JP6691889B2 - Novel tetrapeptide compound derived from shochu distillation residue - Google Patents

Description

TECHNICAL FIELD The present invention relates to a novel biologically active tetrapeptide compound obtained from a shochu distillation residue by-produced in the production of shochu. In the present invention, the tetrapeptide means a compound presumed to have four amino acid residues cyclically linked by a peptide bond.

  Regarding the shochu distillate residual liquid produced as a by-product during the production of shochu, the fatty liver suppressing effect (patent document 1), the antioxidative effect (patent document 2), and the blood pressure of the specific fraction purified from the barley shochu distillate residual liquid are included. Fractions having various physiologically active actions such as a lowering action (Patent Document 3) have been reported. In addition, a tyrosinase inhibitory activity of a specific purified fraction has been reported from the brown sugar shochu distillation residual liquid (Patent Document 4).

  As the cyclic tetrapeptide having physiological activity, a cyclic tetrapeptide having immunosuppressive activity (Patent Document 5), anti-mitotic inhibitory activity (Patent Document 6), and protein tyrosine kinase inhibitory activity (Patent Document 7) are cyclic. Tetrapeptides have been reported.

JP 2001-145472 A Patent No. 4694099 Patent No. 4584611 JP, 2004-248592, A Patent No. 5901543 Japanese Patent No. 4006466 JP 2012-180315 A

The Journal of Immunology (2011) Vol. 186, No. 8, p. 4762-4770

  An object of the present invention is to isolate and purify a novel and useful compound, which has not been known so far, from barley shochu distillation residual liquid from which a plurality of fractions having useful physiological activities have been obtained.

The present inventors, in order to solve the above problems, known useful fractions from barley shochu distillation residual liquid, and as a result of conducting research by creating various fractions by a new purification step, barley shochu distillation A novel tetrapeptide compound contained in the residual liquid was identified, and it was found that this compound has a suppressive effect on the expression of the active oxygen generating gene (DUOX1), leading to the completion of the present invention.

The present invention comprises the following technical matters.
(1) A tetrapeptide compound having a molecular weight of 486 and a molecular formula of C ₂₄ H ₃₀ N ₄ O ₇ or a pharmaceutically acceptable salt thereof, which is contained in an ethanol elution fraction of a synthetic adsorbent adsorption fraction of barley shochu distillation residual liquid.
(2) The tetrapeptide compound or the pharmaceutically acceptable salt thereof according to (1), which has an effect of suppressing the expression of the DUOX1 gene in normal human epidermal keratinocytes treated with cytokine.
(3) A food or drink, a supplement, or a pharmaceutical product containing the tetrapeptide compound or the pharmaceutically acceptable salt thereof according to (1) or (2).

According to the present invention, a novel tetrapeptide compound can be provided from a barley shochu distillation residual liquid. INDUSTRIAL APPLICABILITY The tetrapeptide compound of the present invention has an activity of suppressing the expression of active oxygen generating gene (DUOX1) among active oxygen control mechanisms known to be associated with inflammatory diseases such as atopic dermatitis and asthma. .. Since it is derived from a fermented food called barley shochu distillation residual liquid, it is not toxic, and can be expected to reduce inflammatory diseases, such as alleviation of symptoms of atopic dermatitis and skin beautifying effect due to anti-dermatitis activity. It is expected to be used as a medicine.

FIG. 3 is a graph showing the suppressive effect of tetrapeptide on the DUOX1 gene expression level in cytokine-treated normal human epidermal keratinocytes.

In the present invention, the tetrapeptide compound also includes a pharmaceutically acceptable salt or derivative thereof as long as the tetrapeptide compound of the present invention does not lose the DUOX1 expression inhibitory activity. Examples of the pharmaceutically acceptable salt include alkali metal salts such as sodium salt and potassium salt, organic acid salts such as sulfate and nitrate, and hydrohalide salts such as hydrofluoride and hydrochloride. .. Examples of the derivative include esterification, amidation, acylation, carboxylation, formylation, phosphorylation, phosphorylation and glycosylation.

The tetrapeptide compound of the present invention can be produced by isolation and purification from a barley shochu distillation residue. It can also be isolated and purified from liquid or solid cultures of Aspergillus oryzae, and can also be chemically synthesized by a known method. For example, it can be produced by synthesizing a chain peptide by a solid phase synthesis method.

When the barley shochu distillation residual liquid is used, the barley shochu distillation residual liquid is subjected to solid-liquid separation to obtain a liquid component, and then the liquid component is adsorbed on the synthetic adsorbent. After elution with 20% ethanol among the adsorbed components, the tetrapeptide compound of the present invention can be further isolated and purified from the fraction eluted with a 40% ethanol eluate.

  The barley shochu distillation residual liquid is typically manufactured from barley koji and steamed barley with a yield of 60 to 70% as a raw material, and the barley koji obtained and the starch contained in the steamed barley are the malt of the barley malt. Saccharification by means of alcoholic fermentation with yeast to obtain shochu aged moromi, and when the obtained shochu aged moromi is distilled using a single distillation apparatus such as vacuum distillation or atmospheric distillation, it is a by-product as a distillation residue. It is a distillation residual liquid of barley shochu.

  The barley koji used in the production of barley shochu may be produced under the koji-making conditions that are usually used in the production of barley shochu. As the koji mold strain to be used, white koji mold (Aspergillus kawachii) generally used in the production of barley shochu is used. Is preferred. It is also possible to use a strain of the genus Aspergillus such as Aspergillus awamori used in awamori production. As the yeast used for producing barley shochu, various kinds of yeast for brewing shochu generally used in producing shochu can be used.

  Solid-liquid separation is performed from the barley shochu distillation residual liquid to obtain a liquid content, whereby the water-insoluble fermentation residue derived from the raw barley or barley koji is removed to obtain a clear liquid. Solid-liquid separation can be performed by a screw-press method or a roller-press method. Then, the liquid component is subjected to an adsorption treatment using a synthetic adsorbent to be adsorbed. As the synthetic adsorbent, aromatic, aromatic modified, or methacrylic synthetic adsorbents can be used. Suitable examples include Amberlite FPX66 manufactured by Dow Chemical Co., and Mitsubishi Chemical Co. Examples include SepaBeads SP850 and DIAION HP20 manufactured by Mitsubishi Chemical Corporation.

Then, the synthetic adsorbent adsorption fraction is sequentially eluted with 20 volume% ethanol and 40 volume% ethanol eluent, and the obtained elution fraction is subjected to high performance liquid chromatography (HPLC). In order to further purify the obtained HPLC chromatograph having a molecular weight of 486, it was lyophilized, dissolved in water, centrifuged, washed again with water, dissolved in an acetonitrile solution, and purified again by HPLC. By doing so, the tetrapeptide compound of the present invention can be isolated and purified.

INDUSTRIAL APPLICABILITY The tetrapeptide compound of the present invention can be used in various forms as foods and drinks, supplements and pharmaceuticals for alleviation and treatment of inflammatory diseases, for example, alleviation of atopic dermatitis and beautiful skin due to anti-dermatitis activity. The “food and drink” includes enteral nutritional foods, nutritionally functional foods, foods with functional claims, foods for specified health uses, etc., in addition to ordinary foods and drinks. In addition, the target of "food and drink" and "medicine" is not limited to humans, and also includes pharmaceuticals and feed for mammals such as pets and livestock.

The tetrapeptide compound of the present invention can be used as an oral composition such as tablets, powders, granules, capsules and solutions. Various components and manufacturing methods for manufacturing oral compositions of various dosage forms can be appropriately selected from components known in the field of manufacturing supplements, pharmaceuticals and the like. Excipients, binders, disintegrants, lubricants, other nutrients and the like can be added to the tablet of the present embodiment as various additives for forming the tablet.

Besides oral administration, it can be used in various dosage forms as parenteral administration agents such as injections, infusions, external preparations, suppositories and the like. Further, the effective dose of the tetrapeptide compound of the present invention in the preparation varies depending on the degree of symptoms to be treated or prevented, the condition of the administration subject (including age and sex), dosage form and the like. The daily dose of the tetrapeptide compound may be about 10 to 1000 mg.

Barley shochu was produced for the purpose of providing the following examples. Barley (70% polished) was used as a raw material.
[Production of barley shochu and barley shochu distillation residue]
After barley absorbs 40% (w / w) of water and steams for 40 minutes, it is left to cool to 40 ° C, inoculated with 1 kg of seed malt (white koji mold) per ton of barley, 38 ° C, RH 95% for 24 hours, 32 ° C. , Barley malt was maintained for 20 hours at 92% RH. In the primary charging, to this barley koji (3 tons as barley), 3.6 kL of water and 1 kg (wet weight) of cultured bacterial cells of shochu yeast as yeast were added to obtain a primary mash, and the obtained primary mash was obtained. It was subjected to fermentation for 5 days (first stage fermentation). Next, in the secondary preparation, 11.4 kL of water and steamed barley (7 tons as barley) were added to the primary mash that had undergone the above-mentioned first-stage fermentation, and the fermentation was continued for 11 days (second-stage fermentation). did. The fermentation temperature was 25 ° C. for both the primary charge and the secondary charge. The secondary moromi, which had undergone the second-stage fermentation, was subjected to simple distillation by a conventional method to obtain 10 kL of barley shochu and 15 kL of a barley shochu distillation residual liquid. The barley shochu distillation residue was used in the following examples.
Hereinafter, the present invention will be described in detail based on examples, but the present invention is not limited to these examples.

  The barley shochu distillation residual liquid obtained in the distillation step of barley shochu production was centrifuged under the conditions of 8000 rpm and 10 min to obtain a liquid component of the barley shochu distillation residual liquid, and 2.5 L of the obtained liquid component was used. The column was filled with a synthetic adsorbent Diaion HP20 (resin volume: 1 L), and a synthetic adsorbent adsorption fraction adsorbed to the column was obtained. further. The eluate obtained by contacting 6.25 L of deionized water with the column that adsorbed the adsorbed fraction of this synthetic adsorbent was removed, and then 2.5 L of a 20 (v / v)% ethanol solution, 40 ( 2.5 L of the eluate was collected by sequentially contacting 2.5 L of v / v)% ethanol solution.

The eluate was concentrated under reduced pressure and freeze-dried. The lyophilized product was dissolved in deionized water to a concentration of 0.1 g / mL, and HPLC using a Phenomenex Synergy 4 μm Hydro-RP 80A column (21.2 × 250 mm) (solution A: 0.05% TFA aqueous solution, Solution B: 0.05% TFA acetonitrile solution). The separation conditions were a B concentration of 15%, a flow rate of 10 mL / min, and a detection wavelength of 280 nm. A fraction of the obtained HPLC chromatograph having a molecular weight of about 486 was collected, concentrated again under reduced pressure and lyophilized, then dissolved in deionized water to 0.1 g / mL, and further centrifuged. The resulting white precipitate was washed with deionized water. This was dissolved in a 62.5% acetonitrile solution and purified by HPLC under the same conditions as above.

In order to analyze the structure of the purified product, Fourier transform infrared spectroscopy (FT-IR), nuclear magnetic resonance analysis (NMR), liquid chromatograph / mass spectrometry (LC / MS), and amino acid analysis were performed. As a result, it was revealed that the compound is a novel tetrapeptide compound having a molecular weight of 486, which is presumed to consist of 4 amino acids, one L-tyrosine, one L-glutamic acid and two L-proline.
The solubility was such that it was readily soluble in dimethyl sulfoxide, sparingly soluble in water and methanol, and soluble in 50% methanol, and its molecular formula was C ₂₄ H ₃₀ N ₄ O ₇ .

Further LC / MS / MS analysis was performed to deduce the cyclic tetrapeptide structure consisting of one tyrosine, one glutamic acid and two prolines. As a result, the product ion d (m / z 279) presumed to be derived from the adjacent tyrosine and glutamic acid was detected. Therefore, it was presumed to be one of the following formula I or formula II. Regarding the other product ions, there was no contradiction regardless of the structure.
In order to determine which structure, each structure was assumed and assigned by various two-dimensional NMR spectra. The results of the HMBC spectrum are shown below . Telecorrelation of 1H and 13C in HMBC is usually detected with 2-4 binding. Focusing on the remote correlation between N and H shown below, there are two bonds between N and H of the putative structure (1), while there are five bonds between N and H of the putative structure (2), which is somewhat It was an unnatural attribution.

According to the above structural analysis results, this compound is likely to be the cyclic tetrapeptide represented by the following formula I, but may be the cyclic tetrapeptide represented by the following formula II.

The tetrapeptide compound of the present invention was tested for the activity of suppressing the expression of the DUOX1 gene in cytokine-treated normal human epidermal keratinocytes.
Cytokine treatment of normal human epidermal keratinocytes For culturing normal human epidermal keratinocytes (NHEK: Kurabo), 10 mL petri dish was used, and 10 μg / mL in normal human epidermal keratinocyte basal medium (Humedia KB-2: Kurabo) Insulin, 0.1 ng / mL human recombinant epidermal growth factor (hEGF), 0.4% (v / v) bovine pituitary extract, 0.67 μg / mL hydrocortisone, 50 μg / mL gentamicin, 50 ng / mL A serum-free medium for growing epidermal keratinocytes (HuMedia-KG2) prepared by adding amphotericin B was used. Cryopreserved cells. The cells were seeded at 2500 cells / mL and cultured at 37 ° C., 5% CO 2, for 5 days. Then, it seed | inoculated to a 24 well petri dish so that it might become 2 * 105 cells / mL, and it culture | cultivated at 37 degreeC, 5% CO2 for 48 hours. After confirming that the cells became confluent, the medium was replaced with a normal human epidermal keratinocyte basal medium containing 1.8 mM Ca 2+, and cultured at 37 ° C., 5% CO 2 for 48 hours. After removing the medium, the medium was replaced with 1.8 mM Ca2 + -containing normal human epidermal keratinocyte basal medium containing 100 μg / mL of the tetrapeptide compound of the present invention (hereinafter referred to as “486”), and 37 ° C., 5% CO2, It was cultured for 96 hours. After removing the medium, the medium was replaced with 1.8 mM Ca2 + -containing normal human epidermal keratinocyte basal medium containing 486 and cytokines (50 ng / mL each of IL-4 and IL-13), and cultured at 37 ° C, 5% CO2 for 48 hours. ..

RNA extraction The total RNA was extracted from the cultured cells using TRIzol (registered trademark) Plus RNA Purification Kit (Thermo Fisher Scientific). The operating procedure followed the manufacturer's protocol. The extracted RNA was stored at -80 ° C.
Reverse Transcription Reaction The extracted RNA was subjected to reverse transcription reaction by using ReverseTra Ace (registered trademark) qPCR RT Master Mix with gDNA Remover (Toyobo). Nuclease free water was added to 0.1 μg of RNA template to make 6 μL of the reaction solution, which was reacted at 65 ° C. for 5 minutes, and then stopped at 4 ° C. Next, 2 μL of 4 × DN Master Mix was added, and after the reaction at 37 ° C. for 5 minutes, the reaction was stopped at 4 ° C. Finally, 2 μL 5 × RT Master MixII was added, and the reaction was carried out at 37 ° C. for 15 minutes, 50 ° C. for 5 minutes, and 98 ° C. for 5 minutes. The reaction solution was stored at -20 ° C.

Quantitative RT-PCR
The cDNA prepared by the above reverse transcription reaction was used as a template. The analysis was performed by the Light cycler Nano system using the FastStart Essential DNA Green Master (Roche) and specific primers for each gene (Table 1). The expression level of the gene was comparatively quantified by the Comparative Ct method, and calculated as a relative value using GAPDH as an internal standard. The reaction is 95 ° C, 10 minutes, followed by an amplification reaction in which 95 ° C, 10 seconds, 60 ° C, 10 seconds, 72 ° C, 15 seconds are repeated 45 times, and finally 95 ° C, 30 seconds, 60 ° C, 20 ° C. Second, 95 ° C., 20 seconds reaction. At this time, the amplification of mRNA expression level was measured by monitoring the fluorescence of SYBR Green binding to DNA.

Results The effect of the tetrapeptide compound (486) of the present invention on DUOX1 gene expression when normal human epidermal keratinocytes were treated with IL-4 and IL-13 was examined (FIG. 1). As a result, the DUOX1 gene expression increased in the cytokine-added group as compared with the cytokine-untreated group, but decreased in the group to which 486 was added at 100 μg / mL, as compared to the DMSO-added group.
From the above results, it was found that the tetrapeptide compound contained in the barley shochu distillation residual liquid suppressed the expression level of the DUOX1 gene highly expressed by inflammation to about 3/5 as compared with the case where it was not added.

  In many patients with atopic dermatitis, interleukin 4 (IL-4) and interleukin 13 (IL-13) are excessively produced in skin tissue, which contributes to keratinocyte dyskeratosis and epidermal barrier dysfunction. It is known that According to Hirakawa et al., It has been reported that in human epidermal keratinocytes stimulated with IL-4 / IL-13, expression of DUOX1 involved in active oxygen production is induced and H2O2 productivity is increased (Non-patent Document 1). ), A relationship between atopic dermatitis and active oxygen control mechanism has been suggested.

The test of Example 2 confirmed that the tetrapeptide compound of the present invention significantly suppresses the expression of the DUOX1 gene in normal human epidermal keratinocytes stimulated with IL-4 and IL-13.
The DUOX1 expression-suppressing effect of the tetrapeptide compound of the present invention suggests the possibility of alleviating or treating inflammatory diseases, for example, alleviating the symptoms of atopic dermatitis or asthma, and anti-dermatitis activity may also be expected to be a beautiful skin effect. Therefore, it is expected to be utilized in foods and drinks and pharmaceuticals as skin care for eating.

According to the present invention, a novel and useful tetrapeptide compound can be isolated and provided from a barley shochu distillation residue. This compound may be used in various applications and forms such as foods and drinks, supplements and pharmaceuticals for improving the functions of skin such as inflammatory diseases such as asthma and atopic dermatitis.

Claims (3)

A tetrapeptide compound having a molecular weight of 486 and a molecular formula of C ₂₄ H ₃₀ N ₄ O ₇ or a pharmaceutically acceptable salt thereof, which is contained in an ethanol elution fraction of a synthetic adsorbent adsorption fraction of barley shochu distillation residue.   The tetrapeptide compound or a pharmaceutically acceptable salt thereof according to claim 1, which has an effect of suppressing the expression of the DUOX1 gene in normal human epidermal keratinocytes treated with cytokine.   Food / beverage products, supplements, or pharmaceuticals containing the tetrapeptide compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof.