JP6049200B2 - Mycosporine-like amino acid and method for producing the same, UV protection agent, and antioxidant - Google Patents

Description

  The present invention relates to a mycosporin-like amino acid that can be applied to various fields, a method for producing the same, an ultraviolet protective agent, and an antioxidant.

  Conventionally, a mycosporine-like amino acid (MAA) is known as an amino acid derivative having a structure in which cyclohexenone or cyclohexenimine is a basic skeleton and an amino acid or an amino alcohol is bound thereto. Mycosporine-like amino acids are widely distributed in marine organisms such as seaweeds, marine animals, microalgae, and fungi. In addition, mycosporine-like amino acids are water-soluble compounds that have a maximum absorption wavelength in the UV-A, B region near the wavelength of 300 to 400 nm, and marine organisms, as an ultraviolet absorber in the body, suppress damage caused by ultraviolet rays. It is considered to be possessed to do so.

  As disclosed in Patent Document 1, as mycosporin-like amino acids, for example, sinoline, porphyra-334, parisin and the like are known. Many of these mycosporin-like amino acids are contained in red algae such as seaweed. Patent Document 1 discloses a configuration in which an antioxidant substance is produced from a red algae extract by microbial fermentation and applied to food and drink. Patent Document 2 discloses a configuration in which an anti-oxidant is generated by heating a mycosporin-like amino acid obtained from algae under predetermined conditions and applied to food and drink.

JP-A-10-77472 JP 2008-247901 A JP-A-9-149774

  By the way, as disclosed in Patent Document 3, conventionally, the genus Nostock (genus Nostoc) is known as one of the genus of cyanobacteria. As the genus Nostock, for example, hair vegetables (Nostoc flagelliforme), katsusen rice (Nostoc sphaericum), rainbow jellyfish (Nostoc commune) and the like are known. The genus Nostock contains abundant proteins, vitamins, polysaccharides and various minerals, and has been used for food since ancient times in Asia such as China and Japan, and South America such as Peru.

  The present invention is based on obtaining a new mycosporin-like amino acid from the genus Nostock, a kind of cyanobacteria. An object of the present invention is to provide a mycosporine-like amino acid that can be applied to various fields and a method for producing the same. Another object is to provide an ultraviolet protective agent and an antioxidant that can be applied, for example, to the fields of pharmaceuticals and foods and drinks.

  In order to achieve the above object, according to one embodiment of the present invention, a mycosporine-like amino acid is a compound having a structure represented by the following general formula (1) or a salt thereof.

In another aspect of the present invention, the ultraviolet protective agent contains the mycosporine-like amino acid as an active ingredient. Another aspect of the present invention is characterized in that the antioxidant contains the mycosporine-like amino acid as an active ingredient.

  Moreover, in another aspect of the present invention, there is provided a method for producing a mycosporine-like amino acid that is a compound having a structure represented by the following general formula (1) or a salt thereof, water, a hydrophilic organic solvent, or water and hydrophilicity The method comprises a step of extracting an extract containing the mycosporin-like amino acid from Nostock using a mixed solution with an organic solvent, and a step of isolating the mycosporin-like amino acid from the extract.

  ADVANTAGE OF THE INVENTION According to this invention, the mycosporin-like amino acid which can be applied to various fields, its manufacturing method, a UV protective agent, and an antioxidant can be provided.

NMR data of Compound 1 purified by Test Example 1. 1 is a diagram showing the structure of Compound 1 purified by Test Example 1. FIG.

Hereinafter, embodiments embodying the present invention will be described in detail.
The mycosporine-like amino acid of this embodiment has a structure represented by the following general formula (1).

The novel mycosporin-like amino acid represented by the general formula (1) is 13-O- (β-Galactosyl) -Porphyra-334 in which galactose is β-bonded to position 13 of Porphyra-334 (Porphyra-334), and the molecular formula C ₂₀ H ₃₂ N ₂ O ₁₃ , molecular weight 508.47.

  This novel mycosporin-like amino acid has an excellent ultraviolet protective effect and antioxidant effect. Therefore, it can be preferably applied as an ultraviolet protective agent or an antioxidant for the purpose of obtaining those effects.

  When the novel mycosporine-like amino acids of the present embodiment are used as UV protection agents, these novel mycosporin-like amino acids are used as active ingredients (UV protection materials) of the UV protection agent. When applying a new mycosporine-like amino acid as a UV protection agent, specific formulations include skin external preparations, pharmaceuticals, quasi-drugs, cosmetics, foods and drinks for the purpose of obtaining UV protection effects, It can also be applied as a test / research reagent. The UV protective agent can effectively exert its action and effect, particularly when applied to the skin surface. Therefore, among the above blended forms, it is preferably applied as a cosmetic or external preparation for the purpose of obtaining an ultraviolet protective effect. Moreover, you may comprise as an eye drop for the purpose of prevention of the eye disease which is caused by ultraviolet rays, such as a cataract.

  When an ultraviolet protective agent is applied to a cosmetic, it can be produced by blending it into a cosmetic base material. The form of the cosmetic may be any of emulsion, cream, powder and the like. By applying such cosmetics to the skin, an ultraviolet protection effect can be obtained. Cosmetic bases are generally blended in common with cosmetics, and include, for example, oil, purified water and alcohol as main components, surfactants, moisturizers, antioxidants, thickeners, anti-oxidants. At least one selected from seborrheic agents, blood circulation promoters, whitening agents, pH adjusters, pigments, preservatives, and fragrances may be appropriately blended.

  In the case where the UV protection agent is used as a medicine or quasi-drug, the form of an external preparation for skin is preferably employed. Although it does not specifically limit as a dosage form, For example, an ointment, a liquid agent, a spray agent, a sheet agent, a powder, and a powder are mentioned. Moreover, you may mix | blend an excipient | filler, a base, an emulsifier, a solvent, a stabilizer etc. as an additive. In addition, when UV protection agents are used as pharmaceuticals or quasi-drugs, all administration methods such as subcutaneous injection, intravascular administration, and transdermal administration are adopted in addition to administration by eye drops, taking (oral intake). It is possible. Although it does not specifically limit as a dosage form, For example, a powder, a powder agent, a granule, a tablet, a capsule, a pill, a suppository, a liquid agent, an injection, etc. are mentioned. Moreover, you may mix | blend an excipient | filler, a base, an emulsifier, a solvent, a stabilizer etc. as an additive.

  When an ultraviolet protective agent is applied to a food or drink, the ultraviolet protective agent can be used as a food or drink itself or by blending it with various food materials or beverage materials. The form of the food or drink is not particularly limited, and may be any of liquid, powder, gel, and solid, and the dosage form is any of tablets, capsules, granules, and drinks. May be. Among these, a capsule is preferable because hygroscopicity is suppressed and storage stability is excellent. As said food-drinks, you may mix | blend gelatinizer containing foodstuffs, saccharides, a fragrance | flavor, a sweetener, fats and oils, a base material, an excipient | filler, a food additive, a subsidiary material, a bulking agent etc. suitably as another component.

  When the novel mycosporine-like amino acids of the present embodiment are used as antioxidants, these novel mycosporine-like amino acids are used as active ingredients (antioxidant materials) of the antioxidants. Antioxidants are food / beverage products (beverages) that effectively prevent deterioration of various products (mainly oxidative deterioration) such as oxidative degradation of fats and oils, perfume degradation, pigment degradation, and pigment fading. Alternatively, it can be used by adding it to food) or pharmaceuticals. Moreover, an antioxidant removes active oxygen in the living body ingested orally, and exhibits a health promotion effect by using it in food and drinks or pharmaceuticals such as health foods. Furthermore, antioxidants can be used in cosmetics, external preparations for skin, or quasi-drugs, and are expected to prevent the aging of the skin, oral cavity, etc. in addition to preventing oxidation of the contained components. Is done. Further, it may be applied as a test / research reagent for the purpose of obtaining an antioxidant action. The specific configuration of each form of these foods and drinks can employ the same configuration as the ultraviolet protective agent described above.

Next, the effect | action of the novel mycosporin-like amino acid comprised as mentioned above is demonstrated.
The novel mycosporin-like amino acid of this embodiment is 13-O- (β-Galactosyl) -Porphyra-334 in which galactose is β-bonded to position 13 of porphyra-334 of the general formula (1). Since this novel mycosporin-like amino acid has an excellent ultraviolet protective effect and antioxidant effect, it can be used for various types of applications such as external preparations for skin, cosmetics, pharmaceuticals, foods and drinks, and the like.

Next, the manufacturing method of the novel mycosporin-like amino acid comprised as mentioned above is demonstrated.
The novel mycosporin-like amino acid represented by the above general formula (1) can be obtained through the extraction process and the isolation process using the genus Nostock, which is one of the genus of cyanobacteria, as an extraction raw material. Moreover, you may synthesize | combine using a well-known organic chemical synthesis method (a semi-synthesis is included). Examples of the genus Nostock used as the raw material for extraction include Nodoc flagelliforme, Nostoc sphaericum and Nostoc commune. Among these, Nostoc sphaericum containing a large amount of the component of the general formula (1) is preferably applied as an extraction raw material.

  The method for extracting the novel mycosporin-like amino acid represented by the general formula (1) from the extraction raw material is based on a known extraction solvent such as water, a hydrophilic organic solvent, or a mixture of water and a hydrophilic organic solvent. Examples include a method for extracting a dissolved component and a supercritical extraction method using water, carbon dioxide gas, or the like as a supercritical fluid. Among these, an extraction treatment method using an extraction solvent capable of efficiently extracting a novel mycosporine-like amino acid is preferably applied. As the hydrophilic organic solvent used in the present embodiment, ethers, lower alcohols such as ethanol, methanol, and isopropanol, ketones such as acetone and methyl ethyl ketone, chloroform, ethyl acetate, and hexane are appropriately selected and used. be able to. These hydrophilic organic solvents may be used alone or in combination of two or more. Among these, a lower alcohol with high extraction efficiency of the present application component is preferable. The extraction solvent may contain a small amount of a solvent other than water and a hydrophilic organic solvent, and other additives such as organic salts, inorganic salts, buffers, and emulsifiers may be dissolved. .

  As the extraction method, any known extraction method such as cooling extraction, room temperature extraction, and heating extraction may be used. The extraction temperature can be appropriately set according to the type of solvent, extraction efficiency, deterioration of components, and the like.

  The extraction operation is performed by immersing the Nostock genus as a raw material in an extraction solvent for a predetermined time. At that time, the concentration of the raw material in the extraction solvent is appropriately set according to the type of solvent, extraction efficiency, efficiency of concentration treatment after extraction, and the like. At the time of extraction, the genus Nostock as a raw material may be appropriately pulverized from the viewpoint of increasing the extraction efficiency.

  In such an extraction operation, a treatment such as a stirring treatment, a pressure treatment, and an ultrasonic treatment may be further performed as necessary in order to increase the extraction efficiency. Moreover, extraction operation may be performed only once with respect to the same raw material, and may be performed in multiple times. Then, by performing a solid-liquid separation operation after the extraction operation, the extract (extract) and the raw material residue are separated. As a method of the solid-liquid separation treatment, for example, a known separation method such as filtration or centrifugation can be used. In addition, the obtained extract (extract) may be appropriately concentrated or dried as necessary.

  The isolation step is a step of isolating and purifying the novel mycosporin-like amino acid contained in the extract obtained in the extraction step. The novel mycosporin-like amino acid is isolated by purifying the extract using one or more chromatography. As the chromatography, known chromatography such as liquid chromatography, supercritical fluid chromatography, and thin layer chromatography can be used. As liquid chromatography, column chromatography can be used, for example, and more specifically, high performance liquid chromatography (HPLC) and open column chromatography can be mentioned. Examples of the chromatography carrier include ion exchange chromatography, partition chromatography (normal phase / reverse phase chromatography), adsorption chromatography, and molecular exclusion chromatography. More specifically, activated carbon, celite, silica gel carrier, ODS carrier, silica gel carrier having an octyl group, and the like can be used as a chromatography carrier. The novel mycosporin-like amino acids can be isolated and purified by a known method of use by appropriately combining these chromatographies. The novel mycosporin-like amino acid can be identified by structure determination or by using a purified product as an index. In addition, during the isolation / purification, the above-described ultraviolet protective action and antioxidant action may be used as indicators.

According to the novel mycosporin-like amino acid of the above embodiment, the following effects can be obtained.
(1) The novel mycosporin-like amino acid of the present embodiment is 13-O- (β-Galactosyl) -Porphyra-334 in which galactose is β-bonded to position 13 of porphyra-334 in the general formula (1). Since this novel mycosporin-like amino acid has an excellent ultraviolet protective effect and antioxidant effect, it can be used for various types of applications such as external preparations for skin, cosmetics, pharmaceuticals, foods and drinks, and the like.

  (2) The ultraviolet protective agent containing the novel mycosporin-like amino acid of this embodiment contains the novel mycosporin-like amino acid having a high ultraviolet protective effect as an active ingredient. Therefore, the effect can be effectively exhibited especially by application to the skin surface. Thereby, prevention of various skin diseases caused by ultraviolet rays is expected.

  (3) Moreover, when the ultraviolet protective agent containing the novel mycosporin-like amino acid of the present embodiment is configured as eye drops, prevention of eye diseases caused by ultraviolet rays, such as cataracts and keratitis, is expected. .

  (4) The antioxidant containing the novel mycosporin-like amino acid of this embodiment contains the novel mycosporin-like amino acid having a high antioxidant action as an active ingredient. Therefore, deterioration of components, such as food / beverage products, cosmetics, a skin external preparation, a pharmaceutical, or a quasi-drug, can be prevented and storage stability can be improved. Moreover, the health promotion effect and the antiaging effect can be exhibited by oral ingestion or percutaneous administration.

  (5) The novel mycosporine-like amino acid of this embodiment is preferably a natural material, Nostock, as an extraction raw material. Therefore, since it is derived from a natural component, it can be safely applied to various uses.

In addition, you may change the said embodiment as follows.
-The novel mycosporine-like amino acid of the above embodiment is a non-human animal, for example, domestic animals (non-human mammals) such as horses, cows and pigs, poultry such as chickens, or pets such as dogs, cats, rats and mice. It may be applied to (various domestic animals).

  -The salt of the compound represented by General formula (1) may be used for the novel mycosporin-like amino acid of this embodiment. For example, the alkali metal salt of a carboxyl group is mentioned.

Next, the embodiment will be described more specifically with reference to examples and comparative examples.
<Test Example 1: Purification of a novel mycosporin-like amino acid>
(1) Extraction treatment Nostoc sphaericum belonging to the genus Nostock was used as an extraction raw material. A novel mycosporin-like amino acid was purified as an ultraviolet absorbing substance from the genus Nostock.

  First, 10 g of Nostoc sphaericum was placed in a 500 mL medium bottle. Next, 300 mL of 75% ethanol (distilled water: ethanol (1: 3, v / v)) was added to the medium bottle and stirred overnight. Thereafter, solid-liquid separation was performed by suction filtration, and the solid content was extracted again in the same manner using 75% ethanol. The filtrate obtained by the first and second extraction treatments was placed in a 1 L eggplant flask and evaporated to prevent bumping. After substantially removing the solvent ethanol, it was transferred to a separatory funnel, extracted with butanol 2-4 times, and degreased by taking the lower layer (aqueous layer). Next, the organic solvent was removed with a rotary evaporator, and the residue was lyophilized.

(2) Activated carbon column chromatography First, the freeze-dried residue was dissolved in 10 mL of distilled water to prepare a column processing sample. Next, about 15 g of activated carbon for chromatography (manufactured by Wako Pure Chemical Industries, Ltd.) and 15 g of celite (Celite 545, manufactured by Wako Pure Chemical Industries, Ltd.) are mixed and suspended in ethanol, an Econo column (manufactured by BioRad, 2.5 cm in inner diameter and 30 cm in length) ) And washed with distilled water for 1 L or more.

  Next, the column processing sample was applied to the column, and an eco-gradient pump (manufactured by BioRad) was used. Distilled water at a maximum flow rate of 10 minutes, distilled water-80% ethanol (distilled water: ethanol (2: 8, It was run with a gradient program of linear gradient elution according to v / v)) for 60 minutes and 80% ethanol for 20 minutes. Fractions were taken every 2 minutes and fractions with UV 334 nm absorption were collected in each fraction. The collected sample was lyophilized.

(3) Purification by HPLC First, the freeze-dried sample was dissolved in 10 mL of distilled water to prepare a sample for HPLC processing. Next, Develocil C8 column (particle size 3 μm, inner diameter 4.6 mm × length 250 mm, manufactured by Nomura Chemical Co., Ltd.), mobile phase (0.05% acetic acid, 5% methanol, balance water), flow rate 0.7 mL / min Using a high performance liquid chromatograph according to the separation conditions, the corresponding peak (elution time around 7 to 9 minutes) was collected while monitoring the ultraviolet absorption at 334 nm. Next, using a rotary evaporator, methanol as a solvent was removed from the peak fraction under a temperature condition of 40 ° C. The remaining residue was lyophilized.

  Next, the lyophilized residue was re-purified again under the same conditions as in the HPLC. The peak fraction obtained as described above (hereinafter referred to as Compound 1) was freeze-dried.

<Test Example 2: Determination of structure of novel mycosporin-like amino acid>
The chemical structure of Compound 1 obtained by the above method was clarified using a nuclear magnetic resonance apparatus (hereinafter referred to as NMR).

(1) Sample preparation First, 1 mL of heavy water (99.8% D) solvent was added to the freeze-dried sample (compound 1) purified by the above chromatography and dissolved well. Next, 0.6 mL of the solution was transferred to a 5 mm diameter NMR tube. Next, 0.05 μL of acetone was added as a reference substance for chemical shift, mixed well, the tube was capped, and a parafilm was wound around the cap.

(2) Measurement by NMR As an apparatus, an NMR apparatus with an 800 MHz ( ¹ H) triple resonance ( ¹ H, ¹³ C, ¹⁵ N) type cryogenic probe was used.

First, an NMR sample tube for measurement was inserted into the apparatus. Next, deuterium lock, tuning and shimming were performed. Next, a one-dimensional NMR spectrum and various two-dimensional NMR spectra were acquired using a standard pulse sequence attached to the apparatus. Acetone (δ _H : 2.22 ppm (CH ₃ ), δ _C : 30.89 ppm (CH ₃ )) was used as an internal standard substance. The measurement temperature was 298K. The conditions shown in Table 1 below were applied as measurement conditions for the one-dimensional NMR spectrum and the two-dimensional NMR spectrum.

Data analysis was performed and the results are shown in FIG.

(3) Structural analysis by liquid chromatograph / mass spectrometer The chemical structure analysis and molecular weight of compound 1 were estimated using a liquid chromatograph / mass spectrometer (hereinafter referred to as LC-MS). A liquid chromatograph / quadrupole time-of-flight mass spectrometer (LC-qTOFMS) was used as an apparatus. The conditions shown in Table 2 below were applied as analysis conditions.

From the above results, Compound 1 has a molecular weight of 508.47, a molecular formula of C ₂₀ H ₃₂ N ₂ O ₁₃ , a structure shown in Formula (2) of FIG. 2, and is a novel mycosporin-like amino acid 13-O -(β-Galactosyl) -Porphyra-334 was identified.

<Test Example 3: Measurement of UV protective effect>
The novel mycosporin-like amino acid (13-O- (β-Galactosyl) -Porphyra-334) obtained as described above was measured for the effect of preventing damage to human skin cells by ultraviolet rays (UVA and UVB) according to the following method. . For comparison, synoline, porphyra-334, mycosporin-glycine, and paricin, which are amino acids for mycosporin, were used as positive controls.

(1) Measurement of protective action against injury of human epidermal keratinocytes HaCaT cells by UVA First, human epidermal keratinocytes HaCaT (DS Pharma Biomedical) was used in 10% fetal bovine serum / Dulbecco's modified Eagle medium (DMEM). In culture. Next, the cell concentration was adjusted to 2.5 × 10 ⁵ cells / mL. Next, it seed | inoculated at 100 microliter / well (25000cells / well) to the 96 well microplate. Next, the cells were cultured for 24 hours, and the dose of the irradiator was confirmed before use.

Replaced with 100 μL of PBS (−) containing 100 ng / mL of 8-methoxypsoralen (8-MOPS) and 0.1 μM, 10 μM, 100 μM, and 1000 μM of the test substance, respectively, and UVA (using Toshiba's black light) Was irradiated at 1 J / cm ² .

After 48 hours, 10 μL of Cell Counting Kit-8 (manufactured by Wako Pure Chemical Industries, Ltd.) was added and incubated at 37 ° C. in a 5% CO ₂ incubator for 1-2 hours, and then the absorbance at 450 nm was measured.

Since the absorbance at 450 nm and the number of cells are positively correlated, the cell viability (the number of cells not irradiated with UVA as 100) was calculated with the absorbance at 450 nm as the number of cells. The logarithm of the concentration of the test substance is plotted on the horizontal axis and the cell viability is plotted on the vertical axis, and a regression line is obtained by using the least square method for the portion showing linearity, and when the survival rate becomes 50% The concentration of the test substance (μM) was determined and expressed as EC ₅₀ (μM). The results are shown in Table 3.

(2) Measurement of protective action against damage of human epidermal keratinocyte HaCaT cells by UVB In the same manner as in the above (1) column, human epidermal keratinocytes HaCaT were added to a 96-well microplate at 100 μL / well (25000 cells / well). Next, the cells were cultured for 24 hours, and the dose of the irradiator was confirmed before use.

Next, the test substance was replaced with 100 μL of PBS (−) containing 0.1 μM, 10 μM, 100 μM, and 1000 μM, respectively, and irradiated with UVB (manufactured by Sankyo Electric Co., Ltd., using a UVB lamp) at 50 mJ / cm ² .

Next, the cell viability (the number of cells not irradiated with UVB is defined as 100) is calculated from the number of cells having an absorbance of 450 nm by the same method as in the above column (1), and the viability is 50. The concentration (μM) of the test substance when it was% was determined and expressed as EC ₅₀ (μM). The results are shown in Table 3.

As shown in Table 3, the novel mycosporin-like amino acid (13-O- (β-Galactosyl) -Porphyra-334) of the present invention is significantly more UV protective than other mycosporin-like amino acids such as Porphyra-334. It was confirmed to be excellent.

<Test Example 4: Measurement of antioxidant action>
The novel mycosporin-like amino acid (13-O- (β-Galactosyl) -Porphyra-334) obtained as described above was measured for radical removal ability as an antioxidant action according to the following method. The measurement was performed according to the method described in R. Re et al., Free Radical Biol. Med. 26, 1231-1237 (1999).

First, 7 mM ABTS (2,2'-azino-bis (3-ethylbezothiazoline-6-sulphonic acid)) methanol solution was added with peroxodisulfate dipotassium methanol to a final concentration of 2.45 mM, and reacted. A radical solution was prepared. Next, samples (new mycosporin-like amino acids) prepared at various concentrations were added to the reaction solution, reacted for 1 hour, and the change in absorbance at 734 nm was measured using a spectrophotometer. In addition, as another measurement method, a free radical monitor (JOEL JES-FR30EX) measured by electron spin resonance was used to directly measure the amount of radical reduction. As a positive control, water-soluble vitamin E derivative Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) was used. When the value when distilled water was used as a sample was 100, the concentration (mM) of the test substance when the value was 50% was determined and expressed as IC ₅₀ (mM). The results are shown in Table 4.

As shown in Table 4, it was confirmed that the novel mycosporin-like amino acid (13-O- (β-Galactosyl) -Porphyra-334) of the present invention exerts a radical removing action in a very small amount.

Next, the technical idea that can be grasped from the above embodiment and other examples will be described below.
(A) A skin external preparation, a pharmaceutical, a quasi-drug, a cosmetic, or a food or drink comprising a compound having a structure represented by the general formula (1) or a salt thereof.

Claims (4)

A mycosporin-like amino acid, which is a compound having a structure represented by the following general formula (1) or a salt thereof.
  An ultraviolet protective agent comprising the mycosporine-like amino acid according to claim 1 as an active ingredient.   An antioxidant comprising the mycosporin-like amino acid according to claim 1 as an active ingredient. A method for producing a mycosporine-like amino acid which is a compound having a structure represented by the following general formula (1) or a salt thereof,
Extracting the extract containing the mycosporine-like amino acid from Nostock using water, a hydrophilic organic solvent, or a mixture of water and a hydrophilic organic solvent;
And a step of isolating the mycosporin-like amino acid from the extract.