JP6407848B2 - Defensin production promoter, cathelicidin production promoter, antibacterial agent - Google Patents

Description

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

The existence of an antibacterial substance (antibacterial peptide) in the living body has been known as a living body defense mechanism.
Anti-microbial peptides are natural antibacterial substances that are responsible for innate immune mechanisms in vivo, and have antibacterial activity against various microorganisms including bacteria, fungi, protozoa, and viruses. It is a peptide substance that has the property to induce proper infection suppression and systemic immune response. In general, antibacterial peptides have an amphipathic structure, and as a mechanism of antibacterial action, the cation site of the antibacterial peptide binds to an anionic phospholipid of the microbial cell membrane and destroys the microbial cell membrane. Shows antibacterial activity.
In conventional antibacterial agents 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-biological defense mechanism.

Defensin is one of the most studied among antibacterial peptides, and β-defensin is a peptide substance expressed in mucosal epithelium such as skin, lung, viscera, kidney and genital organs. It is known not only to have antibacterial properties, but also plays an important role in the regulation of systemic immune responses and inflammatory responses, and promotes immune responses and healing of inflammation. (Non-Patent Document 1)
In addition, defensin has the effect of inducing a systemic immune response as well as antibacterial action by promoting its secretion when physical damage or infection of the skin occurs, especially in the skin, Also plays a function of inducing proliferation and healing wounds (Non-Patent Document 2)
There are reports that it is also effective for patients with atopic dermatitis. (Non Patent Literature 3)
Known defensin production promoters include organic acids, yeast-derived polysaccharides, yeast-derived insoluble fractions, yeast-derived mannan-containing components, yogurt, amazake extract, shochu mash, and miso miso concentrate. (Patent Documents 1 to 5)

Cathelicidin exhibits a wide range of antibacterial effects and serves various immunomodulatory functions. LL-37, one of the degradation products of cathelicidin, has a function of regulating inflammation in vivo with a wide range of antibacterial activities. That is, LL-37 exhibits chemotactic properties for eosinophils, mononuclear cells, and T cells, as well as direct antibacterial activity against bacteria, fungi, and viruses, and induces endothelial cell proliferation. In particular, LL-37 present in the skin fulfills the function of antigen suppression by rapidly defending when a foreign antigen penetrates (Non-Patent Document 4).
In fact, the use of prevention or treatment of diseases that cause reduced levels of dermatitis, wound healing, angiogenesis (atherosclerosis, coronary heart disease, stroke, arterial occlusive disease or ulcer) Are known. (Patent Documents 6 to 8)
There are reports that it is also effective for patients with atopic dermatitis. (Non Patent Literature 3)

  Enmeiso (extended herb) is a Japanese folk medicine that is dried from the whole grass of the family Lamiaceae (Isodon japonicus) and has been used for indigestion, loss of appetite, abdominal pain, and so on. Furthermore, uses such as antiallergic agents, antihistamines, anticomplement activators, anti-obesity cosmetics, platelet aggregation inhibitory action, angiogenesis inhibitors, tight junction formation accelerators, and the like are known. (Patent Documents 9 to 14)

Arnica plants belonging to the family Asteraceae have wound healing, antibacterial action, analgesic action, anti-inflammatory action, etc., and are used to improve symptoms such as rheumatism, arthritis, oral mucosal inflammation, bruises and sprains. Arnica montana, Arnica chamissonis, Arnica unalascensis, etc. are currently used for medicinal purposes, and flowers and roots are mainly used as parts.
Furthermore, uses such as whitening, antiallergic agents, hyaluronic acid degradation inhibitors, head external preparations, profilagrin mRNA expression promoters, active oxygen scavengers, and the like are also known. (Patent Documents 15 to 20)

Gardenia (Gardenia jasminoides) is an evergreen shrub belonging to the genus Acanthaceae. The fruit is called Yamakiko or Yuzu and is used as a herbal medicine as anti-inflammatory, hemostatic, antipyretic and sedative.
Applications of ceramide production promoters, matrix metalloprotease inhibitors, hair quality improvers, trypsin inhibitors and the like are also known. (Patent Documents 21 to 24)

Chamomile (Matricaria chamomilla) is also called chamomile or chamomile, and it is called German chamomile when distinguished from roman chamomile.
It has been used for gynecological diseases because it has a healthy stomach, sweating, and anti-inflammatory action in Europe.
Furthermore, uses such as whitening cosmetics, collagen gel contraction promoters, deodorants, collagen gel contraction promoters, skin mite control agents are known. (Patent Documents 25 to 29)

Anjin is a seed of apricot (Prunus armeniaca) and is used as a Chinese medicine for gout, asthma and cough. In addition, apricot tofu with anpricot powder is widely eaten in China and Japan.
Furthermore, uses such as a whitening cosmetic composition, a brain function improving agent, a protease inhibitor, an anticaries agent, and a hair cosmetic are known. (Patent Documents 30 to 34)

International Publication No. 2005/027893 Japanese Patent Laid-Open No. 2003-197 JP 2003-262 A JP 2006-241023 A JP-A-2005-270117 US Patent Application Publication No. 2006/292551 JP-T-2006-518375 European Patent Application No. 1358888 JP 07-196522 A JP 09-087189 A Japanese Patent Laid-Open No. 10-203990 JP 2007-197388 A JP 2012-025777 A JP 2013-056841 A Japanese Patent Laid-Open No. 08-012561 JP 09-087189 A JP-A-10-130162 JP-A-11-228355 JP 2006-016337 A JP 2006-117612 A JP 2000-169359 A JP 2000-319155 A JP 2001-270811 A JP 2006-182732 A Japanese Patent Application Laid-Open No. 07-025742 Japanese Patent Laid-Open No. 10-072336 JP 2002-255776 A JP 2003-176208 A Japanese Patent Laid-Open No. 2005-060320 JP 07-126149 A Japanese Patent Laid-Open No. 07-165589 JP 10-338642 A JP 2001-097875 A JP 2002-173416 A

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 Dermatis”, The England Journal of Medicine, 2002, pp. 3471-1151. Braff MH, Bardan A, Nizet V et al., “Cutaneous defense mechanisms by antimicrobial peptides,” J Invest Dermatol, 2005, 125, 9

  The purpose of the present invention is to promote the production of defensin and cathelicidin, not only antibacterial activity against bacteria, fungi and viruses in the skin, oral cavity and digestive tract, but also immune enhancement, dermatitis, atopic dermatitis, wound healing, inflammation Another object of the present invention is to obtain a preparation effective for the prevention or treatment of diseases that cause a decrease in the level of angiogenesis.

As a result of intensive studies by the present inventors, it has been found that an extract of enamel, a plant extract of the genus Arnica, an extract of gardenia fruit, an extract of chamomile, and an extract of apricot kernel achieve the above object.
Enmeiso, Arnica plants, gardenia fruits, chamomiles, and apricots are dried as necessary, and then extracted after processing such as shredding and pulverization 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 water that has been subjected to various treatments. Examples of the treatment applied to water include purification, heating, sterilization, filtration, ion exchange, adjustment of osmotic pressure, 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, 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.
In addition, when using the mixed solvent of the said water and a hydrophilic organic solvent, in the case of a lower alcohol, it is 1-20 mass parts with respect to 10 mass parts of water, and in the case of a lower aliphatic ketone, it is 10 mass parts of water. On the other hand, it is preferable to add 1 to 15 parts by mass. In the case of a 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. Furthermore, in order to raise extraction efficiency, you may stir or homogenize in an extraction solvent. The extraction temperature is suitably about 5 ° C. to 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 single 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 a plurality of times.
As a result of investigations by the present inventors, a substance that exerts the effect of the present invention is also extracted into water by 80% ethanol. Therefore, when purifying to some extent, after extracting with water, insoluble matters are removed, etc. It has also been found that further extraction may be performed by adding an amount to 5 times the amount of ethanol.
If necessary, purification treatment such as deodorization and decolorization may be further added within a range that does not affect the effect, and concentration can be performed by a vacuum concentrator such as an evaporator or solvent removal by heating.
Further, this extract can be further purified using a synthetic adsorbent (Diaion HP20, Sephabies SP825, Amberlite XAD4, MCIgelCHP20P, etc.), dextran resin (Sephadex LH-20, etc.), ultrafiltration and the like. is there.

The preparation of the present invention exhibits a medicinal effect for any of oral, injection and external use.
The amount of these extracts in the preparation is 0.000001 to 10.0% by weight, preferably 0.00001 to 3.0% by weight, more preferably 0.00005 to 1.0% by weight, as a solid content. is there.

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

  As the surfactant, any of anionic, cationic, nonionic, natural, and synthetic surfactants can be used, but it is preferable to use a nonionic surfactant in consideration of irritation to the skin. 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 sorbite fatty acid ester, polyoxyethylene alkyl ether, polyoxyethylene polyoxypropylene glycol. , Polyoxyethylene polyoxypropylene alkyl ether, polyethylene glycol fatty acid ester, polyoxyethylene castor oil, polyoxyethylene hydrogenated castor oil, alkylglycoside and the like.

  Examples of the oil component include fats and oils, waxes, hydrocarbons, higher fatty acids, higher alcohols, esters, essential oils, silicone oils, and the like. Examples of the fats and oils include soybean oil, nutka oil, jojoba oil, avocado oil, almond oil, olive oil, cacao oil, sesame oil, persic oil, castor oil, coconut oil, mink oil, beef fat, pork fat and the like, and these Hardened oil obtained by hydrogenation of natural fats and oils and synthetic triglycerides such as myristic acid glyceride and 2-ethylhexanoic acid triglyceride; waxes include, for example, carnauba wax, whale wax, beeswax, lanolin and the like; hydrocarbons For example, liquid paraffin, petrolatum, paraffin microcrystalline wax, ceresin, squalane, bristan etc .; higher fatty acids include, for example, lauric acid, myristic acid, palmitic acid, stearic acid, behenic acid, oleic acid, linoleic acid, Linolenic acid, lanolinic acid, isostearic acid, etc .; Examples of the class alcohols include lauryl alcohol, cetyl alcohol, stearyl alcohol, oleyl alcohol, lanolin alcohol, cholesterol, 2-hexyldecanol, etc .; examples of the esters include cetyl octanoate, triglyceride octanoate, myristyl lactate, cetyl lactate, Examples of essential oils include mint oil, jasmine, isopropyl myristate, myristyl myristate, octyldodecyl myristate, isopropyl palmitate, isopropyl adipate, butyl stearate, decyl oleate, cholesterol isostearate, POE sorbite fatty acid ester, etc. Oil, pepper brain oil, cypress oil, spruce oil, ryu oil, turpentine oil, cinnamon oil, bergamot oil, mandarin oil, ginger oil, pine , Lavender oil, bay oil, clove oil, hiba oil, rose oil, eucalyptus oil, lemon oil, thyme oil, peppermint oil, rose oil, sage oil, menthol, cineole, eugenol, citral, citronellal, borneol, linalool, geraniol, Camphor, thymol, spirantol, pinene, limonene, terpene compounds, etc .; examples of silicone oils include dimethylpolysiloxane. These oily components described above 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, petrolatum, paraffin microcrystalline 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 myristate, octyldodecyl myristate, cholesterol isostearate, POE sorbite fatty acid ester, peppermint oil, spruce oil, cinnamon oil, rose Oil, menthol, cineol, eugenol, citral, citronellal, geraniol, pinene, limonene, dimethylpolysiloxane It is preferable to use.

The following components can be further added to the preparation of the present invention, but the components are not limited thereto.
Colors: Yellow color No. 4, Blue No. 1 and Yellow No. 202 are recognized as food additives such as tar color dyes I and II, chlorophyll, riboflavin, crocin, safflower, anthraquinone, etc. Natural pigments.
Vitamins; vitamin A, vitamin C, vitamin D, vitamin E, etc.
Others: bactericides, preservatives, other ingredients necessary for formulation.

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

EXAMPLES Next, an Example is given and this invention is demonstrated in detail.

Example 1
After adding 2 liters of 50% (V / V) aqueous ethanol solution to 50g of 50% (V / V) ethanol solution, it is extracted for 24 hours, filtered (No5C) and evaporated. Was lyophilized.

Example 2
Arnica montana (flower, dried product, shredded product) is added to 50 g of 2-liter 50% (V / V) ethanol aqueous solution, extracted for 24 hours with occasional stirring, and filtered (No5C). After evaporation, this was freeze-dried.

Example 3
Add 1 liter of 30% (V / V) ethanol aqueous solution to 50 g of gardenia fruit (dried product, shredded product), extract for 24 hours with occasional stirring, filter (No5C), evaporate this Lyophilized.

Example 4
Add 50 liters of chamomile (flower, dried product, shredded product) to 50 g of 50% (V / V) ethanol aqueous solution, extract with stirring occasionally for 24 hours, filter (No5C) and evaporate Lyophilized.

Example 5
Add 1 liter of 30% (V / V) ethanol aqueous solution to 50 g of apricot kernel (dried product, shredded product), extract for 24 hours with occasional stirring, filter (No5C), evaporate, and freeze-dry this did.

Confirmation test HuMedia- cultivated human foreskin-derived epidermis cells (Kurabo) in the 2nd passage in HuMedia-KG2 medium (without phenol red) so as to be 50-70% confluent, and the calcium concentration was changed to 1.8 mM the day before. Examples were added to KG2 medium and cultured in a 37 ° C., 5% CO 2 incubator for 2 days.

<Extraction of RNA>
Total RNA was extracted from the cells after exfoliation with trypsin / EDTA and then using 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>
Using 2.5 μg of total RNA, RT reaction was performed according to Toyobo recommended protocol (TOYOBO BIOCHEMICALS FOR LIFE SCIENCE 2008/2009, page 1-42) using MMLV Reverse Transcriptase RNase H- (Toyobo).
Real-time PCR was performed as follows using Applied Biosystems 7500 Real-Time PCR System. Using the SYBR Green method (THUNDERBIRD SYBR qPCR Mix, Toyobo Co., Ltd.) and the 7500 Real-Time PCR System operation manual (Applied Biosystems), gene expression comparison was performed by the Comparative CT (ΔΔCT) method (n = 3). GAPDH was used as an internal standard.
The target genes are defensin 1 and cathelicidin.

The result of the confirmation test is shown in FIG.
Examples 1 to 5 were found to promote defensin 1 and cathelicidin gene expression.

  Moreover, the external preparation which mix | blended the Example was created, and as a result of actually using it, the improvement was seen in dermatitis, atopic dermatitis, and wound healing.

It is a figure which shows the gene expression level change of defensin 1 and cathelicidin by the confirmation test result of Examples 1-5. The vertical axis represents the gene expression level when the gene expression level when no example is added is 1. The working concentration was 0.1% in Example 1, 0.025% in Example 2, 0.1% in Example 3, 0.05% in Example 4, and 0.2% in Example 5.

Claims (2)

A defensin production promoter containing as an active ingredient an extract of apricot kernel extracted with a mixed solvent of water and ethanol . A cathelicidin production promoter containing, as an active ingredient , an extract obtained by extracting apricot kernel with a mixed solvent of water and ethanol .