JP6802537B1 - Dopaquinone derivatives exhibiting hemidesmosome activating action and methods for producing them - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a dopaquinone derivative exhibiting a hemidesmosome activating action and a method for producing the same. A target dopaquinone derivative comprises dopaquinone and a monocyclic carbon compound. This derivative exhibits a hemidesmosome-activating effect on cells. The hemidesmosome activating action of human hair pigment cells increases the black pigment of hair and can be used for gray hair dyeing. It also increases skin keratin. The production method is to cultivate and acclimatize human skin keratinocytes using a fermented solution obtained by fermenting grape buds and rice bran with natto and Benikouji as a medium, and further cultivate and acclimatize human skin fibroblasts. It consists of a step of purifying the acclimatized culture medium. This dopaquinone derivative can be used in health foods, cosmetics such as gray hair dyes, pharmaceuticals, quasi-drugs, and plant activators. [Selection diagram] None

Description

The present invention relates to a dopaquinone derivative exhibiting a hemidesmosome activating action and a method for producing the same.

The white hair of human hair is a morphological change caused by a decrease in pigment cells of hair follicles and a decrease in activity due to aging. Increased gray hair is also associated with hair fragility.

The pigment cells in the hair are located around the hair matrix cells and supply the pigment to the hair, so they are strongly related to the color of the hair. It has been found that intercellular adhesion factors are involved in supplying pigments to hair. Cell-cell adhesion factors include adhesion junctions, tight junctions, desmosomes and hemidesmosomes.

The relationship between pigment cells in human hair and gray hair was studied, and it was found that the decrease in hemidesmosome in the pigment cells was associated with the decrease in pigment cells. Therefore, inventions relating to the activation of hemidesmosome have been made.

Inventions relating to hemidesmosomes include, for example, inventions of cell adhesion stimulated by a cell matrix and composition of hemidesmosomes, which are biocompatible molded articles suitable for use in the living body of mammals and on the molded articles. A manufactured article containing a hemidesmosome formation-promoting protein composition is shown (see, for example, Patent Document 1).

Further, as another invention, in an artificial tooth root for attaching the artificial tooth root body to the alveolar portion of the jaw bone, a flange portion is provided around the base end edge of the artificial tooth root body in contact with the opening edge end surface of the bone hole. There is an invention of an artificial tooth root characterized by the fact that the function of hemidesmosome in an artificial tooth root is mentioned (see, for example, Patent Document 2).

However, the above invention does not show a specific structure for activation of intracellular hemidesmosome.

Although a natural product having few side effects that exerts a hemidesmosome activating effect is desired, there is no invention regarding a useful substance. Moreover, no invention regarding a specific manufacturing method has been reported.

Japanese Patent Application No. 6-522472 Japanese Patent Application No. 3-8559

As described above, there is no invention relating to the hemidesmosome activating action of existing natural products and the invention relating to the method for producing the same, and there is a problem that industrial use is limited. In addition, chemically synthesized substances have safety problems and their use is limited.

Therefore, a substance exhibiting an excellent hemidesmosome activating action with weak side effects and a method for producing the same are desired.

In order to achieve the above object, the invention according to claim 1 relates to a dopaquinone derivative exhibiting a hemidesmosome activating action represented by the following formula (1).

Further, in order to achieve the above object, the invention according to claim 2 relates to a method for producing a dopaquinone derivative exhibiting a hemidesmosome activating action represented by the formula (1).

Since the present invention is configured as described above, the following effects are obtained.

According to the derivative according to claim 1, an excellent hemidesmosome activating action can be exhibited.

According to the method for producing a derivative according to claim 2, a dopaquinone derivative exhibiting a hemidesmosome activating action can be efficiently produced.

Hereinafter, embodiments embodying the present invention will be described in detail.

First, a dopaquinone derivative (hereinafter referred to as a dopaquinone derivative) exhibiting a hemidesmosome activating action represented by the following formula (1) is composed of 24 carbon elements, 33 hydrogen elements, 4 oxygen elements and 1 nitrogen element. Has been done.

That is, it is the chemical formula of C24H33O4N1 and has a molecular weight of about 399.5.

This dopaquinone derivative consists of one molecule of dopaquinone and one molecule of monocyclic carbon compound. The bond between the two molecules is an ester bond, and the carboxyl group of the dopaquinone side chain and the hydroxyl group of the monocyclic carbon compound are ester-bonded.

Since this dopaquinone derivative has few hydroxyl groups, it has low solubility in water, while it is oil-soluble. Since hemidesmosome is bound to the cell membrane, high lipophilicity is preferable because it is easy to approach the vicinity of hemidesmosome and the action is direct.

There are two types of functional groups in this dopaquinone derivative, which are the functions of dopaquinone and a monocyclic carbon compound. Dopaquinone has an excellent antioxidant and reducing action due to the quinone group, and this reducing action stabilizes the construction of hemidesmosome. Hemidesmosome has a three-dimensional structure in which multiple proteins and cell membranes are bound to each other. The reactivity between the cell membrane and the protein is disrupted by oxidation, and the hemidesmosome is disrupted. The reducing action of this dopaquinone derivative stabilizes the cell membrane and protein binding, and also stabilizes the hemidesmosome.

Since the carbon chains of the monoelemental ring carbon compound are arranged three-dimensionally, they enter the cell membrane inside the cell, exhibit a lining action, and stabilize the cell. There are many sebaceous glands, and the oxides of sebum damage cells. The monocyclic carbon compound exhibits a protective action against this oxidative damage.

In this dopaquinone derivative, hemidesmosome is stabilized and activated by the synergistic action of two kinds of functional groups. The action of hemidesmosome increases pigment production in the pigment cell and enhances pigment secretion. In addition, it strengthens the connections between cells and enhances the function and structure of the body. In addition, it enhances the electrical transmission of nerve cells and enhances nerve activity. It has basic functions such as strengthening the blood vessel wall and preventing bleeding.

In the case of pigments, the pigments contain secretions such as proteins and keratins in addition to melanin, and these pigments constitute the color of hair. The increase in pigment will increase the color of the hair. Since the proliferation of pigment cells is delayed by aging and the pigment secretion is reduced, this dopaquinone derivative has a function of promoting pigment secretion from the hair pigment cells of the elderly to darken the color of the hair.

Since this dopaquinone derivative exhibits both lipophilic and water-soluble properties, it can act on both the cell membrane and the cytoplasm, and in terms of affinity with the solvent, it becomes compatible with the solvent of cosmetics and foods, and the solubility is enhanced. Is preferable. In addition, it is also excellent in absorption into the body, and when it is used as a food, it is absorbed into the body from the intestinal tract through the cell membrane of intestinal villous cells. From the skin and scalp, it dissolves in sebum and penetrates into the sebaceous glands, where it is absorbed by the hair follicle tissue. As described above, it is preferable that the dopaquinone derivative has high absorbability.

This derivative itself is decomposed into dopaquinone and a monocyclic carbon compound in the cell, and these substances are highly safe, and as a result, the safety of this dopaquinone derivative is also high, which is preferable. This dopaquinone can be organically synthesized using dopaquinone and a monocyclic carbon compound as raw materials. This organically synthesized derivative is used for analysis and analysis as a standard substance. However, there is a safety drawback that it is difficult to use in industry because chemical production is costly and harmful organic solvents and heavy metals are used.

Regarding the structure of this dopaquinone derivative, the peak positions were 1.63, 1.64, 1 by 600 MHz H-NMR (1H-NMR) analysis (manufactured by Bruker) in deuterated chloroform of the chemically synthesized derivative. .70, 1.71, 1.99, 2.02, 2.06, 2.08, 2.11, 2.13, 2.14, 2.15, 4.05, 4.47, 4.75 It is found at 5.17, 5.18, 5.60, 6.11, 6.87 and 7.29 ppm.

In addition, in C-NMR (13C-NMR) analysis of chemically synthesized derivatives in deuterated chloroform, the peak positions were 16.1, 16.5, 17.9, 25.9, 26.4, 26.9, 39.9, 40.0, 43.6, 60.5, 70.6, 105.7, 119.8, 124.5, 125.4, 127.7, 129.1, 132. It is observed at 6, 136.8, 142.2, 145.5, 148.8, 152.4 and 172.7 ppm.

Since this dopaquinone derivative is naturally derived, it is highly safe and has excellent utility for hair cells, hair root cells, skin cells, nerve cells, vascular cells, cardiomyocytes and the like. If a large amount of this derivative is ingested, it is highly safe because it is decomposed in the living body.

Furthermore, this derivative produces hydrogen gas when powdered and reacted with an aqueous solution. Since the generated hydrogen gas has a function of removing active oxygen, it is preferable because it can remove active oxygen generated by ultraviolet rays and oxidizing substances to maintain a stable living body. Further, hydrogen gas is preferable because it scavenges hydroxyl radicals, exhibits a reducing action, and exerts an antioxidant action.

For example, when this conductor acts on skin epidermal cells, it proliferates skin cells and increases keratin. This dopaquinone derivative activates keratin synthase through hemidesmosome activation and increases the amount of keratin. Keratin is preferable because it strengthens the skin and hair.

Further, this derivative also has a function of suppressing the production of prostaglandins and kinins which are inflammatory substances by a reducing action, and it is preferable to have an anti-inflammatory action.

Methods for producing this dopaquinone derivative include a fermentation method, an enzymatic reaction method, and a chemical synthesis method. For example, as a method for producing this derivative, it can be extracted from grape buds, rice bran and the like. It can also be extracted from various plants. Further, in this extraction method, it is preferable to use a digestive enzyme such as protease or lipase because the extraction efficiency is enhanced.

In particular, it is preferable that the dopaquinone derivative is synthesized intracellularly and secreted extracellularly by culturing keratinocytes and fibroblasts derived from human skin together with fermented grape bud extract.

A fermentation method in which plants such as grape buds, maki berries, turmeric, asai palm, roses, and lavender are fermented with Bacillus natto and Monascus purpureus is also useful as a method for producing this dopaquinone derivative. These fermentation techniques are preferable because they have abundant knowledge in Japan, have a lot of achievements as food, and have high safety.

Further, it is preferable to purify for the purpose of obtaining a high-purity derivative. As a purification method, it is preferable to use a purification operation of extracting with a separation solvent using a separation resin.

It is preferable that this dopaquinone derivative exhibits an excellent hemidesmosome activating effect and is used in cosmetics. In addition, since it exhibits a keratin-increasing effect on skin cells, it can also be used as a basic cosmetic, shampoo, eyelash growth agent, hair restorer, and hair cosmetic. For example, it is preferable to exhibit a growth factor action on human keratinocytes and fibroblasts.

It is preferable to add the extract to the dopaquinone derivative and then disperse it in the fat and oil because the obtained active moiety is stably maintained in the fat and oil. For example, extraction with soybean oil, rice bran oil, grape seed oil, olive oil, jojoba oil is preferable. This derivative is soluble in both water-soluble and oil-soluble solvents. This amphipathic property is preferred as it broadens the use of this derivative.

It is used as a pharmaceutical product as an injection or an oral preparation or a parenteral preparation such as a coating agent, and as a quasi-drug, it is blended in hair growth agents, tablets, capsules, drinks, soaps, coating agents, gel agents, toothpaste, etc. Will be used. In particular, since it is resistant to gastric acid, its contents are protected against gastric acid, so that it is used for enteric-coated preparations.

Examples of the oral preparation include tablets, capsules, powders, syrups, drinks and the like. When mixed with the above tablets and capsules, it can be used together with binders, excipients, leavening agents, leavening agents, sweeteners, flavoring agents and the like. The above tablets can also be coated with shellac or sugar.

Further, in the case of the above-mentioned capsule, the above-mentioned material can further contain a liquid extract carrier. In the case of the above syrups and drinks, sweeteners, preservatives, pigment flavors and the like can be added.

Examples of parenteral preparations include injections in addition to external preparations such as ointments, creams and liquids. Vaseline, paraffin, oils and fats, lanolin, macrogold and the like are used as the base material of the external preparation, and can be an ointment, a cream or the like for introducing an extract by a usual method.

Injections include liquids and lyophilizers. When used, it is aseptically dissolved in distilled water for injection, physiological saline, or the like.

As a food preparation, it is used for foods for the purpose of activating hemidesmosome, health foods for the purpose of activating hemidesmosome, and foods for protecting the skin. In addition, it is preferable to use it as a food with a functional health such as a food with a nutritional function or a food for specified health use.

When the obtained food preparation is used for pets such as dogs and cats and livestock animals, it is used as a feed or a supplement for pets for the purpose of improving hair color and hair gloss.

As a cosmetic, it can be used together with a surfactant, a solvent, a thickener, an excipient and the like according to a conventional method. For example, it can be in the form of cream, hair gel, facial cleanser, beauty essence, lotion, etc., and is a cosmetic that exhibits hemidesmosome activating action and keratin production. The form of the cosmetic is arbitrary and can be used as a solution, a cream, a paste, a gel, a gel, a solid or a powder. This derivative is soluble in both water-soluble and oil-soluble solvents. This amphipathic property is preferred as it broadens the use of this derivative.

In addition, this derivative is also used as a plant activator utilizing the hemidesmosome activating action. That is, this derivative activates the growth of plants by the action of strengthening the adhesion of plant cells, resulting in flowering, fruiting, and an increase in yield. For example, it is preferable to bind a plant vitalizing agent of HB-101 (manufactured by Flora Co., Ltd.) to this derivative because the function of promoting plant growth is enhanced, maintained, and stabilized.

Below, human skin fibroblasts are further cultured in a medium obtained by culturing and acclimatizing human skin keratinocytes using a fermented solution obtained by fermenting grape buds and rice bran with natto and Benikouji. A method for producing a dopaquinone derivative exhibiting a hemidesmosome activating action represented by the formula (1), which comprises a step of purifying the acclimated culture medium to be acclimatized, will be described.

The raw materials used in this production process are grape buds, rice bran, natto bacteria, Benikouji bacteria, human skin keratinocytes, and human skin fibroblasts. The production process is a combination of fermentation and culture, and finally the desired dopaquinone derivative is obtained by purifying the conditioned culture solution. The target dopaquinone derivative is a substance consisting of one molecule of dopaquinone represented by the formula (1) and one molecule of a monocyclic carbon compound.

The raw material grape bud is a fresh bud portion obtained by germinating the seeds of grape (scientific name: Vitaceae). Grape buds are used as raw materials for cosmetics and health foods. The grape buds used may be produced in Japan, Asia, Europe or the United States, and grapes cultivated without pesticides in Japan are particularly preferable because they are fresh and highly safe. It is preferably dried and pulverized, and autoclaved sterilized prior to fermentation is preferred because it facilitates fermentation. A powder having a particle size of 3 micrometers or less is preferable because it facilitates the fermentation process.

In addition, the rice that is the source of rice bran, which is the raw material, is preferably not a genetically modified organism because it is suitable for the Japanese food culture. The production area may be any production area such as Japanese or American. Rice bran is the exodermis / embryo part of rice, and autoclave sterilization before fermentation is preferable because it facilitates fermentation. A powder having a particle size of 3 micrometers or less is preferable because it facilitates the fermentation process.

Of these, rice bran cultivated organically or without pesticides is more preferable because it does not contain harmful pesticides or metals.

When using these raw materials, Nara Kikai Seisakusho Co., Ltd.'s free mill, super free mill, sample mill, goblin, super clean mill, micros, small vacuum dryer manufactured by Toyo Riko Co., Ltd., and Matsui Co., Ltd. It is dried and crushed by a small decompression heat transfer dryer DPTH-40, a crusher such as Clean Dry VD-7, VD-20 manufactured by AKUM Kyushu Technos Co., Ltd., and DM-6 manufactured by Nakayama Institute of Technology. As a result, the fermentation process is likely to proceed efficiently.

The natto bacterium used is the scientific name Bacillus subtilis, which is widely used in the production of natto and food processing in Japan, and is a useful edible bacterium with abundant eating experience. Bacterial species native to Japan such as Okinawa and Kagoshima, and Southeast Asia of China and Taiwan are used. Of these, natto bacteria manufactured by Natto Motoho are preferable because they exhibit high fermentability.

The amount of each of the above fermentation additions is preferably 0.4 to 20% by weight for rice bran and 0.002 to 0.2% by weight for Bacillus natto with respect to 1 weight of grape bud. Pre-culturing Bacillus natto before fermentation is preferable because it shortens the initial fermentation time and shortens the fermentation time.

The fermentation is carried out in a clean culture tank and it is preferred to mix the ingredients with sterilized tap water. Further, the fermented product is carried out and produced by the following steps.

The fermentation step may be either a static method or a stirring method, but the stirring method is preferable because the fermentation can be carried out in a short time. Fermentation is preferably carried out at 40-44 ° C. for 24 to 72 hours. This fermentation of Bacillus natto decomposes dietary fiber and protein, which are the raw materials for fermentation, to reduce the molecular weight.

Next, the above-mentioned Bacillus natto fermented liquid is then fermented by Monascus purpureus. The Monascus purpureus used is a filamentous fungus with the scientific name Monascus purpureus, which is a useful edible bacterium used in food processing in Japan and Asia. The Monascus purpureus used is preferably derived from Japan because of its high fermentation ability and quality, and in particular, the Monascus purpureus manufactured by Benikoji Honpo Co., Ltd. is preferable because it has abundant usage records and good quality.

The amount of each addition for the above fermentation is preferably 0.002 to 0.1 weight by weight of Monascus purpureus with respect to 1 weight of the above natto bacteria fermentation broth. It is preferable to pre-culture the Monascus purpureus before fermentation because it shortens the initial fermentation time and shortens the fermentation time.

The above fermentation may be carried out by either a standing method or a stirring method in a clean culture tank, but the stirring method is preferable because the fermentation can be carried out in a short time. Fermentation is preferably carried out at 38-45 ° C. for 24 to 96 hours. A substitution reaction occurs due to the ester enzyme reaction generated in the fermentation of Monascus purpureus.

The filtrate filtered through a filter cloth or the like is autoclaved and used as a medium for cell culture in the following culture steps.

The above fermentation broth contains proteins, lipids, carbohydrates, vitamins and minerals, has a neutral pH, and is suitable for cell culture. This is used as a medium. However, it is necessary to remove bacterial contamination caused by contamination with Bacillus natto and Monascus purpureus.

The above culture solution is placed in a sterilization tube, and about 1% of collagenase is added thereto to sterilize by filtration. This is used as a cell exfoliating solution. Sterilize human facial skin, which has been confirmed to be free of viral infections such as HIV, with rubbing alcohol. Immerse the above collagenase solution in the facial skin at room temperature for about 5 minutes. Collagenase separates the epidermis and dermis layer.

The epidermis and dermis layers are exfoliated by collagenase and further separated into single cells. The cells are suspended in the above medium to reduce the activity of collagenase. The cells are dispersed and only a single cell is transferred into a sterile centrifuge tube by a cell strainer.

This is centrifuged at 1500 rpm for 5 minutes to centrifuge the single cells, the medium is added thereto, the cells are dispersed again, and the cells are replaced with fresh medium by centrifugation. The cell suspension is transferred to a culture dish coated with an anti-keratin antibody and adhered under 5% carbon dioxide at 37 ° C. for 1 day.

Since keratin is present on the surface of skin epidermal cells, the adhered cells are keratinocytes. Fresh medium is added to the petri dish to which the obtained cells and the medium are attached separately, and the cells are further cultured at 37 ° C. under 5% carbon dioxide gas to proliferate the keratinocytes.

On the other hand, since fibroblasts remain in the culture solution of the supernatant, they are transferred to a culture dish coated with an anti-fibronectin antibody and adhered at 37 ° C. for 1 day under 5% carbon dioxide gas. Adhesion to anti-fibronectin antibody is peculiar to fibroblasts, and this adhesion gives skin fibroblasts. The attached fibroblasts are washed with fresh medium to proliferate the fibroblasts.

Next, the production of the cell conditioned culture solution will be described. The keratinocytes obtained by the above cell step are seeded in a sterile petri dish together with fresh medium. The number of cells is about 10,000, and the medium is about 5 mL. This is cultured under 5% carbon dioxide gas at 37 ° C. for 7 days. The number of days of culture is before becoming confluent. After culturing, the supernatant is collected and sterilized by filtration to prepare a keratinocyte conditioned culture solution.

Next, skin fibroblasts are cultured using the keratinocyte conditioned medium as a medium. 10000 cells are seeded in a sterile petri dish together with 5 mL of the keratinocyte conditioned medium and cultured at 37 ° C. for 7 days under 5% carbon dioxide. After culturing, the supernatant is collected and sterilized by filtration to prepare a keratinocyte and fibroblast conditioned culture solution.

The desired dopaquinone derivative is separated from the above keratinocyte and fibroblast conditioned culture medium by a purification method and purified. As a purification method, a purification operation of extracting with a separation solvent using a separation resin is used.

For example, it is preferable that the material is separated by a separation carrier or a resin and separated. As the separation carrier or resin, a porous dopaquinone, a silicon oxide compound, polyacrylamide, polystyrene, polypropylene, a styrene-vinylbenzene copolymer or the like whose surface is coated as described later is used. Those having a particle size of 0.1 to 300 μm are preferable, and the finer the particle size, the higher the accuracy of separation, but there is a drawback that the separation time is long.

For example, a reversed-phase carrier or a resin whose surface is coated with a hydrophobic compound is used for separating highly hydrophobic substances. Those coated with a cationic substance are suitable for separating anionically charged substances. In addition, those coated with an anionic substance are suitable for separating cationically charged substances. When coated with a specific antibody, it is used as an affinity carrier or resin that separates only specific substances.

The affinity carrier or resin is utilized for the specific preparation of the antigen by utilizing the antigen-antibody reaction. Distributable carriers or resins, such as silica gel (manufactured by Merck & Co., Inc.), are used for isolation of substances when there is a difference in partition coefficient between the substance and the solvent for separation.

Of these, an adsorptive carrier or resin, a distributable carrier or resin, a molecular sieve carrier or resin, and an ion exchange carrier or resin are preferable from the viewpoint of reducing the production cost. Further, a reversed-phase carrier or resin and a distributable carrier or resin are more preferable because the difference in partition coefficient with respect to the separation solvent is large.

When an organic solvent is used as the separation solvent, a carrier or resin resistant to the organic solvent is used. In addition, carriers or resins used for pharmaceutical production or food production are preferable. From these points, Diaion (manufactured by Mitsubishi Chemical Corporation, HP-20 and HP-21) and XAD-2 or XAD-4 (manufactured by Roam and Haas) are used as adsorptive carriers, and Sephadex LH is used as a carrier for molecular sieves. More preferably, -20 (manufactured by Amasham Pharmacia), silica gel as a distribution carrier, IRA-410 (manufactured by Roam & Haas) as an ion exchange carrier, and DM1020T (manufactured by Fuji Silicia) as a reverse phase carrier.

Of these, Diaion HP-20, Sephadex LH-20 and DM1020T are even more preferred.

The resulting extract is dissolved in a separation carrier or solvent for swelling the resin prior to separation. The amount thereof is preferably 1 to 40 times, more preferably 4 to 20 times the weight of the extract from the viewpoint of separation efficiency. The separation temperature is preferably 4 to 30 ° C., more preferably 10 to 25 ° C. from the viewpoint of substance stability.

As the separation solvent, water, a lower alcohol containing water, a hydrophilic solvent, or a lipophilic solvent is used. As the lower alcohol, methanol, ethanol, propanol and butanol are used, but ethanol used for food is preferable.

When Sephadex LH-20 is used, a lower alcohol is preferable as the separation solvent. When silica gel is used, chloroform, methanol, acetic acid or a mixture thereof is preferable as the separation solvent.

When Diaion HP-20 and DM1020T are used, the separation solvent is preferably a lower alcohol such as methanol or ethanol or a mixed solution of lower alcohol and water. Further, it is preferable to collect a fraction containing activity and remove the solvent by drying or vacuum drying to obtain a powder or a concentrated solution because the influence of the solvent can be excluded.

It is preferable to collect a fraction containing the dopaquinone derivative and remove the solvent by drying or vacuum drying to obtain the desired dopaquinone derivative as a powder or a concentrated solution because the influence of the solvent can be excluded.

Further, it is preferable to powder the dopaquinone derivative for the purpose of antiseptic.

Hereinafter, the above embodiment will be specifically described with reference to Examples and Test Examples. These are just examples, and it is possible to change the conditions within the range of common sense according to the difference in the material, raw material, and sample.

Grape seeds (produced in Japan and Nagano prefecture) purchased from Sakata seeds (Kanagawa prefecture) were used. The seeds were washed with tap water and then sown on water-soaked gauze placed in a constant temperature bath at 37 to 39 ° C. This was cultured for 7 days and germinated. The germinated grape buds were crushed with a crusher (Super Free Mill manufactured by Nara Kikai Seisakusho Co., Ltd.) to obtain 500 g of a crushed product.

In addition, rice bran produced in Aichi prefecture was purchased from JA Aichi and used. After washing, this was crushed and 500 g was used for fermentation. These ground pieces were autoclaved.

These were placed in a clean fermentation tank (sterilized round 40-liter tank for fermentation), 10 kg of autoclave-sterilized tap water was added, and the mixture was stirred.

5 g of natto bacteria manufactured by Natto Motopo was added and fermented. The fermentation temperature was 39 to 42 ° C., and fermentation was carried out for 40 hours. Fermentation was controlled by measuring changes in protein by a protein quantification method.

After fermentation, the fermentation broth was filtered to collect 8.7 kg of filtrate. This fermented liquor was fermented by Monascus purpureus. That is, 8 g of a preculture solution of Monascus purpureus (manufactured by Benikoji Honpo Co., Ltd.) was added to 8 kg of the above fermentation solution to ferment. The fermentation temperature was 39-40 ° C. and fermentation was carried out for 38 hours. In this fermentation as well, changes in protein were measured and controlled by the protein quantification method.

After fermentation, the obtained fermentation broth was heated and boiled at 95 ° C. to kill the bacteria. After cooling this, the filtrate was collected by filtration. The filtrate was filtered under reduced pressure with a sterilized membrane filter to collect the fermentation broth. 4.4 kg of the fermentation broth was collected and used as the following medium for cell culture.

As a quality test, it was inspected in advance that it contained proteins, carbohydrates, lipids, vitamins and minerals and that there was no contamination with bacteria, and those with no abnormalities were used as a medium for the following culture operations.

After autoclaving the above medium, it was cooled and placed in a sterilization tube (Falcon, 15 mL), about 1% of collagenase (Nitta Gelatin) was added thereto, and this was sterilized by filtration. This was used as a cell exfoliating solution. The facial skin of a human (50 years old, female) who had been confirmed to be free of viral infections such as HIV was sterilized with disinfectant ethanol.

The cell exfoliating solution consisting of the above collagenase was immersed in the facial skin at room temperature for about 5 minutes. It was confirmed that the epidermis and the dermis layer were separated by collagenase. The isolate was collected in sterile tubes and dispersed into single cells.

After suspending the collected cells in the above medium to reduce the activity of collagenase, the dispersed cells are dispersed, filtered by a cell strainer (manufactured by Falcon), and only the cells are sterilized in a centrifuge tube (manufactured by Falcon). ).

The cells were centrifuged by centrifugation at 1500 rpm for 5 minutes, the above medium for cell culture was added thereto, and the cells were dispersed again and replaced with fresh medium by centrifugation. This cell suspension was transferred to a culture dish (Falcon) coated with an anti-keratin antibody (human type, monoclonal antibody, manufactured by Cosmo Bio, product number NM002276) and cultured at 37 ° C. for 1 day under 5% carbon dioxide gas. ..

The obtained cells and the supernatant were separated. Fresh medium was added to the petri dish to which the cells were attached, and the cells were further cultured at 37 ° C. under 5% carbon dioxide gas to proliferate the keratinocytes.

The supernatant of the remaining culture was transferred to a culture dish coated with an anti-fibronectin antibody (human type, monoclonal antibody, manufactured by Takara Bio, product number M001) for collecting fibroblasts, and at 37 ° C. under 5% carbon dioxide gas. Incubated for 1 day. After washing the attached fibroblasts, they were detached with a 1% trypsin solution and used as fibroblasts in the following cultures.

First, the keratinocytes obtained by the above cell collection step were seeded in a sterile petri dish together with a fresh medium. The number of cells was 10,000, and the medium was about 5 mL. This was cultured under 5% carbon dioxide gas at 37 ° C. for 7 days. The state of the culture was observed under a microscope, and the cells were collected before becoming confluent. Only this supernatant was collected and sterilized by filtration to prepare a keratinocyte conditioned culture solution.

Next, the skin fibroblasts collected by the above anti-fibronectin antibody method were cultured. 10000 fibroblasts were seeded in a sterile petri dish together with 5 mL of the above-mentioned keratinocyte conditioned culture solution and cultured at 37 ° C. for 7 days under 5% carbon dioxide gas. After culturing, before becoming confluent, the supernatant was collected and sterilized by filtration to prepare a keratinocyte and fibroblast conditioned culture solution.

The above keratinocyte and fibroblast conditioned culture solution was freeze-dried with a freeze-dryer (Freeze Trap VA-140S manufactured by Titec) to obtain 20.9 g of the target powder.

20 g of the obtained powder was suspended in 100 mL of purified water and subjected to 500 g of Diaion HP-20 (manufactured by Mitsubishi Chemical Corporation) swollen with 4% ethanol. After washing with 1900 mL of 4% ethanol, further washing with 1000 mL of 10% ethanol.

800 mL of 60% ethanol was added thereto, and the desired dopaquinone derivative was fractionated. This purification operation was carried out three times to obtain the final purified product. The obtained final purified product was dried under a vacuum dryer to obtain 0.4 g of powder. This was designated as sample 1.

The test methods and results for structural analysis of dopaquinone derivatives will be described below.
(Test Example 1)

The sample 1 obtained as described above was dissolved in purified water, filtered, and then analyzed by high performance liquid chromatography (HPLC, Shimadzu Corporation).

Further, the analysis was performed in deuterated chloroform with a 600 MHz nuclear magnetic resonance apparatus (NMR, manufactured by Bruker). As a result of structural analysis, a dopaquinone derivative was identified from Specimen 1. That is, a structure in which one molecule of dopaquinone and one molecule of a monocyclic carbon compound were ester-bonded was analyzed.

According to the H-NHR analysis results, 1.63, 1.64, 1.70, 1.71, 1.99, 2.02, 2.06, 2.08, 2.11, 2.13, 2.14 , 2.15, 4.05, 4.47, 4.75, 5.17, 5.18, 5.60, 6.11, 6.87 and 7.29 ppm.

Furthermore, according to the C-NMR analysis results, 16.1, 16.5, 17.9, 25.9, 26.4, 26.9, 39.9, 40.0, 43.6, 60.5, 70 6.6, 105.7, 119.8, 124.5, 125.4, 127.7, 129.1, 132.6, 136.8, 142.2, 145.5, 148.8, 152.4 And a peak was observed at 172.7 ppm.

The chart of the analysis result of C-NMR is shown below. (The horizontal axis unit is ppm, and the vertical axis unit is peak intensity.)

The above analytical values were the same as the peaks of the organically chemically synthesized dopaquinone derivative, and were identified as the desired dopaquinone derivative. In addition to the above, analysis by thin layer chromatography and the like revealed that the purity of this derivative contained in sample 1 was 99.1%.

Further, when 0.1 g of the obtained sample 1 powder was dissolved in 10 mL of purified water, generation of hydrogen gas was observed. As a result of quantification by gas chromatography (PDD high-sensitivity analysis system manufactured by Shimadzu Corporation), a hydrogen gas concentration of 1.6 ppm was detected.

The test results on hemidesmosome production using human hair pigment cells are shown below.
(Test Example 2)

Hair was collected from the head of a healthy human (50 years old, female), and hair matrix cells were collected from the hair by the collagenase cell dispersion method. That is, after washing the hair, a 1% collagenase solution was added thereto, the hair was collected, and the hair root portion was cut. The tip portion was dispersed with a 1% collagenase solution to collect cells. This was used as a hair root cell.

Pigment-containing pigment cells were collected from these hair root cells. As the medium, the above-mentioned medium was used. The hair root cell suspension was seeded in a sterile petri dish coated with an anti-melanin antibody (monoclonal antibody, manufactured by Funakoshi, product number ACR423BK).

This was cultured under 5% carbon dioxide gas at 37 ° C. for 1 day. After 1 day, the medium was removed and fresh medium was added to the adhered cells. Under a microscope, the presence or absence of pigment was confirmed to confirm that the cells were hair-derived pigment cells. The positive rate of pigment content in the cells was 92%. These were designated as pigment cells.

The above pigment cells were suspended in a medium to prepare the number of cells to 3000. These 3000 pigment cells were cultured with 3 mL of medium in a culture dish (manufactured by Falcon) under 5% carbon dioxide gas at 37 ° C. for 1 day.

After 1 day, only medium was added as sample 1 or solvent control at a concentration of 10%, and the cells were further cultured for 3 days. Three days later, cells were collected in 1% trypsin solution and the live cell counts were counted under a microscope by the trypan blue dye exclusion method.

Further, the cell suspension was dispersed in a phosphate buffer solution and crushed by ultrasonic waves to prepare a cell suspension. The amount of pigment contained in these cells was quantified by the ELISA method using an anti-melanin antibody. This experiment was carried out twice using five petri dishes, and the average value was used for evaluation.

As a result, the number of pigment cells in the sample 1 addition group increased to 166% as compared with the solvent control group. In addition, the amount of dye increased to 241% as compared with the solvent control group. As a result of collecting pigment cells derived from forearm hair and performing the same experiment, the sample 1 did not proliferate the pigment cells, and no increase in the amount of pigment was observed.

In addition, ELISA using the anti-hemidesmosome antibody (manufactured by Cosmo Bio, monoclonal antibody, NU-01-PLN) and HRP-labeled anti-mouse IgG1 antibody (manufactured by Funakoshi) as a cell suspension by collecting the treated pigment cells. Hemides mosome was quantified by the method. As a result, the amount of hemidesmosome in Specimen 1 increased to 355% as compared with the solvent control.

The confirmation test of keratin production using human skin cells will be described below.
(Test Example 4)

The first representative epidermis cultured cells derived from human skin purchased from Cosmo Bio Co., Ltd. were used. The cells were suspended in a special culture medium and pre-cultured to proliferate the cells. The cells were cultured in a carbon dioxide incubator at 37 ° C. under 5% carbon dioxide. Then, the cells in the growth phase were exfoliated with a trypsin-containing medium and used for the experiment. The number of viable cells was counted under a microscope by the trypan blue dye exclusion method. The number of cells was adjusted to 1000 per mL, 5 mL each was seeded in a culture dish, and the cells were further cultured in a medium at 37 ° C. and 5% carbon dioxide. This was irradiated with ultraviolet rays by an ultraviolet irradiation device (Loctite, output 88 MH) to damage the cells. Irradiation was carried out for 1 hour with the petri dish lid removed.

Skin cells were damaged by this UV irradiation, and recovery from this damage was tested. In addition, this method is a method carried out for evaluation of a test substance for skin.

Here, the above-mentioned sample 1 was used as a test substance, and a physiological saline solution was used as a solvent control. This was cultured at 37 ° C. for 3 days, and the number of viable cells was counted under a microscope. Further, the cells were dispersed in purified water to obtain a cell dispersion liquid by an ultrasonic crusher (manufactured by Titec). The amount of keratin contained in this cell dispersion was quantified by the ELISA method using an anti-keratin antibody. In addition, these experiments were carried out twice using 5 petri dishes and evaluated by the average value.

As a result, as a result of determining the ratio by adding the sample 1 with the number of cells of the solvent control as 100%, the number of cells in the sample 1 increased to 122%. The amount of keratin increased to 188% depending on the sample 1 when the value of the solvent control was 100%. In addition, this derivative did not damage human-derived skin cells and increased the number of cells, which was considered to be highly safe.

In addition, ELISA using the above-mentioned treated skin cells as a cell suspension using an anti-hemidesmosome antibody (manufactured by Cosmobio, monoclonal antibody, NU-01-PLN) and an HRP-labeled anti-mouse IgG1 antibody (manufactured by Funakoshi). Hemides mosome was quantified by the method. As a result, the amount of hemidesmosome in Specimen 1 increased to 311% as compared with the solvent control.

The confirmation test for blood leakage using human vascular endothelial cells will be described below.
(Test Example 4)

Human vascular endothelial cells purchased from Cosmo Bio Co., Ltd. were used. 1000 cells were seeded on collagen-coated cell inserts (falcons) and cultured in a carbon dioxide incubator at 37 ° C. under 5% carbon dioxide gas. This was set on a 24-well microplate (manufactured by Falcon), 0.01% sodium dodecyl sulfate (SDS, manufactured by Wako Pure Chemical Industries, Ltd.) was added to the insert side, and solvent control or sample 1 was added thereto at a concentration of 10%. ..

Further, 10 μg of FITC (manufactured by Wako Pure Chemical Industries, Ltd.) was added as a fluorescent marker for detecting leakage. This was cultured under 5% carbon dioxide gas in a carbon dioxide gas incubator for 1 day. One day later, the culture solution in the lower layer was collected and sterilized by filtration, and the amount of leaked FITC was quantified by a spectrofluorometer (Hitachi spectrofluorometer F-2700, excitation wavelength 495 nm, fluorescence wavelength 520 nm). The amount of fluorescence of Sample 1 with respect to the solvent control was determined. In addition, these experiments were carried out twice using 5 petri dishes and evaluated by the average value.

As a result, the amount of FITC was 23% in Sample 1, and 77% suppression was observed. Since SDS penetrates the cell membrane and reduces hemidesmosome, Specimen 1 strengthens hemidesmosome and prevents leakage. This is a leakage model of the blood vessel wall, and sample 1 suppressed the leakage of the blood vessel wall.

Furthermore, vascular endothelial cells were collected and used as a cell suspension by the ELISA method using an anti-hemidesmosome antibody (manufactured by Cosmo Bio, monoclonal antibody, NU-01-PLN) and an HRP-labeled anti-mouse IgG1 antibody (manufactured by Funakoshi). Desmosome was quantified. As a result, the amount of hemidesmosome in Specimen 1 increased to 299% as compared with the solvent control.

The dopaquinone derivative obtained in the present invention exhibits a hemidesmosome activating action and has few side effects, so that it is used for preventing gray hair, and by strengthening the blood vessel wall, the QOL of the people can be improved.

The dopaquinone derivative obtained by the present invention is used as a cosmetic for skin improvement and hair growth, and contributes to the development of the cosmetics industry.

Claims (2)

A dopaquinone derivative exhibiting a hemidesmosome activating action represented by the following formula (1).
Human skin keratinocytes were cultivated and acclimated using a fermented solution obtained by fermenting grape buds and rice bran with natto and Benikouji, and human skin fibroblasts were further cultivated and acclimated. A method for producing a dopaquinone derivative exhibiting a hemidesmosome activating action represented by the formula (1) of claim 1, which comprises a step of purifying the conditioned culture medium.