JP2020083829A - Gene repair action-exhibiting polyphenol derivative - Google Patents

Abstract

To provide a polyphenol derivative that exhibits gene repair action.SOLUTION: The polyphenol derivative is an amphipathic compound composed of polyphenol, a carboxylic acid form of pyridine, and glycerin. The production method comprises the steps of fermenting cowberry and soybean by adding Staphylococcus epidermis, which is a human skin indigenous bacterium, to yield a fermentation solution, and further purifying with Diaion HP-20. The yielded polyphenol derivative exhibits excellent gene repair enzyme inducing action and gene repair, and is suitable for improving cell function. Furthermore, this derivative has applications as water- and oil-soluble cosmetics, foods and plant activators.SELECTED DRAWING: None

Description

This invention relates to a polyphenol derivative exhibiting a gene repairing action.

As cells age, the frequency of gene damage increases. Although cells have the function of gene repair, the damage of genes increases due to aging and aging, so that the damage of genes cannot be kept up and the damage accumulates.

In the process, cell-synthesized protein synthesis is reduced. When many proteins in cells are exhausted for life support, the cells themselves are gradually damaged, die, mutate, and deteriorate in function. In addition, when many cells reach such a state in each organ of the body, the ability of the organ itself is weakened, and gradually it becomes a symptom of a disease. On the other hand, several types of gene repair have been recognized. For example, it has a repair function by the combination of DNA polymerase and ligase. In addition, it is OGG1, which is 8-oxoguanine DNA glycosylase that is a repair enzyme of 8-hydroxyl-2-deoxyguanosine for the damage of the base due to active oxygen.

There is also an SOS repair function via DNA polymerase, and there is a repair function for various disorders. Activating these gene repair functions is preferable because it repairs the damage of genes due to aging and chemical substances and recovers the disease from the genes. Studies have been conducted to activate this gene repair function.

The invention relating to the gene repair function includes, for example, gene repair by in vivo removal of target DNA, but it is a treatment at the cell level and its application is limited to the living body (for example, see Patent Document 1). Further, the invention of target modification of DNA describes the regulation of genes by vectors (for example, refer to Patent Document 2).

Japanese Patent Application No. 2000-59444 Japanese Patent Application No. 2013-541944

Gene repair by existing substances is mild, and there is a problem that their industrial use is limited. Further, chemically synthesized substances have safety problems and their use is limited.

Therefore, a natural product having a weak side effect and an excellent gene repairing action is desired.

In order to achieve the above object, the invention according to claim 1 relates to a polyphenol derivative having a gene repairing action represented by the following formula (1).

Since the present invention is configured as described above, it has the following effects.

The polyphenol derivative according to claim 1 is excellent in gene repair action.

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

The polyphenol derivative exhibiting a gene repairing action has a structure represented by the following formula (1).

The polyphenol derivative exhibiting a gene repairing action as represented by the above formula (1) is composed of 1 molecule of glycerin, 3 molecules of pyridine having 2 molecules of carboxylic acid, and 3 molecules of para-dihydroxybenzene. All of these molecules and their bonds are naturally occurring in nature, and the bonds between the molecules are linked by ester bonds or ether bonds found in nature.

This polyphenol derivative can be obtained by chemical synthesis using glycerol, polyphenols containing hydroxybenzene and pyridine as raw materials. However, in this case, in the chemical synthesis, the loss of raw materials is large and the manufacturing cost is high, so that the industrial use is limited.

On the other hand, the chemically synthesized high-purity polyphenol derivative is used to obtain a standard product for analysis or a small amount of sample product.

Analyzing the structure of this polyphenol derivative is preferable from the viewpoint that the active ingredient can be specified. In addition, it is preferable because it can be used as an index of the content of the active ingredient when it is sold for use in products and formulations.

When a high purity (purity 99.1%) standard product chemically synthesized was used for the structural analysis of this polyphenol derivative and analyzed by H-NMR at 600 MHz in deuterated chloroform, the peak position was 3.25. 3.77, 3.83, 6.45, 6.62, 6.70, 6.95, 7.02, 7.10, 7.11, 7.25, 7.26, 7.27, 7. Found at 28, 7.38, 7.45 and 7.48 ppm.

Further, when analyzed by C-NMR at 500 MHz in deuterated chloroform as a structural analysis of this polyphenol derivative, the peak positions were 41.2, 41.5, 41.7, 99.8, 108.3, 114. .9, 117.1, 119.0, 121.4, 123.6, 125.1, 129.1, 129.2, 129.5, 129.7, 130.6, 130.7, 131.0. , 131.3, 131.4, 131.8, 133.3, 135.6, 135.7, 135.8, 136.0, 138.3, 145.6, 147.4, 147.9, 148. Found at 0.4, 150.3, 154.0, 171.5, 172.3 and 172.4 ppm.

Furthermore, it is preferable that the polyphenol derivative is analyzed by high performance liquid chromatography or the like, and the analysis data is collected to identify the structure. The chemical formula is C42H29N3O15 and the molecular weight is 815.69.

That is, it is composed of 42 carbons, 29 hydrogens, 3 nitrogens and 15 oxygens. Further, the polyphenol derivative which is the constituent component is a naturally occurring compound.

Originally, this polyphenol is a water-soluble phenolic compound widely distributed in animals and plants and microorganisms, and in particular, it is a polyphenol having two hydroxyl groups. This polyphenol is a natural product that exists in nature, and its safety is high. The hydroxyl group of this polyphenol has an antioxidant effect and exhibits a reducing effect. The activated state of polyphenol is related to the state in which the cell membrane is stably maintained.

In this polyphenol derivative, one of the polyphenols in one molecule has an ester bond with two carboxylic acids bonded to one molecule of the pyridine ring. Since this pyridine ring exhibits weak basicity, it is electrically positive, that is, in a positive state. On the other hand, the hydroxyl groups of polyphenols are weakly acidic and thus neutralized to become neutral as a whole.

When this derivative is incorporated into a cell membrane, it has a function of adjusting the electric potential of the cell membrane between plus and minus, and contributes to stabilization and activation of the cell membrane in nerve cells, cardiomyocytes, muscle cells and skin cells. Furthermore, this derivative is amphipathic and serves to maintain the structure of the cell membrane.

This polyphenol derivative has a high absorption rate through the skin and intestinal tract as a result of its electrical activity and amphipathic nature. In particular, in transdermal absorption, the absorption is increased about three times as compared with polyphenol alone.

Furthermore, this polyphenol derivative has an emollient action as a skin moisturizer, and brings moisture to the surface of keratinocytes as a substitute for sebum. It is a preferable condition for the skin that the water content is protected by this polyphenol derivative because the sebum of the skin is easily lost by washing the face.

The polyphenol derivative stabilizes the cell membrane by penetrating extracellularly and intracellularly through the cell membrane via polyphenol. It also activates the receptors present on the cell membrane. Furthermore, the fluidity of the cell membrane is increased to enhance the function of the cell membrane. This polyphenol derivative migrates into cells and stabilizes the nuclear membrane. It is preferred that the gene is protected by the stabilizing action of the nuclear membrane. This polyphenol derivative eliminates gene damage caused by active oxygen at the nuclear membrane stage.

Furthermore, since the carboxylic acid moiety of the polyphenol is weakly acidic, it is preferable that it has strong acid resistance, exhibits resistance to gastric acid when ingested orally, and has a high absorption rate.

Further, since it is weakly acidic, it is preferable that it is not irritating to the skin when applied to the skin.

This polyphenol derivative penetrates the nuclear membrane and activates a gene repair enzyme. The targeted gene repair enzymes are DNA polymerase and 8-oxoguanine DNA glycosylase or 8-hydroxydeoxyguanine DNA glycosidase (both abbreviated as OGG1).

The repair of genes by DNA polymerase is called SOS repair, and it consists of the steps of acting on base changes and adducts to cut and replicate DNA strands. Since the repair by the DNA polymerase is performed 10,000 times or more a day, it is preferable to continuously use this derivative.

Gene repair by OGG1 is a process of eliminating an oxidant of a base such as 8OHdG and incorporating a normal base. This polyphenol derivative exhibits a gene repairing action by carrying out the functions of these gene repairing enzymes and protecting the genes from oxidants and active oxygen. Gene repair enzyme action depends on both activation of enzyme activity and activation of the enzyme at the mRNA transcription level.

The gene repairing action of this polyphenol derivative works on all cells having the gene in the nucleus to repair the gene.

It also repairs genes in response to gene damage caused by all substances such as active oxygen, free radicals, ultraviolet rays, chemical substances, side effects of drugs, metals, and aging.

Further, since this polyphenol derivative exhibits both fat-soluble and water-soluble properties, it passes through the cell membranes of animals and the cell walls of plants and yeasts and is easily absorbed into cells. Further, since it is soluble in both a water-soluble solvent and an oil-soluble solvent, it is preferable that a wide range of solvents can be used.

Furthermore, it easily penetrates the corneal cell membrane of the skin, and maintaining the barrier function of the stratum corneum is preferable from the viewpoint of skin health and beauty. In addition, this polyphenol derivative is preferable because it passes through the cell membrane and activates gene repair in skin cells to promote cell regeneration and function.

For plants, this polyphenol derivative passes through the cell wall and cell membrane of the plant and enters the plant cell, promotes gene repair, promotes flowering, fruiting, and leaf growth to prolong the life of the plant. Is preferred. That is, it has a function as a plant activator.

In addition, this polyphenol derivative, when made into powder, reacts with a water-soluble solvent to generate hydrogen gas and eliminates active oxygen. The amount of hydrogen gas generated is a saturation concentration of 1,6 ppm, and the hydrogen gas is generated for about 1 minute to 2 hours after dissolution. Hydrogen gas has a function of scavenging hydroxy radicals, and an excellent scavenging action of active oxygen has been confirmed.

It is preferable that this polyphenol derivative not only works for gene repair, but also promotes skin cell function by promoting cell proliferation, ceramide, collagen and elastin production.

It also activates intracellular gene repair in nerve cells. Nerve cells are susceptible to gene damage due to active oxygen and amyloid β protein in dementia, Alzheimer's disease, etc., and the genes are difficult to repair. Therefore, gene repair using this polyphenol derivative is preferable for the purpose of restoring the function of nerves and protecting and restoring nerve diseases.

It is also preferable to protect the myelin sheath and promote the release of neurotransmitters from nerve endings to enhance neurotransmission. Furthermore, the generated hydrogen gas is a small molecule and passes through the blood-brain barrier to repair damaged brain cells.

It is preferred to enhance muscle contraction and increase nerve and muscle activity by enhancing the release of acetylcholine from the nerve endings of motor neurons.

Further, it is preferable that this polyphenol derivative exhibits a gene repairing action on skin cells, and activates genes of collagen and elastin synthase to enhance their production. It is preferable because it can be used more as a cosmetic.

In myocardial infarction, it is preferable that the polyphenol derivative activates the activity of the heart by the gene repair action of the cardiomyocytes even in the infarction and ischemia of the coronary arteries to exert the cardiotonic action. Further, the hydrogen gas generated at the same time improves the damage of active oxygen due to ischemia-reperfusion in myocardium.

In particular, in the blood vessel at the infarct site, this polyphenol derivative promotes angiogenesis, improves blood flow, and lowers blood pressure.

In addition, this polyphenol derivative promotes the transport of fat to muscle cells during the exercise of athletes, the exercise of the general public, and when it is desired to strengthen muscles. This is preferred because it is via a pathway that activates energy production at the genetic level. In addition, hydrogen gas generated from this derivative at the time of muscle activity erases active oxygen during exercise, which is preferable because it reduces gene damage of muscle cells due to active oxygen.

In vivo, this polyphenol derivative is decomposed by esterase in the kidney and liver and excreted in urine. It is decomposed into polyphenols, glycerin, etc., which are highly safe constituents. Therefore, the polyphenol derivative is not accumulated in the body, is decomposed by the in-vivo enzyme, and the decomposed product is a natural product, which is highly safe.

Furthermore, since the polyphenol portion has a plant activating effect that promotes plant growth, it is preferable that this polyphenol derivative also promotes plant growth from the viewpoint of industrial use.

In addition, when a plant is infected with bacteria or viruses, the gene may be damaged. It is preferable to activate gene repair against such genetic disorders.

This polyphenol derivative also exists in nature, and is widely present in plants such as Staphylococcus and bilberries, which are types of microorganisms. Staphylococcus is also known as a normal bacterium of the epidermis of the skin, and particularly Staphylococcus epidermidis is preferable because it contributes to the health of the skin as a beneficial bacterium of the skin.

It is possible to extract this polyphenol derivative from the above plants by purification. However, purification requires a large amount of raw materials and uses an organic solvent and the like, so industrial use is limited.

This polyphenol derivative is preferably produced by fermenting Sphingomonas and cowberry. As a fermentation method, it is obtained by fermenting with Sphingomonas and Coleomom. The cells used are resident in the skin, so they are highly safe.

This manufacturing method is preferable because the raw material has food experience and the production amount of the polyphenol derivative is large.

When the obtained polyphenol derivative is used as a pharmaceutical material, it is preferable to purify the target polyphenol derivative because the purity of the target polyphenol derivative is increased and impurities can be removed.

It is used as a pharmaceutical agent as a parenteral agent such as an injection or an oral agent or a coating agent, and as a quasi drug, it is used by being mixed with tablets, capsules, drinks, soaps, coating agents, gel agents, toothpaste, etc. It

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

Further, in the case of the above-mentioned capsule, the above material may further contain a liquid carrier such as oil and fat. In the case of the above-mentioned syrups and drinks, sweeteners, preservatives, coloring and flavoring agents can be added.

Examples of parenteral agents include injections as well as external preparations such as ointments, creams and liquids. As a base material for the external preparation, petrolatum, paraffin, oils and fats, lanolin, macro gold and the like are used, and an ointment, a cream and the like can be prepared by a usual method.

Injections include liquid preparations and other freeze-dried preparations. It is used by aseptically dissolving it in distilled water for injection, physiological saline, etc. before use.

Since it has a gene repairing effect as a food preparation, it is used for energy drinks and tonic foods that work to repair muscle cells. It is used as a food for rehabilitation of nerves through dietary disorders of nerve cells and a meal for learning because it promotes nerve activity. It is also used for beauty foods. It is preferable to use it as a nutritionally functional food or a food for specified health use as a food with health claims.

When the obtained food preparation is used for pets and domestic animals such as dogs and cats, it is used as a feed or a supplement for pets for the purpose of recovery of muscular gene damage, suppression of aging and improvement of exercise capacity.

It can be used as a cosmetic 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, gel for hair, facial cleanser, beauty essence, lotion and the like.

The cosmetic may be in any form and can be used in the form of solution, cream, paste, gel, gel, solid or powder. Since this derivative is soluble in both water-soluble and oil-soluble solvents, it can be used in a wide range of cosmetics. That is, it can be dissolved in an aqueous solution and oil.

The cosmetics produced here are used for the purpose of repairing genes impaired in skin, increasing ceramide, collagen and elastin, and maintaining skin health.

Further, this polyphenol derivative can be used as a toothpaste, mouthwash, toothpaste, etc. for the purpose of maintaining the function of gingival cells in which a gene is damaged.

Further, it can be used as a plant activator for the purpose of leaf setting and increase of yield by recovering the gene damage to plants.

This plant activator can be used for the purpose of promoting the cultivation of flowers such as high-class and rare orchids, Phalaenopsis orchids, and Matsubara, and stabilize the cultivation of leaves, vegetables, and grains. It can also be used for cultivating vegetables and leaves in plant factories, and can increase cultivation efficiency.

Next, a method for producing a polyphenol derivative exhibiting a gene repairing effect, which comprises a step of fermenting cowberry and soybeans by Staphylococcus epidermidis and then purifying with diaion HP20 manufactured by Mitsubishi Chemical, will be described.

The polyphenol derivative as used herein is composed of one molecule of the polyphenol represented by the above formula (1), pyridine having a carboxylic acid, and glycerin. All of these bonds are of a natural type, and the substances are bound to each other via an ester bond and an ether bond.

This polyphenol derivative, polyphenol, pyridine having carboxylic acid, and glycerin are preferable because they are naturally present, have abundant food experience, and are recognized as safe.

This derivative also acts on the skin, nerves, bones, muscles, liver and kidneys, etc., and restores the damaged gene to restore tissue and body function.

This production method comprises a step of fermenting cowberry and soybean by Staphylococcus epidermidis, which is a resident bacteria of the skin. It is also possible to use plants and algae containing polyphenols in place of the cowberry.

The raw materials are cowberry, soybean and Staphylococcus epidermidis.

The cowberry as used herein is a plant of the genus Vaccinium vitis, belonging to the genus Vaccinium vine, whose fruits are edible. Cultivated or grows naturally in Japan, Asia, Europe and America. This cowberry is rich in polyphenols and is preferable as a raw material for the target polyphenol derivative. Fruits and leaves are used as the parts to be used, but fruits that are abundant in food experience are preferable from the viewpoint of safety.

The soybean used may be from Japan, Europe or America, or from China, but soybean from Japan with stable quality is preferable.

The bilberries may be collected from Japan, the United States, Asia and other countries, but from the viewpoint of high quality and price, Japanese products are preferable because they are of good quality.

The peach and soybean are preferably dried and powdered, and autoclave sterilized before fermentation is preferable because the fermentation can be performed smoothly.

A powder having a particle size of 3 micrometers or less is preferable because it facilitates the fermentation process.

The bacterium used for fermentation is Staphylococcus epidermidis, which is a resident human skin bacterium. This bacterium is resident in human skin and protects the skin while suppressing the growth of highly toxic bacteria such as acne bacteria. This bacterium is a Gram-positive bacterium and is a non-pathogenic bacterium.

Staphylococcus epidermidis is also used for food. In particular, as a cosmetic product, a bacterium collected from healthy human skin is preferable because it is strongly activated.

The addition amount of each of the above fermentations is preferably 0.001 to 0.03 weight of Staphylococcus epidermidis per 1 weight of dry powder of cowberry and soybean. It is preferable that the Bacillus natto is pre-cultured before being fermented, because the initial time of fermentation is shortened and the fermentation time is shortened.

It is preferred that the fermentation is carried out in a clean culture tank and that the materials are mixed with sterilized tap water.

Further, this fermentation is heated to 38 to 40° C. and fermented for 2 to 14 days. It is preferable to carry out the process control of fermentation by quantifying the target polyphenol derivative by HPLC or TLC and confirming the growth of the bacterial cells because the production amount is adjusted.

It is preferred that the fermentation is carried out in a clean culture tank and that the materials are mixed with sterilized tap water.

The polyphenol derivative produced by this fermentation process is formed.

It is preferable that the fermented product is extracted with hydrous ethanol because the product can be efficiently recovered, the bacteria can be sterilized, and the next step can be easily performed. In addition, ultrasonic treatment of the obtained fermented product is preferable because the product is easily separated. Concentration by freeze-drying or the like is preferable because the following steps can be carried out in a short time. The purification steps are combined and repeated to increase the purity.

It is preferable to separate the target polyphenol derivative from the reduction reaction product and purify it from the viewpoint that the intake amount can be reduced as a highly pure substance. As a purification method, DIAION HP20 manufactured by Mitsubishi Chemical is used. Moreover, since the purity is increased by repeating this purification, it is preferable to repeat the purification.

The polyphenol derivative of interest having high purity can be obtained by purifying and fractionating with DIAION HP20 manufactured by Mitsubishi Chemical. As the carrier or resin for separation, a porous polysaccharide, a silicon oxide compound, polyacrylamide, polystyrene, polypropylene, a styrene-vinylbenzene copolymer, etc., the surface of which is coated as described later, is used. Those having a particle size of 0.1 to 300 μm are preferable. The finer the particle size, the more accurate the separation is performed, but there is a drawback that the separation time is long.

Furthermore, the reverse phase carrier or resin whose surface is coated with a hydrophobic compound is preferably used for separating highly hydrophobic substances. Those coated with a cationic substance are suitable for separating anionically charged substances. Also, those coated with anionic substances are suitable for separating cationically charged substances. When coated with a specific antibody, it is used as an affinity carrier or resin for separating only a specific substance.

Affinity carriers or resins are used for the specific preparation of antigens using the antigen-antibody reaction. A distributable carrier or resin is used for isolation of substances such as silica gel (manufactured by Merck & Co., Inc.) when there is a difference in the distribution coefficient between the substance and the separating solvent.

Among these, the adsorbent carrier or resin, the distributable carrier or resin, the molecular sieve carrier or resin, and the ion exchange carrier or resin are preferable from the viewpoint that the manufacturing cost can be reduced. Furthermore, the reverse phase carrier or resin and the distributable carrier or resin are more preferable because of a large difference in partition coefficient with respect to the separation solvent.

When an organic solvent is used as the separating solvent, a carrier or resin having resistance 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 Co., Ltd.) and XAD-2 or XAD-4 (manufactured by Rohm and Haas) as an adsorptive carrier, and Sephadex LH-20 (manufactured by Amersham Pharmacia) as a carrier for molecular sieves. More preferably, silica gel is used as a carrier for distribution, IRA-410 (manufactured by Rohm and Haas Company) as an ion exchange carrier, and DM1020T (manufactured by Fuji Silysia Company) as a reverse phase carrier.

Of these, DIAION HP-20 is preferable because it can purify organic acids, peptides and polyphenols from natural products. Further, Sephadex LH-20 and DM1020T are more preferable.

The obtained extract is dissolved in a solvent for swelling the carrier for separation or the resin before separation. From the viewpoint of separation efficiency, the amount thereof is preferably 2 to 40 times the amount of the extract, and more preferably 4 to 20 times. The temperature for separation is preferably 10 to 30°C, more preferably 12 to 25°C from the viewpoint of the stability of the substance.

As the separating solvent, water, or 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, and ethanol used for food is preferable. In addition to the water-soluble solvent, it can be dissolved in oil-soluble solvents such as vegetable oil, fish oil, and animal fats and oils such as lard.

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

When using DIAION HP-20, the separating solvent is preferably a lower alcohol such as methanol or ethanol, or a mixed solution of a lower alcohol and water.

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

Further, it is preferable to carry out the final extraction with an edible oil or an oil or fat used for cosmetics, because the obtained polyphenol derivative is stably maintained. For example, extraction with soybean oil, rice bran oil, grape seed oil, olive oil, jojoba oil is preferable.

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

Hereinafter, the said embodiment is concretely demonstrated using an Example and a test example. It should be noted that these are examples, and the conditions can be changed within the range of common sense according to the difference in the raw material and the sample.

The cowberry cultivated in Gunma Prefecture was purchased from Kenko Village and used. That is, 2 kg of fresh cowberry was washed with tap water and then ground with a grinder (Super Free Mill manufactured by Nara Kikai Seisakusho Co., Ltd.) to obtain 1.0 kg of dried powder of cowberry.

This was put in a clean fermentation tank (sterilized round 40 liter tank for fermentation, manufactured by Endo Kagaku), and 5 kg of sterilized tap water was added and stirred.

Separately, Hokkaido soybeans were purchased, washed and pulverized to obtain 1.0 kg of soybeans. Furthermore, Staphylococcus epidermidis collected from the facial skin of a healthy human (60 years old, male) was cultured and added to the fermentation tank. The Staphylococcus epidermidis used was obtained in advance as a 100% pure colony in a selective medium. Other bacteria and viruses such as Escherichia coli were also examined, but no contamination with other bacteria or viruses was found.

The above-mentioned pre-cultured Staphylococcus epidermidis fermentation preparation liquid and cowberry and soybean dry powder were agitated and heated in a temperature range of 36 to 39° C. for fermentation.

In the fermentation process, the fermentation liquor was sampled while bubbling and stirring by aeration, and the fermentation was carried out for 5 days. After completion of fermentation, the fermented product was taken out from the fermentation tank and sterilized by boiling. The fermented product was filtered with a filter cloth to obtain 3.9 kg of a fermented liquid.

Fermentation was stopped by heating this fermented liquid by an autoclave (121°C, 10 minutes). This was filtered and the filtrate was used as the target polyphenol derivative. This was designated as Sample 1.

Further, this sample 1 was purified using DIAION HP-20 manufactured by Mitsubishi Chemical. That is, 4 L of purified water containing 5% ethanol was added to 200 g of the polyphenol derivative of the specimen 1 described above, and 500 g of DIAION (HP20 type, manufactured by Mitsubishi Chemical) was suspended in a 5% ethanol solution and packed ( (Made by Endo Kagaku).

To this, 5 L of 5% ethanol solution was added for cleaning, and further 1 L of 30% ethanol solution was added for washing. Further, 1 L of a 60% ethanol solution was added to elute the target polyphenol derivative, and the eluate was concentrated and purified. This purification operation was repeated 3 times. The polyphenol derivative purified by three purification operations was distilled under reduced pressure to remove the ethanol portion to obtain an aqueous solution. This was vacuum dried to obtain 94 g of a purified product of the polyphenol derivative, which was designated as Sample 2. The polyphenol derivative had a purity of 99.1% and a yield of about 6.9%. This was a sufficient yield for production from a natural product, and it was confirmed that this production method is an excellent production method.

The test methods and results relating to the structural analysis of the polyphenol derivative will be described below.
(Test Example 1)

The sample 2 obtained as described above was dissolved in deuterated dimethyl sulfoxide (manufactured by Sigma-Aldrich) and analyzed by high performance liquid chromatography (HPLC, Shimadzu Corporation).

Furthermore, as a result of analyzing this with a nuclear magnetic resonance apparatus (600 MHz, NMR, manufactured by Bruker), as a result of H-NMR measurement in deuterated dimethyl sulfoxide of this purified product, peak positions are 3.25 and 3.77. 3.83, 6.45, 6.62, 6.70, 6.95, 7.02, 7.10, 7.11, 7.25, 7.26, 7.27, 7.28, 7. It was observed at 0.38, 7.45 and 7.48 ppm.

Further, when analyzed by 600 MHz C-NMR in deuterated dimethyl sulfoxide as a structural analysis of this polyphenol derivative, the peak positions were 41.2, 41.5, 41.7, 99.8, 108.3, 114.9, 117.1, 119.0, 121.4, 123.6, 125.1, 129.1, 129.2, 129.5, 129.7, 130.6, 130.7, 131. 0, 131.3, 131.4, 131.8, 133.3, 135.6, 135.7, 135.8, 136.0, 138.3, 145.6, 147.4, 147.9, 148.4, 150.3, 154.0, 171.5, 172.3 and 172.4.
Was recognized by.

Below, the chart of the analysis result of 13 C-NMR is shown. (The horizontal axis unit is ppm, and the vertical axis unit is peak intensity.)

In addition, in the analysis of the purified product, Sample 2, by HPLC, the number of main peaks was one and impurities were less than 0.6%. The purity of the target polyphenol derivative in Sample 1 was 61.7%.

From the above analysis results, it was revealed that it has the same structure as the chemically synthesized standard product. It was confirmed from Sample 2 that the target polyphenol derivative was obtained. That is, it was composed of 1 molecule of glycerin, 3 molecules of pyridine having 2 molecules of carboxylic acid, and 3 molecules of para-dihydroxybenzene. The bonds between each molecule were linked by ester bonds or ether bonds. Further, when the sample 2 was pulverized and dissolved in an aqueous solution, generation of hydrogen gas was confirmed by gas chromatography (manufactured by Shimadzu Corporation). The amount of hydrogen gas generated in this case was 1.6 ppm.

The skin action test using human skin epidermal cells is described below. In addition, this test method is a reproducible conventional method that can verify the effects of the components biochemically.
(Test Example 2)

Human-derived epidermal cells (epidermal cell derived from epidermis) purchased from Kurabo Industries were used. 1000 cells cultured using a MEM medium containing 5% fetal bovine serum (manufactured by Sigma) as a culture solution were seeded on a 35 mm culture dish (manufactured by FALCON) and cultured at 37° C. in 5% carbon dioxide gas. This was irradiated with ultraviolet rays of 280 nm for 1 hour by an ultraviolet irradiation device (manufactured by Quark Technology). Specimens 1 and 2 and EGF (Epidermal growth factor, manufactured by Funakoshi Co., Ltd.) as a positive control were added thereto at a final concentration of 0.1 mg/ml. This was cultured and tested for 48 hours.

After collecting the culture solution, the survival rate of epidermal cells was counted by the trypan blue method. Then, a suspension of epidermal cells was prepared. From this, mRNA was extracted with a nucleic acid extraction kit (Funakoshi). According to a conventional method, mRNA of DNA polymerase and 8-oxoguanine DNA glycosylase (abbreviated as OGG1) was quantified by RT-PCR method. At the same time, the amount of 8-OHdG in the cell suspension was quantified with a kit (manufactured by Japan Aging Control Research Institute). It is an ELISA kit using a monoclonal antibody specific for 8-OHdG. In addition, the average value was calculated using five petri dishes. It was compared with a solvent control group to which a solvent was added.

As a result, the number of human-derived epidermal cells increased to 177% as an average value as compared with the solvent control group by the addition of 0.1 mg/ml of Sample 1. Moreover, in the case of the sample 2, the amount increased to 225%. On the other hand, EGF was 169%. As a result, the samples 1 and 2 exhibited a cell proliferation action superior to that of EGF. When the sample 1 and EGF were added at the same time, the cell number was 366%, confirming the synergistic action of the sample 1 and EGF.

The expression level (copy number) of mRNA of DNA polymerase in the cells was 14 copies in the solvent control group, 189 copies in the sample 1 treated group, 566 copies in the sample 2 treated group, and 102 copies in the EGF treated group. In addition, the mRNA expression level of DNA polymerase was remarkable in Sample 1 and Sample 2, and was superior to EGF. This showed the increasing action of the gene repair enzyme at the gene level by the samples 1 and 2. In addition, it was confirmed that 1.6 ppm of hydrogen gas was generated in the culture solution to which the samples 1 and 2 were added one hour after the addition.

The expression level (copy number) of OGG1 mRNA in the cells was 20 copies in the solvent control group, 233 copies in the sample 1 treatment group, 884 copies in the sample 2 treatment group, and 221 copies in the EGF treatment group.

The expression level of OGG1 mRNA was high in Sample 1 and Sample 2, and was higher than that of EGF. This indicates that the sample 1 and the sample 2 have a gene repair enzyme inducing action.

The amount of 8OHdG in the cells was 633 ng in the solvent control group, 108 ng in the sample 1 treatment group, 37 ng in the sample 2 treatment group, and 552 ng in the EGF treatment group.

8OHdG is a mutated state in which the gene is modified by active oxygen and represents a gene disorder. This value was low in Sample 1 and Sample 2, and was superior to the function of EGF. This indicated the gene repairing effect of Sample 1 and Sample 2.

On the other hand, in the skin irritation experiment using EpiSkin (manufactured by SkinEthic) which is artificial skin as part of the safety test, there is no cytotoxicity due to addition of Sample 1 and Sample 2 confirmed. This method has been established as a method that does not use animals as a skin irritation test evaluation method using cells.

The polyphenol derivative obtained by the present invention exhibits a gene repairing action and enhances cell functions such as skin cells and nerve cells. This will improve the people's QOL, increase the healthy working population, and reduce medical costs.

The polyphenol derivative obtained in the present invention exhibits a gene repairing action, thereby increasing the cells of the skin, contributing to the improvement of the skin of those suffering from skin troubles such as wrinkles and tarmi as a cosmetic material and contributing to the development of the cosmetic industry. To do.

Since the polyphenol derivative obtained in the present invention is produced by a fermentation method, it can be used as a functional food and contributes to the development of the food industry and the fermentation industry.

Claims (1)

A polyphenol derivative having a gene repairing action represented by the following formula (1).