JP6885575B2 - Defensin production promoter, antibacterial agent - Google Patents

Description

The present invention relates to a defensin production promoter and an antibacterial agent.

The existence of antibacterial substances (antibacterial peptides) in the living body has been known for a long time as an innate biological defense mechanism.
Anti-microbial peptides are natural antibacterial substances that are responsible for the innate immune mechanism in vivo, and have antibacterial activity against various microorganisms including bacteria, fungi, protozoa, and viruses, and are localized. It is a peptide substance that has the property of suppressing infection and inducing a systemic immune response. Normally, an antibacterial peptide has an amphipathic structure, and as an antibacterial action mechanism, the cation site of the antibacterial peptide binds to an anionic phospholipid possessed by the microbial cell membrane and destroys the microbial cell membrane. Shows antibacterial activity.
With conventional antibacterial agents using various drugs such as antibiotics and synthetic antibacterial agents, resistant microorganisms appear, and the only countermeasure against resistant bacteria is to develop new antibacterial agents.
However, any method that promotes the production of antibacterial peptides does not have the above-mentioned problems and can enhance the self-defense mechanism.

Defensin is one of the most studied antibacterial peptides, and β-defensin is a peptide substance expressed in the mucosal epithelium of the skin, lungs, internal organs, kidneys, genitals and the like. It is known to play an important role not only in antibacterial properties but also in the regulation of systemic immune response and inflammatory response, and promotes immune response and healing of inflammation. (Non-Patent Document 1)
In addition, defensin has the effect of promoting systemic immune response as well as antibacterial action by promoting its secretion when physical damage or infection occurs on the skin. Especially in the skin, keratinocyte differentiation and It also has the function of inducing proliferation and healing wounds (Non-Patent Document 2).
It has been reported that it is also effective for patients with atopic dermatitis. (Non-Patent Document 3)
Known defensin production promoters include organic acids, yeast-derived polysaccharides, yeast-derived insoluble fractions, yeast-derived mannan-containing components, yogurt, amazake extracts, shochu mash, and bran miso concentrate. (Patent Documents 1 to 5)

Sagarame (Eisenia arborea Areschoug) is a seaweed of brown algae, kelp family (Laminariaceae), and genus Arame (Eisenia). It is used as an ingredient.
Further, applications such as an angiogenesis inhibitor, β-glucuronidase inhibitor, AGE production inhibitor, and aquaporin production-enhancing preparation are known. (See Patent Documents 6 to 9)

Black tea is made from tea (Camellia sinensis) leaves and fermented picked tea leaves.
Of course, it is mainly used for drinking, but applications such as a plaque formation inhibitor, a melanin production inhibitor, an allergen inactivating agent, and an OPH activity enhancer are also known. (See Patent Documents 10 to 13)

Clove is a dried flower bud of the Myrtaceae plant Syzygium aromaticum (syn. Eugenia aromatica) and is used as an aromatic stomachic agent.
Also called clove, it is widely used as a spice.
Further, applications such as caries preventives, hyaluronidase inhibitors, hair cosmetics, and antiallergic agents are known. (See Patent Documents 14 to 17)

Panax ginseng is also called ginseng, ginseng, or simply ginseng.
It is a herbal medicine that has been used for a long time and is also described in "Shinko Honsokyo".
Further, applications such as a ceramide production promoter, a hair restorer, a glutathione production promoter, an anti-inflammatory agent, an anti-aging agent, an anti-obesity agent, and an elastin production promoter are known. (Patent Documents 18 to 22)

International Publication No. 2005/027893 Japanese Unexamined Patent Publication No. 2003-197 Japanese Unexamined Patent Publication No. 2003-262 Japanese Unexamined Patent Publication No. 2006-24123 Japanese Unexamined Patent Publication No. 2005-270117 Japanese Unexamined Patent Publication No. 2006-022033 Japanese Unexamined Patent Publication No. 2006-04518 Japanese Unexamined Patent Publication No. 2008-21246 Japanese Unexamined Patent Publication No. 2013-087057 Japanese Unexamined Patent Publication No. 2000-344642 Japanese Unexamined Patent Publication No. 2001-158726 Japanese Unexamined Patent Publication No. 2008-174526 Japanese Unexamined Patent Publication No. 2014-118406 Japanese Unexamined Patent Publication No. 5-255098 Japanese Unexamined Patent Publication No. 6-9371 Japanese Unexamined Patent Publication No. 8-231348 Japanese Unexamined Patent Publication No. 10-36276 Japanese Unexamined Patent Publication No. 2000-169359 Japanese Unexamined Patent Publication No. 2003-238365 Japanese Unexamined Patent Publication No. 2009-132662 Japanese Unexamined Patent Publication No. 2010-155787 Japanese Unexamined Patent Publication No. 2012-056933

Science 1999; 286: 525-528 Niyonsaba F, Ushio H, Nakano N, et al., "Antimicrobial peptides human β-defensins stimulate epidermal keratinocyte migration, proliferation and production of proinflammatory cytokines and chemokines", J Invest Dermatol, 2007 year, the first 127, pp. 594, pp. Ong PY et al., "Endogenous Antimicrobial Peptides and Skin Infections in Atopic Dermatitis", The New England Journal of Medicine, 2002, pp. 347

An object of the present invention is to promote the production of defensin, which is not only antibacterial against bacteria, fungi, and viruses in the skin, oral cavity and digestive organs, but also immunopotentiation, dermatitis, atopic dermatitis, wound healing, inflammation, and blood vessels. The purpose is to obtain an effective formulation for the prevention or treatment of diseases that cause a decrease in the level of newborns.

As a result of diligent studies by the present inventors, it was found that an extract of sagarame, an extract of black tea, an extract of clove, and an extract of Panax ginseng achieve the above-mentioned object.
Sagarame, black tea, clove, and Panax ginseng are extracted after being dried as necessary and then subjected to treatments such as shredding and crushing in consideration of extraction efficiency.
Drying may be performed in the sun or using a commonly used dryer.
As the solvent used for the extraction, water, a hydrophilic organic solvent, or a mixture thereof is used.
Examples of water that can be used as the extraction solvent include pure water, tap water, well water, mineral spring water, mineral water, hot spring water, spring water, fresh water, and the like, as well as those subjected to various treatments. Treatments applied to water include, for example, purification, heating, sterilization, filtration, ion exchange, osmotic pressure adjustment, buffering and the like. Therefore, the water that can be used as the extraction solvent in the present invention includes purified water, hot water, ion-exchanged water, physiological saline, phosphate buffer, phosphate buffered saline and the like.
Examples of the hydrophilic organic solvent include lower alcohols having 1 to 5 carbon atoms such as methanol, ethanol, propyl alcohol and isopropyl alcohol; lower aliphatic ketones such as acetone and methyl ethyl ketone; 1,3-butylene glycol and propylene glycol. Examples thereof include polyhydric alcohols having 2 to 5 carbon atoms such as glycerin, and a mixed solvent of these hydrophilic organic solvents and water can be used.
When a mixed solvent of the water and the hydrophilic organic solvent is used, 1 to 20 parts by mass with respect to 10 parts by mass of water in the case of lower alcohol, and 10 parts by mass of water in the case of lower aliphatic ketone. On the other hand, it is preferable to add 1 to 15 parts by mass. In the case of polyhydric alcohol, it is preferable to add 1 to 20 parts by mass with respect to 10 parts by mass of water.

The amount of the organic solvent used for extraction is preferably about 5 to 100 times, more preferably about 10 to 50 times the amount of the plant as a raw material. In order to further increase the extraction efficiency, stirring or homogenization may be performed in the extraction solvent. It is appropriate that the extraction temperature is from about 5 ° C. to a temperature equal to or lower than the boiling point of the extraction solvent. The extraction time varies depending on the type of extraction solvent and the extraction temperature, but it is appropriate to set it to about 1 hour to 14 days.
The extraction operation is not limited to a one-time operation. A fresh solvent can be added again to the residue after extraction to perform an extraction operation, or the extraction solvent can be brought into contact with the extraction raw material multiple times.
As a result of examination by the present inventors, 80% of ethanol is extracted from water as a substance exhibiting the effect of the present invention. Therefore, when purifying to some extent, after extracting with water, insoluble matter should be removed, etc. It was also found that further extraction should be performed by adding an amount to 5 times the amount of ethanol.
If necessary, further purification treatment such as deodorization and decolorization may be added within a range that does not affect the effect, and the concentration can be achieved by a vacuum concentrator such as an evaporator or solvent removal by heating.
It is also possible to further purify this extract using a synthetic adsorbent (Diaion HP20, Sephadex SP825, Amberlite XAD4, MCIgelCHP20P, etc.), dextran resin (Sephadex LH-20, etc.), ultrafiltration, etc. is there.

The pharmaceutical product of the present invention exhibits a medicinal effect by oral, injectable, or external use.
The amount of these extracts to be added to the formulation is 0.000001 to 10.0% by weight, preferably 0.00001 to 3.0% by weight, and more preferably 0.00005 to 1.0% by weight as a solid content. is there.

In addition to the above ingredients, the formulations of the present invention include surfactants, oily ingredients, moisturizers, polymer compounds, ultraviolet absorbers, anti-inflammatory agents, and bactericidal agents used in various pharmaceutical and cosmetic formulations. Antioxidants, metal ion blockers, preservatives, vitamins, pigments, fragrances, water and the like can be blended.

As the above-mentioned surfactant, any of anionic, cationic, nonionic, natural and synthetic surfactants can be used, but it is preferable to use nonionic surfactants in consideration of skin irritation. Examples of the nonionic surfactant include glycerin fatty acid ester, propylene glycol fatty acid ester, sorbitan fatty acid ester, polyoxyethylene sorbitan fatty acid ester, polyoxyethylene sorbit fatty acid ester, polyoxyethylene alkyl ether, and polyoxyethylene polyoxypropylene glycol. , Polyoxyethylene polyoxypropylene alkyl ether, polyethylene glycol fatty acid ester, polyoxyethylene castor oil, polyoxyethylene hydrogenated castor oil, alkyl glycoside and the like.

Examples of the oily component include fats and oils, waxes, hydrocarbons, higher fatty acids, higher alcohols, esters, essential oils, silicone oils and the like. Examples of fats and oils include soybean oil, nuka oil, jojoba oil, avocado oil, almond oil, olive oil, cacao oil, sesame oil, persic oil, paraffin oil, palm oil, minced oil, beef fat, pork fat and other natural fats and oils. Hardened oil obtained by hydrogenating natural fats and oils from Japan and synthetic triglycerides such as myristic acid glyceride and 2-ethylhexanoic acid triglyceride; Examples of waxes include carnauba wax, whale wax, honey wax, lanolin and the like; hydrocarbons. Examples include liquid paraffin, vaseline, paraffin microcrystalin wax, selecin, squalane, bristan, etc .; and higher fatty acids include, for example, lauric acid, myristic acid, palmitic acid, stearic acid, behenic acid, oleic acid, linoleic acid, etc. Linolenic acid, lanolinic acid, isostearic acid and the like; higher alcohols include, for example, lauryl alcohol, cetyl alcohol, stearyl alcohol, oleyl alcohol, lanolin alcohol, cholesterol, 2-hexyldecanol and the like; esters include, for example, cetyl octanoate. , Octanoic acid triglyceride, myristyl lactate, cetyl lactate, isopropyl myristate, myristyl myristate, octyldodecyl myristate, isopropyl palmitate, isopropyl adipate, butyl stearate, decyl oleate, cholesterol isostearate, POE sorbit fatty acid ester, etc. The essential oils include, for example, peppermint oil, jasmine oil, show brain oil, hinoki oil, peppermint oil, ryu oil, terepine oil, coconut skin oil, bergamot oil, orange oil, shove oil, pine oil, lavender oil, and bay oil. , Clove oil, Hiba oil, Rose oil, Eucalyptus oil, Lemon oil, Thyme oil, Peppermint oil, Rose oil, Sage oil, Mentor, Cineol, Eugenol, Citral, Citroneral, Borneol, Linarol, Geraniol, Kamfer, Timor, Spirantol, Paraffin, limonene, terpene-based compounds and the like; Examples of silicone oils include dimethylpolysiloxane and the like. These above-mentioned oily components can be used alone or in combination of two or more. In the present invention, among these, myristic acid glyceride, 2-ethylhexanoic acid triglyceride, lanolin, liquid paraffin, vaseline, paraffin microcrystalin wax, squalane, lauric acid, myristic acid, palmitic acid, linoleic acid, linolenic acid, isostearic acid. , Cetyl alcohol, stearyl alcohol, oleyl alcohol, cholesterol, cetyl octanoate, triglyceride octanoate, isopropyl myristyrene, octyldodecyl myristate, cholesterol isostearate, POE sorbit fatty acid ester, peppermint oil, paraffin oil, caprylic acid, rose It is preferable to use oil, menthol, cineol, eugenol, citral, citroneral, geraniol, pinen, limonene and dimethylpolysiloxane.

The following components can be further added to the formulation of the present invention, but the components are not limited thereto.
Pigments: Tar dyes specified in the Ordinance of the Ministry of Health and Welfare such as Yellow No. 4, Blue No. 1, Yellow No. 202, etc. Approved as food additives such as pigments in Appendix I and II, chlorophyll, riboflavin, crosin, safflower, and anthraquinone Natural pigments, etc.
Vitamins; vitamin A, vitamin C, vitamin D, vitamin E, etc.
Others; bactericides, preservatives, other ingredients necessary for formulation, etc.

The pharmaceutical product of the present invention can be produced according to a conventional method by adding the optional ingredient to the essential ingredient as necessary.

Example 1
Add 2 liters of 50% (V / V) ethanol aqueous solution to 50 g of sagarame (dried product, shredded product), extract for 24 hours with occasional stirring, filter (No5C), evaporate, and then freeze-dry. did.

Example 2
Add 2 liters of 30% (V / V) ethanol aqueous solution to 30 g of black tea (dried product, shredded product), extract for 24 hours with occasional stirring, filter (No5C), evaporate, and then freeze-dry. did.

Example 3
Add 2 liters of 50% (V / V) ethanol aqueous solution to 20 g of clove (dried product, shredded product), extract for 24 hours with occasional stirring, filter (No5C), evaporate, and then freeze-dry. did.

Example 4
Add 2 liters of 20% (V / V) ethanol aqueous solution to 50 g of Panax ginseng (root, dried product, shredded product), extract for 24 hours with occasional stirring, filter (No5C), and evaporate this. It was lyophilized.

Confirmation test After culturing the second-generation human foreskin-derived epidermal cells (Kurabou) in HuMedia-KG2 medium (without phenol red) so as to be 50-70% confluent, the calcium concentration was changed to 1.8 mM the day before. Examples were added to KG2 medium and cultured at 37 ° C. in a 5% CO2 incubator for 2 days.

<RNA extraction>
Total RNA was extracted from cells by exfoliation with trypsin / EDTA, and then prepared using the illustra RNA Mini RNA Isolation Kit (GE Healthcare) according to the attached manual of GE Healthcare. The RNA concentration was calculated using NanoDrop1000 (Thermo SCIENTIFIC).

<RT reaction and real-time PCR>
An RT reaction was performed using 2.5 μg of Total RNA and MMLV Reverse Transcriptase RNase H- (Toyobo) according to the Toyobo recommended protocol (TOYOBO BIOCHEMICALS FOR LIFE SCIENCE, page 1-42).
Real-time PCR was performed using Applied Biosystems 7500 real-time PCR System as follows. Gene expression by Comparative CT (△△ CT) method (n = 3) using the SYBR Green method (THUNDERBIRD SYBR qPCR Mix, Toyobo Co., Ltd.) and using the 7500 real-time PCR System operation manual (Applied Biosystems). GAPDH was used as the internal standard.
The target gene is defensin 1.

The result of the confirmation test is shown in FIG.
Examples 1 to 4 were found to promote the expression of the defensin 1 gene.

In addition, as a result of creating an external preparation containing the examples and actually using it, not only antibacterial properties against bacteria, fungi, viruses, etc. on the skin, oral cavity, digestive organs, etc., but also immunity enhancement, dermatitis, etc. It was possible to obtain an effective preparation for the prevention or treatment of diseases causing atopic dermatitis, wound healing, inflammation, and a decrease in the level of angiogenesis.

It is a figure which shows the gene expression level change of defensin 1 in the confirmation test result of Examples 1 to 4. The vertical axis is the gene expression level when the gene expression level when the example is not added is 1. The working concentration was 0.05% in Example 1, 0.01% in Example 2, 0.01% in Example 3, and 0.2% in Example 4.

Claims (1)

A defensin production promoter containing a 50% (V / V) ethanol aqueous solution extract of sagarame as an active ingredient (excluding antibacterial applications) .

Images