JP4537024B2 - Inflammatory disease preventive / therapeutic agent - Google Patents

Description

  The present invention relates to an inflammatory disease preventive / therapeutic agent using a physiologically active substance derived from a plant growing in Okinawa, and a functional food or food material for preventing / ameliorating an anti-inflammatory disease containing these as active ingredients.

  Conventionally, excellent natural resources containing active ingredients such as antioxidant activity have been developed for plants growing in Okinawa. For example, processed foods (for example, see Patent Document 1) in which a medicinal turmeric rhizome is applied to improve the taste, and antioxidant plant fibers using plant fibers that are not used as foods and their processed Foods (for example, refer to Patent Document 2), processed foods (for example, refer to Patent Document 3) that use guava leaves, moon peach, mugwort, tea leaves, etc. to improve the medicinal effect by fermentation treatment and improve the taste. Has been developed. Further, a substance for inhibiting α-amylase activity, characterized by comprising as an active ingredient a dry powder or extract of one or more kinds of plants selected from the group consisting of ghetto, red-crowned wrinkles, hiraemon lemon, damselfly and strelitzia (For example, refer patent document 4) etc. are also reported.

  On the other hand, nitric oxide (NO) has been found to have an important effect in mammalian cells. L-citrulline and NO are produced from L-arginine by the catalytic action of NO synthase (NOS). NOS is classified into constitutive NOS (cNOS) and inducible NOS (iNOS), and cNOS has a constant catalytic action that constantly produces low concentrations of NO in vascular endothelial cells and gastric mucosal cells. On the other hand, iNOS is activated by stimulation of lipopolysaccharide (LPS), cytokines or pathogens in cells involved in inflammation such as macrophages and neutrophils, hepatocytes and vascular smooth muscle, and is non-constant It has an inductive catalytic action that produces a large amount of NO. For example, it has been clarified that when macrophages in a living body are stimulated by a pathogen, the expression level of iNOS increases and NO production is promoted from L-arginine. The produced NO acts on an electron transport enzyme in the mitochondrial system of the cell to inhibit its activity (immunity response regulating action) and inhibit infection. Other effective actions of NO in vivo include relaxing blood vessels and reducing blood pressure, or preventing adhesion of multinucleated leukocytes and platelets to prevent platelet aggregation.

  However, when high concentrations of NO are produced from macrophages due to inflammatory diseases such as nephritis, liver damage, ulcerative colitis, rheumatoid arthritis, and allergic diseases, the surrounding tissues are damaged by NO and self- Symptoms similar to the immune phenomenon may occur. For example, when suffering from sepsis, the production of a large amount of NO causes shock symptoms due to myocardial contractility (septic shock). It can also trigger onset. In order to prevent tissue or cell damage due to excessive production of NO or the onset of induced diseases, it may be necessary to suppress the production of NO so that NO is present in the living body at an effective concentration. For this reason, the development of NOS inhibitors that inhibit the action of NOS involved in NO production and NO production inhibitors are being promoted. As typical NOS inhibitors, NG-monomethyl-L-arginine (L -NMMA) and L-arginine derivatives such as NG-nitro-L-arginine methyl ester (L-NAME) (where NG represents that a nitro group is attached to the N atom of the guanidino group). And a compound having a structural formula similar to that of L-arginine as a substrate. Examples of iNOS induction inhibitors include corticosteroids, serine / protease inhibitors, cysteine / protease inhibitors, etc., or a laurel-derived sesquiterpene costunolide having a NO production inhibitory action based on iNOS induction inhibition derived from natural products ( costunolide, dehydrocostus lactone (see, for example, Non-Patent Document 1), rhubarb-derived stilbene, rhapontigenin, piceatannol, resveratrol (see, for example, Non-Patent Document 2) ), A taxi-derived triterpene alisol F (for example, see Non-Patent Document 3) and the like have been reported.

On the other hand, hydroxystilbene present in grapes is a vasodilator (see, for example, Patent Document 5), a therapeutic agent for various heart diseases (see, for example, Patent Document 6), and a mutation and carcinogenesis inhibitor (for example, In addition, drugs and cosmetics (see, for example, Patent Document 8) that are used for stimulating collagen synthesis containing hydroxystilbene are known. Among the hydroxystilbene compounds, resveratrol having a hydroxyl group at the 3, 5, 4 'positions is present in grape skin or wine, and is known to have antitumor and anti-inflammatory effects. Skin cosmetics (see, for example, Patent Document 9) are also known.
Japanese Patent Application Laid-Open No. 11-289973 JP 2002-204673 A JP 2002-330725 A JP 2001-333733 A EP 96-830517 CA2187990 JP-A-6-24967 JP-A-11-322577 JP 2001-199870 Matsuda H., Kageura T., Toguchida I., UedaH., Morikawa T., Yoshikawa M. Life Sciences, 66, p. 2151-2157, 2000 Matsuda H., Kageura T., Morikawa T., Toguchida I., Harima S., Yoshikawa M. Bioorganic & Medicinal Chemistry Letters, 10, 323-327, 2000 Matsuda H., Kageura T., Toguchida I., Murakami T., Kishi A., Yoshikawa M., Bioorganic & Medicinal Chemistry Letters, 9, 3081-3086, 1999

  The ghetto (Alpinia specios K. Schum) distributed from southern Kyushu to southern China to tropical Asia belongs to the family Gingidae (Zingiberaceae), also known as Alpinia. Although it is used as a raw material for ropes, the useful physiologically active substances contained therein are not sufficiently utilized.

  An object of the present invention is to effectively use a ghetto plant having physiological activity, which can be easily obtained as a plant-derived component, has a low side effect on the living body and has high safety, and an anti-inflammatory agent using these. It is to provide a functional food or food material for prevention / amelioration of inflammatory diseases.

  The present inventors aimed at effective utilization of ghetto plants that grow in Okinawa, and as a result of keen research on their physiological activities, found that the extract of ghetto leaves has excellent NO production inhibitory activity, As a result of searching for an effective ingredient for NO production inhibitory activity of ghetto, we found that at least pinosylvin monomethyl ether is involved in NO production inhibitory activity. From MS spectrum etc., it estimated with 3-methoxy-5-hydroxystilbene of the following structure.

The structure and ¹ HNMR measurement values shown in Table 1 are compared with literature values to confirm that the compound having a remarkable NO production inhibitory activity is pinosylvin monomethyl ether, and the present invention is completed based on these findings. Has been reached.

That is, the present invention relates to (1) a preventive / therapeutic agent for inflammatory diseases based on nitric oxide production, comprising a chloroform extract of ghetto leaf as an active ingredient.

The present invention also provides (2) a preventive / therapeutic agent for inflammatory diseases based on nitric oxide production as described in (1) above , wherein the chloroform extract of ghetto leaves contains pinosylvin monomethyl ether About.

  The prophylactic / therapeutic agent for inflammatory diseases of the present invention is intended to make effective use of ghetto plants, and can be easily obtained as a plant-derived component, and can easily prepare an inflammatory disease prophylactic / therapeutic agent that has low side effects on the living body and high safety Thus, a functional food or food material for prevention / amelioration of anti-inflammatory diseases can be easily obtained.

  The agent for preventing / treating inflammatory diseases of the present invention is not particularly limited as long as it contains ghetto and / or a processed product thereof as an active ingredient. Ghetto (Alpinia specios K. Schum) used for the inflammatory disease preventive / therapeutic agent of the present invention is a plant belonging to the family Gingidae (Zingiberaceae), and the use site is the underground part such as root and rhizome, stem, bark, trunk, It may be an aerial part such as a leaf, flower or fruit, or a mixture of two or more of these.

  Examples of the treatment of the processed ghetto can include pulverization, cutting, drying, extraction, roasting, ultrasonic homogenization, freezing, heating, enzyme treatment, fermentation, and the like. Two or more may be combined. For example, a treatment for crushing the ghetto to a particle size of 3 mm or less, preferably 0.5 to 1.0 mm is preferable. By setting the particle size to 3 mm or less, the active ingredient can be easily extracted, and if the particle size is in the range of 0.5 to 1.0 mm, this effect can be obtained more remarkably. Moreover, although the living body of a plant may be pulverized as it is, a pulverization process may be performed on a processed product that has been previously subjected to a cutting process, a drying process, a freezing process, or the like. Conversely, the pulverized product obtained by pulverizing the plant can be subjected to a drying process such as an air drying process at room temperature, a heat drying process at around 40 to 60 ° C., or a sun drying process. Furthermore, an extraction process can also be performed with respect to the dry-processed material after a grinding process, and the ground material after a dry process.

  Examples of the solvent used in the extraction treatment include lower monohydric alcohols such as water, methyl alcohol, and ethyl alcohol, liquid polyhydric alcohols such as glycerin, propylene glycol, and 1,3-butylene glycol, and non-hexane such as hexane. One kind or two or more kinds of polar solvents can be used. As a preferable extraction method, for example, methyl alcohol or ethyl alcohol having a water concentration of 0 to 100% by volume is used, and extraction is performed at 0 to 80 ° C. for 30 minutes to 5 days, preferably using an ethyl alcohol aqueous solution at room temperature for 24 hours. And then filtering.

  As the inflammatory disease preventive / therapeutic agent of the present invention, an lyophilized product obtained by lyophilization after extracting the solvent as it is or after removing the solvent is particularly advantageous. In addition, a plurality of these extracts can be mixed as necessary, or a mixture obtained by mixing with a solvent can be used.

  In addition, as an agent for the prevention and treatment of inflammatory diseases of the present invention, pinosylvin monomethyl which is a stilbene compound contained therein is obtained from an extraction treatment product obtained by the extraction treatment of a ground treatment product of the ghetto leaves or dry leaves. A purified product obtained by further separating and purifying ether (1-styryl-3-hydroxy-5-methoxybenzene) (PME) can be used. As such separation / purification treatment, known separation / purification methods of natural organic compounds can be applied. For example, adsorption / desorption using activated carbon, silica, etc., or chromatography, liquid-liquid extraction, fractional precipitation, etc. The method of separating and purifying pinosylvin monomethyl ether can be mentioned according to the technique. In particular, the separation / purification treatment using medium-pressure column chromatography of the reverse layer system (C18 gel) is effective for removing chlorophyll-based impurities. Since it can remove efficiently, it is preferable. Pinosylvin monomethyl ether separated and purified from ghetto has excellent NO production inhibitory activity, and 2.6 mg can be separated from 100 g of dried ghetto leaves (containing 6.5% by weight of water).

  The prophylactic / therapeutic agent for inflammatory diseases of the present invention comprising the above ghetto and / or processed product thereof as an active ingredient has NO production inhibitory activity. That is, it suppresses the expression of iNOS that is excessively expressed by various factors in vivo and produces excessive NO, thereby suppressing NO production, and for the treatment and prevention of inflammatory diseases that require suppression of NO production. It is effective and particularly useful for treating inflammation caused by macrophage NO production by suppressing macrophage NO production. Inflammation caused by NO production in which the agent for preventing and treating inflammatory diseases of the present invention is effective includes inflammation caused by exogenous biological factors caused by infection with pathogenic bacteria such as bacteria, viruses and parasites, heat, and cooling. Inflammation due to physical factors such as mechanical trauma, ultraviolet rays, radiation, inflammation due to chemical factors such as strong acids, strong alkaline chemicals, toxic substances, etc., cells of immune complexes produced endogenously, for example, in the body And inflammation such as nephritis, hepatic disorder and ulcerative colitis, ventilation, arthritis, rheumatoid arthritis, and the like due to allergic inflammation caused by deposition in tissues and abnormal metabolites generated in the body.

Such NO production inhibitory action can be measured by a chemiluminescence method, an electrode method, an electron spin resonance (ESR) method, a Griess method, or the like. Griess method is a metabolite of NO NO ₂ ^- and Griess reagent the amount by measuring the absorbance for (sulfanilamide and N-(1-naphthyl) ethylenediamine) and red azo compound produced by diazotization coupling reaction detects, NO ₂ from the amount of the azo compound ^- to calculate the amount, NO ₂ in the control ^- NO ₂ by converting the amount of calculated value as 100 ^- the amount of production rate, i.e., in a method for determining the NO generation rate Yes, it is used as an index for evaluating the NO production action of the subject.

  Further, in combination with this, by detecting the number of viable cells by MTT method or the like, the amount of NO produced can be detected, and the degree of NO production inhibitory activity can be determined. In this MTT method, 3- (4,5-dimethyl-2-thiazolyl) -2,5-diphenyl-2H tetrazolium bromide (MTT) is a substrate for intracellular mitochondrial dehydrase, and has high viability. It is a method that utilizes the fact that the amount of MTT that is reduced to a large extent and the resulting yellow to red formazan amount corresponds well to the number of viable cells, and can measure only living cells, see 630 nm The absorbance of the sample at a wavelength of 570 nm is measured as the wavelength, and the cell viability of the sample can be determined by the following calculation formula.

Cell viability = 100 × B / A
In the formula, A indicates a value obtained by dividing the absorbance at 570 nm from the absorbance at 570 nm of the control, and B indicates a value obtained by dividing the absorbance at 570 nm of the sample by the absorbance at 630 nm. The cell viability detected by the MTT method for the NO production inhibitor of the present invention is often higher than that of the control, and it is clear that the cells are proliferating as NO production is suppressed.

  The prophylactic / therapeutic agent for inflammatory diseases of the present invention can be a preparation for internal use in combination with a known pharmaceutical carrier or an external preparation. As a pharmaceutical carrier in such an internal preparation, a liquid or solid carrier that is pharmaceutically acceptable for the active ingredient of the ghetto and / or processed product thereof of the present invention can be applied. The inflammatory disease preventive / therapeutic agent of the present invention includes a solvent, a dispersant, an emulsifier, a buffer, a stabilizer, an excipient, a binder, a disintegrant, a lubricant, a diluent, and a pH buffer as necessary. In addition, a solubilizing agent, an isotonic agent, and the like can be added to form a solid preparation such as a tablet, granule, powder, powder, and capsule, and a liquid preparation such as a normal solution, suspension, and emulsion. These preparations can be administered orally or parenterally. That is, it can be administered orally in commonly used dosage forms, such as powders, granules, capsules, syrups, suspensions, etc., or, for example, in dosage forms such as solutions, emulsions, suspensions, etc. These can be administered parenterally in the form of injections or can be administered intranasally in the form of sprays. The dose can be appropriately selected depending on the type of disease, the weight of the patient, the dosage form, and the like. The dosage of such a preparation is appropriately set according to the preparation form, administration method, purpose of use and age, weight, and symptoms of the patient applied thereto, and is not constant, but in general, the active ingredient contained in the preparation The amount is 10 μg to 200 mg / kg per adult day. Since the dose varies depending on various conditions, an amount smaller than the above dose may be sufficient or may be necessary beyond the range. In addition to being orally administered as it is, the drug of the present invention can be added to any food or drink as a food or food material to be described later and taken on a daily basis. When the inflammatory disease preventive / therapeutic agent of the present invention is used as an external preparation, ethanol or the like and an emulsion, and ghetto extract can be kneaded with petrolatum or the like to obtain an ointment.

  As a method for preparing the preventive / therapeutic agent for inflammatory diseases of the present invention, the processed product such as the above ghetto and freeze-dried product is powdered with a mixer or the like, and the obtained powder is granulated, encapsulated and tableted by a conventional method. Specific examples include a method of preparing a solution by making it into a solution, dissolving, suspending, or dispersing in a solvent.

  The functional food or food material for prevention / amelioration of inflammatory diseases of the present invention comprising ghetto and / or processed product thereof as an active ingredient is a food or drink raw material of one or more selected from ghetto and / or processed product thereof Or can be obtained by adding and blending after the production process or production. Such a functional food is not particularly limited, and it is desirable to take a substance having a moderate NO production inhibitory effect every day for prevention and improvement of inflammation induced by excessive NO, etc. The functional food or food material for preventing / ameliorating anti-inflammatory diseases containing the preventive / treating agent for inflammatory diseases of the present invention is very useful in that respect. The functional food or food material for prevention / amelioration of anti-inflammatory diseases of the present invention is not particularly limited and may be any yogurt, drink yogurt, juice, milk, soy milk, liquor, coffee, Various beverages such as black tea, sencha, oolong tea, sports drinks, baked confectionery such as pudding, cookies, bread, cakes, jelly and rice crackers, Japanese confectionery such as sheep candy, bread and confectionery such as frozen confectionery, chewing gum, udon and soba Noodles, fish paste products such as kamaboko, ham, fish sausage, seasonings such as miso, soy sauce, cooking oil, margarine, lard, butter, dressing, mayonnaise, sweetener, dairy products such as cheese, butter, It can be used as a food by blending it with various side dishes such as tofu, konjac, other boiled rice cakes, dumplings, croquettes and salads. A compounding quantity is not specifically limited, It can select suitably with foodstuffs.

  EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, the technical scope of this invention is not limited to these illustrations.

[Sample preparation]
Ghetto leaves were pulverized to a particle size of 1 mm and dried at 60 ° C. for 1-2 days. 1 g of the obtained dried product was extracted with a 10 mL chloroform solution at room temperature for 1 week and then filtered to obtain an extract. Thereafter, the extract was concentrated under reduced pressure to prepare a 20 mg / mL dimethyl sulfoxide solution, and diluted with dimethyl sulfoxide to prepare samples with final concentrations of 20 μg / mL and 4 μg / mL.

[Separation of pinosylvin monomethyl ether (PME)]
Pinosylvin monomethyl ether was separated by use of reverse pressure (C18 gel) medium pressure column chromatography. Samples were prepared using dimethyl sulfoxide solutions of separated pinosylvin monomethyl ether (PME) at concentrations of 100 μM, 20 μM, 4 μM, and 0.8 μM.

[Measurement of NO production suppression]
Mouse macrophage-derived RAW264.7 cells were added to a 24-well microplate at 2 × 10 ⁵ cells / well and pre-cultured at 37 ° C. for 12 hours. Thereafter, the plate was washed twice with 1 mL of PBS, 0.5 mL of DMEM as a medium was added, and 5 μL of each concentration sample prepared in Examples 1 and 2 was added. For control, 5 μL of DMSO was used. To this, 10 μL of L-arginine (final concentration was 2 mM), 10 μL of LPS (final concentration excluding the blank was 100 ng / mL), 10 μL of IFN-γ (final concentration excluding the blank of IFN-γ) Was added to 100 U / mL) and 0.5 mL of DMEM as a medium, and the plate was shaken well and cultured at 37 ° C. for 24 hours.
[NO ₂ ^- Measurement]
500 μL of Griess reagent having the following composition was added to 500 μL of the culture supernatant, and after stirring, the absorbance at 543 nm was measured with a spectrophotometer (UV-2200AI: manufactured by Shimadzu Corporation). The absorbance is shown in Table 2. NO ₂ The absorbance in the case of control from the absorbance of the sample as 0 ^- seeking the ratio of the amount, as NO production inhibition ratio was calculated by the following equation. The results are shown in Table 1.

NO production inhibition rate = {1- (B / A)} × 100
In the formula, A represents the absorbance of the control, and B represents the absorbance of the sample.

Griess reagent 1% by weight sulfanilamide dissolved in 5% by weight phosphoric acid aqueous solution and 0.1% by weight N- (1-naphthyl) -ethylenediamide dihydrochloride solution are mixed in the same volume ratio. It was.

[MTT method]
To each well of the plate from which the supernatant had been removed, 500 μL of DMEM and 50 μL of MTT reagent were added and cultured at 37 ° C. for 1.5 hours. Thereafter, 1 mL of DMSO was added to each well, and the cells were broken with ultrasonic waves (ultrasonic cleaning device: manufactured by Tokyo Science Instrument Co., Ltd.), and then 500 μL of hydrochloric acid / 2-propanol was added to each well and stirred well, at 630 nm. As a reference wavelength, the absorbance at a wavelength of 570 nm was measured with a spectrophotometer (UV-2200AI: manufactured by Shimadzu Corporation). The absorbance is shown in Table 2. The cell viability was calculated from the absorbance according to the following formula. The results are shown in Table 3.

Cell viability = 100 × B / A
In the formula, A represents the absorbance of the control, and B represents the absorbance of the sample. The absorbance was a value obtained by dividing the absorbance at a wavelength of 630 nm from the absorbance at a wavelength of 570 nm.

Claims (2)

A prophylactic / therapeutic agent for inflammatory diseases based on nitric oxide production, comprising a chloroform extract of ghetto leaves as an active ingredient. Chloroform extract of the leaves of Alpinia zerumbet The preventive or therapeutic agent according to claim 1 inflammatory diseases based on nitric oxide production, wherein the containing pinosylvin monomethyl ether.