JP2018150268A - αMSH(1-8) EXPRESSION INHIBITOR - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel αMSH(1-8) expression inhibitor, and a pharmaceutical composition, food composition, and skin whitening food composition containing the agent.SOLUTION: In order to solve the problem, the inventors have conducted αMSH(1-8) expression inhibition tests on multiple plant extracts to confirm its effectiveness. As a result, it is found out that rotungenic acid included in Ilex rotunda has high αMSH(1-8) expression inhibitory effect.SELECTED DRAWING: None

Description

  The present invention describes a peptide composed of eight amino acids (hereinafter referred to as αMSH (1-8)) composed of serine, tyrosine, serine, methionine, glutamic acid, histidine, phenylalanine, and arginine, which are produced by ultraviolet rays and stress. ) Expression inhibitors, oral administration compositions, and whitening oral administration compositions.

  Pro-opiomelanocortin (hereinafter referred to as POMC) is a polypeptide precursor composed of 241 amino acid residues. A signal peptide sequence having a length of 44 amino acid residues is excised from preproopiomelanocortin (pre-POMC), which is a polypeptide precursor having a length of 285 amino acid residues, by post-translational processing to form POMC. POMC is mainly produced in the anterior and middle lobes of the pituitary gland, but it is also produced from skin tissues and the like, and it is also known that the production amount of POMC is increased by stimulation with ultraviolet rays or the like (see Non-Patent Document 1). ).

  In addition, there are portions in POMC that can be cleaved by processing, and up to 10 active peptides shown below are produced by tissues and converting enzymes. The 10 active peptides include N-terminal peptide proopiomelanocortin (NPP or pro-γ-MSH), γ-melanocyte-stimulating hormone (γ-MSH), adrenal gland Corticotropin (adrenocorticotropic hormone, ACTH), α-melanocyte stimulating hormone (α-MSH), corticotropin-like intermediate peptide (CLIP), β-lipotropic hormone (β-lipotropic hormone , Β-LPH), γ-lipotropic hormone (γ-LPH), β-melanocyte stimulating hormone (β-MSH), β-endorphin methionine-enkephalin ( methionine-enkephalin).

  Further, α-melanocyte stimulating hormone produced by further processing of adrenocorticotropic hormone is widely known as a hormone that induces melanin production. Although it is a hormone derived from adrenocorticotropic hormone involved in stress response, it is recognized as a melanin-synthesizing hormone induced by ultraviolet rays in general dermatology. On the other hand, α-MSH secreted by keratinocytes has a low concentration, and many are reported to be secreted at a high concentration in the state of POMC not subjected to intracellular processing.

  We have found that αMSH (1-8) is generated by extracellular processing from POMC not only by POMC but also by trypsin-like enzyme when the skin of mice is irradiated with ultraviolet rays (see Non-Patent Document 2). Moreover, although this peptide may be described as ACTH (1-8) depending on literature, it is the same peptide.

  Conventionally known POMC processing active peptides, which are processed in keratinocytes, are individually secreted from keratinocytes as active peptides. On the other hand, αMSH (1-8) is produced by extracellular processing after being secreted outside keratinocytes as POMC. From this, it can be said that αMSH (1-8) is a novel active peptide produced by a synthetic route completely different from the existing system. Furthermore, tryptase, which is a kind of serine protease, has been identified as a factor for processing αMSH (1-8) from POMC (see Non-patent Documents 3 and 4).

  In addition, it has been clarified that this αMSH (1-8) binds to the MC1 receptor of melanocytes and induces melanin production (see Non-Patent Document 3). Production suppression effects and melanin production inhibition effects have already been found (see Patent Document 1).

  Thus, it is clear that αMSH (1-8) is deeply involved in the melanin production pathway, but the existence of this peptide is a new biochemical finding, and various researches are currently being conducted. is there. For example, since this peptide is induced by ultraviolet rays, it is highly likely that it functions as a sensor for all reactions in the body starting with ultraviolet rays. That is, it can be considered to act as a factor of skin aging due to adjustment of immune function and drying of wrinkles and sagging.

(Non-Patent Document 1) Aline Primot, Elena Sviderskaya, et al: THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol.279, No.49,: pp.51226-51233 (2004)
(Non-Patent Document 2) Yamane, Yamamoto et al .: The 130th Annual Meeting of the Japanese Pharmaceutical Society, 28SJ-pm15, p. 3-101 (Non-patent Document 3) Oishi, Yamamoto et al. 3-118 (Non-patent Document 4) Yamamoto et al: Sci Rep, 2015 Sep 29; 5: 14579

(Patent Document 1) JP-A-2015-137267

  An object of the present invention is to provide a novel αMSH (1-8) expression inhibitor, an oral administration composition, and a whitening oral administration composition.

  In order to solve the above-mentioned problems, the present inventors conducted αMSH (1-8) expression suppression tests on a large number of plant extracts and confirmed the effectiveness. As a result, the present inventors found a high αMSH (1-8) expression inhibitory effect on rotgenic acid contained in black moth.

  Rotogenic acid suppresses the production of αMSH (1-8) produced in the body by receiving ultraviolet rays and stress. Since αMSH (1-8) binds to the MC1 receptor of melanocytes and induces melanin production, rotgenic acid that showed αMSH (1-8) expression-suppressing action is particularly effective in the prevention of skin whitening, spots and freckles. Is used in pharmaceutical compositions, food compositions, and food compositions for whitening.

It is a figure which shows (alpha) MSH (1-8) expression inhibitory effect of rotgenic acid.

  In addition, the plant: Ilex rotunda (Aquifoliaceae) and Ilex genus (Ilex) is used as the “Acrofoliaceae” used in the present invention.

The compound in the form of rotgenic acid and / or pharmaceutically acceptable salt represented by the following chemical structural formula (formula 1) used in the present invention can be obtained by extraction, synthesis, etc. from plants, animals, fungi and the like. In addition, since kuroganemochi contains a lot of rotgenic acid, purification from the column may be used. It can also be made into a salt by reacting with an alkali usually used in medicines and cosmetics. Examples of such salts include alkali metal salts such as sodium salt and potassium salt, alkaline earth metal salts such as calcium salt and magnesium salt, organic amine salts such as ammonium salt, triethanolamine salt and triethylamine salt, lysine Preferred examples include basic amino acid salts such as salts and arginine salts.

  In producing the αMSH (1-8) expression inhibitor, food composition, and whitening food composition of the present invention, the shape of the rotgenic acid used is any of liquid, solid, powder, paste, etc. A shape may be used, and an optimum shape for carrying out the present invention can be arbitrarily selected. ΑMSH (1-8) is a peptide consisting of eight amino acid sequences of “Ser Tyr Ser Met Glu His Phe Arg”.

  The content of rotgenic acid in the various compositions of the present invention is not particularly limited as long as it can be confirmed that the production of αMSH (1-8) can be suppressed, but is generally 0.0001 to 10% by weight. And is most preferably 0.001 to 1% by weight.

  About utilization of (alpha) MSH (1-8) expression inhibitor of this invention, it can utilize for a pharmaceutical composition, a food-drinks composition, and the food composition for whitening. Any form that can suppress the expression of αMSH (1-8) can be used regardless of whether it is solid or liquid.

  In addition, specific examples of the pharmaceutical composition, food composition (including beauty and health-oriented food and drink), and whitening food composition according to the present invention include soft drinks, carbonated drinks, nutrition drinks, fruit drinks, lactic acid drinks, and the like. Ice cream, ice sherbet, shaved ice and other frozen desserts; buckwheat noodles, udon, harusame, gyoza skin, sweet husk, Chinese noodles, instant noodles and other noodles; Sweets such as biscuits, jelly, jam, cream, baked goods, baked goods; crabs, salmon, clams, tuna, sardines, shrimp, bonito, mackerel, whales, oysters, saury, squid, scallops, scallops, abalone, sea urchin, crabs Fishery products such as kamaboko, ham and sausage; dairy products such as processed milk and fermented milk; salad oil, tempura oil, mar Fats and processed oils such as phosphorus, mayonnaise, shortening, whipped cream, dressing, etc .; seasonings such as sauces and sauces; curry, stew, oyakodon, rice cakes, miscellaneous cooking, Chinese rice cakes, rice cakes, tempura, eel rice, hayashi rice, Retort pouch foods such as oden, mabodorf, beef bowl, meat sauce, egg soup, omelet rice, dumplings, shumai, hamburger, meatballs; various forms of health foods and nutritional supplements; tablets, capsules, drinks, troches, etc. Drugs and quasi drugs are raised.

  In the pharmaceutical composition, food composition, and whitening food composition of the present invention, glucose, fructose, sucrose, maltose, sorbitol, stevioside, rubusoside, corn syrup, as long as the effects of the present invention are not impaired. Lactose, citric acid, tartaric acid, malic acid, succinic acid, lactic acid, L-ascorbic acid, dl-α-tocopherol, sodium erythorbate, glycerin, propylene glycol, glycerin fatty acid ester, polyglycerin fatty acid ester, sucrose fatty acid ester, sorbitan Fatty acid esters, gum arabic, carrageenan, casein, gelatin, pectin, agar, vitamin B, nicotinic acid amide, calcium pantothenate, amino acids, calcium salts, pigments, fragrances, preservatives, etc. Can do.

  Production examples in the rotgenic acid extract used in the present invention will be described in detail below, but the black beetroot extract of the present invention is not limited to the following production examples.

(Production example) Production of rotgenic acid As in Japanese Patent Application No. 2017-41854, in accordance with the previous report (Phytochemistry 28 (5) 1479-1482 (1989)), extraction was performed 3 times with 4 L of methanol against 2 kg of black berries. Concentrate to 500 mL, add 500 mL of water, add 500 mL of diethyl ether, extract and concentrate the ether layer. A solid content of 15.2 g was obtained. Further, 15.2 g of a solid content was dissolved in a mixed solution of methanol and dichloromethane (methanol: dichloromethane = 2: 98), and column purification was performed to obtain 384 mg of a solid content. Thereafter, column purification was performed with a mixed solution of water and methanol to obtain 24.5 mg of rotgenic acid.

(Test 1) Confirmation of rotgenic acid 13.0 mg of rotogenic acid obtained in the above production example was dissolved in C ₅ D ₅ N (600 μL), and ¹³ C-NMR measurement was performed. Further, the above rotogenic acid solution was diluted ₆ times with C ₅ D ₅ N, and ¹ H-NMR measurement was also performed.

(Test results)
The results show the chemical shift values shown in Table 1 and are almost identical to the values described in the previous report (Phytochemistry 28 (5) 1479-1482 (1989)), and thus confirmed to be rotgenic acid.
¹³ CNMR physical properties of rotgenic acid

(Test 2) Preparation of rotgenic acid salt Rotogenic acid obtained in the above production example was prepared in the usual manner to prepare sodium and calcium salts of rotogenic acid.

(Test 3) Expression inhibition evaluation test of αMSH (1-8) of rotgenic acid Normal human skin keratinocytes (Kurabo) and normal human skin melanocytes (Kurabo) are cultured at a ratio of 10: 1. After irradiating the cells with 2 mJ ultraviolet rays (UV-B), the cells are cultured in a serum-free medium (OPTI-MEM, Gibco) containing a test sample (using rosmarinic acid as a control for rotgenic acid). After 24 hours, the culture supernatant was collected, reacted by adding recombinant tryptase (Promega, rhSkin, β-tryptase), and stopped by adding trifluoroacetic acid. Then, Yamamoto et al: Sci Rep , 2015 Sep 29; 5: 14579), and then roughly purified by gel filtration chromatography, separated and detected by reverse phase HPLC, and the concentration was measured. The analysis conditions are as follows.
(Gel filtration chromatography)
Column: Sephadex G-75 fine column (1.0 × 100cm)
Solvent: A fraction near 1.1 kDa in terms of 1M dextran acetate was collected and injected into HPLC.
(HPLC)
Analytical instrument: SLC-6B (Shimadzu)
Detector: SPD-7A
Column: ODS column (4.6 x 150 mm, Myfhtsil RP-GP 5 μm, manufactured by Kanto Chemical Co., Inc.)
Flow rate: 1.0 mL / min
Solvent: Liquid A: Acetonitrile, Liquid B: 0.01 M hydrochloric acid gradient;
0 min;% A solution /% B solution = 100/0 → 30 min;% A solution,% B solution = 40/60

(Test results)
FIG. 1 shows the results of Test 3. Although rosmarinic acid did not show an inhibitory effect on the expression of αMSH (1-8), rotgenic acid showed an inhibitory effect on the expression of αMSH (1-8). In addition, the same effect was obtained when the test 3 was performed for the sodium salt and calcium salt of rotgenic acid obtained by the test 2.

(Prescription example)
Although the formulation example is described below with respect to rotgenic acid, it is not limited to the formulation of the present invention.

(Formulation example 1) Tablet type confectionery weight%
1. Lactose 2
2. Vitamin C 3
3. Citric acid 1.5
4). Gelatin 1% aqueous solution 4
5. Rotogenic acid 0.001
6). Sucrose fatty acid ester 1
7). Powdered strawberry flavor 0.5
8). Total powdered sugar to 100

(Formulation example 2) Tablet weight%
1. Microcrystalline cellulose 30
2. Carboxymethylcellulose calcium 24
3. Polyvinylpyrrolidone 10
4). Rotogenic acid 0.001
5. Talc 5
6). Corn starch to 100

(Formulation example 3) Tablet health food weight%
1. Salmon nasal cartilage extract containing proteoglycan 4
(Proteoglycan F, manufactured by Ichimaru Falcos)
2. Chondroitin sulfate 2
3. Hyaluronic acid 0.5
4). N-acetylglucosamine 40
5. Collagen extract powder 7.5
6). Rotogenic acid 0.001
7). Crystalline cellulose 9
8). Sucrose fatty acid ester 3
9. Fine silicon oxide 2
10. Make all maltitol 100

(Formulation example 4) Cookie confectionery Weight%
1. Light flour 35
2. Whole egg 10
3. Rotogenic acid 0.001
4). Butter 20
5. Sugar 12
6). Salt 10
7). Baking powder 0.3
8). Make all water 100

(Prescription Example 5) Capsule Tablet Weight%
1. Lactose 49
2. Rotogenic acid 0.001
3. Magnesium stearate 1
4). Corn starch to 100

(Formulation example 6) Chewing gum weight%
1. Rotogenic acid 0.001
2. Sugar 53.0
3. Gum base 20.0
4). Minamata 16.0
5. Fragrance 0.5
6). Glucose to 100

(Formulation example 7) Gummy weight%
1. Rotogenic acid 0.001
2. Reduced water tank 40.0
3. Granulated sugar 20.0
4). Glucose 20.0
5. Gelatin 4.7
6). Ume Juice 4.0
7). Ume Flavor 0.6
8). Dye 0.02
9. Make all water 100

(Prescription Example 8) Soft drink weight%
1. Fructose dextrose solution 20.0
2. Liquid yogurt 30.0
3. Emulsifier 0.5
4). Sodium hyaluronate (molecular weight 50,000-150,000) 0.1
5. Rotogenic acid 0.001
6). Perfume appropriate amount 7. Purify water to 100

(Test 4) Single-dose toxicity test Ddy mice (male and female, 1 group, 5 mice) fasted for 16 hours before the test at a dose of 6 g / kg (body weight) of rotgenic acid and rotogenic acid sodium salt (5 weeks old) was orally administered, and the onset, degree, etc. of toxic symptoms were observed over time. As a result, no abnormality was observed in all mice for 14 days, and as a result of dissection, it was confirmed that there was no abnormality.

(Test 5) Use Effect Test Based on Formulation Example 8 of the present invention, soft drinks of Formula A and Formula B as shown in Table 2 below were prepared. Ten adult males and females drank 500 ml of the prepared soft drink per day, and conducted a use test for one month. In addition, the test subject was divided into five persons, and the spots and pigmentation were evaluated as compared with before use.

(Test results)
As a result, as shown in Table 3 below, it was confirmed that the prescription B containing rotgenic acid was more effective than the prescription A without prescription.
Effective: Blemish and pigmentation improved compared to before use.
Slightly effective: Blemishes and pigmentation were slightly improved than before use.
Invalid: No change before use.

Claims (4)

  An αMSH (1-8) expression inhibitor comprising rotgenic acid or a salt thereof as an active ingredient.   A pharmaceutical composition containing rotgenic acid or a salt thereof.   A food composition containing rotgenic acid or a salt thereof.   A whitening food composition containing rotgenic acid or a salt thereof.

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