JP6704106B2 - Phenolic derivatives exhibiting cytoplasmic fluidity increasing action - Google Patents

Description

The present invention relates to a phenol derivative exhibiting a cytoplasmic fluidity increasing action.

Functionality and normal growth of cells involve normal gene status, normal energy transfer in cells and normal intracellular signal transduction system, substance transfer and maintenance of cytoskeleton. There is. In particular, protoplasmic fluidity is involved in the maintenance of these cell functions.

Protoplasmic fluidity mainly involves tubulin proteins that control the cytoskeleton, microtubules, intermediate filaments, actin, and myosin, and in addition to these, ATP production, mitochondrial function, and ions such as endoplasmic reticulum and calcium ions. In addition to fluidity, it is closely related to the total function including water.

The discovery of protoplasmic fluidity itself was in the 20th century and is an old research result, but there are many unclear points about its functionality. Therefore, the relationship between cell function and protoplasmic fluidity has not been utilized for research and industry. In addition, identification of activators of protoplast fluidity and substances that act on suppression has not been achieved.

Among them, for example, as an invention relating to a cytoplasm of a cell, there is a composition containing microvesicles derived from a protoplast of a cell, but there is no explanation about the cytoplasmic fluidity in the present invention (for example, Patent Document 1). See 1.).

Further, as an invention relating to a drug discovery screening method and device, there has been invented a method of distinguishing protoplast flow from Brownian movement and determining the activity of cells based on the protoplast flow. It is not an invention relating to the above, but a substance relating to its activation is not described (for example, refer to Patent Document 2).

Special table 2013-534830 Patent No. 4507060

There is a problem that existing substances are not effective in increasing cytoplasmic fluidity and their industrial use is limited, and chemically synthesized substances have safety problems and their use is limited. ..

Therefore, a natural product having weak side effects and an excellent protoplast fluidity increasing action is desired.

In order to achieve the above object, the invention described in claim 1 relates to a phenol derivative represented by the following formula (1) and having a protoplast fluidity increasing action.

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

The phenol derivative according to claim 1 is excellent in the cytoplasmic fluidity increasing action.

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

The phenol derivative exhibiting the protoplast fluidity-increasing action has a structure represented by the following formula (1).

It consists of one molecule of hydroxysesamol, one molecule of erythritol, and one molecule of tyrosine as shown in the above formula (1). All of these bonds are of the natural type and are linked via ester and ether bonds.

This phenol derivative can be obtained by chemical synthesis using hydroxysesamol, erythritol, tyrosine and the like as raw materials. However, its chemical synthesis results in significant loss of raw materials, which limits its industrial application. Chemical synthesis is preferable in order to obtain a standard product or a small amount of sample of this phenol derivative.

Analyzing the structure of this phenol derivative is preferable in 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.

As an example of the structural analysis of this phenol derivative, for example, by H-NMR of 400 MHz in deuterated chloroform, the peak positions are 3.079, 3.432, 3.781, 3.814, 5.727, 6 It is found at 0.021, 6.138, 6.302, 6.549, 6.952, 7.137, 7.376, 7.462, 7.484, 7.846 and 7.901 ppm.

Furthermore, this phenol derivative is analyzed by high performance liquid chromatography or a mass spectrometer to identify its structure.

Hydroxysesamol, which is a constituent, is a hydroxy form of sesamol, which is one of the constituents of sesamin contained in plants such as sesame, and is a phenol that is used in plants and animals. Its chemical formula is C7H6O4.

This hydroxysesamol has hydroxyl groups at the 1- and 3-positions of the complex benzene ring, and has strong antioxidant, antibacterial and enzyme activating effects due to the two phenolic hydroxyl groups. In this phenol derivative, the two hydroxyl groups of hydroxysesamol are both bound, but they bind to tubulin, actin, and myosin, which are related to protoplasmic fluidity, and act to increase the actin-myosin binding and ATP utilization rate. It has an effect of increasing plasma fluidity.

In this phenol derivative, one of the hydroxyl groups of hydroxysesamol is ester-bonded with the carboxyl group of tyrosine. The other hydroxyl group has an ether bond with the hydroxyl group of erythritol.

Hydroxysesamol itself is less toxic and exhibits both water-soluble and lipophilic properties as compared to sesamol. Therefore, it is preferable to exhibit both properties of cell membrane permeation and cytoplasmic lysis.

In addition, it is preferable that the hydroxysesamol portion can pass through the mitochondrial membrane to eliminate active oxygen generated during ATP production from the viewpoint of suppressing cell aging and oxidation. This antioxidant power results in increased cytoplasmic fluidity as well as cell stabilization.

Further, it also has a function of producing useful substances and promoting fermentation of yeasts and useful microorganisms used for fermentation by increasing their protoplast fluidity. This function of promoting fermentation can be utilized in the fermentation process with low reactivity and in the production of substances by fermentation. That is, it is preferable that the production of the rare substance can be promoted by promoting the fermentation having poor reactivity.

Erythritol, which is a constituent component, is a kind of sugar alcohol having the chemical formula C4H10O4. It is a natural, low-calorie sweet substance contained in fruits such as melons, vegetables and natto, and fermented products such as sake and soy sauce.

This erythritol is a natural product and preferably has high safety. Furthermore, since the hydroxyl group of erythritol exhibits a reducing action, it is preferable from the viewpoint of the stability of the components that it contributes to the removal of active oxygen and the stability of the substance.

In this phenol derivative, the phenolic hydroxyl group of hydroxysesamol and the hydroxyl group of erythritol are ether-bonded.

The constituent tyrosine has a molecular formula of C9H11NO3, is of L type, and is of natural type. Tyrosine is a form in which one phenolic hydroxyl group is bonded to phenylalanine and is classified as a nonessential amino acid.

Tyrosine is involved in the action on nerve cells because it is induced by the neurotransmitters dopa, dopamine, noradrenaline and adrenaline. That is, the tyrosine portion is preferable from the viewpoint of protection of peripheral nerves and brain functions.

The phenol derivative tyrosine exerts an antioxidant effect by the phenolic hydroxyl group and is necessary for increasing the plasma fluidity.

Since this phenol derivative exhibits both fat-soluble and water-soluble properties, it passes through the cell membrane and cell wall and is absorbed into cells. This phenol derivative binds to tight junction proteins such as tubulin, actin and myosin, and occludin in the cytoplasm and cell membrane lining proteins to increase protoplasmic fluidity.

This phenol derivative promotes the binding of actin and myosin to promote contraction and relaxation of the cell skeleton, thereby increasing protoplast fluidity. That is, the ATP reaction site of actin and myosin is activated at the binding site of tyrosine and hydroxysesamol of this phenol derivative.

In addition, the structures of the phenol derivatives hydroxysesamol and erythritol increase the sliding and loosening of actin and myosin, strengthen the relaxation and contraction of actin and myosin, and increase protoplasmic fluidity.

Furthermore, the hydroxyl group of erythritol and the amino group of tyrosine activate the active center of the ATP-producing enzyme to increase glycolytic ATP production, thereby increasing the amount of local ATP which is a driving force for the increase of protoplasmic fluidity.

Increased cytoplasmic fluidity causes intracellular ATP, vitamins, minerals, water, proteins, lipids, carbohydrates and other nutrients, enzymes and growth factors, and intracellular AMP components such as cyclic AMP and cyclic Move GMP etc. This migration of nutrients and growth factors increases the reactivity of cells and activates cell functions.

Increased cytoplasmic fluidity also increases cell membrane fluidity, normalizing growth factor receptor migration and reactivity. Furthermore, it is preferable to activate the movement of genes such as transcription factors, enzymes and mRNAs that move in the nuclear pores and to contribute to the proper transmission of gene information, since this leads to normalization and maintenance of cell functions. ..

Since the cytoplasmic fluidity is abnormal in pathological cells and cancer cells, the increase in cytoplasmic fluidity due to this phenol derivative normalizes the cells and improves diseases such as cancer at the cellular level. Phenol derivatives are preferred.

It is also suitable for excretion of toxins and waste products in epithelial cells and liver cells of kidney cells. By increasing protoplasmic fluidity, waste products such as waste products and oxidative substances can be efficiently removed extracellularly. Excrete. This phenol derivative is preferable because it has a detoxifying effect that cells are purified by this excretion.

Furthermore, by increasing the cytoplasmic fluidity of macrophages, monocytes, Langerhans cells, mesangial cells, Kupffer cells, and other phagocytic cells, it promotes the migration and degradation of phagocytic tissues and foreign substances eaten by bacteria and viruses. This phenol derivative is preferable from the viewpoint of increasing immune function and suppressing allergic reaction.

In nerve cells, since axonal transport is associated with protoplasmic fluidity, growth factors such as NGF are transported, and active neurotransmission is preferable because it normalizes the function of nerve cells. This phenol derivative is also preferable in terms of activation of neurotransmission in cerebral nerve cells such as peripheral neuropathy and dementia and excretion of amyloid accumulated in the brain.

In addition, since this phenol derivative has an excellent hydrogen gas generating action and antioxidant ability because it contains abundant hydroxyl groups, it suppresses the production of melanin and brings about a skin whitening action, which makes it useful as a cosmetic. It is preferable because it increases. That is, the powder of the phenol derivative reacts with the water-soluble solvent to generate hydrogen gas. The hydrogen gas concentration of this aqueous solution is 1.6 ppm.

In the myocardial infarction, this phenol derivative exerts an effect in preventing and treating myocardial infarction by activating the mass transfer of myocardial cells even in the coronary infarction or ischemic state. Particularly, in the vascular smooth muscle at the infarct site, this phenol derivative exerts a relaxing action to improve blood flow and blood pressure. It is preferable to generate hydrogen gas because hydrogen gas removes active oxygen generated during ischemia reperfusion.

Further, it is preferable that this phenol derivative enhances muscle activity during exercise by producing ATP through glycolysis in the apnea state. Particularly, when the athlete wants to strengthen the muscle, it is preferable because it enhances the intracellular movement of glucose, vitamins, minerals, and oxygen in muscle cells, transports it to mitochondria, and excretes waste products, carbon dioxide, and active oxygen. Further, since active oxygen is generated in mitochondria, it is preferable that the mitochondria be eliminated by hydrogen gas.

This phenol derivative is decomposed by esterase in the kidney and liver in the body and excreted in urine. It is decomposed into the highly safe components hydroxysesamol, erythritol and tyrosine. Therefore, this phenol 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.

This phenol derivative easily penetrates the fat cell membrane, moves the neutral fat of adipocytes, enhances this decomposition, and also decomposes carbohydrates. Since sugar is consumed, it is also preferable for diabetes prevention and diet control.

Furthermore, this phenol derivative also suppresses inflammation of skin epithelial cells and suppresses the formation of wrinkles. In addition, by stabilizing the keratinocytes, the barrier function of the skin keratin is maintained and the invasion of foreign substances, irritants and bacteria is suppressed. This function can be used as cosmetics.

This phenol derivative activates cells such as macrophages and lymphocytes due to an increase in cytoplasmic fluidity, and thus can be used as an immunostimulant or antiallergic agent. Further, this phenol derivative acts on nerve cells and enhances the electric potential of the cell membrane and nerve conduction, and is thus suitable as a therapeutic agent for dementia, Alzheimer's disease, and Parkinson's disease.

In addition, this phenol derivative is a substance whose decomposed constituents are all present in nature, and its safety has been confirmed because it has abundant experience in eating and use as cosmetics. Furthermore, this phenol derivative activates the cytoskeleton of corneal cells, lens cells, and retinal cells of the eye to restore and accelerate the function of cells due to conjunctivitis, cataract, glaucoma, and retinal detachment, and is used for restoration of visual acuity. It

This phenol derivative exists in nature, and it is found in trace amounts in sesame seeds and the like. It is possible to extract this phenol 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 phenol derivative is preferably used to increase sesame seeds by a fermentation method or the like. As a fermentation method, it is obtained by mixing it with soybeans and fermenting it with Bacillus natto or Beniococci. This method is preferable because it has experience in eating and produces a large amount of phenol derivatives.

When the obtained phenol derivative is used as a pharmaceutical material, it is preferable to purify the target phenol derivative because the purity of the target phenol 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.

As a food formulation, supplements that activate cell function and increase immunity by increasing cytoplasmic fluidity, detox and nourishing tonic foods, beauty supplements that maintain whitening and skin health, nerves, liver and kidneys. It is used in health foods that improve functions, health foods and beauty foods that are used for diets that strengthen muscles and decompose fat. In addition, it is preferable to use it as a nutritionally functional food or a food for specified health care as a food with health claims.

When the obtained food preparation is used for pets such as dogs and cats and domestic animals, it is used as a feed and a supplement for pets for the purpose of strengthening detox, epithelial tissues and muscles of the whole body, and bone cells.

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.

It is preferable that the obtained cosmetic composition promotes the proliferation of skin cells by enhancing the plasma fluidity even in a low oxygen condition, and promotes the production of keratin and collagen to prevent wrinkles and sagging. Furthermore, the whitening effect is exhibited by suppressing the production of melanin by the antioxidant effect.

Further, this phenol derivative exerts an antibacterial action and an antioxidant action due to the phenolic hydroxyl group, and can be used as a toothpaste, mouthwash, toothpaste, etc. for the purpose of suppressing inflammation and growing gingival cells.

Furthermore, it can be used as a fermentation activator that activates fermentation by increasing the protoplasmic fluidity of useful microorganisms and yeasts. With this fermentation activator, it is possible to activate a fermentation stage having low reactivity to produce useful ingredients and foods. For example, in the fermentation process of pharmaceutical raw materials, the production process can be improved at low cost by making the fermentation process efficient.

Further, it can be used as a plant activator that activates the growth of plants by increasing the protoplasmic fluidity of plant cells. This plant activator can be used to grow rare orchids and flowers and activates the cultivation of fruits, vegetables and grains. It can also be used for cultivating vegetables and fruits in plant factories, and can increase cultivation efficiency.

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

Black sesame seeds, which were cultivated in pesticide-free in Kagawa prefecture, were purchased from Michibachi Ichibitei and used. 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 1.1 kg of sesame seed dry powder pulverized product.

Soybeans produced in Hokkaido were subjected to a mixer (made by Cuisinart) to obtain 1.1 kg of pulverized soybeans. The sesame seed and soybean pulverized product were subjected to an autoclave and sterilized at 121° C. for 20 minutes.

These were put in a clean fermentation tank (sterilized round 40 liter tank for fermentation), and 5 kg of sterilized tap water was added and stirred.

Separately from this, 11 g of the powdered Natto bacterium produced by Natto Honpo was provided in a small fermentation tank to prepare a sterilized soybean powder and a pre-fermentation preparation liquid.

The above-mentioned precultured fermentation preparation liquid of Bacillus natto, sesame seed dry powder, and soybean were added to a fermentation tank, and after stirring, the mixture was heated in a temperature range of 40 to 42° C. and fermented.

In the fermentation process, the fermentation liquor was sampled while bubbling and stirring was performed by aeration and fermented for 3 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 1.1 kg of a fermented liquid of Bacillus natto. To 1 kg of this fermented solution, 10 g of Beniojiji bacteria manufactured by Beni-Koji Honpo was added and fermented at 41° C. for 6 days.

Ethanol was added to this fermented product to sterilize it by boiling. This was filtered, and the filtrate was freeze-dried to obtain 0.56 kg of the desired phenol derivative. This was designated as Sample 1. Further, when 0.1 g of the sample 1 was dissolved in 10 mL of purified water, it was measured by gas chromatography (Shimadzu) that 1.6 ppm of hydrogen gas was generated.

Furthermore, a purified product was obtained for the purpose of structural analysis and experiments. That is, 1 L of purified water containing 5% ethanol was added to 200 g of the phenol derivative of the above-mentioned specimen 1, and 500 g of DIAION (manufactured by Mitsubishi Chemical) was suspended in a 5% ethanol solution and applied to a glass column.

To this, 3 L of 5% ethanol solution was added for cleaning, and further, 2 L of 70% ethanol solution was added to elute the desired phenol derivative, followed by concentration and purification. The ethanol portion was removed from the purified phenol derivative by vacuum distillation to obtain an aqueous solution. 22 g of a purified product of the phenol derivative was obtained from this and used as the sample 2. Further, it was confirmed by gas chromatography (Shimadzu Corporation) that 1.6 ppm of hydrogen gas was generated in the aqueous solution when 0.1 g of the sample 1 was dissolved in 5 mL of purified water.

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

The sample 2 obtained as described above was dissolved in ethanol and analyzed by high performance liquid chromatography with a mass spectrometer (HPLC, Shimadzu Corp.).

As a result of analyzing this by a nuclear magnetic resonance apparatus (400 MHz, H-NMR, manufactured by Bruker), a phenol derivative consisting of one molecule each of hydroxysesamol, erythritol, and tyrosine was detected in the sample 2.

That is, in deuterated chloroform of H-NMR, 3.079, 3.432, 3.781, 3.814, 5.727, 6.021, 6.138, 6.302, 6.549, 6. Signals were observed at 952, 7.137, 7.376, 7.462, 7.484, 7.846 and 7.901 ppm.

It was found that the above analysis results have the same structure as the chemically synthesized standard product. That is, it was confirmed from Sample 2 that the target phenol derivative was one ester of hydroxysesamol, one molecule of erythritol, and one molecule of tyrosine.

The confirmation test using human skin epithelial cells is described below.
(Test Example 2)

Human-derived skin epithelial cells purchased from Kurabo Co., Ltd. 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. under 5% carbon dioxide gas. This was irradiated with ultraviolet rays by an ultraviolet ray irradiation device (made by Eye Graphics Co., Ltd.). Further, the above-mentioned samples 1 and 2 and EGF (Funakoshi Co., Ltd., human type) as a positive control were added at a final concentration of 0.1 mg/ml. This was cultured and tested for 48 hours.

The observation of protoplasmic fluidity was performed by analyzing the moving image with a high-sensitivity differential interference microscope and a video system. That is, while the cells treated with the specimen were placed in a culture dish, a cell image was photographed with a high-sensitivity camera at a magnification of 100 times using a microscope (ORCA-Flash 4.0V2) manufactured by Hamamatsu Photonics Co., Ltd. and a Nikon microscope.

The intracellular cytoplasmic fluidity of these cells was observed for 1 minute and edited into a video, and the image was analyzed at low speed to count the cytoplasmic fluidity from the migration distance and migration frequency of intracellular substances. 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 skin epithelial cells was increased to 143% as an average value by the addition of 0.1 mg/ml of Sample 1 as compared with the solvent control group. Moreover, in the case of the sample 2, the amount increased to 211%. On the other hand, with EGF, the increase was 140%, and Sample 1 and Sample 2 were superior.

The protoplasmic fluidity was increased to 166% by the sample 1 as compared with the solvent control group. In addition, the addition of Sample 2 was found to be 334% of the solvent control, which was an increase. With EGF, it was 105%, and the cytoplasmic fluidity-increasing effect of Sample 1 and Sample 2 was remarkable.

Since the cytoplasmic fluidity increasing action is also an index of cell activity, the fact that the glycolytic activity was increased by the treatment of Sample 1 and Sample 2 was confirmed to be the cell activating action of Sample 1 and Sample 2.

The intracellular keratin amount was increased to 155% by the sample 1 as compared with the solvent control group. Further, the addition of the sample 2 resulted in 199% of the solvent control. EGF was 151%, and Sample 1 and Sample 2 were superior to the increase in keratin production. It was confirmed that 1.6 ppm of hydrogen gas was generated even in the culture solution.

The confirmation test using the human nerve cell disorder model is described below.
(Test Example 3)

Human nerve cells (Human Neurons (HN)) purchased from Cosmo Bio were used. As the culture medium, 1000 cells cultured using a dedicated culture medium (neuronal cell growth medium) were seeded on a 35 mm culture dish and cultured at 37° C. under 5% carbon dioxide gas. A 1% acrylamide aqueous solution was added to this to weaken the nerve cells.

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

After culturing, protoplasmic fluidity was measured by microscopic observation as described above in a state where nerve cells were placed in a culture dish. In addition, the number of cells was counted microscopically. 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 addition of 0.1 mg/ml of Sample 1 increased the number of nerve cells to 133% as an average value as compared with the solvent control group. Moreover, in the case of the sample 2, it increased to 193%. On the other hand, with NGF, the increase was 130%, and the samples 1 and 2 were superior.

The cytoplasmic fluidity of nerve cells was increased to 169% by the sample 1 as compared with the solvent control group. In addition, the addition of Sample 2 increased the amount to 266% of the solvent control. With NGF, it was 153%, and Sample 1 and Sample 2 were superior to NGF in the cytoplasmic fluidity increasing action. Furthermore, it was confirmed that 1.6 ppm of hydrogen gas was generated in this culture solution.

The phenol derivative obtained in the present invention increases cytoplasmic fluidity of cells, enhances detox function by generating hydrogen gas, reduces cancers such as skin, liver and nerves, reduces inflammatory diseases and health disorders, and Since there are few side effects, it is possible to improve the QOL of the people, increase the healthy working population, and reduce medical expenses.

Since the phenol derivative obtained in the present invention has an action of improving skin inflammation, it contributes to the improvement of the skin of a person suffering from skin troubles such as wrinkles and sagging as a cosmetic and contributes to the development of the cosmetic industry.

Claims (1)

A phenol derivative represented by the following formula (1), which exhibits a cytoplasmic fluidity increasing action.