions of the skin and joints, the amount of biological hyaluronic acid decreases, resulting in dry skin, rough skin, firmness, decreased elasticity, increased spots and wrinkles Or joint pain due to deterioration of the wettability of the joint.
In order to improve such a condition, cosmetics containing biological components such as hyaluronic acid and natural moisturizing factor are applied to the skin, and hyaluronic acid is directly injected into the joint. However, even if hyaluronic acid is given from the outside, the fundamental function is not improved and a sufficient effect cannot be expected. In particular, there is a problem that hyaluronic acid is hardly absorbed from the skin.
For this reason, instead of simply replenishing hyaluronic acid itself from the outside, by utilizing the self-healing power of the living body inherently possessed by humans, by promoting the hyaluronic acid producing ability of human skin fibroblasts themselves, Development of substances that fundamentally improve the functions of living bodies is expected. Therefore, attempts have been made to extract substances having hyaluronic acid production promoting action from natural products (see, for example, Patent Documents 3 to 5).

The epidermis has a protective function of protecting the skin from various stimuli from the outside by constantly creating new keratinocytes through the division and subsequent differentiation of keratinocytes. In particular, in the differentiation process of keratinocytes, a protein such as involucrin is expressed from the spinous layer to the granule layer, is crosslinked by the action of the enzyme transglutaminase-1, and is an insoluble cell membrane-like structure that wraps around keratinocytes. It forms a fido envelope (hereinafter referred to as “CE”) and contributes to the stability of the keratinocyte cytoskeleton and structure.
However, when the amount of transglutaminase-1 or involucrin produced in the epidermis decreases due to various factors, CE formation becomes incomplete and keratinization cannot be performed normally. As a result, it is considered that the keratin barrier function and the skin moisturizing function are lowered, and skin symptoms such as rough skin and dry skin are exhibited.
For this reason, by increasing the production of involucrin and transglutaminase-1 in the epidermis of keratinocytes and promoting the formation of CE to normalize keratinization, the skin barrier associated with external stimuli such as drying and ultraviolet rays It is considered that the reduction of function can be suppressed and various skin symptoms such as dry skin and rough skin can be prevented and improved.

  However, so far, it is excellent in safety, environment, and productivity, and does not adversely affect the quality of the addition target in terms of taste, smell, feeling of use, etc., skin cosmetics, cosmetic foods and drinks, pharmaceuticals In addition, melanin production inhibitors, glutathione production promoters, hyaluronic acid production promoters, and involucrin production promoters that can be widely used as research reagents, etc. have not been provided yet, and their prompt provision is strongly demanded. Is the current situation.

JP 2006-241062 A JP 2006-347934 A JP 2000-143527 A JP 2003-192567 A JP 2004-51533 A

An object of the present invention is to solve the conventional problems and achieve the following objects. That is, the first object of the present invention is to provide a melanin production inhibitor that has an excellent melanin production inhibitory action and is excellent in safety, environmental properties, and productivity.
A second object of the present invention is to provide a glutathione production promoter that has an excellent glutathione production promoting action and is excellent in safety, environmental performance, and productivity.
In addition, a third object of the present invention is to provide a hyaluronic acid production promoter that has an excellent hyaluronic acid production promoting action and is excellent in safety, environmental performance, and productivity.
A fourth object of the present invention is to provide an involucrin production promoter that has an excellent involucrin production promoting action and is excellent in safety, environmental properties, and productivity.
In addition, the present invention fifthly has an excellent melanin production inhibitory action, glutathione production promoting action, hyaluronic acid production promoting action, and involucrin production promoting action, and has excellent safety, environmental friendliness, and productivity. The purpose is to provide a fee.

  As a result of intensive studies by the present inventors in order to solve the above problems, the following knowledge was obtained. That is, rutinose and alkylrutinose have an excellent melanin production inhibitory effect and glutathione production promoting action, methylrutinoside has an excellent hyaluronic acid production promoting action, and alkylrutinose has an excellent involucrin production promoting action. As a result, the present invention has been completed.

The present invention is based on the above findings of the present inventors, and means for solving the above problems are as follows. That is,
<1> A melanin production inhibitor characterized by containing at least one of rutinose and alkylrutinose.
<2> A melanin production inhibitor containing at least one of rutinose, methylrutinoside, ethylrutinoside, and rutinosylglycerin.
<3> A glutathione production promoter characterized by containing at least one of rutinose and alkylrutinose.
<4> A glutathione production promoter comprising at least one of rutinose, methylrutinoside, ethylrutinoside, and rutinosylglycerin.
<5> A hyaluronic acid production promoter characterized by containing methylrutinoside.
<6> An involucrin production promoter characterized by containing an alkyl rutinose.
<7> An involucrin production promoter characterized by containing at least one of methylrutinoside, ethylrutinoside, and rutinosylglycerin.
<8> A skin cosmetic characterized by containing at least one of rutinose and alkylrutinose.
<9> A skin cosmetic containing at least one of rutinose, methylrutinoside, ethylrutinoside, and rutinosylglycerin.

According to the melanin production inhibitor of the present invention, it is possible to solve conventional problems, and to provide a melanin production inhibitor that has excellent melanin production inhibitory action and is excellent in safety, environmental performance, and productivity. be able to.
According to the glutathione production promoter of the present invention, it is possible to solve conventional problems, and to provide a glutathione production promoter that has an excellent glutathione production promoting action and is excellent in safety, environmental performance, and productivity. be able to.
According to the hyaluronic acid production promoter of the present invention, the hyaluronic acid production promoter that can solve various conventional problems, has an excellent hyaluronic acid production promoting action, and is excellent in safety, environmental performance, and productivity. Can be provided.
According to the involucrin production promoter of the present invention, it is possible to solve the conventional problems, and to provide an involucrin production promoter that has an excellent involucrin production promoting action and is excellent in safety, environment and productivity. be able to.
According to the skin cosmetics of the present invention, conventional problems can be solved, and have excellent melanin production inhibiting action, glutathione production promoting action, hyaluronic acid production promoting action, involucrin production promoting action, safety, environment Skin cosmetics excellent in productivity and productivity can be provided.

(Melanin production inhibitor, glutathione production promoter, hyaluronic acid production promoter, involucrin production promoter)
The melanin production inhibitor of the present invention contains at least one of rutinose and alkylrutinose, and further contains other components as necessary. There is no restriction | limiting in particular as said alkyl rutinose, Although it can select suitably according to the objective, Methyl rutinoside, ethyl rutinoside, and rutinosyl glycerol are preferable.
The glutathione production promoter of the present invention contains at least one of rutinose and alkylrutinose, and further contains other components as necessary. There is no restriction | limiting in particular as said alkyl rutinose, Although it can select suitably according to the objective, Methyl rutinoside, ethyl rutinoside, and rutinosyl glycerol are preferable.
The hyaluronic acid production promoter of the present invention contains at least methylrutinoside, and further contains other components as necessary.
The involucrin production promoter of the present invention contains at least alkyl rutinose, and further contains other components as necessary. There is no restriction | limiting in particular as said alkyl rutinose, Although it can select suitably according to the objective, Methyl rutinoside, ethyl rutinoside, and rutinosyl glycerol are preferable.

<Lucinose>
The rutinose is a compound represented by the following structural formula (1).

There is no restriction | limiting in particular as a manufacturing method of the said rutinose, According to the objective, it can select suitably, For example, it can manufacture by making a rutin degradation enzyme act on rutin.
The rutin is one of flavonol glycosides widely distributed in natural plants such as Enju (Leguminosae), Buckwheat (Tapaceae), Henruda (Rutaceae), and is useful for preventing arteriosclerosis and hypertension. It is known as an active substance, and has a structure in which quercetin (aglycone) and rutinose (disaccharide) are bound as shown in the following structural formula (X).

The rutin-degrading enzyme is not particularly limited as long as it is an enzyme capable of decomposing rutin and cutting out rutinose, and can be appropriately selected according to the purpose. For example, it can be applied to plants such as Enju, buckwheat, and Henruda. And enzymes derived from microorganisms such as bacteria belonging to the genus Penicillium . Among these, rutin-degrading enzyme contained in tartary buckwheat seed is preferable in terms of high activity and high reliability in terms of safety.
The rutin degrading enzyme may be purified or unpurified. That is, a plant or microorganism extract containing the rutin-degrading enzyme may be used as it is, or a plant body or a microorganism itself may be used.
Further, for example, in the case of rutin-degrading enzyme contained in the tartary buckwheat seed, extract the tartary buckwheat seed or buckwheat flour prepared by grinding this seed to obtain an extract (crude enzyme solution), which is further purified It is preferable to use the purified enzyme obtained in this way. However, because the enzyme contained in tartane soba has high activity, it functions well even if the extract is used as it is.

  The method for extracting the rutin-degrading enzyme from plants and microorganisms is not particularly limited, and a method usually used for extracting the enzyme can be appropriately selected. For example, a method of stirring and extracting in an acetate buffer can be mentioned.

  The conditions for the treatment with the rutin degrading enzyme are not particularly limited and may be appropriately selected depending on the intended purpose. However, the pH is preferably 3 to 9, and the temperature is preferably 80 ° C. or less, and the pH is 5 and the temperature is 40 ° C. Is more preferable.

  Since the rutinose has a melanin production inhibitory action and a glutathione production promotion action, it can be used as a melanin production inhibitor and a glutathione production promoter using these actions.

<Alkyl rutinose>
The alkyl rutinose is a compound represented by the following general formula (1).
However, in said reaction formula, R represents an alkyl group.

There is no restriction | limiting in particular as a manufacturing method of the said alkyl rutinose, Although it can select suitably according to the objective, The method of making the said rutin degradation enzyme act on the said rutin in presence of alcohol is preferable. Thereby, as shown in the following reaction formula, transfer of rutinose contained in rutin to an alkyl group occurs, and alkylrutinose can be generated.
However, in said reaction formula, R represents an alkyl group.

  Examples of the alcohol include methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, 1-pentanol, 1-hexanol, 1-heptanol, 1-octanol, 1-nonanol, 1-decanol, Examples include 2-methyl-1-propanol, 2-methyl-2-propanol, phenylmethanol, 2-phenoxyethanol, 1,2-ethanediol, and 1,2,3-propanetriol, depending on the target product. Can be selected as appropriate. For example, when methanol is used, methylrutinoside can be produced, when ethanol is used, ethylrutinoside can be produced, and when 1,2,3-propanetriol is used, rutinosylglycerin can be produced.

  Moreover, as a manufacturing method of the said alkyl rutinose, you may just make it react by adding an alcohol solution to tartane buckwheat seed or the buckwheat flour which milled it, for example. Dattan buckwheat seeds contain much more rutin than normal buckwheat seeds (about 1300 mg% compared to about 14 mg% for normal buckwheat seeds; (It represents the amount of mg of rutin contained).

  In addition, when using a rutin-degrading enzyme derived from a microorganism, in addition to extracting the enzyme from the microorganism itself, if the microorganism secretes the enzyme outside the cell, the culture solution in which the microorganism is cultured is used as a crude enzyme solution. It is also possible to produce the alkylrutinose by adding rutin and alcohol directly to the culture and reacting them.

  The reaction conditions by the rutin degrading enzyme are not particularly limited and may be appropriately selected depending on the intended purpose. However, the pH is preferably 3 to 9, and the temperature is preferably 80 ° C. or less, and the pH is 5 and the temperature is 40 ° C. Is more preferable.

-Methylrutinoside-
The methylrutinoside is a compound represented by the following structural formula (2).

  Since the methylrutinoside has a melanin production inhibitory action, a glutathione production promoting action, a hyaluronic acid production promoting action, and an involucrin production promoting action, using these actions, a melanin production inhibitor, a glutathione production promoter, It can be used as a hyaluronic acid production promoter and an involucrin production promoter.

-Ethyl rutinoside-
The ethylrutinoside is a compound represented by the following structural formula (3).

  Since the ethylrutinoside has a melanin production inhibitory action, a glutathione production promoting action, and an involucrin production promoting action, using these actions as a melanin production inhibitor, a glutathione production promoter, and an involucrin production promoter. Can be used.

-Rutinosylglycerin-
There is an isomer in the rutinosylglycerin. Examples of the isomers of rutinosylglycerin include 1-rutinosylglycerin (compound represented by the following structural formula (4)) and 2-rutinosylglycerin (compound represented by the following structural formula (5)). .

  Since the rutinosylglycerin has a melanin production inhibitory action, a glutathione production promotion action, and an involucrin production promotion action, the melanin production inhibitor, the glutathione production promoter, and the involucrin production promotion are utilized by using these actions. It can be used as an agent.

  As an active ingredient of the melanin production inhibitor, at least one of the rutinose and alkylrutinose may be contained, and a pharmacologically acceptable salt thereof may be contained. In addition, a hydrate or solvate of at least one of the rutinose and alkylrutinose or a pharmacologically acceptable salt thereof may be contained. In addition, at least one of the rutinose and alkylrutinose may be modified or substituted as long as the melanin production inhibitory action is not impaired.

  As an active ingredient of the glutathione production promoter, at least one of the rutinose and alkylrutinose may be contained, and a pharmacologically acceptable salt thereof may be contained. In addition, a hydrate or solvate of at least one of the rutinose and alkylrutinose or a pharmacologically acceptable salt thereof may be contained. Further, at least one of the rutinose and alkylrutinose may be modified or substituted as long as the glutathione production promoting action is not impaired.

  As an active ingredient of the hyaluronic acid production promoter, the methylrutinoside itself may be contained, or a pharmacologically acceptable salt thereof may be contained. Moreover, the hydrate or solvate of the said methylrutinoside or its pharmacologically acceptable salt may be contained. The methylrutinoside may be modified or substituted as long as the hyaluronic acid production promoting action is not impaired.

  As an active ingredient of the involucrin production promoter, the alkylrutinose itself may be contained, or a pharmacologically acceptable salt thereof may be contained. Moreover, the hydrate or solvate of the said alkyl rutinose or its pharmacologically acceptable salt may be contained. In addition, at least one of the alkylrutinose may be modified or substituted as long as the involucrin production promoting action is not impaired.

  The pharmacologically acceptable salt is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include hydrochloride, sulfate, hydrobromide, nitrate, hydrosulfate, and phosphoric acid. Salt, acetate, lactate, succinate, citrate, maleate, hydroxymaleate, tartrate, fumarate, methanesulfonate, p-toluenesulfonate, camphorsulfonate, sulfamine , Mandelate, propionate, glycolate, stearate, malate, ascorbate, pamonate, phenylacetate, glutamate, benzoate, salicylate, sulfanilate, 2 -Acetoxybenzoate, ethanedisulfonate, oxalate, isethionate, formate, trifluoroacetate, ethyl succinate, lactobionate, glucone Salt, glucoheptonate, 2-hydroxyethanesulfonate, benzenesulfonate, paratoluenesulfonate, lauryl sulfate, aspartate, adipate, hydroiodide, nicotinate, oxalate , Picrate, thiocyanate, undecanoate and the like.

The melanin production inhibitor of the present invention may be composed only of the rutinose, may be composed only of the alkylrutinose, or is composed of the rutinose and the alkylrutinose. It may also contain other components. Moreover, the alkyl rutinose may contain 2 or more types.
There is no restriction | limiting in particular as content of the said rutinose and alkyl rutinose in the said melanin production inhibitor, According to the objective, it can select suitably.

The glutathione production promoter of the present invention may be composed only of the rutinose, may be composed only of the alkylrutinose, or is composed of the rutinose and alkylrutinose. It may also contain other components. Moreover, the alkyl rutinose may contain 2 or more types.
There is no restriction | limiting in particular as content of the said rutinose and alkyl rutinose in the said glutathione production promoter, According to the objective, it can select suitably.

The hyaluronic acid production promoter of the present invention may consist only of the methylrutinoside or may contain other components.
There is no restriction | limiting in particular as content of the said methyl rutinoside in the said hyaluronic acid production promoter, According to the objective, it can select suitably.

The involucrin production promoter of the present invention may be composed of the above alkylrutinose, or may contain other components. Moreover, the alkyl rutinose may contain 2 or more types.
There is no restriction | limiting in particular as content of the said alkyl rutinose in the said involucrin production promoter, According to the objective, it can select suitably.

<Other ingredients>
The rutinose and alkylrutinose may be any of powder, granule, tablet, liquid, etc. according to a conventional method using a pharmaceutically acceptable carrier such as dextrin and cyclodextrin, or any other auxiliary agent. In addition to being able to be used in combination with other compositions (for example, external preparations for skin), it can be used as an ointment, a liquid for external use, a patch, and the like. In this case, as an auxiliary agent, for example, an excipient, a binder, a disintegrant, a lubricant, a stabilizer, a flavoring agent and the like can be used.

  The melanin production inhibitor, glutathione production promoter, hyaluronic acid production promoter, and involucrin production promoter of the present invention include a melanin production inhibitory action, a glutathione production promotion action, a hyaluronic acid production promotion action, and an involucrin as needed. A natural extract or the like having a production promoting action can be used in combination.

The melanin production inhibitor exerts a melanin production inhibitory action by the action of at least one of rutinose and alkylrutinose contained as an active ingredient.
The glutathione production promoter exhibits a glutathione production promoting action by the action of at least one of rutinose and alkylrutinose contained as an active ingredient.
The hyaluronic acid production promoter exhibits hyaluronic acid production promoting action by the action of methylrutinoside contained as an active ingredient.
The involucrin production promoter exerts an involucrin production promoting action by the action of alkylrutinose contained as an active ingredient.

  According to the melanin production inhibitor of the present invention, it is possible to prevent and improve, for example, skin darkness (skin pigmentation) and obtain a whitening effect through an excellent melanin production inhibitory action. However, the melanin production inhibitor of this invention can be used for all the uses meaningful in exhibiting a melanin production inhibitory effect besides these uses.

  According to the glutathione production promoter of the present invention, through an excellent glutathione production promoting action, for example, the protection against oxidative stress that declines with aging is increased, and the damage to oxidative stress caused by ultraviolet rays is suppressed, and the prevention and improvement of skin aging, Alternatively, pigmentation such as spots can be improved, and a whitening effect can be obtained. However, the glutathione production promoter of the present invention can be used for all purposes other than these uses that are meaningful for exerting a glutathione production promoting action.

  According to the hyaluronic acid production promoter of the present invention, it is possible to produce a large amount of hyaluronic acid that is effective in preventing aging of human skin, prevention and treatment of arthritis, etc. through an excellent hyaluronic acid production promoting action. It can be suitably used for materials. However, the hyaluronic acid production promoter of the present invention can be used for all purposes that are meaningful for exerting hyaluronic acid production promoting action in addition to these uses.

  According to the involucrin production-promoting agent of the present invention, through the excellent involucrin production-promoting action, for example, the formation of CE, which is an insoluble cell membrane-like structure enveloping keratinocytes, is promoted to normalize keratinization. It is possible to prevent and improve various skin symptoms such as dry skin and rough skin, and to prevent and improve skin aging. . However, the involucrin production-promoting agent of the present invention can be used for all uses that are meaningful for exerting an involucrin production-promoting action in addition to these uses.

  The melanin production inhibitor, glutathione production promoter, hyaluronic acid production promoter, and involucrin production promoter of the present invention have an excellent action and are excellent in usability and safety when applied to the skin. Suitable for blending into skin cosmetics.

Further, since the melanin production inhibitor, glutathione production promoter, hyaluronic acid production promoter, and involucrin production promoter of the present invention have excellent actions and are not expected to be digested in the digestive tract, they are used for cosmetic purposes. It is suitable for blending into food and drink or medicine.
Further, since the melanin production inhibitor, glutathione production promoter, hyaluronic acid production promoter, and involucrin production promoter of the present invention have excellent actions, research on the functions of melanin, glutathione, hyaluronic acid, and involucrin, It can be suitably used as a reagent for studying diseases related to melanin, glutathione, hyaluronic acid, and involucrin.

(Skin cosmetic)
The skin cosmetic of the present invention contains at least one of rutinose and alkylrutinose, and further contains components appropriately selected as necessary.
At least one of the rutinose and the alkyl rutinose may be in the form of a melanin production inhibitor, a glutathione production promoter, a hyaluronic acid production promoter, and an involucrin production promoter.
The skin cosmetic includes scalp cosmetics.
There is no restriction | limiting in particular as said alkyl rutinose, Although it can select suitably according to the objective, Methyl rutinoside, ethyl rutinoside, and rutinosyl glycerol are preferable.

The use of the skin cosmetic is not particularly limited and can be appropriately selected from various uses. For example, ointments, creams, emulsions, lotions, packs, jellies, lip balms, lipsticks, bath preparations, astringents, hair tonics. , Hair cream, hair liquid, shampoo, pomade, rinse, and the like.
There is no restriction | limiting in particular as a compounding quantity of at least any one of the said rutinose and an alkyl rutinose, Although it can select suitably according to the objective, It converts into at least any one of the said lutinose and an alkyl rutinose, and is 0. 0.0001% by mass to 10% by mass is preferable, and 0.001% by mass to 1% by mass is more preferable.

Various main agents and auxiliaries usually used in the production of skin cosmetics and other components can be added to the skin cosmetics as needed, as long as the purpose and action and effects of the present invention are not impaired. .
The other components are not particularly limited as long as they do not hinder the melanin production inhibitory action, glutathione production promoting action, hyaluronic acid production promoting action, and involucrin production promoting action of the present invention, and are appropriately selected according to the purpose. For example, whitening agents, astringents, bactericides, antibacterial agents, ultraviolet absorbers, cell activators, fats and oils, waxes, hydrocarbons, fatty acids, alcohols, esters, surfactants, Fragrance, etc.

  The melanin production inhibitor, glutathione production promoter, hyaluronic acid production promoter, involucrin production promoter, and skin cosmetic of the present invention are preferably applied to humans. As long as the effect is exhibited, it can be applied to animals other than humans.

  EXAMPLES Hereinafter, although a manufacture example and an Example are shown and this invention is demonstrated concretely, this invention is not restrict | limited to the following manufacture example and Example. In the following examples, “%” is “mass%” unless otherwise specified.

(Production Example 1: Preparation of crude enzyme solution (rutin-degrading enzyme-containing solution))
After adding 1.5 L of 20 mM acetate buffer (pH 5) to 50 g of tartary buckwheat flour, the mixture was stirred and extracted for 1 hour. 1 The crude enzyme liquid 1.41L was obtained by filtering with a filter paper.

(Production Example 2: Production of rutinose)
Rutin (300 μg) and 20 mM acetate buffer (pH 5) (300 μL) were mixed, and 100 μL of the crude enzyme solution prepared in Production Example 1 was further added to prepare a reaction solution. After holding this reaction solution at 40 ° C. for 3 minutes, 75% acetonitrile was used as a solvent, and it was passed through an Asahipak NH ₂ column (manufactured by Asahi Kasei Co., Ltd.) at a flow rate of 1 mL / min. (Retention time) = A peak was detected at 11 minutes.
When the substance of the peak was subjected to structural analysis by ¹³ C-NMR, it was confirmed to be rutinose. The assignment data for rutinose is shown in Table 1.

(Production Example 3: Production of methylrutinoside)
Rutin (300 μg), methanol (60 μL), and 20 mM acetate buffer (pH 5) (240 μL) were mixed, and 100 μL of the crude enzyme solution prepared in Production Example 1 was further added to prepare a reaction solution. After holding this reaction solution at 40 ° C. for 3 minutes, 75% acetonitrile was used as a solvent, and it was passed through an Asahipak NH ₂ column (manufactured by Asahi Kasei Co., Ltd.) at a flow rate of 1 mL / min. (Retention time) = A peak was detected at 6.77 minutes.
When the substance of the peak was subjected to structural analysis by ¹³ C-NMR, it was confirmed to be methylrutinoside. The assignment data for methylrutinoside is shown in Table 2.

(Production Example 4: Production of ethyl rutinoside)
Rutin (300 μg), ethanol (60 μL), and 20 mM acetate buffer (pH 5) (240 μL) were mixed, and 100 μL of the crude enzyme solution prepared in Production Example 1 was further added to prepare a reaction solution. After holding this reaction solution at 40 ° C. for 3 minutes, 75% acetonitrile was used as a solvent, and it was passed through an Asahipak NH ₂ column (manufactured by Asahi Kasei Co., Ltd.) at a flow rate of 1 mL / min. (Retention time) = A peak was detected at 6.1 minutes.
When the substance of the peak was subjected to structural analysis by ¹³ C-NMR, it was confirmed to be ethylrutinoside.

(Production Example 5: Production of rutinosylglycerin)
Rutin (300 μg), 1,2,3-propanetriol (60 μL), and 20 mM acetate buffer (pH 5) (240 μL) were mixed, and 100 μL of the crude enzyme solution prepared in Production Example 1 was added to prepare a reaction solution. After holding this reaction solution at 40 ° C. for 3 minutes, 75% acetonitrile was used as a solvent, and it was passed through an Asahipak NH ₂ column (manufactured by Asahi Kasei Co., Ltd.) at a flow rate of 1 mL / min. Peaks were detected at (retention time) = 9.6 minutes and 10.6 minutes.
About each substance of said peak, after fractionating by HPLC, when structural analysis was performed by ¹³ C-NMR, a peak at RT = 9.6 minutes was 2-lutinosylglycerin, a peak at RT = 10.6 minutes Was confirmed to be 1-rutinosylglycerin. Table 3 shows the attribution data of 1-lutinosylglycerol and Table 4 shows the attribution data of 2-rutinosylglycerol.
In each test of Examples described later, 1-lutinosylglycerin was used.

(Example 1: Test for inhibiting melanin production)
Using the rutinose of Production Example 1, methylrutinoside of Production Example 2, ethylrutinoside of Production Example 3 and rutinosylglycerin of Production Example 4 as test samples, the melanin production inhibitory action was tested by the following test method.

B16 melanoma cells were cultured at 37 ° C. under 5% CO ₂ using Dulbecco's MEM medium containing 10% by mass FBS, and then cells were collected by trypsin treatment. The collected cells were diluted with Dulbecco's MEM medium containing 10% by mass FBS and 1 mmol / L theophylline to a concentration of 24.0 × 10 ⁴ cells / mL, and then seeded at 300 μL per well in a 48-well plate at 37 ° C. The cells were cultured for 6 hours under 5% CO ₂ . After completion of the culture, 300 μL of a test sample (sample concentration: 100 μg / mL, 200 μg / mL, 400 μg / mL) dissolved in Dulbecco's MEM medium containing 10% by mass FBS and 1 mmol / L theophylline was added to each well, Incubated for 4 days under 5% CO ₂ . After completion of the culture, the medium was removed from each well, 200 μL of 2 mol / L NaOH solution was added, the cells were disrupted with an ultrasonic crusher, and the absorbance at a wavelength of 475 nm was measured to obtain the amount of melanin produced.
In addition, in order to measure the cell viability, after culturing in the same manner, each well was washed with 400 μL PBS (−), and neutral red dissolved in Dulbecco's MEM medium containing 10% by mass FBS at a final concentration of 0.05 mg / mL was added to each hole. 200 μL was added. After culturing at 37 ° C. under 5% CO ₂ for 2.5 hours, the neutral red solution is discarded, and 200 μL of an ethanol / acetic acid solution (ethanol: acetic acid: water = 50: 1: 49) is added to each well. Extracted. After extraction, the absorbance at a wavelength of 540 nm was measured.
As a blank test, cells cultured only in Dulbecco's MEM medium containing 10% by mass FBS and 1 mmol / L theophylline were tested in the same manner.
The results are shown in Table 5.
In addition, the calculation method of a melanin production inhibitory effect and a cell survival rate is as follows.
Melanin production inhibition rate (%) = {1− (B / D) / (A / C)} × 100
Cell viability (%) = (D / C) × 100
In the above formula, A is “absorbance at 475 nm without addition of test sample”, B is “absorbance at 475 nm with addition of test sample”, C is “absorbance at 540 nm without addition of test sample”, and D is “ It represents “absorbance at 540 nm with addition of test sample”.

From the results in Table 5, it was confirmed that rutinose, methylrutinoside, ethylrutinoside, and rutinosylglycerin have a melanin production inhibitory effect. Among these, ethyl rutinoside showed the strongest melanin production inhibitory action.

(Example 2: Glutathione production promotion inhibitory effect test <B16 melanoma cell>)
The glutathione production promoting action was tested by the following test method using the rutinose of Production Example 1, methylrutinoside of Production Example 2, ethylrutinoside of Production Example 3, and rutinosylglycerin of Production Example 4 as test samples.

B16 melanoma cells were cultured at 37 ° C. under 5% CO ₂ using Dulbecco's MEM medium containing 10% by mass FBS, and then cells were collected by trypsin treatment. The collected cells were diluted with Dulbecco's MEM medium containing 10% by mass FBS to a concentration of 10 × 10 ⁴ cells / mL, and then seeded at 200 μL per well in a 48-well plate, and then incubated at 37 ° C. under 5% CO _2. Cultured overnight. After completion of the culture, 200 μL of a test sample (sample concentration: 12.5 μg / mL, 50 μg / mL, 200 μg / mL) dissolved in Dulbecco's MEM medium containing 1% by mass of FBS was added to each well, and the culture was performed at 37 ° C., 5% CO. ₂ and cultured for 24 hours. After completion of the culture, the medium was removed from each well, washed with 400 μL of PBS (−), and cells were lysed using 150 μL of M-PER (registered trademark) (manufactured by PIERCE). The total glutathione was quantified using 100 μL of this.
That is, 100 μL of lysed cell extract, 50 μL of 0.1 M phosphate buffer, 25 μL of 2 mM NADPH, and 25 μL of glutathione reductase (final concentration: 17.5 units / mL) were added to a 96-well plate and heated at 37 ° C. for 10 minutes. Then, 25 mM of 10 mM 5,5′-dithiobis (2-nitrobenzoic acid) was added, and the absorbance (OD) at a wavelength of 412 nm until 5 minutes was measured to obtain ΔOD / min. The total glutathione concentration was calculated based on a calibration curve prepared using oxidized glutathione. The obtained value was corrected to the glutathione amount per total protein amount, and the glutathione production promotion rate was calculated according to the following formula. The results are shown in Table 6.
Glutathione production promotion rate (%) = (F / E) × 100
However, in the above formula, E represents “the amount of glutathione per total protein in the cells to which the test sample is not added (control)”, and F represents “the amount of glutathione per total protein in the cells to which the test sample was added”. .

From the results shown in Table 6, it was confirmed that rutinose, methylrutinoside, ethylrutinoside, and rutinosylglycerin have a glutathione production promoting action. Among these, rutinosylglycerin showed the strongest glutathione production promoting action.

(Example 3: Glutathione production promoting action test <fibroblast>)
The glutathione production promoting action was tested by the following test method using the rutinose of Production Example 1, methylrutinoside of Production Example 2, ethylrutinoside of Production Example 3, and rutinosylglycerin of Production Example 4 as test samples.

Human normal skin fibroblasts (NB1RGB) were cultured at 37 ° C. under 5% CO ₂ using 10% by mass of FBS-containing α-MEM medium, and then cells were collected by trypsin treatment. The collected cells were diluted with α-MEM medium containing 10% by mass of FBS to a concentration of 2.0 × 10 ⁵ cells / mL, and then seeded at 200 μL per well in a 48-well plate at 37 ° C., 5% CO _2. Incubate overnight under. After completion of the culture, 200 μL of a test sample (sample concentration: 12.5 μg / mL, 50 μg / mL, 200 μg / mL) dissolved in 1% by mass of FBS-containing D-MEM medium was added to each well at 37 ° C., 5% They were cultured for 24 hours in CO ₂ under. After completion of the culture, the medium was removed from each well, washed with 400 μL of PBS (−), and cells were lysed using 150 μL of M-PER (registered trademark) (manufactured by PIERCE). Using 100 μL of this, total glutathione was quantified.
That is, 100 μL of the lysed cell extract, 50 μL of 0.1 M phosphate buffer, 25 μL of 2 mM NADPH, and 25 μL of glutathione reductase (final concentration 17.5 units / mL) were added to a 96-well plate and heated at 37 ° C. for 10 minutes. After that, 25 μL of 10 mM 5,5′-dithiobis (2-nitrobenzoic acid) was added, and the absorbance (OD) at a wavelength of 412 nm until 5 minutes was measured to obtain ΔOD / min. The total glutathione concentration was calculated based on a calibration curve prepared using oxidized glutathione. The obtained value was corrected to the amount of glutathione per total protein amount, and the glutathione production promotion rate was calculated according to the following formula. The results are shown in Table 7.
Glutathione production promotion rate (%) = (H / G) × 100
In the above formula, G represents “the amount of glutathione per total protein in a cell to which a test sample is not added (control)”, and H represents “the amount of glutathione per total protein in a cell to which a test sample is added”. .

From the results in Table 7, it was confirmed that rutinose, methylrutinoside, ethylrutinoside, and rutinosylglycerin have a glutathione production promoting action.

(Example 4: Hyaluronic acid production promoting test)
Using methylrutinoside of Production Example 2 as a test sample, hyaluronic acid production promoting action was tested by the following test method.

Human normal neonatal skin epidermal keratinocytes (NHEK) were cultured at 37 ° C. under 5% CO ₂ using human normal neonatal epidermal keratinocyte medium (KGM), and then cells were collected by trypsin treatment. The collected cells were diluted with KGM to a concentration of 1 × 10 ⁵ cells / mL, then seeded at 100 μL per well in a 96-well plate, and cultured at 37 ° C. under 5% CO ₂ for 24 hours. After completion of the culture, 100 μL of a test sample dissolved in KGM (sample concentration: 1.56 μg / mL, 6.25 μg / mL, 25 μg / mL, 100 μg / mL) was added to each well, and at 37 ° C. under 5% CO _2. For 7 days. After completion of the culture, the amount of hyaluronic acid in the medium in each well was measured by a sandwich method using hyaluronic acid binding protein (HABP). The results are shown in Table 8.
In addition, the calculation method of hyaluronic acid production promotion rate and cell viability is as shown in the following formula.
Hyaluronic acid production promotion rate (%) = (I / J) × 100
Cell viability (%) = (K / L) × 100
In the above formula, I is “hyaluronic acid amount when test sample is added”, J is “hyaluronic acid amount when test sample is not added”, K is “absorbance at 540 nm in cells to which test sample is added”, L Represents “absorbance at 540 nm in cells to which no test sample was added”.

From the results in Table 8, it was confirmed that methylrutinoside has a significant hyaluronic acid production promoting effect depending on the concentration.

(Example 5: Involucrin production promoting action test)
Using methylrutinoside of Production Example 2, ethylrutinoside of Production Example 3 and rutinosylglycerin of Production Example 4 as test samples, the involucrin production promoting effect was tested by the following test method.

Human normal neonatal skin epidermal keratinocytes (NHEK) were cultured at 37 ° C. under 5% CO ₂ using human normal neonatal epidermal keratinocyte medium (KGM), and then cells were collected by trypsin treatment. The collected cells were diluted with KGM to a concentration of 1 × 10 ⁵ cells / mL, then seeded in a 48-well plate at 200 μL per well, and cultured overnight at 37 ° C. and 5% CO ₂ . After completion of the culture, the medium was removed, and 200 μL of a test sample (sample concentration: 12.5 μg / mL, 50 μg / mL) dissolved in KGM was added to each well and cultured at 37 ° C. under 5% CO ₂ for 48 hours. . After completion of the culture, the medium was removed, the cells were fixed to the plate, and the amount of involucrin expressed on the cell surface was measured by ELISA using a monoclonal anti-human involucrin antibody (manufactured by SIGMA) (Plate reader made by Bio-Tech Instruments). . From the obtained measurement results, the involucrin production promotion rate (%) was calculated by the following formula. The results are shown in Table 9.
Involucrin production promotion rate (%) = (M / N) × 100
In the above formula, M represents “absorbance at a wavelength of 405 nm when a test sample is added”, and N represents “absorbance at a wavelength of 405 nm when no test sample is added (control)”.

From the results of Table 9, it was confirmed that methylrutinoside, ethylrutinoside, and rutinosylglycerin have an involucrin production promoting action.

(Formulation example 1)
-Hair tonic-
A hair tonic having a hair growth action having the following composition was produced by a conventional method.
・ Pyridoxine hydrochloride ... 0.1g
・ Resorcin ・ ・ ・ 0.01g
・ D-pantothenyl alcohol ... 0.1g
・ Dipotassium glycyrrhizinate ... 0.1g
・ L-Menthol 0.05g
・ 1,3-Butylene glycol ・ ・ ・ 4.0g
・ Assembly extract ... 0.1g
・ Carrot extract ... 0.1g
・ Cudin extract ... 0.1g
・ Methylrutinoside (Production Example 3) 0.2g
-Fragrance-appropriate amount-Purified water-the balance (total: 100.00 g)

The melanin production inhibitor, glutathione production promoter, hyaluronic acid production promoter, and involucrin production promoter of the present invention have an excellent melanin production inhibitory effect, glutathione production promoting action, hyaluronic acid production promoting action, and involucrin production promoting action. Since it has, it can utilize suitably as skin cosmetics, cosmetics and drinks, medicine, and a research reagent.

Claims (9)

  A melanin production inhibitor containing at least one of rutinose and alkylrutinose.   A melanin production inhibitor comprising at least one of rutinose, methylrutinoside, ethylrutinoside, and rutinosylglycerin.   A glutathione production promoter comprising at least one of rutinose and alkylrutinose.   A glutathione production promoter containing at least one of rutinose, methylrutinoside, ethylrutinoside, and rutinosylglycerin.   Hyaluronic acid production promoter characterized by containing methylrutinoside.   An involucrin production promoter comprising an alkyl rutinose.   An involucrin production promoter comprising at least one of methylrutinoside, ethylrutinoside, and rutinosylglycerin.   A skin cosmetic comprising at least one of rutinose and alkylrutinose. A skin cosmetic comprising at least one of rutinose, methylrutinoside, ethylrutinoside, and rutinosylglycerin.