JP2006022002A - Nitrogen monoxide production inhibitory composition and chronic arthritis inhibitory composition - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new nitrogen monoxide production inhibitory composition and chronic arthritis inhibitory composition by using a natural material having a high safety as its raw material. <P>SOLUTION: This extract from the leaves of citrus fruit trees to begin with the leaves of Citrus hystrix used as a spice in a Thai food, especially the extract obtained by extraction of a hot water extract residue of the leaves of the citrus fruit trees by using an organic solvent such as ethanol has an excellent nitrogen monoxide production inhibitory activity and a chronic arthritis inhibitory activity, and is extremely useful as an additive for a food, feed, medicine, etc., having a preventing or treating effect of diseases associated with the excessive production of the nitrogen monoxide. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a composition for inhibiting nitric oxide production containing a specific plant extract as an active ingredient, and an arthritis inhibiting composition.

  It was clarified that nitric oxide (hereinafter abbreviated as NO) was first produced in vivo as an endothelial cell-derived smooth muscle relaxing factor in the 1980s. Since then, various studies have been carried out, and NO acts not only as a vasodilator and a neurotransmitter, but also as a mediator of infection, inflammation, and immune response via NO itself or peroxynitrite generated from NO. It has been known to have both good and bad effects, such as cell cytotoxicity.

  In vivo, NO is known to be produced by an enzyme using L-arginine as a substrate. It has been confirmed that there are three types of enzymes that catalyze the reaction: neural nNOS, vascular endothelial eNOS, and induced iNOS. NO produced by nNOS and eNOS is used to maintain homeostasis, but NO produced excessively by iNOS induced by infection and inflammation is not a physiological function but a variety of diseases Is deeply involved in.

Currently, diseases that are known to involve excessively produced NO include various digestive diseases associated with chronic inflammation, diseases such as arthritis, rheumatism, type 1 diabetes and diabetic complications, bronchial asthma, etc. Respiratory disease, cancer development due to DNA damage. (See Non-Patent Document 1.)

  In chronic arthritis such as rheumatism, it is known that inflammatory mediators produced in the proliferated joint synovium advance cartilage and bone destruction. In recent years, it has been reported that NO production is increased in the local area of joint inflammation, and it is known that NO is deeply involved in the onset and progression of chronic arthritis. (See Non-Patent Document 2.)

  Therefore, suppressing excessive NO production is very useful in preventing various diseases, and in recent years, many NO production inhibitors derived from synthetic products and natural products have been proposed. Examples of those derived from natural products include those derived from plants of the genus Salacia (see Patent Document 1), those derived from various herbal medicines and herbs (see Patent Document 2), and those derived from Indonesian folklore medicinal plants ( (See Patent Document 3).

Moreover, it is known that the extract of the kumomikan leaf which is citrus has the antioxidant property (refer patent document 4) and the carcinogenesis promotion inhibitory effect (refer patent document 5) in the auto-oxidation of linoleic acid. However, it has never been known to have an NO production inhibitory effect and an arthritis inhibitory effect.
JP 2003-063974 A JP 2002-087975 A JP 2002-047181 A JP-A-11-199427 Japanese Patent No. 3298723 Illustrated Medicine & Science Series "NO Physiology and Disease", Yodosha, 1999 Latest medical supplement "Rheumatic 2000"

  Finding compositions that can prevent or treat various diseases, especially chronic arthritis such as rheumatism, by using highly safe natural products and suppressing NO produced excessively in the body. It is to provide supplements or medicines.

  The present inventors examined NO production inhibitory effect and chronic arthritis inhibitory effect using a plant material which is a highly safe natural product. As a result, the present inventors have found that a citrus leaf extract including Komikankan leaves has a strong NO production inhibitory effect and a chronic arthritis inhibitory effect, and has completed the present invention.

Furthermore, in the present invention, the extract obtained by extracting the residue obtained by extracting hot water from citrus leaves such as kumikakan leaves with an organic solvent such as ethanol is effective as an NO production inhibitor and a chronic arthritis inhibitor. I found it.

  By compounding into food, feed, pharmaceuticals, etc., it effectively suppresses NO production of macrophages, and various diseases associated with excessive NO production, such as various digestive diseases associated with chronic inflammation, type 1 diabetes and diabetes It is useful for the treatment and prevention of complications, respiratory diseases such as bronchial asthma, etc., and especially chronic arthritis such as rheumatism. In addition, the use of natural products does not show any harmful effects on the human body and animals, and is extremely safe.

  The present invention is described in detail below.

  Citrus leaves used in the present invention refers to the leaves of the genus Citrus (Rutaceae), Kumquat, Karatachi, for example, Satsuma mandarin, Natsumikan, Kumquat, Yuzu, Hassaku, Iyokan, Navel, Ponkan, Orange, Examples include Daidai, Grapefruit, Lime, Lemon, Kobumikan, Sudachi, Kabosu, Karachi and other leaves. More preferably, the leaves of Kumomikan, Natsumikan, Yuzu, and Kumquat are mentioned. As these citrus leaves, fresh leaves can be used for extraction as they are, but those subjected to processing such as crushing, drying and pulverization may be used according to a conventional method.

In the present invention, the citrus leaves subjected to the hot water extraction treatment are citrus fruits recovered from the extract solution after being subjected to the extraction treatment for several seconds to several days, preferably several minutes to several hours, using water of 40 ° C. or higher. Leaves. The amount of solvent used for extraction is suitably 1 to 10,000 parts by weight, more preferably 1 to 1000 parts by weight, per part by weight of dry raw material. Citrus leaves after the hot water extraction treatment can be recovered from the extract by a known method. For example, filtration using a cloth, paper, etc., centrifugation, etc. are mentioned.

In the present invention, a desired extract can be obtained by performing an extraction operation on the citrus leaves after the hot water extraction treatment using an organic solvent or the like. The solvent that can be used for extraction with an organic solvent is not particularly limited as long as it can dissolve water-insoluble or insoluble components contained in citrus leaves after hot water extraction treatment. Alcohols such as methanol, ethanol, propyl alcohol, isopropyl alcohol, butanol, isobutanol, etc., or polyhydric alcohols such as propylene glycol and 1,3-butylene glycol, acetone, dioxane, methyl ethyl ketone, acetonitrile, ethyl acetate, butyl methyl ketone, Examples include diethyl ether, dichloromethane, xylene, trichloroethylene, carbon tetrachloride, benzene, chloroform and toluene.

The above solvents may be used alone, or two or more kinds may be mixed and used, or water and an organic solvent may be used in combination. When the above lower alcohols and polyhydric alcohols are used as hydrous alcohols, the water content is preferably 60% or less. In particular, ethanol and hydrous ethanol are preferable.

The extraction conditions with the organic solvent described above can be efficiently extracted by stirring or sonicating at a temperature of 10 ° C. or more for several minutes to several days, preferably tens of minutes to several tens of hours. . The amount of solvent used for extraction is suitably 1 to 10,000 parts by weight, more preferably 1 to 1000 parts by weight, per part by weight of dry raw material. The extract after the extraction treatment can be recovered from the extract by a known method. For example, filtration using a cloth, paper, etc., centrifugation, etc. are mentioned.

An extract can be obtained by concentrating the extract thus obtained and then drying. Depending on the solvent used, the extract can be used as it is. For example, an extract using ethanol or the like can be used without removing the solvent.

The disease involving excessive NO in the present invention refers to a disease accompanied by symptoms caused by cell damage via NO itself excessively produced by iNOS or peroxynitrite produced from NO, for example, Examples include chronic arthritis such as rheumatism, various digestive diseases associated with chronic inflammation, type 1 diabetes and diabetic complications, respiratory diseases such as bronchial asthma, and cancer associated with DNA disorders.

  The activity of the NO production inhibitor of the present invention can be confirmed by an iNOS induction test using the established macrophage cell RAW264.7, which is a known method. NO produced by iNOS induced by the action of lipopolysaccharide and interferon-γ on RAW264.7 cells is quantified as a nitrite ion by the grease method. At this time, the activity of the inhibitor can be measured by measuring the amount of NO production to be suppressed in the presence of the test substance.

  Confirmation of the effect on chronic arthritis in the present invention can be confirmed by an adjuvant arthritis suppression test which is a known animal model experimental system for rheumatoid arthritis. In this test, an adjuvant prepared from Mycobacterium tuberculosis is injected subcutaneously into the footpad of rats to cause rheumatoid arthritis symptoms, and the improvement or alleviation of symptoms by the test substance is scored and measured.

  In any case where the composition of the present invention is used as a NO production-suppressing composition or an arthritis-suppressing composition, an extract of a plant body may be taken as it is, but an excipient (sucrose, lactose, sugar Alcohol, starch, dextrin, gelatin, celluloses, etc.) may be added and taken as a solid preparation such as a tablet, granule or capsule. Furthermore, a binder, a disintegrant, a surfactant, a lubricant, a fluidity promoter, a corrigent, a colorant, a fragrance and the like can be blended.

  The dose of the NO production-suppressing composition or arthritis-suppressing composition of the present invention varies depending on the administration route, the degree of the disease, the age of the recipient, and the like. The amount may be about 1 mg to 1000 mg / kg body weight. The number of doses is not particularly limited, but the daily dose may be divided into 1 to 5 doses.

Furthermore, in the present invention, the above plant body extract may be added to various foods, feeds, pharmaceuticals and the like according to a conventional method.
In the above-described food, feed, pharmaceutical, etc., particularly in the raw material blending step, the present invention is the same as the normal manufacturing step by adding an appropriate amount of the above plant extract and mixing and stirring to adjust the raw material. A processed product containing the drug can be obtained. The addition amount may be appropriately selected so as to replace a part thereof within the range not deteriorating the flavor of the food, using the oral dose as a guide.

  EXAMPLES Next, although an Example is given and this invention is demonstrated in more detail, this invention is not restrict | limited at all by these Examples.

  250 g of a mixed solution of chloroform / methanol (2: 1) was added to 5 g of kojimikan powder that had been freeze-dried and pulverized as the raw material for extraction, and extracted by sonication for 30 minutes to recover the supernatant. As for the residue, 45 ml of the same solvent was added, and sonication was performed once again to recover the supernatant again. The collected supernatants were combined and dried under reduced pressure to obtain 0.4 g of Extract A.

In the extraction method of Example 1, the chloroform / methanol mixed solution was replaced with ethanol and the same operation was performed to obtain 0.2 g of extract B.

250 ml of distilled water was added to 5 g of freeze-dried Komikan leaf powder, heated to 80 ° C., stirred and extracted for 60 minutes, and then the supernatant was collected. After adding 45 ml of distilled water to the residue and washing at 80 ° C. for 5 minutes, the supernatant was collected again. The collected supernatants were combined and dried under reduced pressure to obtain 1.6 g of Extract C.

Extraction with ethanol similar to Example 2 was performed using the residue of hot water extraction in Example 3 as an extraction raw material, and 0.2 g of extract D was obtained.

The NO production inhibitory activities of Kumika mandarin leaf extracts with different extraction conditions were compared. Mouse macrophage cell RAW264.7 was used for measurement of NO production inhibitory activity. 2 × 10 ⁵ cells of RAW264.7 cells were planted in a 24-well microplate and cultured in a carbon dioxide incubator under 5% CO ₂ conditions at 37 ° C. for about 16 hours, and then the medium was replaced with 1 ml of colorless D-MEM. Then, 5 μl of DMSO solution of the test sample, L-arginine (final concentration 2 mM), interferon-γ (final concentration 100 U / ml) and lipopolysaccharide (final concentration 100 ng / ml) are added as inducers, and carbon dioxide gas is added again. The cells were cultured at 37 ° C. for 24 hours under 5% CO ₂ in an incubator. After culturing, NO produced in the culture solution was colorimetrically determined by the Grease method as nitrite and compared. The viability of the cells used for the assay was measured by the MTT method. The NO production suppression rate and the cell viability were determined from the following formulas.

NO production inhibition rate (%) = {((X−C) − (Y−C)) / (X−C)} × 100
Cell viability (%) = (Y / X) × 100
X: Measured value when only the inducer is added (Positive control) Y: Measured value when the inducer and the test sample are added C: Measured value when only the cells are cultured (Control)

  The NO production inhibitory activities of the extracts A, B, C and D obtained in Examples 1 to 4 were compared by the above methods. As shown in FIG. 1, it was found that the extract D has a particularly high activity.

  In the extraction method of Example 4, the extraction operation was performed with the temperature at the time of water extraction as the pretreatment being 45 ° C., the residue was recovered, and ethanol extraction was performed in the same manner. 0.24g of extract D-2 was obtained per 5g of dried komikankan leaves.

  In the extraction method of Example 4, the temperature at the time of water extraction as a pretreatment was set to 20 ° C., and the extraction operation was performed. 0.24 g of extract D-3 was obtained per 5 g of dried kubomikan leaves.

  In order to compare the influence on the NO production inhibitory activity given by the pre-treatment water extraction temperature in the extraction method of Example 4, the NO production inhibitory activity of the extracts of Examples 4, 6 and 7 was used in Example 5. The method was compared. As shown in FIG. 2, it was confirmed that the NO production inhibitory activity of the extract D and the extract D-2 obtained by treating the extraction with water, which was a pretreatment of ethanol extraction, at 45 ° C. or higher was high.

  An extract was prepared by the method of Example 4 using kumquat leaves as an extraction raw material. 18 mg of Extract 1 was obtained per 1 g of fresh leaves.

  An extract was prepared by the method of Example 4 using yuzu leaves as an extraction raw material. 11 mg of extract 2 was obtained per 1 g of fresh leaves.

  An extract was prepared by the method of Example 4 using Natsumikan leaves as an extraction raw material. 12 mg of extract 3 was obtained per 1 g of fresh leaves.

The NO production inhibitory activity of the extracts D, 1, 2, and 3 obtained in Examples 4, 9, 10, and 11 was compared by the method of Example 5. As shown in FIG. 3, it was confirmed that each citrus leaf extract has almost the same NO production inhibitory activity.

Using the extract D prepared by the method of Example 4, the inhibitory effect on rat-adjuvant arthritis was confirmed. Lewis female rats (8-week-old, SPF) were preliminarily raised for 7 days as 5 mice per group and used for the experiment. As the sensitizing adjuvant, M. tuberculosis H37Ra killed fine powder was prepared as a 2 mg / ml liquid paraffin suspension. The rats were fixed on a fixed base under ether anesthesia, and 0.1 ml of the prepared adjuvant was injected into the footpad skin of the right hind limb to induce arthritis.
As a test sample, a composition containing 30 mg / ml of Extract D in an aqueous solution of 0.5% (V / W) sodium carboxymethylcellulose (CMC) was prepared. In the test group, this composition was sensitized on day 0 to day 21 (22 days) for 6 days in a week, 1 ml / 100 g in the morning b. w. Gavage was administered at the rate of. On the other hand, in the control group, only 0.5% CMC aqueous solution was similarly administered by gavage.

During the study period, body weight, foot volume and arthritis score were measured at day 0, 3, 8, 13, 16 and 21. The arthritis score is determined by macroscopically observing the degree of redness, swelling and tonicity of the right forelimb, left forelimb and left hindlimb excluding the right hindlimb of the sensitized site, and a score of 0 to 4 points according to the criteria shown below. A total of up to 12 points was evaluated.
0: nil
1: mild
2: moderate
3: moderately-severe
4: severe

  The obtained body weight, arthritis score and paw volume were shown as an average value ± standard error for each group. Statistical tests were performed using analysis software (Stat View, Abacus Inc., USA) in order to test statistically significant differences between the groups. Body weight and volume data were subjected to analysis of variance (ANOVA) to confirm that they were equivariant, and then subjected to a multiple comparison test using Fisher's PLSD method to compare between groups. Moreover, the score data compared between groups using the Mann-Whitney U test. In all cases, p <0.05 was determined to be statistically significant.

  From the results on the 21st day after sensitization, the test group to which the composition containing Extract D was administered showed significant suppression and insensitivity of arthritis score (Table 2) and sensitized limb (right limb) volume (Table 3). The suppression tendency of the limb (left limb) volume (Table 4) was shown.

  From the above results, it was suggested that Komikankan leaf extract is effective against rat adjuvant arthritis, which is one of the animal models of rheumatoid arthritis.

  As described above, the results of Examples 5, 8 and 12 proved that the extract of the present invention has an NO production inhibitory action. Moreover, it was proved from the results of Example 13 that the composition of the present invention has an effect of suppressing chronic arthritis.

The figure which compared the influence which the extraction solvent and extraction operation have on the nitric oxide production suppression activity of an extract in the Komi orange leaf extraction method. (Example 5) The figure which compared the influence which the temperature of the water in the pre-treatment in the method for extracting Komikankan leaves exerts on the nitric oxide production inhibitory activity of the extract. (Example 8) The figure which confirmed the nitric oxide production inhibitory activity of various citrus leaf extracts. Example 12

Claims (4)

  A nitric oxide production inhibitor comprising as an active ingredient an extract obtained by extracting citrus leaves after extraction with water heated to 40 ° C. or more with an organic solvent such as ethanol. 2. The nitric oxide production inhibitor according to claim 1, wherein the citrus leaf is a leaf of Citrus hystrix . A composition for treating or preventing a disease involving excessive production of nitric oxide, comprising the nitric oxide production inhibitor according to claim 1 or 2 as an active ingredient. The composition for treatment or prevention according to claim 3, wherein the disease associated with excessive production of nitric oxide is chronic arthritis.

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