JP2010208972A - Endothelin expression inhibitor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an endothelin expression inhibitor. <P>SOLUTION: This endothelin expression inhibitor contains Rosmarinus officinalis L. or an extract thereof as an active ingredient. Since having an excellent endothelin-1 expression inhibiting action, the endothelin expression inhibitor is useful for medicines, quasi-drugs, cosmetics or foods which exhibit effects such as the prevention and/or treatments of various diseases, the improvement of glucose transport in intestinal tracts, and the improvement of analgesia and the like, caused by the physiological actions of the endothelin. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to an endothelin expression inhibitor.

Endothelin is a peptide consisting of 21 amino acid residues and is a physiologically active substance that continuously and indirectly contracts vascular and non-vascular smooth muscle. Endothelin is known to have three types of endothelin (endothelin-1, endothelin-2, endothelin-3) as a family peptide with similar structure in animals including humans. It is known to have a contracting action and a pressurizing action.
Increased action of endothelin causes persistent vasoconstriction in peripheral, renal and cerebral blood vessels, so hypertension, myocardial infarction, stroke, acute renal failure, Raynaud syndrome, atherosclerosis, asthma, prostate cancer It is thought to cause various diseases. Endothelin-1 has been reported to inhibit glucose transport in the small intestine (Non-Patent Document 1). Furthermore, endothelin-1 is considered to be involved in pain regulation because the threshold of pain sensation decreases in endothelin-1 KO mice (Non-patent Document 2).

It has been shown that the physiological action of endothelin is triggered by binding of endothelin to its receptor. Therefore, substances that inhibit the binding of endothelin to receptors and substances that suppress the expression of endothelin itself improve glucose transport in the small intestine and improve pain sensation in addition to the prevention and treatment of various diseases as described above. It is thought that it is useful for such.
For this reason, search for substances that suppress the action of endothelin has been conducted so far, and it has been reported that, for example, bosentan hydrate, thiadiazole derivatives, cyclic peptides, propolis compositions, etc. antagonize the endothelin receptor (non- Patent Document 3, Patent Documents 1 to 3).

On the other hand, rosemary is a Labiatae or evergreen small shrub native to the Mediterranean region, and its leaves and flowers are widely used mainly for cooking, medicinal purposes, and fragrances. Rosemary contains many polyphenol components such as carnosic acid, carnosol, and rosmarinic acid, and is known to have an excellent antioxidant effect. For example, carnosic acid and carnosol have been reported to increase the resistance of human fibroblasts to hydrogen peroxide and suppress apoptosis (Non-patent Document 4). Rosemary is known to have a deodorizing action, an antibacterial action, and the like.
However, the effect of rosemary on the expression of endothelin in epidermal cells has never been known so far.

JP-A-6-298971 JP-A-8-225595 JP 2004-161664 A

Journal of Physiology, 499.2, 391-402 (1997) Neuroscience, 130, 349-358 (2005) Journal of Pharmacology and Experimental Therapeutics, 270, 228-235 (1994) Fragrance Journal, 8, 55-60 (2004)

  The present invention relates to providing an endothelin expression inhibitor.

  As a result of repeated research on substances that suppress endothelin expression, the present inventor has found that rosemary has an excellent inhibitory effect on the expression of endothelin-1.

  That is, this invention provides the endothelin expression inhibitor which uses rosemary or its extract as an active ingredient.

  Since the endothelin expression inhibitor of the present invention has an excellent endothelin-1 expression inhibitory action, it has effects such as prevention and / or treatment of various diseases resulting from the physiological action of endothelin, improved glucose transport in the intestinal tract, and improved pain reduction. It is useful as a medical product, a quasi-drug, a cosmetic, and a food product.

It is a figure which shows the endothelin-1 gene expression level of the epidermal cell by hydrogen peroxide. It is a figure which shows the endothelin-1 expression inhibitory effect of a rosemary extract.

  In the present invention, rosemary means rosemary of the family Lamiaceae (Rosmarinus officinalis L.). Benenden blue, creeping, marine blue, Mallorca pink, Tuscana blue, Rex and the like are known as rosemary varieties, and any of these varieties can be used in the present invention.

  Rosemary can be used in the above-ground parts such as leaves, stems, buds, flowers and branches; underground parts such as roots; seeds and the like, but leaves and flowers are preferably used. These can be used as they are or after dry pulverization.

  As the extract of rosemary, an extract obtained by extracting the above-mentioned use site, preferably leaves, as they are or after being dried and appropriately sized or pulverized, and further separated A fraction (component) having higher activity obtained by purification is included. The extraction method may be any of immersion, decoction, leaching, reflux extraction, supercritical extraction, ultrasonic extraction, microwave extraction, and the like.

As the extraction solvent, either a polar solvent or a nonpolar solvent can be used, and these can also be mixed and used. For example, water; alcohols such as methanol, ethanol, propanol and butanol; polyhydric alcohols such as ethylene glycol, propylene glycol and butylene glycol; ketones such as acetone and methyl ethyl ketone; esters such as methyl acetate and ethyl acetate; tetrahydrofuran Linear and cyclic ethers such as diethyl ether; polyethers such as polyethylene glycol; halogenated hydrocarbons such as dichloromethane, chloroform and carbon tetrachloride; hydrocarbons such as hexane, cyclohexane and petroleum ether; benzene and toluene Aromatic hydrocarbons such as; pyridines; supercritical carbon dioxide; oils, waxes, other oils, etc., which can be used alone or in combination of two or more, and are repeated by changing the solvent It is also possible to carry out.
In the present invention, the rosemary extract is extracted with one or more polar solvents selected from water, alcohols, polyhydric alcohols and ketones from the viewpoint of endothelin-1 expression inhibitory action. It is preferable to use it. Moreover, the extract of rosemary can also use a commercial item.

  The rosemary extract used in the present invention may be a crudely purified product as long as it conforms to food and pharmaceutical acceptable standards and exhibits the effects of the present invention. The purity of the product may be increased by appropriately combining known separation and purification methods. Examples of the purification means include organic solvent precipitation, centrifugation, ultrafiltration membrane, high performance liquid chromatograph, column chromatograph and the like.

  The rosemary extract of the present invention may be used as it is, or as a diluted solution diluted with an appropriate solvent, or as a concentrated extract or dry powder. A paste prepared may be used.

The rosemary extract of the present invention significantly suppressed the expression of the endothelin-1 gene in an in vitro test system using epidermal cells, as shown in Examples below.
Here, it is known that endothelin-1 is overproduced in human epidermal cells by ultraviolet irradiation (Photochem Photobiol. 1996 63 (1): 1-10), and generally in human epidermal cells as test substances. The endothelin-1 production inhibitory effect is evaluated by whether or not the production of endothelin-1 in cells irradiated with ultraviolet rays is suppressed. On the other hand, the present inventors have found that the expression of endothelin-1 gene in human epidermal cells is increased by hydrogen peroxide (Reference Example 1). From this, exposure to hydrogen peroxide is considered to have an effect equivalent to that of ultraviolet irradiation on human epidermal cells, and the endothelin of the test substance is used as an indicator of the endothelin-1 gene expression inhibitory action in the in vitro test system. It is thought that the expression suppression effect can be evaluated.

  Therefore, the rosemary and the extract thereof are an endothelin expression inhibitor, various diseases that are considered to be useful for suppressing the expression and action of endothelin, such as hypertension, myocardial infarction, stroke, acute renal failure. , Raynaud's syndrome, atherosclerosis, asthma, prostate cancer and other preventive / therapeutic agents, intestinal glucose transport improvers, pain-reducing agents (hereinafter "endothelin expression inhibitors" etc.) Can be used to produce the agent.

  Such an endothelin expression inhibitor or the like has an endothelin expression inhibitory effect and various diseases that are considered to be useful for inhibiting endothelin expression and its action, such as hypertension, myocardial infarction, stroke, acute renal failure, Raynaud Human or veterinary drugs, quasi-drugs, foods, and functionalities that demonstrate effects such as prevention and treatment of syndrome, atherosclerosis, asthma, prostate cancer, etc., improved glucose transport in the intestinal tract, and improved pain reduction Can be used as food and cosmetics. The endothelin expression inhibitor is, for example, various diseases that are considered to be useful in suppressing the expression and action of endothelin, such as hypertension, myocardial infarction, stroke, acute renal failure, Raynaud's syndrome, atherosclerosis. It can be applied to foods, functional foods, foods for the sick, and foods for specified health use with the concept of prevention / treatment of infectious diseases, asthma, prostate cancer, etc., improved glucose transport in the intestinal tract, and improved pain reduction.

  When the endothelin expression inhibitor of the present invention is used as a medicine, it can be administered in any dosage form. Examples of the dosage form include oral, enteral, transmucosal, injection and the like. Examples of the dosage form of the preparation for oral administration include tablets, capsules, granules, powders, and syrups. Examples of parenteral administration include intravenous injection, intramuscular injection, suppository, inhalation agent, transdermal absorption agent, eye drop, nasal drop and the like.

  In such a preparation, one or more kinds selected from the rosemary of the present invention and the extract thereof may be used in combination with a pharmaceutically acceptable carrier. Such carriers include, for example, excipients, binders, disintegrants, lubricants, diluents, osmotic pressure regulators, pH regulators, emulsifiers, preservatives, stabilizers, antioxidants, colorants, ultraviolet rays. Absorber, moisturizer, thickener, brightener, activity enhancer, anti-inflammatory agent, bactericidal agent, corrigent, flavoring agent, extender, surfactant, dispersant, buffer, preservative, fragrance, coating agent Etc.

  Of these dosage forms, oral administration is preferred, and when used as a preparation for oral administration, the content of the rosemary of the present invention or the extract thereof in the preparation (in terms of the dry product of the extract) is usually It is 0.01 mass%-100 mass% of mass, Preferably, it is 0.1 mass%-70 mass%, More preferably, it is 0.5 mass%-50 mass%.

  The dosage of the formulation may vary according to the patient's condition, weight, gender, age or other factors, but the daily dosage per adult for oral administration is usually rosemary (extract 1 to 2000 mg, preferably 10 to 1000 mg, and more preferably 50 to 500 mg in terms of dry matter. Moreover, although the said formulation can be administered according to arbitrary administration schedules, it is preferable to administer once to several times a day.

  When the endothelin expression inhibitor of the present invention is used as a food, the form may be solid, semi-solid or liquid. Examples of food include breads, noodles, confectionery, jelly, dairy products, frozen foods, instant foods, processed starch products, processed meat products, other processed foods, beverages, soups, seasonings, nutritional supplements, etc. , And their raw materials. Further, like the above-mentioned oral administration preparation, it may be in tablet form, pill form, capsule form, liquid form, syrup form, powder form, granule form and the like.

  To prepare various forms of food, at least one selected from rosemary and its extract, other food materials, solvents, softeners, oils, emulsifiers, preservatives, fragrances, stabilizers, coloring An agent, an ultraviolet absorber, an antioxidant, a humectant, a thickener and the like can be used in appropriate combination.

  In addition, the content of rosemary or the extract thereof in the food (converted to the dried product) is usually 0.001 to 100% by mass, and 0.002 to 60% by mass, depending on the form of use. Is preferable, and 0.005-40 mass% is more preferable.

  When the endothelin expression inhibitor of the present invention is used as a quasi-drug or cosmetic, it can be used as a topical skin preparation, cleaning agent, makeup cosmetic, etc., depending on the method of use, lotion, emulsion, gel , Creams, ointments, powders, granules and the like. Such quasi-drugs and cosmetics in various dosage forms are blended with one or more selected from the rosemary of the present invention and extracts thereof, and quasi-drugs, skin cosmetics and cleansing agents. Oily ingredients, moisturizers, powders, pigments, emulsifiers, solubilizers, detergents, UV absorbers, thickeners, drugs (eg anti-inflammatory agents, bactericides, antioxidants, vitamins, fat metabolism promoting action Alternatively, it can be prepared by appropriately combining a drug or natural product known to have an effect of promoting uncoupling protein expression), a fragrance, a resin, an antibacterial / antifungal agent, a plant extract, an alcohol or the like.

  In general, the content of the rosemary of the present invention or the extract thereof in the quasi-drug and cosmetics (in terms of dry matter of the extract) is preferably 0.00001 to 20% by mass. It is more preferable to set it to 0001-10 mass%.

Reference Example 1 Effect of hydrogen peroxide on endothelin-1 gene expression in epidermal cells

1) Method Human epidermal cells (NHEK (F) derived from neonatal foreskin, Kurabo Industries) are seeded at a rate of 2500 cells / cm ^{2 in} EpiLife ^TM , the epidermal keratinocyte growth medium, and the cell density becomes subconfluent at 37 ° C. Until cultured. After washing with PBS (−), 2.0 ml PBS (−) containing ₅ to 100 μM H ₂ O ₂ was added and cultured at 37 ° C. for 1 hour. The untreated control was washed with PBS (−), added with 2.0 ml PBS (−), and cultured at 37 ° C. for 1 hour.
After washing with EpiLife ^TM , the epidermal keratinocyte growth medium, 2.0 ml of the growth medium was newly added and the culture was resumed. After 6, 12, and 24 hours, respectively, using ISOGEN (Wako Pure Chemical Industries, Ltd.) Then, the total RNA of the cells was collected, and cDNA was prepared using this as a template using Superscript III First Strand Synthesis Supermix (Invitrogen). Using this cDNA, the expression level of endothelin-1 gene was measured by real-time PCR using TaqMan (registered trademark) Gene Expression Assays as primers and probes (equipment used: ABI PRISM (registered trademark) 7000 sequence detection system).

The gene expression level in each sample was expressed as a relative value with respect to the standard cDNA, and the relative expression level of the RPLP0 gene as an internal standard gene was measured and normalized. The IDs of TaqMan (registered trademark) Gene Expression Assays used for examining the expression levels of endothelin-1 and RPLP0 genes are Hs00174961_m1 and Hs99999902_m1, respectively.
Significant difference was tested by t-test, and the significance level was 5%. When performing a multiple test, the significance level was calculated by Bonferroni correction, and this was used for determination.

2) Results As is apparent from FIG. 1, the expression of endothelin-1 gene in human epidermal cells was increased by hydrogen peroxide. In addition, the expression fluctuation | variation of the endothelin-1 gene in FIG. 1 was represented as a relative ratio (%) when the endothelin-1 gene expression level of an untreated control is set to 100. Therefore, using the endothelin-1 gene expression inhibitory action in cells under the hydrogen peroxide load condition as an index, it is considered possible to evaluate the endothelin-1 expression inhibitory effect of the test substance.

Example 1 Examination of endothelin-1 gene expression inhibitory action by rosemary extract 1) Method [cell culture]
Cultured human epidermis cells (NHEK (F) derived from neonatal foreskin, Kurabo) are added to EpiLife ^TM , a medium for epidermal keratinocyte growth, with growth additives (insulin 0.5 ml, hEGF 0.5 ml, hydrocortisone 0.5 ml, antibacterial agent 0.5 ml, BPE) 2 ml) was added (hereinafter referred to as growth medium). For passage, 0.025% trypsin / 0.01% EDTA was used.

[Hydrogen peroxide treatment]
After removing the medium and washing twice with PBS (−), 2.0 ml of PBS (−) containing 100 μM H ₂ O ₂ was added, followed by incubation at 37 ° C. for 1 hour. The untreated control was washed with PBS (−) in the same manner as the hydrogen peroxide treatment, and then 2.0 ml of PBS (−) was added and cultured at 37 ° C. for 1 hour.

2) Evaluation method 2500 cells / cm ^{2 of} seedling cells were seeded in a 6-well plate and cultured in a 2.0 ml growth medium. After confirming that the cell density reached subconfluence, the medium was replaced with a medium supplemented with the sample. The sample-added medium was prepared by adding Falco Rex Rosemary E (Ichimaru Falcos Co., Ltd., extraction solvent 50% ethanol aqueous solution) to the growth medium to a residue of 0.00075% final concentration.
After culturing in a sample-added medium for 24 hours, the above-described hydrogen peroxide treatment was performed, and after washing with a growth medium, 2.0 ml of a growth medium was newly added and cultured for 12 hours. The cytotoxicity of the sample was determined by observing the morphology of the cells and determining whether there was any abnormality. As a solvent control, a 50% ethanol aqueous solution was used instead of the sample.
6 hours and 12 hours after the resumption of culture, the total RNA of the cells was recovered using ISOGEN (Wako Pure Chemical Industries, Ltd.), and Superscript III First Strand Synthesis Supermix (Invitrogen) was used as a template. CDNA was created. Using this cDNA, the expression level of endothelin-1 gene was compared and examined by real-time PCR using TaqMan (registered trademark) Gene Expression Assays as primers and probes as in Reference Example 1.

3) Results FIG. 2 shows the endothelin-1 gene expression level when the rosemary extract was added and the endothelin-1 gene expression level when the 50% ethanol aqueous solution (solvent control) was added. The expression variation of the endothelin-1 gene in FIG. 2 was determined as a relative ratio (%) when the untreated control endothelin-1 gene expression level was 100.
As is clear from FIG. 2, rosemary extract significantly suppressed endothelin-1 gene expression from epidermal cells under hydrogen peroxide load conditions. From this, it was confirmed that rosemary extract has an endothelin-1 expression inhibitory effect.

Claims (1)

  An endothelin expression inhibitor comprising rosemary or an extract thereof as an active ingredient.

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