JP2020138927A - Hydroxypyran derivative exhibiting endothelial cell proliferating action and methods for producing the same by culturing human buccal cells - Google Patents

Abstract

To provide a hydroxypyran derivative exhibiting endothelial cell proliferation action and production methods thereof.SOLUTION: The hydroxypyran derivative of interest is constituted from hydroxypyran, dihydroxybenzene and leucine. The derivative exerts endothelial cell proliferating action and increases keratin in the skin cells. VEGF participates in the proliferating action. The production method thereof comprises culturing human buccal cells in a culture medium to obtain the culture fluid, where the culture medium is obtained by fermenting okra sprouts and rice bran using Monascus purpureus and Bacillus natto. Use of buccal cells stabilizes the derivative. This hydroxypyran derivative can be used for health food, cosmetics, pharmaceuticals, quasi drugs and plant vitalizing agents.SELECTED DRAWING: None

Description

The present invention relates to a method for producing a hydroxypyran derivative exhibiting a vascular endothelial cell proliferation action, which comprises a step of culturing human oral cells.

Endothelial cells are located one layer inside the blood vessels and regulate the smooth flow of blood. It also has an anticoagulant effect to prevent blood from coagulating. Furthermore, it is involved in the contraction and relaxation of blood vessels and exhibits a blood pressure regulating effect.

Damage to vascular endothelial cells causes blood to coagulate and form thrombi. It is also involved in the development of atherosclerosis together with vascular smooth muscle cells. Disorders of vascular endothelial cells are serious disorders related to blood pressure regulation, immune function, arteriosclerosis, lipid metabolism, kidney disease, liver disease and skin health. In addition, VEGF is known as a vascular endothelial growth factor.

Therefore, measures to prevent damage to vascular endothelial cells and to improve the damage are being researched. Has been developed. For example, there is an invention of an agent for promoting the production of vascular endothelial growth factor, and it has been reported that the growth of vascular endothelial cells composed of a component derived from Lepidium Mayanywalp is promoted. (See, for example, Patent Document 1.) VEGF has a side effect of promoting angiogenesis due to cancer, and the present invention lacks the safety of this factor.

As VEGF-related inventions for vascular endothelial growth agent, there are a method for producing a vascular endothelial growth factor (VEGF) production promoter, a method for producing a hair papilla cell activator, and a method for producing a hair cosmetic. (See, for example, Patent Document 2.)

Furthermore, there is an invention relating to a vascular endothelial growth factor production promoter, and an active ingredient of a plant extract of the genus Pandanus is recognized. (See, for example, Patent Document 3.) However, here, it is not an invention relating to a detailed substance, but merely the proliferation of vascular endothelial cells via VEGF production, and this function is limited.

As described above, although a natural product that exhibits a vascular endothelial cell proliferation effect and has few side effects is desired, there is no invention regarding a useful substance.

Japanese Patent Application No. 2015-097572 Japanese Patent Application No. 2014-258409 Japanese Patent Application 2002-205071

As mentioned above, the vascular endothelial growth effect of existing natural products is mild, and there is a problem that industrial use is limited, and chemically synthesized substances have safety problems and can be used. limited.

Therefore, a substance having a weak side effect and exhibiting an excellent vascular endothelial cell proliferation action 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 hydroxypyran derivative exhibiting a vascular endothelial cell proliferative 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 hydroxypyran derivative exhibiting a vascular endothelial cell proliferation action represented by the formula (1) according to claim 1. It includes a step of culturing human oral cells.

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

According to the hydroxypyran derivative according to claim 1, an excellent vascular endothelial cell proliferation action can be exhibited.

According to the method for producing a hydroxypyran derivative according to claim 2, the hydroxypyran derivative can be efficiently produced.

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

First, the hydroxypyran derivative exhibiting the vascular endothelial cell proliferation action represented by the following formula (1) is composed of 28 carbon elements, 27 hydrogen elements, 1 nitrogen element and 9 oxygen elements.

That is, it is the chemical formula of C28H27N1O9. The constituents are hydroxypyran, dihydroxybenzene and leucine.

As shown in the formula (1), this hydroxypyran derivative is composed of one molecule of hydroxypyran, an ethylene side chain, dihydroxybenzene and leucine. Hydroxypyran is bound to dihydroxybenzene, which is bound to an ethylenic carboxylic acid with dihydroxybenzene via a hydroxyl group. Furthermore, the hydroxyl group of dihydroxybenzene is bonded to the carboxylic acid of L-leucine.

Structurally, the centrally located benzene ring and the hydrophobic portion of leucine are hydrophobic, while hydroxypyran, amino groups and hydroxyl groups are water soluble. Due to this structure, this derivative has both hydrophilicity and hydrophobicity. That is, it has both water-soluble and fat-soluble properties. This derivative easily passes through the lipophilic part of the cell membrane due to its hydrophobic functional group. In addition, it also passes through the nuclear envelope portion and acts directly on vascular endothelial cell proliferation, and also exhibits a vascular endothelial cell proliferation effect by increasing mitochondrial ATP production.

That is, this derivative directly proliferates vascular endothelial cells without mediated by VEGF (Vascular Endothelial Growth Factor) or VEGF receptor. This proliferative property does not cause side effects such as angiogenesis and cancer growth due to VEGF deficiency or increased VEGF.

The mechanism of vascular endothelial growth by this derivative is to enhance the binding and reactivity of the transcription factor of nuclear STAT1 and to promote ATP production in mitochondria. Furthermore, in mitochondria, it binds to the mitochondrial membrane and stabilizes the membrane. As mentioned above, it is not involved in the function of VEGF.

However, coexistence with VEGF exerts a synergistic effect on the proliferative potential of vascular endothelial cells. That is, since VEGF and this derivative have different action sites, the effect of VEGF is not eliminated and is not suppressed, and they exert a synergistic proliferative action.

This derivative is highly safe because it is an ester bond of existing substances such as hydroxypyran, leucine, and dihydroxybenzene.

This hydroxypyran derivative can be organically synthesized using hydroxypyran, leucine, and dihydroxybenzene 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 hydroxypyran derivative, the peak positions were 1.13, 1.87, 1.97, according to the 600 MHz H-NMR (1H-NMR) analysis (manufactured by Bruker) in deuterated chloroform of this derivative. 2.19, 3.02, 3.12, 3.14, 3.27, 3.39, 3.80, 3.97, 4.41, 5.10, 5.12, 7.50, 8. It is found at 24, 9.66 and 13.98 ppm.

In addition, in the C-NMR (13C-NMR) analysis of this derivative in deuterated chloroform, the peak positions were 17.4, 29.4, 35.3, 37.4, 53.2, 55.4, 65. 8.8, 67.1, 72.6, 74.3, 87.0, 90.6, 97.0, 109.8, 113.6, 113.7, 117.5, 121.7, 121.9 , 131.2, 142.8, 143.8, 158.7, 165.2, 170.0, 171.9, 190.7 and 193.4 ppm.

Since this hydroxypyran derivative is naturally derived, it is highly safe and has excellent intracellular and nuclear translocation properties. However, even if a large amount of this derivative is ingested, the excess amount is decomposed in the living body, so that the safety is high.

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.

In addition, this derivative acts on vascular endothelial cells to proliferate. It also acts on the mitochondria of vascular endothelial cells to produce ATP. This action of increasing ATP production is due to the stabilizing action of the mitochondrial membrane. The mitochondrial membrane is impaired by the respiration that occurs during ATP production, reducing the ability to produce ATP. Active oxygen is involved here, but this derivative has the function of removing the generated active oxygen.

This derivative improves peripheral circulation by proliferating vascular endothelial cells in peripheral blood vessels and producing ATP. By improving peripheral circulation, the function of the skin, heart, nerves, liver, kidneys and digestive organs is maintained and made healthy. For example, it increases skin cells and improves wrinkles by strengthening peripheral blood vessels in the skin and improving blood flow. In addition, it promotes the excretion of melanin and improves spots and blemishes.

Furthermore, in the heart, it strengthens the coronary blood vessels and has a good effect on the heart. Furthermore, in nerves, it exerts an improving effect on cerebrovascular accidents.

Examples of the extraction method or production method of this hydroxypyran derivative include a fermentation method, an enzymatic reaction method and a chemical synthesis method. For example, as a method for producing this hydroxypyran derivative, it can be extracted from okra buds, rice bran, soybeans and the like. It can also be extracted from various plants and plant callus. 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, a fermentation method in which okra and rice bran are fermented with Bacillus natto and Monascus purpureus, or a production method in which human oral cells or skin epidermal cells are cultured to obtain a culture solution are efficient production steps for this derivative.

In particular, one of the production methods for this hydroxypyran derivative is to culture human oral cells using a fermented solution obtained by fermenting okura buds and rice bran with Bacillus natto and Monascus purpureus as a medium to obtain the culture solution. There is an efficient manufacturing method. These fermentation techniques and culture techniques are preferable because they have abundant experience and knowledge in Japan and are highly safe. Since human oral cells contain ester-binding enzymes and transesterifying enzymes, the method using human oral cells is specific.

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.

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 hydroxypyran, 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 more preferable because they have high separation efficiency and have been used in many cases.

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 23 ° 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 that this hydroxypyran derivative is used in cosmetics that exert an excellent vascular endothelial cell proliferation effect, act on peripheral blood vessels of the skin, and improve melanin excretion and skin blood flow. That is, it can be used as a cosmetic, shampoo, eyelash growth agent, hair restorer, and hair cosmetic. Furthermore, it can be used as a cosmetic that improves blood flow and increases skin turnover by synergistic action with VEGF.

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 used by being blended in tablets, capsules, drinks, soaps, coating agents, gel agents, toothpaste, etc. Toothpaste.

Examples of the oral preparation include tablets, capsules, powders, syrups, drinks and the like. When mixed with the above-mentioned tablets and capsules, it can be used together with a binder, an excipient, a leavening agent, a lubricant, a sweetener, a flavoring agent and the like. The above tablets can also be coated with shellac or sugar.

Moreover, in the case of the above-mentioned syrup 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. As the base material of the external preparation, petrolatum, paraffin, oils and fats, lanolin, macrogold and the like are used, and ointments, creams and the like can be prepared 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 as a food for the purpose of vascular endothelial growth, a health food for the purpose of vascular endothelial growth, and a food for skin protection. In addition, it is preferable to use it as a food with a health function in 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 domestic animals, it is used as feed and pet supplement for the purpose of effective utilization of nutrients by strengthening blood flow and blood vessels of the skin.

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 vascular endothelial growth effect. 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 vascular endothelial cell proliferation action. It is preferred that this derivative improves the circulation of nutrients in the plant, activates growth and results in flowering, fruiting and increased yield. For example, this derivative is preferable because it enhances, maintains, and stabilizes the function of promoting plant growth together with the plant vitalizer of HB-101 (manufactured by Flora Co., Ltd.).

Hereinafter, a method for producing a hydroxypyran derivative exhibiting a vascular endothelial cell proliferation action represented by the formula (1), which includes a step of culturing human oral cells, will be described. Specifically, it comprises a production process of culturing human oral cells using a fermented solution obtained by fermenting okra buds and rice bran with Bacillus natto and Monascus purpureus as a medium to obtain the culture solution. This production process consists of two production steps: a production process obtained by fermenting okra buds and rice bran with Bacillus natto and Monascus purpureus, and culturing human oral cells using this fermented solution as a medium.

The raw materials used are okra buds and rice bran, Bacillus natto and Monascus purpureus, and human oral cells.

Okra buds are sprouts sprouted from the seeds of okra, which belongs to the genus Okras of the Malvaceae family and has the scientific name Abelmoschus Esculentus. Okra contains abundant nutrients, especially viscous substances such as mucopolysaccharides, mucopolysaccharide proteins and mucins. In addition to mucopolysaccharides, okra buds contain nutrients that promote cell growth. These nutrients are preferred for the growth of human oral cells. The okra buds used here may be from Japan, Asia, the United States, etc., but Japanese okra buds are preferable because they have stable quality and are less contaminated with pesticides. In particular, okra buds manufactured by Salad Cosmo Co., Ltd. are cultivated without pesticides and are preferable because of their high safety and quality.

Okra sprouts are preferably dried and pulverized, and autoclaved sterilization prior to fermentation is preferred for smooth fermentation. A powder having a particle size of 3 micrometers or less is preferable because it facilitates the fermentation process.

The rice bran used as a raw material may be produced in any of domestic, Asian, and American production areas, but Japanese rice bran collected from pesticide-free rice is preferable. In particular, pesticide-free, organic JAS-certified rice bran manufactured by Marukawa Miso Co., Ltd. is preferable because of its high quality.

When using these raw materials, Nara Kikai Seisakusho Co., Ltd.'s free mill, super free mill, sample mill, goblin, super clean mill, micros, Toyo Riko's small vacuum dryer as a vacuum dryer, Matsui Co., Ltd. It is dried and crushed by a small decompression heat transfer dryer DPTH-40, Clean Dry VD-7, VD-20 manufactured by AKUM Kyushu Technos Co., Ltd., DM-6 manufactured by Nakayama Institute of Technology, etc. 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 0.3 to 5% by weight for rice bran and 0.004 to 0.05% by weight for Bacillus natto with respect to 1 weight of okra buds. Pre-culturing Bacillus natto before fermentation is preferable because it shortens the initial fermentation time and shortens the fermentation time.

The above fermentation is carried out in a clean culture tank and it is preferable to mix the above materials with sterilized tap water. First, okra sprouts and rice bran are fermented by Bacillus natto. Fermentation is preferably carried out at 37-42 ° C. for 24 to 96 hours. If the temperature is low and the time is short, fermentation will not proceed, and if the temperature is high and the time is long, there is a risk of spoilage. The fermented product is filtered and the filtrate is subjected to the following steps.

Fermentation conditions are the same as the above fermentation method. Fermentation of Bacillus natto decomposes polyphenols and proteins to reduce their molecular weight.

This fermented filtrate is fermented by Monascus purpureus. The Monascus purpureus used has a scientific name of Monascusae, 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, Monascus purpureus manufactured by Monascus purpureus is preferable because it exhibits high fermentability.

The amount of each addition for fermentation is preferably 0.001 to 0.03 weight for Monascus purpureus with respect to 1 weight of the fermentation broth of Bacillus natto. Pre-culturing Monascus before fermentation is preferable because it shortens the initial fermentation time and shortens the fermentation time.

In addition, this fermentation is heated to 35 to 48 ° C., and the fermentation is carried out for 4 to 14 days.

After fermentation, heating at about 90 ° C. kills Bacillus natto and Monascus purpureus, and fermentation is stopped. This fermentation broth is filtered, and the filtrate is used as a medium for culturing human oral cells. Therefore, it is autoclaved. This is used as a medium for human oral cells.

Human oral cells are cultured using this fermented solution as a medium. Since human oral cells contain ester-binding enzymes and transesterifying enzymes, the method using human oral cells is excellent. That is, human oral cells are collected from the oral cavity of an adult human who has been confirmed to be free of viruses and bacteria based on ethical consent. That is, after infiltrating the above-mentioned medium for human oral cells with a sterilized cotton swab, the oral tissue is collected by rubbing the oral cavity. Friction is performed non-invasively so that the sampler is not physically burdened. This sample collection is not a medical practice.

The cells of the rubbed oral tissue are dispersed by the 1% collagenase solution contained in the above-mentioned medium for human oral cells. That is, human oral tissue is placed in a 15 mL sterilization test tube (manufactured by FALCON, etc.), 10 mL of 1% collagenase solution contained in a medium for human oral cells is added, and the mixture is heated at 37 ° C. for 5 minutes and stirred. .. After heating, the cells are cooled to 4 ° C., and the dispersed cells are collected by a cell separator (such as a cell strainer manufactured by FALCON). The above-mentioned medium for human oral cells is added to the cells to wash the cells, the cells are seeded in a 35 mm diameter culture dish (manufactured by FALCON, etc.) at a cell count of 1000 cells per mL, and cultured at 37 ° C. under 5% carbon dioxide gas. Will be done.

The culture is carried out in a carbon dioxide gas incubator (for example, BNA600 manufactured by Yamato Scientific Co., Ltd.). The cells are cultured for 5 to 10 days while observing the state of the cells under a microscope. Before becoming confluent, the culture is stopped, the culture solution is collected with a sterile pipette (manufactured by FALCON, etc.) and transferred to a sterile centrifuge tube (manufactured by FALCON, etc.). This is centrifuged at 1500 rpm for 5 minutes to centrifuge the remaining cells, and the supernatant portion is collected. The supernatant is sterilized by boiling at 100 ° C. for 5 minutes, cooled, and then microfiltered through a membrane filter and transferred to a clean container to prepare a solution containing the desired hydroxypyran derivative.

Separation and purification of the desired hydroxypyran derivative from the above-mentioned reaction product by the purification method described above is preferable from the viewpoint that the intake amount can be reduced as a highly pure substance. In particular, the step of purifying by column chromatography using Diaion HP-20 is preferable because the purity is high and the recovery rate is high. By repeating this purification step about 3 to 5 times, a highly pure refined product can be obtained.

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

Further, it is preferable to powder the hydroxypyran derivative from the viewpoint of antiseptic and stability.

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.

Okra buds purchased from Salad Cosmo Co., Ltd. were used. This was crushed with a crusher (Super Free Mill manufactured by Nara Kikai Seisakusho Co., Ltd.) to obtain 1 kg of crushed dry powder of okra buds.

In addition, pesticide-free rice bran purchased from Marukawa Miso Co., Ltd. was used. It was crushed by a crusher and about 1 kg was used for fermentation.

1 kg of crushed okra buds and 1 kg of crushed rice bran were placed in a clean fermentation tank (sterilized round 40 liter tank for fermentation), 10 kg of sterilized tap water was added, and the mixture was stirred. This was sterilized by boiling and cooled while maintaining the sterilized state.

Separately, 10 g of Bacillus natto purchased from Natto Motoho was placed in a small fermentation tank, and a pre-cultured culture solution was prepared together with sterilized soybean powder.

The above-mentioned solution of pre-cultured Bacillus natto was added to the fermentation tank containing the above-mentioned okra buds and rice bran powder, and after stirring, the mixture was heated in a temperature range of 39 to 42 ° C. and fermented.

In the fermentation process, the fermentation broth was sampled while bubbling and stirring by aeration. The state of fermentation was monitored by quantification of the eluted protein (Buret method).

After completion of fermentation, the supernatant of the obtained fermentation broth was roughly filtered through a filter cloth to obtain a filtrate.

5 kg of this fermentation filtrate was placed in a clean fermentation tank, and 10 g of Monascus purpureus manufactured by Monascus purpureus was pre-cultured and fermented. The temperature range was 38-42 ° C. and fermentation was carried out for about 7 days.

After fermentation, Bacillus natto and Monascus purpureus were killed by heating at 90 ° C., and fermentation was stopped. This fermented liquid was suction-filtered with a filter paper (No. 2) of Toyo Filter Paper, and the filtrate was autoclaved and used as a medium for human oral cells.

Human oral cells were collected from the oral cavity of an adult (Japanese male, 60 years old) who was confirmed not to be infected with a virus or bacteria based on ethical consent. After infiltrating the above-mentioned medium for human oral cells into a sterilized cotton swab, the oral cavity was rubbed. This friction was performed so as not to put a physical load on the sample collector. In addition to the above males, 10 males (Japanese, 22-72 years old) and 8 females (Japanese, 23-80 years old) were similarly subjected to the same procedure, and similar results were obtained. Got

The oral tissue obtained by rubbing was placed in a 15 mL sterilization test tube (manufactured by FALCON), 10 mL of the 1% collagenase solution contained in the above medium for human oral cells was added, and the mixture was heated at 37 ° C. for 5 minutes. Stirred.

After heating, the cells were cooled to 4 ° C., and the dispersed cells were collected as a filtrate by a cell separator (cell strainer manufactured by FALCON). The above medium for human oral cells was added to the cells to wash the cells, and the number of cells was adjusted to 1000 per mL. This 1 mL was inoculated in a petri dish for culture having a diameter of 35 mm (manufactured by FALCON, etc.) and cultured at 37 ° C. under 5% carbon dioxide in a carbon dioxide culture solution (BNA600 manufactured by Yamato Scientific Co., Ltd.). Observation under a microscope was performed and the culture was stopped before becoming confluent.

This culture broth was collected with a sterile pipette (manufactured by FALCON) and transferred to a sterile centrifuge tube (manufactured by FALCON). This was centrifuged at 1500 rpm for 5 minutes to centrifuge the remaining cells, and the supernatant portion was collected.

The supernatant was sterilized by boiling at 100 ° C. for 5 minutes, cooled, and then microfiltered through a membrane filter and transferred to a clean container to prepare a solution containing the desired hydroxypyran derivative. This was designated as sample 1. About 50 petri dishes were used for culturing, and 41 g of the solution of sample 1 was collected in total.

30 g of the obtained sample 1 was suspended in 100 mL of purified water and subjected to a glass column (3 cm diameter x 90 cm in length) containing 500 g of Diaion HP-20 (manufactured by Mitsubishi Chemical Corporation) swollen with 3% ethanol. After washing with 1800 mL of 3% ethanol, further washing was performed with 1000 mL of 15% ethanol.

To this was added 700 mL of 50% ethanol to separate the fractions. The target hydroxypyran derivative was detected by fractionation by 50 mL by the HPLC method, and the fraction of the hydroxypyran derivative was collected. This purification operation was carried out 5 times to obtain a final purified product. This liquid was distilled under reduced pressure by an evaporator to evaporate ethanol to obtain a purified hydroxypyran derivative solution. This was designated as sample 2.

The test method and results for structural analysis of the hydroxypyran derivative will be described below.
(Test Example 1)

Specimen 1 and Specimen 2 obtained as described above were diluted with purified water, filtered, and analyzed by high performance liquid chromatography (HPLC, Shimadzu Corporation).

Further, the samples 1 and 2 were analyzed in deuterated chloroform with a 600 MHz nuclear magnetic resonance apparatus (NMR, manufactured by Bruker). As a result of the structural analysis, a target derivative in which one molecule of hydroxypyran, one molecule of leucine, two molecules of ethylene group, and two molecules of dihydroxybenzene were bound was detected from Specimen 2 and Specimen 1.

According to the results of H-NHR analysis at 600 MHz, 1.13, 1.87, 1.97, 2.19, 3.02, 3.12, 3.14, 3.27, 3.39, 3.80, 3 Peaks were observed at .97, 4.41, 5.10, 5.12, 7.50, 8.24, 9.66 and 13.98 ppm.

Furthermore, according to the C-NMR analysis results, 17.4, 29.4, 35.3, 37.4, 53.2, 55.4, 65.8, 67.1, 72.6, 74.3, 87 .0, 90.6, 97.0, 109.8, 113.6, 113.7, 117.5, 121.7, 121.9, 131.2, 142.8, 143.8, 158.7 , 165.2, 170.0, 171.9, 190.7 and 193.4 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 hydroxypyran derivative, and were identified as the desired hydroxypyran derivative. This derivative contained in Specimen 2 had a purity of 98.2%, that is, a purity of 98.2%.

The purity of this derivative in Specimen 1 was 78.2%.

The confirmatory tests on the proliferativeness and ATP production of human-derived vascular endothelial cells are described below.
(Test Example 2)

Human-derived vascular endothelial cells (adult-derived, cutaneous microvessel type, D10017) were purchased from Takara Bio and used. These vascular endothelial cells are derived from adult skin and are suitable for evaluating blood vessels in the skin. These human-derived vascular endothelial cells were cultured in a special culture solution (manufactured by Takara Bio Inc.) at 37 ° C. under 5% carbon dioxide gas. The cells in which proliferation was observed were detached with a trypsin EDTA solution, dispersed in a culture solution, and 2000 cells were seeded in a 35 mm diameter petri dish (Falcon tissue culture dish 3001, manufactured by Falcon) together with 1 mL of the culture solution.

This was cultured for another day to attach the cells to the petri dish. Specimen 1, Specimen 2 and VEGF collected in Examples were added thereto so as to have a final concentration of 10 mg / mL (1% concentration). The control VEGF was a highly active recombinant VEGF (manufactured by Humanzyme) expressed in human cells, which was purchased from Funakoshi and used.

In addition, a group was also set up to investigate the synergistic effect of adding sample 2 and VEGF at the same time. A group was provided in which only the culture solution was added as a solvent control. The experiment was carried out on 5 petri dishes, the average value was calculated, and the result was indicated by the ratio with the solvent control.

Two days after the sample treatment, the number of living cells was exfoliated with trypsin and EDTA solution, dispersed in the culture solution, and counted under a microscope by the trypan blue dye method using a hemocytometer. In addition, the cells were destroyed by hypotonic treatment. The amount of ATP contained in this cell dispersion was quantified with an ATP quantification kit (manufactured by Caiman) by chemiluminescence. It was determined by the HPLC method (manufactured by Shimadzu Corporation) that VEGF was not contained in the normal culture solution. As the measurement conditions of VEGF, Simpack SCR was used, and 20% acetonitrile containing 0.1% acetic acid was used as the mobile phase, and the measurement was performed at a column temperature of 37 ° C. and a wavelength of 290 nm. The above VEGF was used as a reference substance.

As a result of this experiment, the number of endothelial cells in Specimen 1 was 182%, that in Specimen 2 was 241%, and that in VEGF was 173% as compared with the solvent control. Furthermore, the simultaneous addition of Specimen 2 and VEGF resulted in 569% compared to the solvent control. From this result, it was considered that Specimen 1 and Specimen 2 exhibited an endothelial cell proliferative action superior to that of VEGF. Furthermore, Specimen 2 showed synergistic action with VEGF.

The ATP content was 134% in sample 1, 202% in sample 2, and 114% in VEGF as compared with the solvent control. Furthermore, the simultaneous addition of Specimen 2 and VEGF resulted in 433% compared to the solvent control. From this result, it was considered that Specimen 1 and Specimen 2 showed a superior effect on ATP production than VEGF.

A semi-quantitative amount of the active phosphorylated STAT was quantified by seeding endothelial cells on a 96-well microplate (manufactured by Falcon) using a Phosph-STATCell-Based ELISA kit (R & D system, purchased from Funakoshi). Compared with the solvent control, it was 154% by the addition of the sample 1, 232% by the addition of the sample 2, and 111% by the addition of VEGF. As a result, it was concluded that Specimen 1 and Specimen 2 increase STAT containing STAT1. On the other hand, the effect of increasing STAT by VEGF was mild, and Specimen 1 and Specimen 2 were superior.

The confirmatory tests on the proliferation and ATP production of human-derived skin epidermal cells are described below.
(Test Example 3)

Human-derived skin epidermal cells (adult-derived, epidermal keratinocytes, KGM-Gold) were purchased from Lonza Japan Co., Ltd. and used. These cells are of adult skin origin and are suitable for assessing the proliferation of epidermal cells. These human-derived skin epidermal cells were cultured in a special serum-free culture solution (manufactured by Lonza Japan) at 37 ° C. under 5% carbon dioxide gas. Proliferated cells were exfoliated with trypsin EDTA solution (manufactured by Wako Pure Chemical Industries, Ltd.), dispersed in the culture medium, and 5000 cells were seeded in a 35 mm diameter petri dish (Falcon tissue culture dish 3001, manufactured by Falcon) together with 1 mL of the culture solution. did.

This was cultured for another 2 days to attach the cells to the petri dish. To this, Specimen 1 and Specimen 2 collected in Examples and EGF as a positive control were added so as to have a final concentration of 10 mg / mL (1% concentration). As the control EGF, a human recombinant EGF (product code 47061000) manufactured by Oriental yeast was purchased and used.

Furthermore, a group in which Specimen 2 and EGF were added at the same time was also set. A group was provided in which only the serum-free culture solution was added as a solvent control. The experiment was carried out on 5 petri dishes, the average value was calculated, and the result was indicated by the ratio with the solvent control.

Two days after the sample treatment, the cells were exfoliated with trypsin and EDTA solution, dispersed in the culture solution, and the number of living cells was counted under a microscope by the trypan blue dye method using a hemocytometer. In addition, the cells were destroyed by hypotonic treatment. The amount of ATP contained in this cell dispersion was quantified with an ATP quantification kit (manufactured by Caiman) by chemiluminescence. It was confirmed that the serum-free culture medium used did not contain EGF.

As a result of this experiment, the number of skin epidermal cells in Specimen 1 was 163%, that in Specimen 2 was 231%, and that in EGF was 155% as compared with the solvent control. Furthermore, the simultaneous addition of Specimen 2 and EGF resulted in 491% compared to the solvent control. From this result, it was considered that Specimen 1 and Specimen 2 exhibited an endothelial cell proliferative action superior to that of EGF. Furthermore, Specimen 2 showed a synergistic effect with EGF.

The ATP content was 141% in sample 1, 191% in sample 2, and 109% in EGF as compared with the solvent control. Furthermore, the simultaneous addition of Specimen 2 and EGF resulted in 266% compared to the solvent control. From this result, it was considered that Specimen 1 and Specimen 2 showed a superior effect on ATP production than EGF.

Further, the cells were dispersed in purified water to obtain a cell dispersion liquid by an ultrasonic crusher. The amount of keratin contained in this cell dispersion was quantified by the ELISA method (Cosmo Bio Co., Ltd.). As a result, the amount of keratin was 138% in sample 1, 192% in sample 2, and 128% in EGF, with the value of the solvent control as 100%. Since keratin is a useful component for skin tissue and protects the skin, Specimen 1 and Specimen 2 showed a better skin protective effect than EGF.

The hydroxypyran derivative obtained by the present invention exhibits a vascular endothelial cell proliferative effect and has few side effects, so that it is used for the treatment and prevention of vascular disorders such as arteriosclerosis, myocardial infarction and cerebrovascular disorders, and improves the QOL of the people. it can.

The hydroxypyran derivative obtained in the present invention is also used as a cosmetic for skin improvement and contributes to the development of the cosmetics industry.

Claims (2)

A hydroxypyran derivative exhibiting a vascular endothelial cell proliferation action represented by the following formula (1).
It is shown in the formula (1) according to claim 1, which comprises a manufacturing process of culturing human oral cells using a fermented solution obtained by fermenting okla buds and rice bran with Bacillus natto and Monascus purpureus as a medium to obtain the culture solution. A method for producing a hydroxypyran derivative exhibiting a vascular endothelial cell proliferative effect.