JP6490997B2 - Jewenol A derivative exhibiting ATP increasing action and method for producing the same - Google Patents

Description

The present invention relates to a Gewenol A derivative exhibiting an ATP increasing action and a method for producing the same.

ATP is a cell energy source, and is a low-molecular phosphate compound that regulates cell maintenance, cell reinforcement, intracellular transport of substances, protoplasmic flow, gene expression, intracellular signal transduction, and the like. It is produced from lipids, carbohydrates and proteins via enzymes in the cytoplasm or mitochondria.

ATP is also found in epithelial cells such as skin, liver, gastrointestinal tract, nerves and muscles, and these epithelial cells also exert cell functions using ATP.

ATP is also a raw material for cAMP and is also involved in intracellular signal transduction. A decrease in ATP means a decrease in cell function. Further, the lack of the action of metabolizing calories to produce ATP causes calories to change to fat, resulting in accumulation of neutral fat and obesity.

Increasing ATP enhances cell function, increases cell functionality, and leads to health maintenance and dieting. Therefore, various studies have been conducted on substances that increase ATP. There is an invention of an ATP production promoter composed of tricaffeoylquinic acid to promote ATP production in nerve cells (see, for example, Patent Document 1), and ATP production of tricaffeoylquinic acid is shown. However, its degree is mild and its use in industry is limited.

Further, there are ATP production promoters and epidermal cell activators (see, for example, Patent Document 2), in which turmeric, hibermata, licorice, kina, apricot, quince, clara, gardenia, sanshishi, kumazasa, grapefruit, gennoshouko, Black tea, green tea, cordyceps, burdock, rice bran, shiitake mushroom, red snapper, perilla, bodaiju, ginger, firewood, dodami, birch, horsetail, horned lye, tamakitsutsu rafuji, mulberry, tokinsenka, dairyo, matsuhodo, bucha An ATP production promoter characterized by containing one or more extracts selected from the group as an active ingredient has been proposed, but the degree of activity is mild.

Japanese Patent No. 5403582 Japanese Patent Application No. 2008-109372

The effect of increasing ATP by existing substances is mild, and there is a problem that the industrial use is limited. In addition, chemically synthesized substances have a problem in safety and are limited in use.

Therefore, a natural product that exhibits an excellent ATP increasing action with low side effects and a production method for efficiently producing the natural product are desired.

In order to achieve the above object, the invention described in claim 1 relates to a Jewenol A derivative having an ATP increasing action represented by the following formula (1).

In order to achieve the above object, the invention described in claim 2 relates to a method for producing a Jewenol A derivative having an ATP increasing action.

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

The Gewenol A derivative according to claim 1 is excellent in ATP increasing action.

According to the manufacturing method of Claim 2, a jewenol A derivative can be manufactured efficiently.

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

The Jewenol A derivative exhibiting an ATP increasing action has a structure represented by the following formula (1).

As shown in the above formula (1), it consists of one molecule of Jewenol A and two molecules of phloroglucinol. These bonds are all natural types, and are bonds via hydroxyl oxygen.

Jewenol A is a natural product classified as a diterpene, and Jewenol B is an analog. The molecular formula of Jewenol A is C20H34O4 and the molecular weight is 338.49. Phloroglucinol is a kind of polyphenol that is present in plants and exhibits antioxidant and antibacterial effects. It is also used as a medicine and exerts antispasmodic action. The molecular formula is C6H6O3 and the molecular weight is 126.11.

This Jewenol A derivative shows a shape in which the phenolic hydroxyl group of phloroglucinol is bonded to the alcoholic hydroxyl group of Jewenol A. It has 4 hydroxyl groups and exhibits a strong antioxidant effect. In addition, it is easily dissolved in water, alcohol, oil and organic solvents, and is easy to use industrially.

This Jewenol A derivative easily penetrates into cell membranes, mitochondrial membranes and nuclear membranes and has a function of stabilizing the cell membranes. In particular, it has a high affinity for the mitochondrial membrane, prevents mitochondrial oxidation, and suppresses oxidation of vitamins, thereby increasing ATP production.

If the amount of this Jewenol A derivative is small, it can be synthesized by a chemical synthesis route. Organic synthesis is possible while protecting the hydroxyl groups of Jewenol A and Phloroglucinol. However, since the cost is high and the influence of the solvent is likely to occur, this production method is limited in industrial use. However, it can be used for collecting reference materials.

This Jewenol A derivative was identified by structural analysis of a standard product, and the chemical shift was 0.28 to 1.4, 2.22 to 2.908, 3 in heavy water in the 400 MHz H-NMR of the standard product chemically synthesized. .134, 3.308, 3.640, 3.779, 6.541, 6.616, 7.028, etc., and a characteristic peak is observed.

This Gewenol A derivative easily penetrates into muscle cells, increases ATP production in muscle cells, and enhances muscle activity. Since calories are consumed, it reduces sugars and fats and exhibits a dieting effect.

In addition, this Jewenol A derivative increases ATP production of skin epithelial cells and promotes skin differentiation and proliferation. By increasing ATP of skin epithelial cells, wrinkles and tarmi are suppressed. It exerts an excellent cosmetic effect by strengthening muscle cells and proliferating skin epithelial cells.

This Jewenol A derivative also acts on nerve cells and enhances nerve cell proliferation and transmission by activating nerve cells through an increase in ATP. This function is also suitable for dementia, Alzheimer's disease, and Parkinsonism.

This Jewenol A derivative is decomposed and metabolized to Jewenol A and phloroglucinol by esterases present in the liver and kidney, so that even if an excessive amount is ingested or applied, there are few side effects due to the excess.

In addition, this Jewenol A derivative is a substance that exists in nature, and its safety has been confirmed because of its abundant experience in food use and cosmetics.

Furthermore, this Jewenol A derivative reinforces the structure by increasing the ATP of corneal cells, lens cells, muscle tissue and optic nerve of the eye, and is used to restore visual acuity.

ATP production by these Jewenol A derivatives is preferable in that it can be used in combination with other pharmaceuticals and raw materials by a mechanism that stabilizes and antioxidants the mitochondrial membrane and enhances the electron transport system by NADH, FAD and iron.

This Jewenol A derivative also exists in nature and is found in trace amounts on the leaves of Akamegasiwa. It is possible to extract this Jewenol A derivative from the above plant by purification.

However, since refining requires a large amount of raw materials, its use as an industrial method is limited.

Although this Jewenol A derivative can be obtained from plants such as red-leafed wrinkles, it is preferable to increase it by fermentation or the like because of its low content. As a fermentation method, mixing with soybean and fermenting it with natto or benichomycete is preferable because it has a dietary experience and has a large increase.

When the obtained Jewenol A derivative is used as a pharmaceutical material, it is preferable to separate and purify the target Jewenol A derivative from the viewpoint of increasing the purity of the target Jewenol A derivative and removing impurities.

Used as pharmaceuticals, parenteral preparations such as injections, oral preparations and coatings, and quasi-drugs used in tablets, capsules, drinks, soaps, coatings, gels, toothpastes, etc. Is done.

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 also 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.

Supplements that provide functionality by increasing ATP production as food preparations, nutritional and tonic foods, beauty supplements that maintain skin health, health foods that improve nerve, liver and kidney functions, muscle strengthening, fat It is used in health foods and beauty foods for the purpose of dieting to break down food. Moreover, as a health functional food, it is preferable to use it for a nutrition functional food or a food for specified health.

When the obtained food preparation is used for pets such as dogs and cats and livestock animals, it is used as a feed or supplement for the purpose of strengthening epithelial tissues, muscles, and bone cells due to an increase in ATP.

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.

It is preferable that the obtained cosmetic increases the ATP of skin epithelial cells to strengthen the skin structure, prevent wrinkles, and prevent sagging.

Moreover, it can utilize for a dentifrice, a bath agent, etc., when a Gewenol A part exhibits an antibacterial action.

Next, a method for producing a Jewenol A derivative exhibiting an ATP increasing action consisting of a step of fermenting a fermented liquid obtained by adding a fermented soybean powder, soybean powder and natto bacteria with Beniculium fungi will be described.

The Jewenol A derivative here is a substance composed of one molecule of Jewenol A and two molecules of phloroglucinol, and these bonds are all natural types and are bonds through oxygen of hydroxyl groups. Jewenol A derivatives promote ATP production by stabilizing mitochondrial membranes and antioxidants of vitamins.

The Jewenol A derivatives Jewenol A and phloroglucinol are preferable because they exist naturally, have abundant food experience, and are recognized as safe.

This derivative acts on epithelial cells such as skin, nerves, bones and muscles, increases ATP, and strengthens tissues.

This production method comprises a step of fermenting a fermented liquid obtained by adding fermented leaves, soybean powder and natto bacteria and fermenting them with B. niger.

The raw materials are red leaves, soybean powder, Bacillus natto, and B. niger.

Akamegasiwa here is the scientific name Mallotus Japanicus and is also described as red buds and red buds. It is a deciduous tree belonging to the genus Akamegasiwa, and the bark is also used as a raw material for pharmaceuticals. This leaf is used as an edible food and becomes a seasoning. In particular, young leaves are suitable for food.

Akamegawashi leaves contain pigments, polyphenols and Jewenol A, which is preferable as a raw material for producing this Jewenol A derivative.

The leaves of Akagasiwa may come from any country such as Japan, China, Taiwan and the United States. In particular, those produced in Japan with low pesticides or reduced pesticides are preferred.

It is preferable that the leaves of Akamegasiwa are dried and pulverized, and sterilization by autoclave 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.

The soybean powder used as a raw material can be used in soybeans of any origin such as Japanese, Chinese, American, and Russian, but is preferably Japanese because of its reliable traceability and clear producers.

Of these, soybeans cultivated organically or without agricultural chemicals are more preferred because they do not contain harmful agricultural chemicals or metals.

When using soybeans, Nara Machinery Co., Ltd. free mill, super free mill, sample mill, goblin, super clean mill, micros, small vacuum dryer manufactured by Toyo Riko as vacuum dryer, small size manufactured by Matsui 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.

Furthermore, it is preferable to sterilize the leaves and soybeans of red algae after pulverization with an autoclave or the like because they can prevent the propagation of various bacteria.

The Bacillus natto used 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.

This Bacillus natto promotes the binding of Jewenol A and Phloroglucinol, which are made of red leaves and soybeans.

As for each addition amount regarding the said fermentation, 0.05 to 4 weight of soybean powder and 0.001 to 0.03 weight of Bacillus natto are preferable with respect to 1 weight of dry powder of the leaves of Akagasiwa. 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 39-44 degreeC, and fermentation is performed for 30 days from 2 days. It is preferable to carry out the process control of the fermentation by quantifying the target Jewenol A derivative by HPLC or TLC and confirming the growth of the cells.

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

Although Jewenol A and phloroglucinol are bound by this fermentation process, the next fermentation by Beniculium fungus is performed because the bond is unstable.

The fungus used is a filamentous fungus of the scientific name Monasuc 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.

As for each addition amount regarding the said fermentation, 0.0001-0.004 weight is preferable with respect to 1 weight of said fermented products. It is preferable to pre-cultivate B. subtilis before fermentation 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 37-42 degreeC, and fermentation is performed for 15 days from 1 day. This fermentation process stabilizes the bond between jewenol A and phloroglucinol by the reducing 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.

Separating and purifying the target Jewenol A derivative from the reduction reaction product is preferable 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.

For example, the target Jewenol A derivative is 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 (Mitsubishi Chemical Co., Ltd.) and XAD-2 or XAD-4 (Rohm and Haas) are used as the adsorptive carrier, and Sephadex LH-20 (Amersham Pharmacia) is used as the molecular sieve carrier. Silica gel as the distribution carrier, IRA-410 (Rohm and Haas) as the ion exchange carrier, and DM1020T (Fuji Silysia) as the reverse phase carrier are more preferable.

Of these, Diaion, 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 1 to 30 times the weight of the extract from the viewpoint of separation efficiency, and more preferably 4 to 25 times. The separation temperature is preferably 6 to 32 ° C, more preferably 10 to 22 ° 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.

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 Jewenol A derivative and remove the solvent by drying or vacuum drying to obtain the target Jewenol A derivative as a powder or a concentrated liquid because the influence of the solvent can be excluded.

Moreover, it is preferable to carry out the final extraction with fats and oils used for edible oils and cosmetics, since the obtained Jewenol A derivative is stably maintained. For example, extraction with soybean oil, rice bran oil, grape seed oil, olive oil or jojoba oil is preferred.

Further, it is preferable to powder this Gewenol A 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.

Akamegashiwa leaves grown in Nagasaki Prefecture with reduced pesticides and organic fertilizers were purchased from Kurama Next Generation Council and used. The leaves 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 a dried powder pulverized product of Akega wrinkle leaves.

Soybeans from Hokkaido were used in a mixer (manufactured by Kuisinart) to obtain 1.7 kg of soybeans. The pulverized leaves and soybeans were put in an autoclave and sterilized at 121 ° C. for 20 minutes.

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

Separately, 12 g of powdered natto bacteria manufactured by Natto Honpo was used in a small fermentation tank to prepare a sterilized soybean powder and a precultured culture solution.

It added to the fermentation tank which put the culture solution of the said natto bacteria previously cultured, the dried powder of the leaf of a red kegweed, and soybean, and after stirring, it heated and fermented in the temperature range of 40-42 degreeC.

In the fermentation process, the fermentation liquor was sampled while bubbling and stirring by aeration. After completion of fermentation, the fermented product was taken out from the fermentation tank and sterilized by boiling. The fermented product was filtered through a filter cloth to obtain 4.1 kg of a fermented solution of Bacillus natto. To 1 kg of this fermented solution, 12 g of Benikouji fungus made by Kurisu Honpo was added and fermented at 37 ° C. for 2 days.

Ethanol was added to the fermented product and sterilized by boiling. This was filtered to obtain a target Jewenol A derivative. This was designated as Sample 1.

Furthermore, a purified product was obtained for the purpose of structural analysis and experiment. That is, 1 L of 7% ethanol-containing purified water was added to 50 g of the above-mentioned Jewenol A derivative of Sample 1, and this was applied to a glass column filled with 500 g of Diaion (manufactured by Mitsubishi Chemical) suspended in 7% ethanol solution. .

To this, 2 L of 7% ethanol solution was added for cleaning, and 1 L of 60% ethanol solution was added to elute the target Jewenol A derivative, followed by concentration and purification. The purified Jewenol A derivative was distilled under reduced pressure to remove the ethanol portion to obtain an aqueous solution. This was designated as Sample 2 by 13 g of a purified product of Gewenol A derivative.

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

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

However, as a result of analysis with a nuclear magnetic resonance apparatus (400 MHz, H-NMR, manufactured by Bruker), a Jewenol A derivative comprising Jewenol A and phloroglucinol was detected from the specimen 2.

That is, the chemical shift of H-NMR is 0.286, 1.14, 2.227, 2.573, 2.908, 3.134, 3.308, 3.64, 3.779, 6.335, The peaks at 6.541, 6.616, 6.753, 6.906, 7.028 and 7.141 ppm were exhibited.

Since the above analysis results were found to exhibit the same structure as a chemically synthesized standard product, it was confirmed that Sample 2 was a Jewenol A derivative in which Jewenol A1 molecule and two phloroglucinol molecules were ether-bonded. .

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

Human skin epithelial cells purchased from Kurabo Industries Co., Ltd. were used. As a culture solution, 1000 cells cultured using 5% fetal calf serum-containing MEM medium (manufactured by Sigma) were seeded in a 35 mm culture dish, and cultured at 37 ° C. under 5% carbon dioxide gas. To this, specimen 1 obtained in Example 1 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 for 48 hours.

After detaching the cells, the number of cells was counted, the cell suspension was adjusted, and the amount of ATP in the cells was measured by an ELISA method using an anti-ATP antibody (low molecule, acidic type). In addition, the petri dish calculated the average value using five sheets.

As a result, the addition of 0.1 mg / ml of Specimen 1 increased the number of epithelial cells to an average value of 167% compared to the solvent control group. In Sample 2, it increased to 233%. On the other hand, EGF increased by 160%, and Sample 1 and Sample 2 were superior.

About ATP amount, it increased to 254% by the sample 1 compared with the solvent control group. Further, the addition of specimen 2 resulted in 426% of the solvent control. In EGF, it was 231%, and the amount of ATP produced in Sample 1 and Sample 2 was remarkable.

The confirmation test using human liver cells is described below.
(Test Example 3)

Human liver cells purchased from Kurabo Industries Co., Ltd. were used. As a culture solution, 1000 cells cultured using a dedicated culture solution were seeded in a 35 mm culture dish, and cultured at 37 ° C. under 5% carbon dioxide gas. To this, 1% ethanol was added to weaken human liver cells.

To this, Specimen 1 obtained in Example 1 and HGF (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 detaching the cells, the number of cells was counted, the cell suspension was adjusted, and the amount of ATP in the liver cells was measured by ELISA using an anti-ATP antibody (low molecule, acidic type). In addition, the petri dish calculated the average value using five sheets.

As a result, the addition of 0.1 mg / ml of Sample 1 increased the number of liver cells to 166% as an average value compared to the solvent control group. In Sample 2, it increased to 228%. On the other hand, the increase in HGF was 155%, and Sample 1 and Sample 2 were superior.

About ATP amount, it increased to 170% by the sample 1 compared with the solvent control group. In addition, the addition of specimen 2 resulted in 338% of the solvent control. With HGF, it was 156%, and the amount of ATP produced in Sample 1 and Sample 2 was remarkable.

The Jewenol A derivative obtained in the present invention strengthens epithelial cells and epithelial tissues through an increase in ATP, reduces health problems such as skin, liver, nerves, etc. and has few side effects. Improve, increase the healthy workforce, and reduce medical costs.

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

Since the Jewenol A derivative obtained in the present invention can also be used as food, it contributes to the development of the food industry.

Claims (2)

A Jewenol A derivative exhibiting an ATP increasing action represented by the following formula (1).
A method for producing a Jewenol A derivative having an ATP increasing action comprising a step of fermenting a fermented liquid obtained by adding a fermented soybean powder and soybean powder and Bacillus natto and fermenting with B. niger.