JP2017141497A - Manufacturing method of methyl lanthionine derivative exhibiting elastin increasing effect - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a manufacturing method of a methyl lanthionine derivative exhibiting an elastin increasing activity.SOLUTION: The target methyl lanthionine derivative is constituted by 3-methyl lanthionine, trisaccharide and dipicolinic acid. The methyl lanthionine binds a hydroxyl group of the trisaccharide and exhibits elastin synthesis promotion and elastin decomposition inhibitory activity. The manufacturing method consists of a process for adding Nemacystus decipiens and soybean, adding branched cyclodextrin to a fermentation liquid fermented by Bacillus natto to conduct a protease treatment and then alkali reducing a filtrate. The derivative reacts with a water-soluble solvent to generate a hydrogen gas. It has an elastin production and antiinflammatory activity and is used for cosmetics and foods.SELECTED DRAWING: None

Description

The present invention relates to a method for producing a methyllanthionine derivative exhibiting an elastin increasing action.

Elastin plays an important role in the construction of tissues as part of the extracellular matrix. For example, in blood vessels it works as part of the elastic fiber.

Elastin gives skin tissue elasticity, and the decrease is believed to be the cause of wrinkles. Since elastin decreases with aging, many cosmetics appeal for an increase in elastin.

During inflammation, an elastin degrading enzyme called elastase is induced, which degrades elastin and exacerbates inflammation.

Therefore, cosmetic and pharmaceutical ingredients that increase elastin have been studied, and some have been put into practical use.

For example, there is an invention of a novel plant-derived elastin-like peptide and its peptidomimetic (see, for example, Patent Document 1).

An invention relating to prevention of loss of collagen and / or elastin has also been recognized (see, for example, Patent Document 2).

Furthermore, there is an invention relating to the use of vitamin C or a derivative thereof or an analog thereof for promoting the synthesis of skin elastin (for example, see Patent Document 3).

Chemically synthesized components have a problem that side effects are observed.

On the other hand, a substance derived from a natural product is highly safe, but has a disadvantage that its elastin increasing action is mild and its effect is weak, and its industrial use is limited.

Japanese Patent Application No. 2008-506820 Japanese Patent Application No. 2005-518232 Japanese Patent Application No. 8-519647

As described above, elastin-increasing action by existing natural products is mild, and there is a problem that its industrial use is limited. Also, chemically synthesized substances have safety problems and their use is limited. ing.

Therefore, there is a demand for a production method for efficiently producing a natural product having weak side effects and exhibiting an excellent elastin-increasing action.

In order to achieve the above object, the invention described in claim 1 relates to a method for producing a methyllanthionine derivative exhibiting an elastin increasing action.

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

According to the manufacturing method of Claim 1, the methyllanthionine derivative which exhibits an elastin increase effect | action efficiently can be manufactured.

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

Addition of mozuku and soybean, fermented fermented natto with fermented cyclodextrin and protease treatment, followed by alkali reduction of the filtered filtrate, increase in elastin represented by the following formula (1) A method for producing a methyllanthionine derivative exhibiting an action will be described.

The methyllanthionine derivative here is a conjugate comprising one molecule of methyllanthionine, one molecule of trisaccharide and one molecule of dipicolinic acid. Methyllanthionine is 3-methyllanthionine, and all of these components are contained in natural plants, and their safety has been confirmed.

Methyllanthionine is a kind of fermented product that exhibits antibacterial and anti-inflammatory effects produced by microorganisms, and is a compound modified by binding threonine and cysteine, which are amino acids.

Methyllanthionine acts on membrane parts of bacteria and cells and regulates cell functions. In addition, these amino acids are all components present in the body, and their safety has been confirmed.

All trisaccharides are derivatives of glucose and also contain uronic acid.

Furthermore, dipicolinic acid is a natural product contained in fermented products such as Bacillus natto and has a function of regulating antibacterial action and cell function. This methyllanthionine derivative has high water solubility due to its large number of hydroxyl groups. On the other hand, since it has an affinity for alcohol, it is soluble in ethanol, glycols, glycerins and the like and is industrially useful.

This methyllanthionine derivative has two action mechanisms for increasing elastin. One is the activation action of elastin synthase. The trisaccharide moiety acts on the active center of elastin synthase and promotes elastin production.

The other is the inhibitory action of elastase, an elastin degrading enzyme. This inhibits the degradation of elastin by the methyllanthionine moiety hindering contact with elastin.

These two actions work synergistically to produce and maintain elastin, increasing the amount of elatin. A small amount of this methyllanthionine derivative can be synthesized chemically. The materials are methyllanthionine and amino acid. This chemically synthesized methyllanthionine derivative serves as a production index as a standard substance.

Furthermore, the methyllanthionine derivative powder reacts with a water-soluble solvent to generate hydrogen gas. Hydrogen gas is a low molecular gas that removes active oxygen and exhibits an anti-inflammatory effect. It has high permeability and is used in medicine. Generation of this hydrogen gas is a preferable action.

Analysis of this methyllanthionine derivative by H-NMR is a conventional method for structural analysis, and the peak of chemical shift at 90 MHz is 7.505, 6.608, 5.1, 4.8, 3.8, 3. 6-3.1, 2.97, 2.67, 2.63, 1.4-1.2 ppm.

In addition, this methyllanthionine derivative is decomposed by esterase localized in the cells to be decomposed into a peptide and methyllanthionine, and therefore has no persistence and is highly safe.

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

It is used as an injectable or parenteral agent such as an oral agent or a coating agent as a pharmaceutical, and as a quasi-drug, it is used in a tablet, capsule, drink, soap, coating agent, gel, toothpaste, 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 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 foods intended to increase elastin. 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 feed or supplement for the purpose of maintaining blood pressure.

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 is used as a solution, cream, paste, gel, gel, solid or powder.

The obtained cosmetic maintains the skin function by increasing and maintaining elastin, and is used for prevention and improvement of wrinkles.

This production method comprises the steps of adding mozuku and soybeans, adding a branched cyclodextrin to the fermented liquid fermented with natto bacteria, treating with protease, and then alkali reducing the filtered filtrate.

The raw materials are mozuku powder, branched cyclodextrin, and protease. Mozuku is a general term for seaweeds belonging to the family Mozuku and Nagamatsumidae, among the brown algae class, and is a branched filamentous algae.

In particular, in Japan, the Okinawa mozuku belonging to the department of the family Nagamatsu, the scientific name Cladosiphon Okamaranus and the scientific name Sphaerotricia Divaricata are used for food.

Mosuku's main production areas are Okinawa Prefecture in Japan, and Tonga, USA, Taiwan, and Asia in general.

Since this mozuku leaf or tuberous part is used for food, the leaf or tuberous root is washed with water and then chopped by a cutting machine to obtain a pulverized product. This pulverized product is dried to obtain mozuku powder.

This mozuku powder contains carbohydrates, lipids, proteins and peptide components, as well as methyllanthionine such as methyllanthionine and cinnamic acid. In particular, it is preferable to use the leaf because the leaf portion is rich in methyllanthionine.

The mozuku powder is suspended with the addition of clean water. 10 to 20 liters of water is added to 10 g of mozuku powder and stirred.

The soybean to be used is preferably any of Japanese, American, Chinese, etc. The non-GMP type is preferred because the genetically modified type is difficult to use in Japan.

Mozuku and soybeans are boiled and sterilized and added to the fermentation tank. Soybeans are preferably 8 to 18 g for 10 g of mozuku because fermentation can be performed efficiently.

Fermentation may be either a stationary method or a stirring method, but a stirring method is preferred because fermentation can be performed in a short time.

Fermentation is preferably performed at 39 to 49 ° C. for 12 to 36 hours. When the temperature is low and the time is short, fermentation does not proceed, and when the temperature is high and the time is long, the target methyllanthionine derivative may be decomposed.

The fermentation broth is preferably filtered with a filter cloth or the like because the following steps can be easily performed.

Branched cyclodextrin is added to the filtrate.

A branched cyclodextrin is one of cyclic glucose, and is preferable because glucose is bound cyclically and used in foods and cosmetics. Since this branched cyclodextrin has a hydrophobic portion in the lumen, it is easy to adsorb highly hydrophobic substances. Branched cyclodextrins manufactured by Shimizu Minato Sugar Co., Ltd. are preferred because of their high quality.

The added branched cyclodextrin is preferably 0.5 to 5 g per 1 g of mozuku. This branched cyclodextrin encapsulates the peptide in peptide and methyllanthionine.

The suspension with the branched cyclodextrin is preferably stirred.

Protease is added to the suspension of mozuku and branched cyclodextrin. As the protease to be used, the quality of protease A “Amano” SD, protease M “Amano” SD or protease P “Amano” 3SD, which are food processing proteases manufactured by Amano Enzyme, is preferable because of its stable use.

The protease to be added is preferably 0.02 to 0.1 g per 1 g of mozuku. It is preferable that the protease is suspended in purified water because the reaction proceeds.

This suspension is preferably warmed and stirred to promote the reaction. As heating, 30-45 degreeC is preferable. Moreover, stirring is preferably 10 to 30 times per minute.

This protease reaction solution is filtered. Efficient filtration is achieved by using filter paper or membrane filter. It is preferable because unreacted components and raw materials can be excluded by filtering to obtain a filtrate.

The obtained reaction product is subjected to alkali reduction treatment. The alkali reduction treatment is preferably performed by an alkali reduction device or an alkali reduction water conditioner.

For example, a continuous generator of alkaline reduced water and strong oxidized water “Protech ATX-501” manufactured by Zemitis, an alkali reduced water production apparatus “Techno Super 502” manufactured by NCI, “Mineria CE-212” manufactured by Maltaca, manufactured by Crescent It is more preferable to use devices such as “Acura Blue” and “Mineral Reduction Water Conditioner” manufactured by Nippon Kosen Co., Ltd.

By electrolysis, a conjugate of the desired methyllanthionine derivative, trisaccharide and dipicolinic acid is obtained as a solution from the cathode side. This alkali reduction stabilizes the bond between methyllanthionine and the trisaccharide. In particular, the hydroxyl group of the trisaccharide is protected.

It is preferable because the reaction is enhanced by repeating the alkali reduction treatment 2 to 10 times. The obtained conjugate is powdered by lyophilization and used.

It is preferable to separate and purify the target methyllanthionine derivative from the reduction reaction product from the viewpoint that the intake can be reduced as a highly pure substance. As a purification method, it is preferable to use a purification operation such as a separation resin.

For example, the target methyllanthionine 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 5 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 a methyllanthionine derivative and remove the solvent by drying or vacuum drying to obtain the desired methyllanthionine derivative as a powder or a concentrated liquid because the influence of the solvent can be excluded.

In addition, it is preferable to powder this methyllanthionine 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.

We purchased 10 kg of Okinawa-grown Okinawa Mozuku from the Okinawa Prefecture Mozuku Promotion Council. This was washed with water and then pulverized with purified water in a pulverizer (Super Free Mill manufactured by Nara Machinery Co., Ltd.) to obtain 9 kg of pulverized product.

This pulverized product was dried with a drier to obtain mozuku powder. 120 g of this mozuku powder was transferred to a clean stainless steel cylinder, and 10 L of purified water was added and suspended.

Furthermore, 120 g of Japanese soybean was purchased, washed, added to the above-mentioned cylinder, and sterilized by boiling at 95 ° C. for 1 hour. These were transferred to a stirring tank (FP211) manufactured by Yokogawa Electric Corporation with a capacity of 100 kg and fermented at 42 ° C. for 18 hours.

The obtained supernatant of the fermentation broth was roughly filtered through a filter cloth to obtain a filtrate.

To this filtrate, 120 g of branched cyclodextrin manufactured by Shisui Minato Sugar Co., Ltd. was added and stirred.

Furthermore, 12 g of protease M “Amano” SD manufactured by Amano Enzyme was added, heated to 37 ° C. and stirred for 1 hour.

Stirring was performed for 3 hours at room temperature using a stirrer. The obtained reaction solution was subjected to suction filtration with a filter paper of Toyo filter paper to obtain a filtrate.

The obtained reaction solution was subjected to Pearl Water DX-7000 and electrolyzed to obtain an alkali-reduced solution from the cathode side.

This solution was lyophilized to obtain 20 g of the desired powder. This was designated as Sample 1. As a result of dissolving 0.1 g of Sample 1 in purified water and quantifying it with gas chromatography (manufactured by Shimadzu Corporation), 1.6 ppm of hydrogen gas was generated.

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

The specimen 1 obtained as described above was dissolved in purified water 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 Bruker make, AC-250). As a result of structural analysis, a conjugate in which 3-methyllanthionine, trisaccharide and dipicolinic acid were bound was detected from specimen 1.

As a result of analysis of 90 MHz H-NMR, 1.213, 1.485, 2.633, 2.678, 2.924, 2.979, 3.052, 3.061, 3.122, 3.275, Peaks of 3.28, 3.656, 3.67, 3.879, 4.821, 5.102, 6.608 and 7.505 ppm were observed.

Moreover, the structure by 3-methyllanthionine, dipicolinic acid, and a trisaccharide was identified by the MASS spectrum and IR analyzer (made by Shimadzu Corporation).

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

The elastin production test using human skin cells will be described below. This test is widely used as a method for observing a vasorelaxation response using the action of calcium channels as an index. Normal human-derived skin cells were cultured in a dedicated culture solution. To this, 0.1 mg, 0.3 mg, and 1 mg of sample 1, methyllanthionine, and arginine obtained in Example 1 were added, respectively, and cultured at 37 ° C. for 1 hour.

After counting the number of cells, the cell suspension was sonicated to prepare a cell suspension. The amount of elastin contained in this solution was quantified by ELISA.

As a result, in the 0.1 mg, 0.3 mg and 1 mg of Example 1, the amount of elastin relative to the solvent control group was 156%, 233% and 330%, respectively, and a significant increase was recognized as compared with the control group. In addition, 1 mg of methyllanthionine alone was 100% of the value of the solvent control group, and there was no change.

On the other hand, the number of viable cells was not changed by Sample 1, and toxicity to vascular cells was not observed.

The elastin degradation test using human skin cells is described below.
(Test Example 3)

Purified elastin was purchased from Sigma. Elastin was dissolved in Tris buffer (pH 7.4). This was treated with elastase to decompose elastin, and the degradation rate of elastin was counted using the change in absorbance at 280 nm as an index.

Under this condition, a 1 mg / mL solution of Specimen 1 was added and the degradation rate of elastin was measured.

As a result, when the 1 mg / mL solution of Sample 1 was added as compared with the solvent control, the degradation rate of elastin was reduced to 46%. Specimen 1 was found to have an elastin degradation inhibitory effect.

The methyllanthionine derivative obtained in the present invention exhibits an elastin increasing action and has few side effects. Therefore, it can improve public QOL as an anti-inflammatory agent and reduce medical costs.

Since the method for producing a methyllanthionine derivative obtained in the present invention can also be used as a food, it contributes to the development of the food industry.

The methyllanthionine derivative obtained in the present invention is used for improving wrinkles as a cosmetic and contributes to the development of the cosmetic industry.

Claims (1)

Addition of mozuku and soybean, fermented fermented natto with fermented cyclodextrin and protease treatment, followed by alkali reduction of the filtered filtrate, increase in elastin represented by the following formula (1) A method for producing a methyllanthionine derivative exhibiting an action.