JP2017014173A - INDOLE DERIVATIVE EXHIBITING NF-κB CLASS II SUPPRESSION ACTION AND METHOD FOR PRODUCING THE SAME - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an indole derivative exhibiting an NF-κB class II suppression action, and a method for producing the same.SOLUTION: An indole derivative is a derivative composed of 3-indole ethanol, gluconic acid and adipic acid. The production method is made of a process where the fruit of jujuba and a fermentation liquid obtained by adding Bacillus natto made of Natto Hompo to soybean powder are fermented with Monascus fungus. The obtained indole derivative shows an excellent effect of suppressing NF-κB class II, and suppresses inflammation and cancer. This is applied to cosmetics, food preparations and medicines. In the cosmetics, the NF-κB class II of skin cells is reduced, and it is effective for the control of wrinkles. Further, it guards plants from fungus and virus, and is utilized as a plant controller suppressing inflammation or a plant activator.SELECTED DRAWING: None

Description

The present invention relates to an indole derivative exhibiting an NF-κB class II inhibitory action and a method for producing the same.

In order for cells to exert their functions normally, gene activation and suppression must be controlled. By transcribing and translating gene information, the biological reaction is switched on, but factors that control gene expression are transcription factors and transcription repressors. Examples of this biological reaction are inflammatory reactions and collagen production and loss.

A typical transcription factor related to inflammation and cancer is NF-κB, that is, Nuclear Factor-kappa B, which is also called nuclear factor κB. Various studies and developments have been conducted for the purpose of suppressing and regulating NF-κB. This NF-κB is widely distributed in eukaryotic cells and is involved in cell proliferation and survival.

In many tumor cells, NF-κB is constitutively activated. In particular, NF-κB class II is involved in inflammation. However, development and identification of this NF-κB class II inhibitor and a substance that acts in the control have not been achieved.

As an invention related to NF-κB, for example, although there is a description of a screening method for a compound that inhibits formation of STAT3, NF-κBp65 and p300 complex, no specific component is mentioned (for example, Patent Document 1). reference.).

In addition, there is an invention of an anti-inflammatory composition, in which a myocardial infarction, viral encephalitis, arthritis, psoriasis, inflammatory bowel disease via NF-κB activation suppression, comprising feruloyl serotonin, Pulmonary hypertension, sepsis, type II diabetes, liver fibrosis, nephrosclerosis, endometriosis, AIDS, adult T-cell leukemia, herpes virus infection, EB virus infection, malignant tumor metastasis, bronchial asthma, allergic rhinitis Although a composition for treating or preventing sepsis, autoimmune disease or allograft rejection has been proposed, side effects due to the chemical synthesis are considered and its industrial use is limited. (For example, refer to Patent Document 2).

Japanese Patent No. 4509656 Japanese Patent No. 5141553

NF-κB class II inhibitory action by existing substances is mild, and there is a problem that its use in industry is limited. Also, chemically synthesized substances have problems in safety and their use is limited. Yes.

Therefore, a natural product exhibiting an excellent NF-κB class II inhibitory effect with weak side effects and a production method for efficiently producing the same are desired.

In order to achieve the above object, the invention according to claim 1 relates to an indole derivative having an NF-κB class II inhibitory activity 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 an indole derivative having an NF-κB class II inhibitory action.

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

The indole derivative according to claim 1 is excellent in NF-κB class II inhibitory action.

According to the production method of claim 2, it is possible to efficiently produce an indole derivative exhibiting an NF-κB class II inhibitory action.

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

The indole derivative exhibiting an NF-κB class II inhibitory action has a structure represented by the following formula (1).

As shown in the above formula (1), an indole derivative exhibiting an NF-κB class II inhibitory action is composed of one molecule of 3-indoleethanol, one molecule of sinapinic acid, and one molecule of gluconic acid. All of these bonds are natural types, and each molecule is bonded through an ester bond and an ether bond.

This indole derivative can be obtained by chemical synthesis using 3-indoleethanol, sinapinic acid, gluconic acid and the like as raw materials by chemical synthesis. However, in the chemical synthesis, the loss of raw materials is large and the cost is high, so that the utilization in industry is limited. Chemical synthesis is preferable to obtain a standard product or a small amount of a sample of this indole derivative.

It is preferable to analyze the structure of this indole derivative from the viewpoint that an 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 an example of the structural analysis of this indole derivative, for example, by 90 MHz H-NMR in deuterated dimethyl sulfoxide, the peak positions are 2.949, 3.287, 3.621, 3.653, 3.845, 3.919, 4.024, 5.485, 6.272, 6.658, 6.835, 7.146, 7.167, 7.512, 7.567 and 8.627 ppm.

Furthermore, this indole derivative is analyzed by high performance liquid chromatography or a mass spectrometer, and its structure is identified.

The constituent 3-indoleethanol is an ethanol derivative of indole, which is a naturally occurring compound and is also referred to as tryptohol. It is an analogue of indole acetic acid used in plants and animals. Its chemical formula is C10H11NO and the molecular weight is 161.2.

This 3-indoleethanol is a nitrogen-containing five-membered complex benzene ring, and this part exhibits hydrophobicity. In this indole derivative, the 6-position hydroxyl group of gluconic acid is ether-bonded to the nitrogen part of the 5-membered ring of 3-indoleethanol. This bond imparts a carbohydrate-derived hydroxyl group to the indole derivative, increasing water solubility.

In this indole derivative, the hydroxyl group of the ethanol portion of 3-indoleethanol and the carboxylic acid portion of sinapinic acid are ester-bonded. This binding makes this indole derivative exhibit an NF-κB class II inhibitory action.

In addition, since the 3-indole ethanol moiety has a plant activating action for promoting plant growth, it is preferable from the viewpoint of industrial use that this indole derivative itself can also promote plant growth.

In addition, this indole derivative suppresses NF-κB class II to suppress bacterial infection because NF-κB class II is involved in changes in which plants are infected with bacteria and viruses to cause inflammation and decrease growth. The point of controlling and protecting the plant from infection is preferable.

Gluconic acid, which is a constituent component, is a kind of natural carboxylic acid having the chemical formula C6H12O7, and in this case, is D-form. It is contained in fermented products such as miso and soy sauce, and exhibits antiseptic action due to abundant hydroxyl groups.

Further, it is preferable that the water solubility is increased by the hydroxyl group and the mobility in blood is improved.

This gluconic acid is a natural product and preferably has high safety. Furthermore, since it is more easily absorbed than glucose, it is preferable that this indole derivative is also well absorbed into the body.

Sipinic acid as a constituent component has a molecular formula of C11H12O5 and a molecular weight of 224.21, and is a kind of natural phenylpropanoids. Another name is synaptic acid, which is excellent in antioxidant and skin beautifying effects.

Sinapic acid is also related to the formation of lignin and has excellent binding properties to plant cell walls and cell membranes.

It is a site that increases the permeability of the cell membrane and promotes the movement of this indole derivative.

The sinapinic acid part of this indole derivative exhibits an antioxidant action and is necessary for suppressing NF-κB class II.

Since this indole derivative exhibits both fat-soluble and water-soluble properties, it passes through the cell membrane and cell wall and is absorbed into the cell. This indole derivative passes through the cell membrane, binds to NF-κB class II in the cell, and suppresses the function of NF-κB class II.

Further, this indole derivative passes through the cell wall and cell membrane of the plant and suppresses NF-κB class II to suppress the aging and inflammation of the plant, thereby enhancing the function of the cell. It is also preferable to protect cells from inflammation and stabilize the cell membrane.

This indole derivative binds to type II of NF-κB. That is, the function of NF-κB class II is suppressed. NF-κB class II type is deeply related to transcriptional factors relating to cell growth called RelA and RelB, and it is preferable to suppress the growth of cancer cells and inflammatory cells.

Furthermore, NF-κB class II type II promotes transcription of inflammatory cytokine mRNA. Inflammatory cytokines include interleukin (IL) -1, IL-6, IL-8, TNFα, and the like, and are proteins that are the main components of the inflammatory reaction.

The increase in inflammatory cytokines induces matrix metalloprotease and collagenase to degrade skin collagen and elastin, causing wrinkles and tarmi.

It is preferable that this indole derivative suppresses the induction of inflammatory cytokine because various inflammations and skin wrinkles are improved.

Since NF-κB class II is abnormal in pathological cells and cancer cells, the normalization of the cells by suppression of NF-κB class II by this indole derivative improves cancer and other diseases at the cellular level. Therefore, this indole derivative is preferable.

Indole derivatives are also effective against active oxygen and oxidative reactions in epithelial cells and liver cells of kidney cells due to their antioxidant action. That is, it has an anti-inflammatory action and a cell defense function with different mechanisms for reducing the cause of cell damage by antioxidant power and suppressing cytokine production by NF-κB class II.

Furthermore, it is preferable that the indole derivative reduces atopy and allergy by suppressing NF-κB class II of cells having immunomodulating action such as macrophages, monocytes, Langerhans cells, mesangial cells, and Kupffer cells.

In nerve cells, NF-κB class II is activated by stress, and nerve function decreases. It is preferable that the indole derivative enhances nerve function by suppressing NF-κB class II.

In addition, it is preferable that this indole derivative exhibits an excellent antioxidant power and suppresses the production of melanin to bring about a skin whitening effect because of its increased use as a cosmetic.

This indole derivative improves the damage caused by NF-κB class II and active oxygen in myocardial infarction and ischemic state in myocardial infarction. In particular, in the vascular smooth muscle at the infarct site, this indole derivative exhibits anti-inflammatory and antioxidant effects to improve blood flow and lower blood pressure.

In addition, this indole derivative improves the excretion of waste products and carbon dioxide and the damage caused by active oxygen by suppressing the activation of NF-κB class II in muscle cells and improving inflammation when athletes want to strengthen muscles. This is preferable.

This indole derivative is degraded in vivo by kidney and liver esterases and excreted in urine. It is decomposed and decomposed into highly safe 3-indoleethanol, gluconic acid and sinapinic acid as constituents. Therefore, this indole derivative is not accumulated in the body, is decomposed by an in vivo enzyme, and the decomposed product is also a natural product, which is highly safe.

This indole derivative easily penetrates into the fat cell membrane, moves the adipose cell neutral fat, enhances this degradation, and also degrades carbohydrates. Since carbohydrates are consumed, it is preferable for diabetes prevention and diet countermeasures.

Furthermore, this indole derivative also suppresses inflammation of skin epithelial cells and suppresses the formation of wrinkles. In addition, it stabilizes the keratinocytes to maintain the skin keratin barrier function and suppress the entry of foreign substances, irritants and bacteria. This function can be used as a cosmetic.

This indole derivative also exists in nature and is found in trace amounts in jujube fruits.

This indole derivative can be extracted from the above plant by purification. However, refining requires a large amount of raw materials and uses organic solvents, which limits the industrial use.

It is preferable that this indole derivative increases jujube fruit by fermentation or the like. As a fermentation method, it is obtained by mixing with soybeans and fermenting with Bacillus natto or Bacillus subtilis.

This method is preferred because it has dietary experience and the production of indole derivatives is large.

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

Supplements that suppress inflammation and prevent allergies by suppressing NF-κB class II as food preparations, detox and nourishing tonic foods, increased collagen, beauty supplements that maintain whitening and skin health, nerves, liver It is used in health foods that improve the function of the kidneys, health foods for the purpose of dieting to strengthen muscles and break down fats, and beauty foods. Moreover, it is preferable to use 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 a supplement for pets for the purpose of suppressing inflammation caused by bacterial infection, allergens and foreign substances.

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.

The obtained cosmetic suppresses inflammation by suppressing NF-κB class II, which is a cause of acne and wrinkles due to aging, and promotes production of keratin and collagen. It is preferable to prevent wrinkles and sagging by this. Furthermore, the whitening effect by suppressing the production of melanin by the antioxidant action is exhibited.

This indole derivative exhibits an antibacterial action and an antioxidant action by an indole hydroxyl group, and can be used for a dentifrice, a mouthwash, a toothpaste and the like for the purpose of suppressing inflammation and proliferation of gingival cells.

Moreover, by inhibiting NF-κB class II in plant cells, it can be used as a plant defense against bacterial infections and viruses that are external to plants.

This plant defense agent can be used to protect rare orchids and flowers, 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.

Next, an indole derivative exhibiting an NF-κB class II inhibitory action consisting of a step of fermenting a fermented liquid obtained by adding jujube fruit, soybean powder, and Natto Honpo natto bacteria and fermenting with Benikouji bacteria manufactured by Benito Honpo The manufacturing method will be described.

The indole derivative here is composed of one molecule of 3-indoleethanol, one molecule of sinapinic acid and one molecule of gluconic acid. These bonds are all natural types, and the substances are bonded through an ester bond and an ether bond.

The indole derivatives 3-indoleethanol, gluconic acid and sinapinic acid are preferable because they exist in nature, have abundant food experience, and are recognized as safe.

This derivative also acts on skin, nerves, bones, muscles, liver, kidney, and the like, and enters the cells to suppress NF-κB class II, thereby suppressing inflammation and maintaining cell function.

This production method comprises a step of fermenting a fermented liquid obtained by adding and fermenting jujube fruit, soybean powder and natto honto natto bacteria with Benikouji bacterium from red rice honpo.

The raw materials are jujube fruit, soybean powder, natto bacterium from Natto Honpo, and Benikouji fungus from Benito Honpo.

The jujube here is written as cocoon, and the scientific name is Zizihus Jujuba, which is a tree of the family Aceraceae. The fruits are used as herbal medicines, edible and cosmetic ingredients, and have abundant food experience. Dates are also expressed as date palms.

There is Daegu as a traditional Chinese medicine using jujube fruits, and it exhibits tonicity and sedation. It also shows complementary and descending effects. It is also used in traditional Chinese medicines such as Kakkonto and Akan Otsuyu. Jujube fruit contains organic acids, indoles, and sugars, and is therefore preferred as a raw material for producing this indole derivative.

The fruit of jujube may come from any country such as Japan, China, Taiwan, USA, Australia. In particular, those produced with low pesticides or reduced pesticides are preferred. For example, the fruit of an Australian jujube is sold at Slow Food Kitchen Co., Ltd.

It is preferable that the jujube fruit is dried and pulverized by removing the seeds, and sterilized by autoclave before the fermentation because the fermentation is carried out 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 jujube fruits and soybeans by pulverization and autoclaving or the like because they can prevent the propagation of germs.

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 binding reaction of 3-indoleethanol, sinapinic acid and gluconic acid consisting of jujube fruits and soybeans.

The amount of each of the above-mentioned fermentations is preferably 0.05 to 5 wt. For soybean 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 40-44 degreeC, and fermentation is performed for 1 to 7 days. It is preferable to carry out the process control of the fermentation by quantifying the target indole 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.

Since the indole derivative produced by this fermentation process is unstable in its binding, the next fermentation with Benikouji fungus made by Kurisu Honpo is performed to stabilize the binding of the desired indole derivative.

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 fungus made by Kurisu Honpo has excellent fermentation efficiency.

As for each addition amount regarding the said fermentation, 0.0003-0.03 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 1 to 7 days. This fermentation process stabilizes the structure of the indole derivative 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 indole 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 indole 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 (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 thereof is preferably 2 to 40 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 39 ° C., more preferably 12 to 37 ° 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 an indole derivative and remove the solvent by drying or vacuum drying to obtain the desired indole 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 indole derivatives obtained are stably maintained. For example, extraction with soybean oil, rice bran oil, grape seed oil, olive oil or jojoba oil is preferred.

In addition, it is preferable to powder this indole 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.

Jujube fruits grown in Australia without chemicals were purchased from a slow food kitchen and used. The fruit was 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 pulverized product of jujube fruit.

Hokkaido soybeans were supplied to a mixer (manufactured by Kuisinart) to obtain 1.0 kg of soybean pulverized material. The jujube of the said jujube fruit and soybean was used for the autoclave, and it sterilized for 20 minutes at 121 degreeC.

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, 10 g of powdered natto bacteria manufactured by Natto Honpo was used in a small fermentation tank, and a sterilized soybean powder and a precultured preparation liquid were prepared.

It added to the fermentation tank which put the fermented preparation liquid of the said Bacillus natto cultured above, the dried powder of jujube fruit, and soybeans, and after stirring, it heated and fermented in the temperature range of 41-42 degreeC.

In the fermentation process, the fermentation liquor was sampled and fermented for 3 days while bubbling and stirring by aeration. After completion of fermentation, the fermented product was taken out from the fermentation tank and sterilized by boiling. This fermented product was filtered through a filter cloth to obtain 0.9 kg of a fermented solution of Bacillus natto. To 1 kg of this fermentation broth, 10 g of Benikouji fungus made by Kurisu Honpo was added and fermented at 40 ° C. for 3 days.

Ethanol was added to the fermented product and sterilized by boiling. This was filtered to obtain a target indole derivative as a filtrate. 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 6% ethanol-containing purified water was added to 200 g of the indole derivative of Sample 1 described above, and 500 g of Diaion (manufactured by Mitsubishi Chemical) was suspended in a 6% ethanol solution and packed into a glass column.

2 L of 6% ethanol solution was added thereto for cleaning, and 1 L of 65% ethanol solution was added to elute the desired indole derivative, followed by concentration and purification. The purified indole derivative was distilled under reduced pressure to remove the ethanol portion to obtain an aqueous solution. From this, 46 g of a purified product of indole derivative was obtained, and this was used as Sample 2.

Hereinafter, test methods and results relating to the structural analysis of indole derivatives will be described.
(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 analyzing this with a nuclear magnetic resonance apparatus (90 MHz, H-NMR, manufactured by Bruker), an indole derivative consisting of one molecule each of 3-indoleethanol, gluconic acid and sinapinic acid was detected from specimen 1 and specimen 2. It was done.

For example, the results of H-NMR in deuterated dimethyl sulfoxide are 2.949, 3.287, 3.621, 3.653, 3.845, 3.919, 4.0024, 5.485, 6.272. , 6.658, 6.835, 7.146, 7.167, 7.512, 7.567, and 8.627 ppm.

The above analysis results were found to exhibit the same structure as a chemically synthesized standard product. That is, it was confirmed from Sample 2 that the molecule was a target indole derivative in which one molecule of 3-indoleethanol, one molecule of gluconic acid, and one molecule of sinapinic acid were ether-bonded and ester-bonded.

The NF-κB class II inhibitory action test using human skin epithelial cells is described below. This test method is a reproducible conventional method that can verify the action of components biochemically.
(Test Example 2)

Human-derived skin epithelial cells purchased from Kurabo Industries Co., Ltd. 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 by an ultraviolet irradiation device (manufactured by Eye Graphics Co., Ltd.) to give oxidative stress. To this, specimen 1 and specimen 2 and human-derived EGF (epidermal growth factor, Funakoshi Co., Ltd., human type) were added at a final concentration of 0.1 mg / ml as a positive control. This was cultured for 48 hours and tested.

Quantification of NF-κB class II was performed by the RT-PCR method. That is, the cells were detached with a trypsin solution and homogenized with a hypotonic solution. MRNA was extracted from this cell disruption solution. Micro-Fast Track 2.0 mRNA Isolation Kit was used for extraction.

  Quantification of NF-κB class II was performed by RT-PCR (Prime Script RT-PCR kit, manufactured by Takara Bio Inc.) using this mRNA as a material.

Furthermore, the amount of interleukin-6 (IL-6) in the cell suspension was quantified using a sandwich ELISA quantification kit manufactured by Takara Bio.

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 Specimen 1 increased the number of skin epithelial cells to 152% as an average value compared to the solvent control group. In Sample 2, it increased to 188%. On the other hand, EGF increased by 140%, and Sample 1 and Sample 2 were superior.

NF-κB class II was reduced to 33% by specimen 1 compared to the solvent control group. In addition, the addition of Sample 2 showed a decrease of 18% of the solvent control. EGF was 105%, and no change was observed.

Since the inhibitory action of NF-κB class II is also an indicator of genetic inflammation, the suppression of NF-κB class II in the treatment of specimen 1 and specimen 2 was confirmed. The amount of mRNA of class I type NF-κB class II did not change in either sample 1 or sample 2.

The amount of intracellular IL-6 was reduced to 51% by Sample 1 compared to the solvent control group. Moreover, it became 22% of the solvent control by the addition of the sample 2. EGF was 101%, and Sample 1 and Sample 2 were more excellent in reducing IL-6.

The following describes an inflammation suppression test using a human nerve cell injury 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. To this, 1% acrylamide aqueous solution was added to stimulate nerve cells.

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 microscopically. Furthermore, the amount of NF-κB class II and IL-6 was quantified according to the method described above in a state where the cells were placed in a culture dish. 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 Specimen 1 increased the number of neurons to 128% as an average value compared to the solvent control group. In Sample 2, it increased to 188%. On the other hand, NGF increased by 126%, and Sample 1 and Sample 2 were superior.

NF-κB class II of nerve cells was reduced to 46% by specimen 1 compared to the solvent control group. In addition, the addition of specimen 2 decreased to 26% of the solvent control. NGF was 102%, and Sample 1 and Sample 2 were more excellent in NF-κB class II inhibitory action than NGF.

The amount of mRNA of class I type NF-κB class II did not change in either sample 1 or sample 2.

The indole derivative obtained in the present invention suppresses NF-κB class II of cells, exhibits an anti-inflammatory action, and reduces cancers such as skin, liver and nerves, inflammatory diseases and health disorders. Furthermore, since it is derived from natural products and has few side effects, it can improve the QOL of the people, increase the healthy working population, and reduce medical costs.

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

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

Claims (2)

An indole derivative exhibiting an NF-κB class II inhibitory action represented by the following formula (1).
A method for producing an indole derivative exhibiting an NF-κB class II inhibitory action comprising a step of fermenting a fermented liquor, fermented with jujube fruit, soybean powder and natto honto from Natto Honpo with Benikouji bacterium from Benito Honpo .