JP2018070558A - Glycerol derivative showing gene restoration action - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a glycerol derivative showing a gene restoration action.SOLUTION: Provided is a glycerol derivative being an amphiphilic compound composed of one molecule of glycerol, two molecules of gallic acid and two molecules of phenol. A method of producing the same is, e.g., composed of a process where a fermentation liquid obtained by adding natto bacteria made by Natto Honpo to the seeds of Sachainchi and rice bran powder is fermented with Monascus bacteria. The glycerol derivative shows an excellent gene restoration action and is suitable for improving cell functions. Further, the derivative is applied as water-soluble and oil-soluble cosmetics, foods and plant activators.SELECTED DRAWING: None

Description

The present invention relates to a glycerol derivative exhibiting a gene repair action.

There are various oxidizing substances, toxins and environmental pollutants in the living environment, and the genes of the living body are damaged. Genes are also damaged by oxidants and saccharified substances produced with aging.

Several types of gene repair methods are recognized. There is a repair function by the combination of DNA polymerase and ligase. Further, there is a repair mechanism by OGG1, that is, 8-oxoguanine DNA glycosylase, which is a repair enzyme of 8-hydroxyl-2-deoxyguanosine, against base damage due to active oxygen.

Furthermore, an SOS repair function via DNA polymerase is also recognized, and various repair functions for genetic disorders exist. Activating these gene repair functions is preferable because it repairs a disorder of a gene caused by aging or a chemical substance and recovers the disease from the gene. Research has been conducted to activate this gene repair function.

As an invention relating to a gene repair function, for example, there is gene repair by in vivo removal of a target DNA, but it is a treatment at a cellular level and its application to living bodies is limited (for example, see Patent Document 1). In addition, in the invention of DNA target modification, gene preparation by a vector is described (for example, see Patent Document 2).

Japanese Patent Application No. 2000-597444 Japanese Patent Application No. 2013-541944

The gene repair action by existing substances is mild, and there is a problem that the industrial use is limited, and the chemically synthesized substance has a problem in safety and its use is limited.

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

In order to achieve the above object, the invention described in claim 1 relates to a glycerol derivative exhibiting a gene repair action represented by the following formula (1).

Since this invention is comprised as mentioned above, there exist the following effects.

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

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

A glycerol derivative exhibiting a gene repair action has a structure represented by the following formula (1).

A glycerol derivative exhibiting a gene repairing action is composed of two molecules of gallic acid and two molecules of a phenol group in one molecule of glycerol as in the above formula (1).

These molecules and their bonds are all naturally occurring in nature, and are crosslinked by ester bonds and ether bonds.

This glycerol derivative can be obtained by chemical synthesis using glycerol, gallic acid, ethanol and phenol as raw materials. However, in the chemical synthesis, the loss of raw materials is large, and the production cost is high, so that the industrial use is limited.

Chemically synthesized high-purity glycerol derivatives are used to obtain analytical standards and trace samples.

It is preferable to analyze the structure of the glycerol derivative from the viewpoint that the active ingredient can be specified. Further, it is preferable because it can be used as an index of the content of an active ingredient when it is used and sold in products and preparations.

As a structural analysis of this glycerol derivative, a high-purity product (purity 98.1%) chemically synthesized using glycerol and gallic acid as raw materials was analyzed by 300 MHz H-NMR in deuterated chloroform (CDCl3) as a standard product. The peak positions are 1.32, 1.39, 1.67, 2.29, 3.55, 3.78, 3.99, 5.80, 5.84, 6.04, 6.13, 6 .16, 6.63, 7.09, 7.24 and 7.53 ppm.

Further, when this high-purity product (purity 98.1%) of this glycerol derivative was analyzed by 300 MHz C-NMR in deuterated chloroform (CDCl3) as a standard product, the peak positions were 16.7, 20.1. 28.6, 43.1, 56.4, 60.9, 61.3, 73.6, 83.4, 84.8, 101.9, 102.0, 112.6, 116.8, 118. 0.0, 120.2, 128.5, 129.3, 132.0, 133.9, 135.0, 137.2, 142.3, 146.0, 148.4, 150.1, 152.5 , 165.1 and 169.8 ppm.

Furthermore, this glycerol derivative is analyzed by high performance liquid chromatography or a mass spectrometer, and its structure is identified. Its chemical formula is C33H32O11. That is, it is composed of 33 carbon elements, 32 hydrogen elements, and 11 oxygen elements.

This component, glycerol, is a naturally occurring compound. The chemical formula for glycerol is C3H8O3. Originally, glycerol is a source of energy that exists as a component of glucose metabolism and triglycerides. Glycerol is also involved in intracellular signal transduction and has an effect on genes and cell membranes.

In this glycerol derivative, glycerol and gallic acid are ester-bonded. That is, the alcoholic hydroxyl groups at positions 1 and 3 of glycerol and the carboxyl group of gallic acid are ester-bonded.

Gallic acid is a naturally occurring polyphenol and has an excellent antioxidant effect. Moreover, it also has a tannin action and is excellent in antibacterial action. Because of its high water solubility, gallic acid alone is thought to be difficult to penetrate into cells.

On the other hand, in this glycerol derivative, gallic acid becomes easy to pass through the cell membrane by binding to glycerol and phenoxy groups. In other words, it reaches into the cell or nucleus. By reaching this, it exhibits an antioxidant effect. In other words, the damaged gene or DNA, that is, the oxidized DNA, works on the oxidized DNA to repair the DNA, which is a gene.

Further, in this glycerol derivative, two molecules of gallic acid exist symmetrically, and each has two hydroxyl groups, a total of four hydroxyl groups, facing each other.

When this hydroxyl group is used as a powder, it exhibits a strong reducing power and generates hydrogen gas when an aqueous solution is added to the powder. The hydrogen gas generated here is not persistent, is transient, and disappears immediately after being used in a living body. That is, the duration of hydrogen gas generation is about 30 minutes.

Since hydrogen gas has a function of damaging genes and scavenging hydroxyl radicals that destroy cells, this glycerol derivative has a function of repairing genes.

In this glycerol derivative, an ethyl group is further bonded to gallic acid, and a phenol group is further bonded to an ether bond. It is preferable for this glycerol derivative that phenol originally has an antiseptic action.

The constituent phenol is a molecular formula of C6H6O1, and is characterized by having a phenolic hydroxyl group. This phenol exists in nature.

The phenol group imparts hydrophobicity to the glycerol derivative and increases the permeability to the cell membrane. As a result, absorption from intestinal cells and permeability to skin cells can be enhanced to exert a gene repair action, which is preferable.

In other words, hydrophobicity and water solubility due to the hydroxyl group of gallic acid are added, and absorption is enhanced by exhibiting amphipathic properties. In the absorption of the intestinal tract, the absorption increases about 3 times as compared with glycerol alone, gallic acid alone or phenol alone.

This glycerol derivative has a function of protecting the gene. It also contributes to gene stabilization. Furthermore, it exhibits radical scavenger action and active oxygen scavenging action to scavenge radicals and active oxygen, thereby reducing gene damage and exerting gene repair action.

Furthermore, it is preferable that acid resistance is generated by the hydroxyl group and phenolic part of the gallic acid of this glycerol derivative, exhibiting resistance to gastric acid and increasing the absorption rate when ingested orally. Moreover, since it is weakly acidic, when applied to the skin, it is preferable that it is not irritating to the skin.

Moreover, since this glycerol derivative is amphiphilic, it is easy to reach | attain in a cell and a nuclear membrane, It is preferable that this glycerol derivative acts directly on the impaired gene. In addition, this glycerol derivative hydrophobically penetrates the nuclear membrane and activates a gene repair enzyme present in the vicinity of the damaged gene. The target gene repair enzymes are DNA polymerase and 8-oxoguanine DNA glycosylase or 8-hydroxyl deoxyguanine DNA glycosidase (all abbreviated as OGG1), and activate these gene repair enzymes.

Among these, gene repair by DNA polymerase is also called SOS repair, which works on damaged bases and adducts of bases, cleaves damaged DNA strands, and replicates normally. Since this DNA polymerase repair is performed 10,000 times or more per day, it is preferable to continue taking and using this glycerol derivative as needed.

Gene repair by OGG1 eliminates oxidized bases such as 8OHdG. Moreover, it integrates in the site | part which excluded the normal base. This glycerol derivative repairs these genes and exerts a gene repairing action by two mechanisms of protecting the genes from oxidizing substances and active oxygen.

This gene repair action by the glycerol derivative works on all cells in which the gene is present in the nucleus and repairs the gene. It also repairs genes in response to gene damage caused by all substances such as active oxygen such as hydroxy radicals, free radicals, ultraviolet rays, chemical substances, pharmaceutical side effects, metals, and aging.

Moreover, since this glycerol derivative exhibits both fat-soluble and water-soluble properties, it easily passes through the cell membrane of animals and the cell walls of plants and yeasts and is easily absorbed into cells. Moreover, since it melt | dissolves in both a water-soluble solvent and an oil-soluble solvent, the point which can utilize a wide solvent is preferable.

Furthermore, it is preferable from the viewpoint of skin health and beauty to maintain the barrier function of the stratum corneum easily through the skin's keratinocyte membrane. This glycerol derivative is preferable because it passes through the cell membrane and activates gene repair in skin cells to promote cell regeneration and function. This glycerol derivative also works against mitochondrial genetic disorders and repairs mitochondrial genetic disorders.

For plants, this glycerol derivative passes through plant cell walls and cell membranes and enters plant cells, promoting the repair of damaged genes, promoting flowering and fruiting, and leaf growth, resulting in plant life. Is preferably extended. In addition, the gallic acid part exhibits an antioxidant action, and the glycerol part promotes plant growth as an energy source. That is, this glycerol derivative functions as a plant activator.

When this glycerol derivative is powdered, it reacts with a water-soluble solvent to generate hydrogen gas and eliminate active oxygen. The amount of hydrogen gas generated is a saturated concentration of 1,6 ppm, and is generated for about 1 minute to 2 hours after dissolution. Hydrogen gas has a function of scavenging hydroxy radicals, and an excellent active oxygen scavenging action has been confirmed.

In addition to acting on gene repair for human skin cells, this glycerol derivative preferably promotes skin cell function by promoting cell growth, collagen and elastin production.

It also activates gene repair in neurons. Nerve cells suffer from dementia, Alzheimer's disease, etc., and are susceptible to gene damage due to active oxygen and amyloid β protein, and the genes of nerve cells are difficult to repair. Therefore, gene repair with this glycerol derivative is preferable for the purpose of restoring the function of nerves and protecting and recovering neurological diseases. It is preferred to enhance neurotransmission by promoting the release of neurotransmitters from nerve endings. In addition, the hydrogen gas generated from this derivative is a small molecule that passes through the blood-brain barrier and restores the function of damaged brain cells and nerve cells.

Furthermore, it is preferable to increase the activity of nerves and muscles by increasing the release of acetylcholine from the end of nerve cells to normalize peripheral nerve transmission by increasing the release of acetylcholine from peripheral nerve cells and motor nerve damage. . The glycerol derivative exhibits a gene repair action on skin cells, and preferably protects collagen and elastin genes to increase their production. It is preferable because use as a cosmetic is increased.

In the case of myocardial infarction, it is preferable that this glycerol derivative activates the activity of the heart by the gene repair action of cardiomyocytes and exerts a cardiotonic action even in the infarct or ischemic state of the coronary artery. Moreover, it is preferable that the hydrogen gas generated at the same time improves the disorder of active oxygen due to ischemia / reperfusion in the myocardium. In particular, in the blood vessel at the infarct site, this glycerol derivative promotes angiogenesis, improves blood flow, and lowers blood pressure.

Further, this glycerol derivative is preferable because it activates energy production in muscle cells during athlete activity, general exercise, and when muscle strengthening is desired. Also, hydrogen gas generated from this derivative during muscle activity is preferred because it eliminates active oxygen during exercise.

This glycerol derivative is decomposed in vivo by kidney and liver esterases and excreted in urine. After being decomposed, it is decomposed into the highly safe components glycerol, gallic acid and phenol. Therefore, this glycerol derivative does not accumulate in the body, is decomposed by in vivo enzymes, and the decomposed product is also a natural product, so it is highly safe. Furthermore, since the glycerol moiety has a plant activating action for promoting plant growth, it is preferable from the viewpoint of industrial use that this glycerol derivative can also promote plant growth.

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

This glycerol derivative is also present in nature, and is found in trace amounts in plants such as sacha inch, orchid and soybean, and seaweeds such as kelp.

It is possible to extract this glycerol derivative from the above plants and algae by purification. However, refining requires a large amount of raw materials and uses organic solvents, which limits the industrial use.

This glycerol derivative is preferably produced by fermenting a seed of Sacha Inch. As a fermentation method, it is preferable to obtain it by mixing it with rice bran and fermenting it with natto or benichow. Since the microbial cells to be used can be used for food, they are highly safe.

This fermentation method is preferred because it has a dietary experience and the production and yield of glycerol derivatives are large.

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

Used as pharmaceuticals as parenterals such as injections, oral preparations or coatings, and quasi-drugs used in tablets, capsules, drinks, soaps, coatings, gels, toothpastes, etc. The

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

Moreover, in the case of the said capsule, liquid carriers, such as fats and oils, can be further contained in said material. In the case of the above syrup and drink, sweeteners, preservatives, pigment flavoring agents 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, fats and oils, lanolin, macro gold, etc. are used as a base material for external preparations, and can be made into ointments, creams, and the like by ordinary methods.

Injections include liquids, and other lyophilization agents. This is used aseptically dissolved in distilled water for injection or physiological saline at the time of use.

As a food preparation, it exhibits gene repairing action, so it is used for energy drinks and nourishing foods. Because it promotes neural activity, it is used for food for nerve rehabilitation through a disorder of a neuronal gene or a meal for learning. It is also used for beauty foods. It is preferable to use it as a health functional food for a nutritional functional food or a food for specified health use.

When the obtained food preparation is used for pets and livestock animals such as dogs and cats, it is used as a feed or pet supplement for the purpose of recovering the damage of animal muscle genes, suppressing aging and improving exercise ability. The

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, gel for hair, facial cleanser, cosmetic liquid, lotion and the like.

The form of the cosmetic is arbitrary, and can be used as a solution, cream, paste, gel, gel, solid or powder. Since this derivative dissolves 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 damaged genes in the skin, increasing collagen and elastin, and maintaining the health of the skin.

This glycerol derivative can also be used in dentifrices, mouthwashes, toothpastes and the like for the purpose of maintaining the function of gingival cells whose genes are impaired.

In addition, it can be used as a plant activator for the purpose of promoting flowering and improving fruit set and yield by restoring genetic damage to plants.

This plant activator can be used for the purpose of promoting the cultivation of flowers such as high-grade and rare orchids, phalaenopsis, roses and pine baluns, and stabilizes the cultivation of fruits, vegetables and cereals. It can also be used for cultivation of vegetables and fruits in plant factories, thus increasing cultivation efficiency.

It is characterized by the process of fermenting the seeds of Sacha Inch. It consists of the process of fermenting the fermented liquor fermented with the addition of Sacha Inch seeds, rice bran powder and Natto Honpo natto bacteria, using Benikouji fungus from Benito Honpo. A method for producing a glycerol derivative represented by the above formula (1) exhibiting a gene repair action will be described.

Here, the glycerol derivative represented by the formula (1) is composed of one molecule of glycerol, one molecule of gallic acid, and two molecules of phenol. These bonds are all natural types, and the substances are bonded through an ester bond or an ether bond.

The glycerol derivatives glycerol, gallic acid, and phenol are preferable because they exist in nature, have abundant food experience, and are recognized as safe.

This derivative also works on skin, nerves, bones, muscles, liver and kidneys, and restores damaged genes and restores tissue and body functions.

This production method comprises a step of fermenting fermented liquor fermented by adding fermented natto fungus from Sacha Inch seeds, rice bran powder and natto Honpo, with Benikouji fungus made from Kurisu Honpo.

The raw materials are Sacha Inch seed, rice bran powder, natto honto natto bacteria, and koji honpo benichouji.

Sachainchi here is the name of Sacha Inchi, the scientific name is Plukenetia volubilis, a perennial plant, and its seeds are used for food. Sacha Inch is native to the Peruvian Amazon rainforest and was cultivated by indigenous people. It has high nutritional value due to its high protein content and lipid content, and also contains gallic acid and phenolic substances, which are antioxidant components, to protect the body.

The raw material used is the seed part of Sacha Inch. Sacha Inch seeds may be any of those collected in Japan, South America, the United States, Asia, and other countries, but the quality is high, and from the viewpoint of price, South American products are preferred because of their good quality.

For example, Peruvian Sacha Inch seeds are imported and used. For example, Sacha Inch Seeds imported by Kunan Service Co., Ltd., Miyakonojo City, Miyazaki Prefecture are preferred because of their high quality.

The seeds of Sacha Inch are preferably dried and pulverized, and autoclaving before fermentation is preferable because fermentation can be performed smoothly.

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

Moreover, the rice bran powder used as a raw material can also be used in rice bran collected from rice (scientific name: Oryza Sativa) of any origin such as Japanese, Chinese, American and Russian. It is preferable to produce in Japan, where traceability is reliable, the use situation of agricultural chemicals can be grasped, and the producer is clear.

Among these, rice bran cultivated organically or without agrochemicals is more preferable because it does not contain harmful agrochemicals and metals.

When using rice bran, a free mill, super free mill, sample mill, goblin, super clean mill, micros, vacuum dryer from Toyo Riko Co., Ltd. It is pulverized by a pulverizer such as a vacuum heat transfer dryer DPTH-40, clean dry VD-7, VD-20 manufactured by AKM Kyushu Technos Co., Ltd., DM-6 manufactured by Nakayama Technical Research Institute. Thereby, the process of fermentation tends to advance efficiently.

Further, it is preferable to sterilize the seeds and rice bran of Sacha Inch after pulverization with an autoclave or the like because they can prevent propagation of various bacteria.

The natto bacterium produced by Natto Honpo is the scientific name Bacillus subtilis, which is widely used in the production of natto in Japan. Bacteria species from Japan such as Okinawa and Kagoshima, and from Southeast Asia such as China and Taiwan are used. The Bacillus natto used is manufactured by Natto Honpo and exhibits high fermentability.

This Bacillus natto promotes the enzymatic binding reaction between glycerol, gallic acid and phenolic substances contained in Sacha Inchi seeds and rice bran.

The amount of each of the above-mentioned fermentations is preferably 0.01 to 4 wt. For rice bran powder and 0.001 to 0.04 wt. It is preferable to pre-cultivate natto bacteria before being fermented, because the initial time of fermentation is shortened and the fermentation time is shortened.

The fermentation is carried out in a clean culture tank, and it is preferable to mix the materials with sterilized tap water.

Moreover, this fermentation is heated at 41-43 degreeC, and fermentation is performed for 14 days from 1 day. It is preferable to quantify the target glycerol derivative by HPLC or TLC, and it is preferable to control the fermentation process by confirming the growth of the bacterial cells because the production amount is adjusted.

The fermentation is carried out in a clean culture tank, and it is preferable to mix the materials with sterilized tap water.

The glycerol derivative produced by this fermentation process is unstable in the formation of natto bacteria and is easily decomposed. Therefore, the next fermentation with Benikouji fungus from Hongpo Honpo is carried out to bind the desired glycerol derivative. Stabilize.

Benikouji fungus made by Benihon Honpo to be used is a filamentous fungus of the scientific name Monascus purpureus and has long been used in fermented foods such as red sake and tofu in Japan, China and Taiwan. In addition, bacterial species from Japan such as Okinawa and Kagoshima, and from Southeast Asia in China and Taiwan are used. Benikouji bacteria manufactured by Kurisu Honpo have excellent fermentation efficiency and high safety.

As for each addition amount regarding the said fermentation, 0.0005-0.06 weight is preferable with respect to 1 weight of said fermented products. It is preferable to pre-cultivate Benikouji fungus made by Kurisu Honpo before fermentation because it shortens the initial time of fermentation and shortens the fermentation time.

The fermentation is carried out in a clean culture tank, and it is preferable to mix the materials with sterilized tap water.

Moreover, this fermentation is heated at 40-44 degreeC, and fermentation is performed for 22 days from 2 days. This fermentation process stabilizes the structure of the glycerol derivative by oxidation and reduction action of Aspergillus niger.

It is preferable that the fermented product is extracted with water-containing ethanol because the product can be efficiently recovered, the bacteria can be sterilized, and the next step can be easily performed. Moreover, since the product is easy to isolate | separate, it is preferable to ultrasonically treat the obtained fermented material. Moreover, it is preferable to concentrate by freeze drying or the like because the following steps can be performed in a short time.

It is preferable to separate and purify the target glycerol derivative from the reaction product from the viewpoint that the intake can be reduced as a highly pure substance. As a purification method, it is preferable to use a purification operation such as a separation resin. In addition, refinement | purification with high purity is obtained by implementing repeatedly or combining refinement | purification.

For example, the target glycerol derivative can be obtained by separation with a separation carrier or resin and fractionation. As the separation carrier or resin, porous polysaccharides, silicon oxide compounds, polyacrylamide, polystyrene, polypropylene, styrene-vinylbenzene copolymers, etc., whose surfaces are coated as described later, are used. Those having a particle size of 0.1 to 300 μm are preferred. The finer the particle size, the higher the accuracy of the separation, but the longer the separation time.

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

The affinity carrier or resin is used for specific preparation of an antigen using an antigen-antibody reaction. A partitionable carrier or resin is used for isolation of a substance such as silica gel (manufactured by Merck) if there is a difference in partition coefficient between the substance and the solvent for separation.

Among these, an adsorbent carrier or resin, a dispersible carrier or resin, a molecular sieve carrier or resin, and an ion exchange carrier or resin are preferable from the viewpoint of reducing production costs. Furthermore, the reverse phase carrier or resin and the dispersible carrier or resin are more preferable because the difference in the distribution coefficient with respect to the separation solvent is large.

When an organic solvent is used as the separation solvent, a carrier or resin having resistance to the organic solvent is used. Moreover, the carrier or resin used for pharmaceutical manufacture or food manufacture is preferable.

From these points, Diaion (HP-20 or HP-21, manufactured by Mitsubishi Chemical Corporation) and XAD-2 or XAD-4 (Rohm and Haas) are used as the adsorptive carrier, and Sephadex LH is used as the molecular sieve carrier. More preferred is -20 (manufactured by Amersham Pharmacia), silica gel as a carrier for distribution, IRA-410 (manufactured by Rohm and Haas) as an ion exchange carrier, and DM1020T (manufactured by Fuji Silysia) as a reverse phase carrier.

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

The obtained extract is dissolved in a solvent for swelling the carrier for separation or the resin before separation. The amount is preferably 2 to 50 times the weight of the extract from the viewpoint of separation efficiency, and more preferably 4 to 20 times the amount. The separation temperature is preferably 10 to 25 ° C., more preferably 12 to 20 ° C. from the viewpoint of the stability of the substance.

As the separation 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 water-soluble solvents, it can be dissolved in animal fats such as vegetable oil, fish oil, lard and the like which are oil-soluble solvents.

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

When Diaion 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.

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

In addition, it is preferable to carry out the final extraction with fats and oils used for edible oils and cosmetics because the glycerol derivative obtained is stably maintained. For example, it is preferable to extract with rice bran oil, rice bran oil, grape seed oil, olive oil or jojoba oil.

In addition, it is preferable to powder this glycerol derivative for the purpose of preserving.

Hereinafter, the embodiment will be specifically described with reference to examples and test examples. These are merely examples, and conditions can be changed within the range of common sense according to differences in materials, raw materials, and specimens.

The seeds of Sacha Inch (scientific name Plukenetia volubilis) cultivated in Peru were purchased from Kunan Service Co., Ltd. in Miyakonojo City, Miyazaki Prefecture. The seeds were washed with tap water, dried in the sun, and pulverized with a pulverizer (Super Free Mill manufactured by Nara Machinery Co., Ltd.) to obtain 1.0 kg of dried powder of Sacha inch seeds.

Also, rice bran (scientific name Oryza Sativa) from Hokkaido was supplied to a mixer (manufactured by Kuisinart) to obtain 1.0 kg of ground rice bran. The sachet seeds and rice bran crushed material were subjected to an autoclave (SDL-320, manufactured by Tommy) and sterilized at 121 ° C. for 20 minutes.

These were put into a clean fermentation tank (sterilized round 40-liter tank for fermentation, manufactured by Endo Kagaku), and 4 kg of sterilized tap water was added and stirred.

Separately, 8 g of powdered natto bacteria (Bacillus subtilis) manufactured by Natto Honpo was supplied to the fermentation tank, and a sterilized rice bran powder and a pre-cultured fermentation preparation liquid were prepared.

It added to the fermentation tank which put the fermented preparation liquid of the said Bacillus natto culture | cultivated above, the dry powder of the seed of sacha inch, and the rice bran, and after stirring, it heated and fermented in the temperature range of 40-42 degreeC.

During the fermentation process, the fermentation liquor was sampled while bubbling and stirring by aeration, and fermented for 6 days. After completion of fermentation, the fermented product was taken out from the fermentation tank and sterilized by boiling. This fermented product was filtered with a filter cloth to obtain 1.2 kg of a fermented solution of Bacillus natto. To 1 kg of this fermentation broth, 10 g of Benikouji fungus (scientific name: Monascus purpureus) manufactured by Kurisu Honpo was added and fermented at 40-42 ° C. for 9 days.

The fermentation was stopped by adding ethanol to the fermented product. Furthermore, it was sterilized by boiling. This was filtered to obtain 0.9 kg of a filtrate, which was the target glycerol derivative. This was also designated as Sample 1.

Furthermore, a purified product was obtained for the purpose of structural analysis and functional experiments. That is, 2 L of purified water containing 5% ethanol was added to 200 g of the above-mentioned glycerol derivative of specimen 1, and 500 g of Diaion (HP-20, manufactured by Mitsubishi Chemical) was suspended and filled in a 5% ethanol solution. It used for the glass column (product made from Endo Kagaku).

To this, 10 L of 5% ethanol solution was added for cleaning, and further, 1 L of 20% ethanol solution was added for washing. Further, 1 L of 50% ethanol solution was added to elute the target glycerol derivative, and this eluate was concentrated. This purification operation was repeated three times, and the ethanol portion was removed from the finally purified glycerol derivative by distillation under reduced pressure to obtain an aqueous solution. This was vacuum dried to obtain 22 g of a purified product of glycerol derivative, which was designated as Sample 2. The yield was about 11%, which was a sufficient yield for production from natural products, and it was confirmed that this production method was an excellent production method.

Below, the test method and result regarding the structural analysis of a glycerol derivative are demonstrated.
(Test Example 1)

The specimen 2 obtained as described above was analyzed by high performance liquid chromatography with a mass spectrometer (HPLC, Shimadzu Corporation). As a result, the detected peak was a single peak and had a purity of 98.8%. In Sample 1, a peak was also observed at the same position, and the purity was 67.8%.

Further, the sample 2 was dissolved in deuterated chloroform (manufactured by Sigma Aldrich) and analyzed by a nuclear magnetic resonance apparatus (300 MHz NMR, manufactured by Bruker). As a result, one sample of glycerol, one molecule of gallic acid, and phenol 1 were obtained from the sample 1 and sample 2. A glycerol derivative consisting of molecules was detected.

That is, as a result of H-NMR measurement, the peak positions are 1.32, 1.39, 1.67, 2.29, 3.55, 3.78, 3.99, 5.80, 5.84, 6 0.04, 6.13, 6.16, 6.63, 7.09, 7.24 and 7.53 ppm. This was the same as the position of the H-NMR peak of the standard product.

Further, when this glycerol derivative was analyzed by 300 MHz C-NMR (13C-NMR) in deuterated chloroform (CDCl 3) using a high purity product (purity 98.1%) as a standard product, the peak position was 16. 7, 20.1, 28.6, 43.1, 56.4, 60.9, 61.3, 73.6, 83.4, 84.8, 101.9, 102.0, 112.6, 116.8, 118.0, 120.2, 128.5, 129.3, 132.0, 133.9, 135.0, 137.2, 142.3, 146.0, 148.4, 150. 1, 152.5, 165.1 and 169.8 ppm. This was also the same as the standard C-NMR result.

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

From the above analysis results, it was found that it had the same structure as the chemically synthesized standard product. Further, as a result of structural analysis such as HPLC, it was confirmed that the target glycerol derivative was obtained by binding one molecule of glycerol, two molecules of gallic acid and two molecules of phenol. Further, when the specimen 2 was powdered, it was dissolved in an aqueous solution, and as a result, 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 effect test using human skin epidermal cells is described below. This test method is a reproducible conventional method that can verify the effects of components biochemically.
(Test Example 2)

Human-derived epidermis cells (derived from epidermis, epider cells) purchased from Kurabo Industries were used. 1000 cells cultured using 5% fetal calf serum-containing MEM medium (manufactured by Sigma) as a culture solution were seeded in a 35 mm culture dish (manufactured by FALCON), and cultured at 37 ° C. under 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). Here, EGF (manufactured by Funakoshi, epidermal growth factor) as a positive control was added at a final concentration of 0.1 mg / ml. This was cultured for 48 hours and tested.

After collecting the culture solution, the survival rate of the epidermal cells was counted under a microscope by trypan blue method. Thereafter, 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. At the same time, the amount of 8-OHdG in the cell suspension was quantified with a kit (manufactured by Japan Aging Control Laboratory). This is an ELISA kit using a monoclonal antibody specific for 8-OHdG. In addition, the petri dish calculated the average value using five sheets. It compared with the solvent control group which added the solvent.

As a result, the addition of 0.1 mg / ml of Sample 1 increased the number of human-derived epidermal cells to 166% as an average value compared to the solvent control group. In Sample 2, it increased to 199%. On the other hand, EGF was 150%. As a result, Specimen 1 and Specimen 2 exhibited a cell activation effect superior to EGF. In addition, when Sample 1 and EGF were added simultaneously, the number of cells was 355%, confirming the synergistic action of Sample 1 and EGF.

The mRNA expression level (copy number) of DNA polymerase in the cells was 12 copies in the solvent control group, 150 copies in the sample 1 treatment group, 433 copies in the sample 2 treatment group, and 101 copies in the EGF treatment group.

The mRNA expression level of DNA polymerase was remarkably higher in Sample 1 and Sample 2, and was superior to EGF. This showed the activation action of the gene repair enzyme by Sample 1 and Sample 2. In addition, in the culture solution to which the specimen 1 and the specimen 2 were added, generation of 1.6 ppm hydrogen gas was confirmed 1 hour after the addition.

The OGG1 mRNA expression level (copy number) in the cells was 19 copies in the solvent control group, 122 copies in the sample 1 treatment group, 663 copies in the sample 2 treatment group, and 79 copies in the EGF treatment group.

The expression level of OGG1 mRNA was high in Sample 1 and Sample 2, and superior to EGF. This indicated that Specimen 1 and Specimen 2 exhibited a gene repair action.

The amount of 8OHdG in the cells was 602 ng in the solvent control group, 90 ng in the sample 1 treatment group, 42 ng in the sample 2 treatment group, and 449 ng in the EGF treatment group. 8OHdG is a mutated state in which the gene is modified with active oxygen, and represents a disorder of the gene. This value was low in Sample 1 and Sample 2, which was superior to the action of EGF. This indicated a gene repair action by Sample 1 and Sample 2.

On the other hand, in a skin irritation experiment using EpiSkin (manufactured by SkinEthic), which is artificial skin, as part of the safety test, no irritation was observed by the addition of Sample 1 and Sample 2, and safety was confirmed. This method has been established as a method for evaluating skin irritation using cells without using animals.

The following describes tests for damage using a human neuronal cell damage model. This test method is a reproducible conventional method that can verify the action of components biochemically.
(Test Example 3)

Human neurons (Human Neurons (HN)) purchased from Cosmo Bio were used. 1000 cells cultured using a dedicated culture solution (neural cell growth medium) as a culture solution were seeded in a 35 mm culture dish and cultured at 37 ° C. in 5% carbon dioxide gas. Nerve cells were stimulated by adding an aqueous acrylamide solution of 1% neurotoxin.

Sample 1 and sample 2 obtained in Example 1 above and NGF (Funakoshi Co., Ltd., human type) as a positive control were added at a final concentration of 0.1 mg / ml. This was cultured for 48 hours.

After completion of the culture, the number of cells was counted under a microscope by trypan blue method. Furthermore, the function of gene repair was verified by the same method as described above. That is, a cell suspension was prepared, and mRNA was extracted therefrom using a nucleic acid extraction kit (manufactured by Funakoshi). According to a conventional method, mRNA of DNA polymerase and 8-oxoguanine DNA glycosylase (abbreviated as OGG1) was quantified by RT-PCR. At the same time, the amount of 8-OHdG in the cell suspension was quantified with a kit (manufactured by Japan Aging Control Laboratory). This is an ELISA kit using a monoclonal antibody specific for 8-OHdG.

As a result, the addition of 0.1 mg / ml of Specimen 1 increased the number of neurons to 139% as an average value compared to the solvent control group. In Sample 2, it increased to 192%. On the other hand, NGF was 135%. As a result, Sample 1 and Sample 2 exhibited a cell activation action superior to NGF. Further, the combined use of specimen 1 and NGF resulted in a cell number of 339%, confirming the synergistic action between specimen 1 and NGF.

The mRNA expression level (copy number) of DNA polymerase in the cells was 18 copies in the solvent control group, 144 copies in the sample 1 treatment group, 633 copies in the sample 2 treatment group, and 88 copies in the NGF treatment group.

The mRNA expression level of DNA polymerase was high in Sample 1 and Sample 2, and superior to NGF. This showed the activation action of the gene repair enzyme by Sample 1 and Sample 2.

The OGG1 mRNA expression level (copy number) in the cells was 20 copies in the solvent control group, 88 copies in the sample 1 treatment group, 329 copies in the sample 2 treatment group, and 44 copies in the NGF treatment group.

The expression level of OGG1 mRNA was high in Sample 1 and Sample 2, and superior to NGF. This showed the activation action of the gene repair enzyme by Sample 1 and Sample 2.

The amount of 8OHdG in the cells was 410 ng in the solvent control group, 104 ng in the specimen 1 treatment group, 39 ng in the specimen 2 treatment group, and 277 ng in the NGF treatment group.

8OHdG is a mutated state in which a gene is modified with active oxygen, and represents a disorder of the gene. This value was low in Sample 1 and Sample 2, which was superior to the function of NGF. This indicated a gene repair action by Sample 1 and Sample 2. In addition, in the culture solution to which the specimen 1 and the specimen 2 were added, generation of 1.6 ppm hydrogen gas was confirmed 1 hour after the addition.

The glycerol derivative obtained in the present invention exhibits a gene repair action and promotes cell functions such as skin cells and nerve cells. This can improve the people's QOL, increase the healthy workforce, and reduce medical costs.

The glycerol derivative obtained in the present invention exhibits a gene repair action, thereby increasing skin cells and contributing to the development of the cosmetic industry by contributing to the improvement of the skin suffering from skin problems such as wrinkles and tarmi as cosmetics. To do.

Since the glycerol derivative obtained by 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.

Since the glycerol derivative obtained in the present invention exhibits a gene repair action on plant cells, it is used as a plant activator that promotes plant growth even in harsh environments, and contributes to agricultural development by growing agricultural products. To do.

Claims (1)

A glycerol derivative exhibiting a gene repair action represented by the following formula (1).