JP6241672B2 - Ellagic acid derivative exhibiting antiviral action and method for producing the same - Google Patents

Description

The present invention relates to an ellagic acid derivative exhibiting an antiviral action and a method for producing the same.

A virus is a parasite of about 0.02 μm. Its replication depends on the cell. Viruses have protein and sometimes lipid envelopes and RNA or DNA cores.

To cause an infection, the virus first attaches to the host cell. The viral DNA or RNA is then separated from the envelope and replicated in the host cell.

Examples of viral infections include serious life-threatening infections such as influenza, hepatitis, epidemic cold, AIDS, Ebola fever and shingles.

Anti-inflammatory drugs and antipyretic analgesics are used as symptomatic treatments for viral infections, and antiviral drugs and vaccines are used as causal therapies. The problem with causal therapy is that there are many types of viruses and they are mutated.

As an invention relating to an antiviral agent, for example, there are inventions of an antiviral powder material and an antiviral extract, and an antiviral agent using urushi is provided (for example, see Patent Document 1), but its action is mild. There is a problem of being.

In addition, there is glycolipid AH-2445 having antiviral activity and a method for producing the same. Here, glycolipid exhibiting antiviral activity is described (for example, see Patent Document 2), but its function is still limited. There is a problem that.

Furthermore, the invention of antiviral pharmaceutical composition describes a pharmaceutical composition containing dipyridamole or a pharmaceutically acceptable salt thereof (for example, see Patent Document 3), but is a chemically synthesized substance. There is a problem that side effects are serious.

Japanese Patent No. 2738908 Japanese Patent No. 4203161 No. 2770911

The antiviral effect of existing substances is mild, and there is a problem that their use in industry is limited. In addition, chemically synthesized substances have problems in safety and their use is limited.

Thus, a natural product having a weak side effect and an excellent antiviral effect and a production method for efficiently producing the natural product are desired.

In order to achieve the above object, the invention according to claim 1 relates to an ellagic acid derivative having an antiviral 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 an ellagic acid derivative having an antiviral activity represented by the formula (1) of claim 1 .

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

The ellagic acid derivative according to claim 1 is excellent in antiviral action.

According to the manufacturing method of Claim 2, an ellagic acid derivative can be manufactured efficiently.

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

An ellagic acid derivative exhibiting an antiviral action has a structure represented by the following formula (1).

As shown in the above formula (1), it has a structure in which one molecule of ellagic acid, one molecule of adenine and one molecule of catechol are bonded.

Originally, ellagic acid is a kind of polyphenol contained in plant fruits and the like, and is composed of 14 carbon elements, 6 hydrogen elements and 8 oxygen elements, and has a molecular weight of 302.19 and CAS No. 476-66-4. is there.

It is preferable that ellagic acid is rich in hydroxyl groups and exhibits its antioxidant power and antibacterial action because this derivative can be stably maintained. In this derivative, a cyclic structure containing oxygen is opened to form a carboxylic acid, which increases the reactivity.

Adenine, which is a constituent component, is a base that exists in a gene and exhibits a function of transmitting genetic information. It is composed of 5 carbon elements, 5 hydrogen elements and 5 nitrogen elements, and has a molecular weight of 135.13.

In this derivative, the nitrogen element at the 9-position of adenine is bonded to the phenolic hydroxyl group of ellagic acid. This bond between oxygen and nitrogen is a covalent bond.

The catechol bonded to this derivative is a polyphenol having two hydroxyl groups at the ortho position of the benzene ring, and is composed of six carbon elements, six hydrogen elements and two oxygen elements. It is a naturally occurring polyphenol with a molecular weight of 110.1.

In this derivative, catechol is bonded to the hydroxyl group of the carboxyl group opened in ellagic acid.

Since catechol exhibits a strong antioxidant action and exhibits an antioxidant action, the derivative is preferably maintained stably.

This ellagic acid derivative can be synthesized organically and used as a standard product. The structure was analyzed by H-NMR, and the peak of chemical shift was 2.48 to 2.5, 3.31 to 3.85, 5.48 to 6.72, 7.09 to 8.9 by 90 MHz NMR measurement. 13 and 10.67 to 12.8 ppm.

That is, this ellagic acid derivative is naturally derived and is preferred because its safety has been confirmed. Furthermore, when humans come into contact with or drink excessive amounts of this ellagic acid derivative, it is decomposed by enzymes such as esterase in the body and decomposed into ellagic acid, adenine, and catechol molecules, thus increasing safety. high.

This ellagic acid derivative is preferable because it is easily decomposed by microorganisms in the soil, has no burden on the environment, and does not accumulate.

Since this ellagic acid derivative passes through the cell membrane and further passes through the nuclear membrane and acts on the gene, it is preferable because its action is direct and efficient.

Since this ellagic acid derivative has a high affinity for viruses and is easily incorporated into viruses, it is preferable that the antiviral action is increased.

Furthermore, the antiviral action inhibits the synthesis of viral RNA and DNA, thus suppressing the growth of both RNA viruses and DNA viruses. Therefore, it is preferable because it works on all types of viruses.

On the other hand, since it is decomposed by intracellular enzymes, particularly elastase, in human cells, it is preferable that safety to humans is high.

This ellagic acid derivative works on skin cells and maintains the skin cells stably by antioxidant and antibacterial effects.

Furthermore, this ellagic acid derivative activates the proliferation of skin epithelial cells. The proliferation of skin cells by this ellagic acid derivative is due to the activation of the EGF receptor on the cell membrane.

This ellagic acid derivative is preferable because it stabilizes cells, activates the turnover of skin cells, and causes proliferation of skin cells.

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

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.

It is used as a food preparation for health foods and beauty foods for the purpose of beauty with antiviral and antioxidant effects. 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 maintaining the virus-removing action and skin health.

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 cosmetics maintain skin health by antiviral action, and are used for whitening action and anti-keratosis barrier formation of atopic dermatitis by antioxidant action.

Next, ellagic acid that exhibits an antiviral effect represented by the formula (1), which comprises fermenting with Dokdami leaves, rice bran and Bacillus natto made by Natto Motopo and Benikouji fungus made by Kurisu Honpo and further protease treatment. A method for producing the derivative will be described.

Here, the ellagic acid derivative represented by the formula (1) is the aforementioned derivative in which one molecule of ellagic acid, one molecule of adenine and one molecule of catechol are bonded. Since this ellagic acid derivative is decomposed and excreted by enzymes in vivo, it is highly safe.

This ellagic acid derivative, ellagic acid, exists naturally, has abundant food experience, and is preferred because of its safety.

This derivative suppresses gene replication against viruses and exerts antiviral effects. In addition, it exhibits skin beautifying action due to its antioxidant action.

This production method comprises a step of fermenting with Dokdami leaves, rice bran and Natto Motopo natto bacteria and Benikouji fungus made of Kurisu Honpo, followed by protease treatment.

The raw materials are dokudami leaf, rice bran, natto, benichomycete and protease.

Dokudami has the scientific name Houttunia cordadata, a plant belonging to the genus Dokudami, which grows naturally in Japan and Asia. It is used as a traditional medicine and a folk medicine, and it is also used as tea by drying leaves.

The name of the crude drug is Zyuyaku, which is known for its diuretic action, arteriosclerosis-preventing action, etc., but is not known for its antiviral action. Dokudami leaves are rich in useful ingredients, are used, and are highly safe.

The dokudami may be from either Japan or Asia, and those produced with low pesticides or reduced pesticides are preferred.

It is preferable that the leaf of Dokudami is dried and pulverized, and it is preferable to sterilize by autoclave before fermentation because the fermentation is performed smoothly.

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

Rice bran that is used as a raw material is also used in rice bran obtained from rice in any production area such as Japanese, Chinese, American and Russian. It is preferable to produce in Japan where the traceability is reliable and the producer is clear.

Among these, rice-derived rice bran cultivated organically or without agricultural chemicals is more preferable because it does not contain harmful agricultural chemicals or metals.

In use, rice bran is a free vacuum mill manufactured by Nara Machinery Co., Ltd., a super free mill, a sample mill, a goblin, a super clean mill, a micros, a vacuum vacuum dryer manufactured by Toyo Riko, and a vacuum vacuum manufactured by Matsui Corporation. It is pulverized by a pulverizer such as a 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 dokudami leaf and rice bran by autoclaving after pulverization because it can prevent the propagation of various bacteria.

Natto Bacillus natto is a Bacillus natto commercially available from Natto Motopo, which is preferable in terms of excellent quality and fermentation efficiency. The scientific name is Bacillus subtilis, and it has been used for natto in Japan, China and Taiwan since ancient times, and has abundant food experience.

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

Furthermore, it is fermented by Benikouji fungus made by Kurisu Honpo. Ellagic acid derivatives are formed by fermentation with Bacillus natto and Bacillus subtilis.

The Benikouji fungus made by Benito Honpo to be 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. Benikouji fungus made by Kurisu Honpo has excellent fermentation efficiency.

The amount of each addition related to fermentation is 0.1 to 1.2 weight for rice bran, 1 part for 0.0002 to 0.007 weight for B. cerevisiae, and 0.0001 to 0.005 weight for B. niger. preferable. It is preferable to pre-culture the natto bacteria of Natto Motopo and Benikouji fungus made of Kurisu Honpo 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 38-42 degreeC, and fermentation is performed for 15 days from 1 day. The target ellagic acid derivative is formed by this fermentation process.

After fermentation, it is treated with protease. Proteases are hydrolytic enzymes that break down proteins to produce peptides and amino acids, and are also used as food. Amano's protease N is preferred because of its high enzyme activity.

By adding a protease to the fermented product and heating it, ellagic acid and protein are decomposed into an ellagic acid derivative.

As for the addition amount of protease, 0.003-0.08 weight is preferable with respect to 1 weight of fermented material. The heating temperature is preferably 38 to 44 ° C. The heating time is preferably 1 to 6 hours.

It is preferable that the protease-treated decomposition product is extracted with water-containing ethanol because the product can be efficiently recovered, the protease can be deactivated, 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 ellagic acid derivative from the above reduction reaction product is preferable because 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 ellagic acid 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 is preferably 1 to 30 times, more preferably 3 to 20 times the weight of the extract from the viewpoint of separation efficiency. The separation temperature is preferably 4 to 30 ° C., more preferably 10 to 25 ° 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 ellagic acid derivative and remove the solvent by drying or vacuum drying to obtain the target ellagic acid 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 because the resulting ellagic acid 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 ellagic acid derivative for the purpose of preserving.

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

The leaves from Ehime Prefecture were purchased from Yanagido Pharmacy and used. This was washed with purified water and then put into a dryer to dry.

A koshihikari rice bran from Aichi Prefecture was used in a mixer (Cuisinart) to obtain a rice bran pulverized product. The pulverized product of the leaf and rice bran was 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 12 kg of sterilized tap water was added and stirred.

Here, 10 g of powdered natto bacteria purchased from Natto Motopo was pre-cultured and added to the fermentation tank.

Furthermore, the solution of 7 g of Beniculium bacteria purchased from the pre-cultivated red rice cake main office was added to the fermentation tank, and after stirring, the solution was heated in a temperature range of 40 to 45 ° C. and fermented for 5 days. In the fermentation process, the fermentation liquor was sampled while bubbling and stirring by aeration.

10 g of Amano Protease N was added to 1 kg of the obtained fermented product and heated at 39 to 40 ° C. for 3 hours.

This treated product was heated and ethanol was added to obtain 781 g of the target ellagic acid derivative-containing extract.

1 L of purified water containing 7% ethanol was added to 500 g of the ellagic acid derivative-containing extract described above, and 200 g of Diaion (manufactured by Mitsubishi Chemical) was suspended in a 9% ethanol solution and packed in a column.

To this, 3 L of 9% ethanol solution was added for cleaning, and 2 L of 60% ethanol solution was added to elute the desired ellagic acid derivative for purification. The ethanol part was removed from the purified ellagic acid derivative by distillation under reduced pressure to obtain an aqueous solution. This was designated as specimen 1 of an ellagic acid derivative.

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

The specimen 1 obtained as described above was dissolved in ethanol and analyzed by high performance liquid chromatography with a mass spectrometer (HPLC, Shimadzu Corporation).

Furthermore, it analyzed with the nuclear magnetic resonance apparatus (NMR, the product made from Bruker). As a result of the structural analysis, an ellagic acid derivative was detected from Sample 1. Further structural analysis identified the structure of this ellagic acid derivative.

That is, 1 molecule of ellagic acid, 1 molecule of adenine and 1 molecule of catechol were observed.

From the result of analysis of 90 MHz H-NMR, 2.48, 2.49, 2.50, 3.31, 3.85, 5.48, 5.54, 6.47, 6.60, 6.72. , 7.09, 7.45, 8.11, 8.13, 10.67, and 12.8 ppm, chemical shift peaks were observed. This coincided with the peak of the standard substance, and the target ellagic acid derivative was confirmed.

That is, it was confirmed that the 9-position bond between the hydroxyl group of ellagic acid and adenine and the carboxyl group of ellagic acid opened with catechol was opened.

The confirmation test using human skin epithelial cells is described below.

The cells used were MDCK cells purchased from ATCC. As the culture solution, a 10% FCS-added MEM medium was used. Cells in proliferative phase were used for experiments. The virus used was the influenza virus AoRP8 strain distributed from ATCC.

The virus was grown in MDCK cells, and the supernatant obtained by centrifugation was used as a virus solution (30000 pfu / ml) in the experiment.

It was made to adsorb | suck to the MDCK cell by which petri dish culture | cultivated at 37 degreeC for 40 minutes. A 10% FCS-added MEM medium containing 0.1 ml was added thereto, and cultured at 37 ° C. under 5% carbon dioxide gas for 10 hours. After completion of the culture, the cells were collected and stored at -80 ° C.

As a control, EME medium supplemented with 10% FCS was used. A solution dissolved in 10% FCS-added MEM medium was used so that the final concentration of the specimen 1 was 0.1 mg / ml. As a positive control, zanamivir (manufactured by GlaxoSmithKline) was used at the same concentration.

Virus was quantified by RT-PCR. The aforementioned cells were thawed at room temperature, sonicated, and centrifuged, and then RNA was extracted from the supernatant. RNA extraction / purification was carried out using an RNA extraction / purification kit (Funakoshi).

The extracted RNA was performed with a fluorescence spectrophotometer. That is, a Biorad cDNA kit (iScript) was used for the RT reaction.

After the reaction using the kit, the amount of viral RNA was quantified by real-time PCR using 2 μL of the reaction solution. The experiment was performed 5 times, and the average value of 5 times was obtained and evaluated.

As a result, Sample 1 decreased to 33% compared to the solvent control. This indicates that virus growth was suppressed. On the other hand, zanamivir as a control decreased to 50% as compared with the solvent control, and Sample 1 was superior in antiviral activity.

Next, a confirmation test using human skin epithelial cells will be described.
(Test Example 3)

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 above and EGF (epidermal growth factor) at a final concentration of 0.1 mg / ml as a control were added. This was cultured for 48 hours.

After detaching the cells, the number of cells was counted, a cell suspension was prepared, and the amount of keratin in the cells was measured by ELISA (Wako Pure Chemical Industries). 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 skin epithelial cells to an average value of 388% compared to the control group. In EGF, the increase was 161%, and Sample 1 was superior in the action of increasing skin cells.

The ellagic acid derivative obtained in the present invention contributes to the maintenance of the national health through antiviral action.

Since the ellagic acid derivative obtained in the present invention has the action of improving skin cells, it contributes to the improvement of those suffering from inflammation, atopy and skin trouble as a cosmetic, and contributes to the development of the cosmetic industry.

Since the ellagic acid derivative obtained in the present invention can be used as a food, it contributes to the development of the food industry.

Claims (2)

An ellagic acid derivative exhibiting an antiviral action represented by the following formula (1).

A method for producing an ellagic acid derivative exhibiting an antiviral action represented by the formula (1) of claim 1, further comprising a step of fermenting with Dokudami leaves, rice bran, Bacillus natto, and Benicorii fungus and further protease treatment.