JP6037548B2 - Anti-inflammatory agent - Google Patents

Description

The present invention relates to anti-inflammatory agents via the inducible nitric oxide synthase containing reduced form Sukitonemi in as an active ingredient.

  In general, nitric oxide (NO) has high reactivity because it has unpaired electrons, and is known to be involved in various reaction systems in vivo. NO is synthesized from alginic acid and oxygen by NO synthase in vivo. There are three known isoforms of NO synthase: neural (nNOS), inducible (iNOS) and endothelial (eNOS). Among them, iNOS is expressed by interferon-γ (IFNγ) and interleukin-1 (IL-1) induced by infection and the like, and its expression is induced at the transcription level, and monoxide plays an important role in the inflammatory reaction. It synthesizes nitrogen and activates a series of biological defense mechanisms.

  Usually, the inflammatory reaction is controlled by mediators such as lipopolysaccharide (LPS) and prostaglandin (PG) in addition to IFNγ and IL-1. However, acute inflammation due to chronic inflammation or microbial infection is harmful to tissues and may cause cell damage. For example, as disclosed in Non-Patent Document 1, excessive production of NO by sustained expression of iNOS can cause inflammatory diseases such as sepsis, cardiovascular disease, rheumatoid arthritis, bronchitis, and cancer, and autoimmune diseases. May cause. Currently, there is an urgent need for the development of preventive and therapeutic drugs for worsening diseases such as inflammatory diseases mediated by iNOS.

Heo SJ, Yoon WJ, Kim KN, Ahn GN, Kang SM, Kang DH, Affan A., Chulhong O., Jung WK, Jeon YJ (2010) .Evaluation of anti-inflammatory effect of fucoxanthin isolated from brown algae in lipopolysaccharide- stimulated RAW 264.7 macrophages. Fo od. Chem. Toxicol., 48, 2045-2051.

Special Table 2007-526768

  By the way, Ishi jellyfish is known as an algae that makes a macroscopically sized colony on the surface of school ground or the like. For example, as disclosed in Patent Document 1, it has been demonstrated that Ishi jellyfish exhibits useful effects on the living body such as cholesterol lowering action, antiviral action, and anticancer action.

The present inventors have found, as a result of intensive research, with the reduced form Sukitonemi down isolated from Nostoc commune, it was found to have an effect excellent nitric oxide production suppression.
The present invention has been made based on the above findings, and an object of the present invention is to provide an anti-inflammatory agent that exhibits an excellent anti-inflammatory action.

Anti-inflammatory agents against bacterial inflammation via inducible nitric oxide synthase of the invention described in claim 1 in order to achieve the above object, comprises a reduced form Sukitonemi in as an active ingredient.

According to the anti-inflammatory agent of the present invention, an excellent anti-inflammatory action can be exhibited.

Explanatory drawing of the isolation process of reduced type skitonemin. Explanatory drawing of the isolation process of skitonemin. The graph which shows the influence on NO production | generation of LPS / IFN (gamma) induction RAW264 cell by a reduced form skitonemin and a skitonemin. (A) Results of Western blot analysis showing the effect of reduced skitonene on iNOS and COX-2 expression in LPS / IFNγ-induced RAW264 cells. (B) Electrophoresis results after RT-RCR showing the effect of reduced skitonene on iNOS mRNA expression in LPS / IFNγ-induced RAW264 cells. (A) Results of Western blot analysis of MAP kinase (p38, SAPK / JNK, ERK) in intracellular signal transduction associated with NO production in LPS / IFNγ-induced RAW264 cells of reduced form of skitonemin. (B) Western blot analysis of IκBα and STAT1 in intracellular signal transduction associated with NO production in LPS / IFNγ-induced RAW264 cells of reduced skitonemin. The figure which shows the influence of MAP kinase (p38, JNK, ERK), I (kappa) -B, STAT1, etc. in the inflammatory signal in LPS / IFN (gamma) induction RAW264 cell of reduced type | mold skitonemin.

Hereinafter, the embodiment which materialized the nitric oxide production inhibitor of the present invention is described in detail. Hereinafter, uses other than anti-inflammatory agents for bacterial inflammation are used as reference examples.
Reduced scytonemin (Reduced scytonemin) and scitonemin (Scytonemin) contained as active ingredients in the nitric oxide production inhibitor of the present embodiment have the structures shown by the following general formula (1) and general formula (2), respectively. . Of these, reduced-type skitonemin is preferred because of its excellent inhibitory effect on nitric oxide production. Hereinafter, skitonemin is used as a reference example.

In addition, as the reduced-type skitonemin and the skitonemine contained in the nitric oxide production inhibitor, for example, purified products derived from natural materials, biochemical products, and chemically synthesized products can be applied. In the case of applying a refined product from a natural material, examples of the raw material of the natural material include cyanobacteria (Cyanobacteria) and Nostoc commune, which is a kind of terrestrial cyanobacteria belonging to the genus Nenjumo. And reduced form skitonemin and skitonemin can be obtained by performing an extraction process and an isolation process by using Ishijelly. Hereinafter, a method for obtaining reduced-type skitonemin and skitonemin from Ishijelly will be described.

  The mushroom jellyfish used as a raw material may be a naturally occurring algal body or an artificially cultured algal body. In addition, it is industrially preferable to use an artificially cultured alga body from the viewpoint that stable supply is possible and quality maintenance is easy. In addition, the jellyfish as a raw material may be in any state of being collected, in a state of being crushed after being collected, in a state of being dried after being collected, and in a state of being crushed and dried after being collected.

  The extraction step is a step of extracting an extract containing reduced-type skitonemin and skitonemine from the medusa. As the extraction solvent used in the extraction step, water, a hydrophilic organic solvent, or a mixed solvent of water and a hydrophilic organic solvent can be used. Examples of the hydrophilic organic solvent include lower alcohols such as methanol and ethanol, acetone, and ethyl acetate. As the extraction method, any known extraction method such as cold water extraction, hot water extraction, hot water extraction, and steam extraction may be used.

  As the extraction operation, the jellyfish is immersed in the extraction solvent for a predetermined time. In extraction operation, in order to improve extraction efficiency, you may further perform processes, such as a stirring process, a pressurization process, and an ultrasonic treatment, as needed. After the extraction operation, for example, by performing a solid-liquid separation operation using a known separation method such as filtration or centrifugation, the extract and the raw material residue are separated. At this time, the obtained extract (extract) is concentrated as necessary.

  The isolation step is a step of isolating and purifying reduced skitonemin and skitonemin contained in the extract obtained in the extraction step. Reduced skitonemin and skitonemin are isolated by purifying the extract using one or more chromatography. As the chromatography, known chromatography such as liquid chromatography, supercritical fluid chromatography, and thin layer chromatography can be used. As liquid chromatography, column chromatography can be used, for example, and more specifically, high performance liquid chromatography (HPLC) and open column chromatography can be mentioned. Examples of the chromatography carrier include ion exchange chromatography, partition chromatography (normal phase / reverse phase chromatography), adsorption chromatography, and molecular exclusion chromatography. More specifically, it is preferable to use a silica gel carrier or an ODS carrier as partition chromatography from the viewpoint of separation efficiency.

  By combining the above various chromatographs as appropriate, the reduced skitonemin and the skitonemin can be isolated and purified by a known method of use. As a method of using the above various chromatographies, known methods can be appropriately employed. The above-mentioned reduced form of skitonemin and skitonemin can be identified by determining the structure or using purified products as indicators.

  Specific blending forms of the nitric oxide production inhibitor of the present embodiment include, for example, additives such as health foods and foods such as foods, pharmaceuticals, quasi drugs, skin external preparations, and cosmetics. The form of the food or drink is not particularly limited, and may be any of liquid, powder, gel, solid and the like, and the dosage form is any of tablets, capsules, granules, and drinks. There may be. As said food-drinks, you may mix | blend gelatinizer containing foodstuffs, saccharides, a fragrance | flavor, a sweetener, fats and oils, a base material, an excipient | filler, a food additive, a subsidiary material, a bulking agent etc. suitably as another component.

  In the case of use as a pharmaceutical, in addition to taking (oral intake), intravascular administration, transdermal administration, intraperitoneal administration, any administration method such as application to the affected area or direct administration to the affected area can be adopted. . As the dosage form, a dosage form suitable for each administration method can be appropriately adopted. For example, powder, powder, granule, tablet, capsule, pill, suppository, liquid, injection, external preparation for skin Etc. If necessary, excipients, bases, emulsifiers, solvents, stabilizers and the like may be added as additives.

  When the nitric oxide production inhibitor of the present embodiment is applied to a cosmetic, it can be produced by blending into the cosmetic base material. The form of the cosmetic may be any of emulsion, cream, powder and the like. By applying such a cosmetic to the skin, an effect of suppressing nitric oxide production can be obtained. Cosmetic bases are generally blended in common with cosmetics, and include, for example, oil, purified water and alcohol as main components, surfactants, moisturizers, antioxidants, thickeners, anti-oxidants. At least one selected from a seborrheic agent, a blood circulation promoter, a whitening agent, a pH adjuster, a pigment, a preservative, and a fragrance is appropriately blended. In addition, when applying the nitric oxide production inhibitor of the present embodiment as an external preparation for skin, cosmetics, and food and drink, it is intended to obtain the above actions and effects in order to distinguish it from conventional products. It is preferable to attach a display.

Next, an effect | action of the nitric oxide production inhibitor comprised as mentioned above is demonstrated.
The nitric oxide production inhibitor of the present embodiment exerts a high nitric oxide production inhibitory action due to reduced skitonemin or skitonemine blended as an active ingredient. More specifically, it exerts an inhibitory effect on nitric oxide production via inducible nitric oxide synthase. Reduced skitonemin and skitonemin suppress the expression of mRNA corresponding to inducible nitric oxide synthase and reduce the amount of inducible nitric oxide synthase.

  Overproduction of nitric oxide by sustained expression of inducible nitric oxide synthase is known to cause inflammatory diseases such as sepsis, cardiovascular disease, rheumatoid arthritis, bronchitis, and cancer, and autoimmune diseases. It has been. Therefore, the nitric oxide production inhibitor of this embodiment can be preferably applied as a prophylactic or therapeutic agent for inflammatory diseases such as sepsis, cardiovascular disease, rheumatoid arthritis, bronchitis, and cancer, and autoimmune diseases.

  Further, the nitric oxide production inhibitor of the present embodiment is about MAP kinase (p38, SAPK / JNK, ERK), IκBα, STAT1 activated by intracellular signal transduction accompanying LPS / INFγ-induced nitric oxide production, Inhibits each activity. Thereby, the production of nitric oxide causing an inflammatory response is reduced. Therefore, the nitric oxide production inhibitor of this embodiment can be preferably applied as an anti-inflammatory agent.

Next, the effect in this embodiment is described below.
(1) The nitric oxide production inhibitor of this embodiment contains reduced-type skitonemin or skitonemine as an active ingredient. Therefore, the application of reduced skitonemin or skitonemin can effectively exert the inhibitory effect on nitric oxide production via inducible nitric oxide synthase.

  (2) The nitric oxide production inhibitor of the present embodiment exerts an action of suppressing the production of nitric oxide via an inducible nitric oxide synthase with reduced-type skitonemin or skitonemin as an active ingredient. Therefore, prophylactic and treatment of inflammatory diseases such as sepsis, cardiovascular disease, rheumatoid arthritis, bronchitis, and cancer, and autoimmune diseases associated with overproduction of nitric oxide via inducible nitric oxide synthase It can be suitably used for medicine.

  (3) In the nitric oxide production inhibitor of this embodiment, preferably, reduced-type skitonemin or skitonemine, which is an active ingredient, is derived from Ishi jellyfish. Therefore, there are few side effects and it can apply more safely with respect to a biological body.

In addition, the nitric oxide production inhibitor of the present embodiment can be modified and embodied as follows.
-The nitric oxide production inhibitor of this embodiment exhibits a nitric oxide production inhibitory action by reduced-type skitonemin or skitonemine, which is an active ingredient. Thereby, the inhibitory effect of inflammation related to nitric oxide can be effectively exhibited. Therefore, you may comprise as an anti-inflammatory agent which uses reduced-type skitonemin or a skitonemin as an active ingredient.

  -The nitric oxide production inhibitor of this embodiment is a MAP kinase (p38, SAPK / activated by intracellular signal transduction associated with LPS / INFγ-induced nitric oxide production, by reduced-type skitonemin or skitonemin as an active ingredient. JNK, ERK), IκBα, and STAT1 activities are suppressed. Therefore, it may be configured as an activity inhibitor of MAP kinase, IκBα, or STAT1 containing reduced-type skitonemin or skitonemin as an active ingredient.

  -The nitric oxide production inhibitor of this embodiment is preferably applied to patients with inflammatory diseases and autoimmune diseases associated with, for example, excessive production of nitric oxide via inducible nitric oxide synthase. However, not only for therapeutic use, but also for healthy people may take in order to prevent these various symptoms.

  -The nitric oxide production inhibitor of the present embodiment is a non-human animal, for example, domestic animals such as horses, cows and pigs (non-human mammals), poultry such as chickens, dogs, cats, rats and mice, etc. May be administered to other pets (various domestic animals).

  -You may apply the nitric oxide production inhibitor of this embodiment as a reagent for experiment and research. It can be used for the purpose of elucidating the mechanism of physiological action related to the production of nitric oxide.

Next, a technical idea that can be grasped from the above embodiment and another example will be described.
(I) An activity inhibitor of MAP kinase, IκBα, or STAT1 containing reduced-type skitonemin or skitonemin as an active ingredient.

(B) A therapeutic or prophylactic agent for autoimmune diseases comprising reduced-type skitonemin or skitonemin as an active ingredient.
(C) A method for producing a nitric oxide production inhibitor or an anti-inflammatory agent, which comprises the reduced form of skitonemin or schitonemin from water jellyfish using water, a hydrophilic organic solvent, or a mixed solvent of water and a hydrophilic organic solvent. A method for producing a nitric oxide production inhibitor or anti-inflammatory agent, comprising: an extraction step for extracting an extract; and an isolation step for isolating the reduced-type skitonemin or skitonemine from the extract.

Next, the above embodiment will be described more specifically with reference to examples.
<1. Extraction and isolation of reduced-type skitonemin and skitonemine>
Ethanol (4 L) was added to dried powder of jellyfish (400 g), stirred at room temperature for 2 hours and allowed to stand for 30 minutes, and then the supernatant was collected. In addition, ethanol (4 L) was added to the precipitate, stirred at room temperature for 2 hours and allowed to stand for 30 minutes, and then the supernatant was collected. The re-extraction operation for this precipitate was repeated a total of 10 times. All the obtained supernatants were filtered with a filter paper, and the filtrate was concentrated under reduced pressure to obtain Ishi jellyfish extract (20 g).

  The reduced schitonemin and schitonemin were isolated by combining a plurality of chromatographic fractions on the jellyfish extract. With reference to FIG. 1, the reduced-type skitonemin and the isolation process of skitonemin are demonstrated.

  The Ishi jellyfish extract (18 g) was fractionated into 8 fractions by performing suction liquid chromatography using neutral silica gel, and the fraction containing reduced skitonemin and skitonemin based on comparison with the standard by TLC (NC-5) was recovered. Fraction NC-5 was fractionated into 5 fractions by performing liquid column chromatography using neutral silica gel, and a fraction containing reduced skitonemin and skitonemin based on comparison with a standard by TLC ( NC-5-4, NC-5-5) were recovered.

  Fraction NC-5-4 was further fractionated into 6 fractions by liquid column chromatography using neutral silica gel. Further, the fraction NC-5-5 was further fractionated into 4 fractions by liquid column chromatography using neutral silica gel. And based on the comparison with the sample by TLC, reduced fractions containing skitonemin and skitonemin (NC-5-4-4, NC-5-4-5, NC-5-5-3) are collected. At the same time, these were combined into one fraction NC-5-A.

  Fraction NC-5-A was fractionated into 8 fractions by liquid column chromatography using Sephadex LH-20. Then, based on the comparison with the standard by TLC, the fraction containing reduced skitonemin (NC-5-A-7) is recovered, and the ODS carrier is used for the fraction NC-5-A-7. The reduced form of skitonemin was isolated by performing the liquid column chromatography used.

As shown in FIG. 2, skitonemine was further purified from the fraction of NC-5-A-1. First, NC-5-A-1 (19.8 mg) was fractionated into two fractions (NC-5-A-1-1, NC-5-A-1-2) by Sephadex LH-20 column chromatography. did. Among them, NC-5-A-1-1 was fractionated by Sephadex LH-20 column chromatography while confirming the degree of purification by NMR spectrum measurement and TLC test. In addition, ODS C ₁₈ column chromatography was repeated to isolate skitonemin.

  In this example, the anti-inflammatory effect of the above-mentioned reduced form of skitonemin and skitonemin isolated from Ishikura jellyfish was examined using an LPS / IFNγ-induced inflammatory reaction system modeled on macrophages (mouse macrophage cell line RAW264 cell line) during bacterial infection. did.

<2. Cell culture>
Mouse macrophage RAW264 cells were obtained from RIKEN (Tsukuba City, Ibaraki Prefecture). RAW264 cells are DMEM (Dulbecco's modified Eagle's medium) containing 10% fetal bovine serum, 100 U / mL penicillin, 100 μg / mL streptromycin (above Invitrogen, CA, USA); Thermo Fisher Scientifics, KK, MA, USA ) at, and cultured under 95% air -5% CO ₂ environment.

<3. Effects of reduced form of skitonemin and scytonemin on RAW264 cells>
The RAW264 cells were prepared to 2 × 10 ⁵ cells / mL, and 500 μL each was seeded on a 24-well multiplate (Thermo Fisher Scientifics). After sowing, reduced-type skitonemin and skitonemin were added at respective concentrations so that the final concentrations were 0.5 μM, 1 μM, and 2.5 μM, respectively, and precultured for 2 hours. After the pretreatment, LPS (final concentration: 200 ng / mL) and IFNγ (final concentration: 25 ng / mL) were added and treated for 24 hours. After 24 hours, the produced nitric oxide (NO) was developed with Griess reagent (Promega, WI, USA) and detected at 540 nm with an absorptiometer (Corona Electric, Ibaraki). The results are shown in FIG. The data was expressed as mean ± standard error. For comparison between the two groups, Student's t-test was performed. 5% significance was marked * and 1% significance was marked **. Further, in the figure, in the columns of LPS and IFNγ, + indicates that there is an addition and − indicates that there is no addition.

As shown in FIG. 3, the reduced-type skitonemin treatment significantly suppressed the production of NO in each concentration treatment as compared with the skitonemin treatment.
<4. Western blotting>
The effect of reduced skitonemin on intracellular signal transduction associated with LPS / IFNγ-induced NO production was examined by Western blotting. We investigated COX-2, a nuclear transcription factor for iNOS and inflammation-related genes, in the NO production signal induced by LPS / IFNγ.

<4-1. Collection of cell lysis sample>
The number of cells was adjusted to 2 × 10 ⁵ cells / mL, and 1 mL each was seeded on a 6-well multiplate. After overnight culture, reduced-type skitonemin was added to a final concentration of 1 μM, and pre-culture was performed for 2 hours. After the pretreatment, LPS (final concentration: 200 ng / mL) and IFNγ (final concentration: 25 ng / mL) were added and treated for 24 hours. After 24 hours of stimulation, the plate was washed with PBS (-) (0.5 mL x 2), containing 2% protease inhibitor, 2% Phosphatase, RIPA Buffer (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP- The cells were lysed using 40, 1% sodium deoxycholate, 0.1% SDS), and the cells were collected with a cell scraper. The protein concentration in the sample solution was measured with a Bio Rad DC protein assay kit (Bio Rab, Hercules, CA, USA). A sample buffer containing 10% mercaptoethanol was added to the sample solution with the adjusted protein concentration, heated at 98 ° C. for 5 minutes, and subjected to Western blotting.

<4-2. SDS-PAGE>
12% gel was set in the electrophoresis tank, and electrophoresis buffer (Bio Rad Tris / Glycine Buffer) was added to one third of the electrophoresis tank. 10 μL of sample was applied to each lane of the gel (equal amount of protein molecular weight marker was applied to the first unused lane), and the sample protein was separated by electrophoresis at 120 V for 1 to 1.5 hours.

<4-3. Western blotting>
After electrophoresis, carefully remove the gel from the glass plate, cut out the unnecessary concentrated gel, and filter paper soaked in a solution consisting of 10% blotting buffer (Tris / Glycine Buffer, Bio Rad), 20% methanol, and 70% ultrapure water. Moved to the top. A PVDF (Polyvinylidene difluoride) membrane (Santa Cruz Biotechnology, CA, USA) was immersed in methanol for 1 minute, hydrophilized, and then immersed in a blotting buffer for equilibration. The gel obtained by SDS-PAGE and a PVDF membrane are sandwiched between a sponge and filter paper soaked in blotting buffer well, set in the electrophoresis tank, and then run for 90 minutes at 60 V while cooling with ice water to transfer the protein to the PVDF membrane. .

  After completion of blotting, the PVDF membrane was immersed in 5% non-fat dry milk (Difco Laboratories Inc, MI, USA) -0.1% Tween 20-containing Tris buffered saline (T-TBS) for blocking for 1 hour. The PVDF membrane was washed 3 times with T-TBS for 5 minutes, immersed in a solution containing each monoclonal antibody (primary antibody) corresponding to iNOS and COX-2, and shaken at 4 ° C. overnight. The next day, it was washed with T-TBS with shaking (10 minutes × 3 times), immersed in a secondary antibody (T-TBS solution containing 5% nonfat dry milk) and shaken for 1 hour. Thereafter, the PVDF membrane was washed with T-TBS (10 minutes × 3 times), and a chemiluminescence detector (Davinch-Chemi, Wako Pure Chemical Industries, Ltd.) using ECL Plus (GE Healthcare UK Ltd, Amersham Place, England) Manufactured by Osaka). In this experiment, β-actin was used as a standard marker for protein expression level. The results are shown in FIG. In the figure, in the LPS / IFNγ column and R-scytonemin column, + indicates addition and-indicates no addition.

<5. iNOS mRNA expression>
The effect of iNOS mRNA on intracellular signal transduction associated with LPS / IFNγ-induced NO production of reduced skitonemin was examined by RT-PCR.

<5-1. Total RNA extraction and PCR>
RAW264 cells adjusted to 2 × 10 ⁵ cells / mL were seeded in a 6-well multiplate and pre-cultured for 12 hours. Then, after stimulation with LPS / IFNγ for 12 hours, the plate was washed 3 times with PBS (−). After washing, Trizol (invitogen) was added at 1 mL / well to lyse cells, and then transferred to a 1.5 mL Eppendorf tube. Next, 200 μL of chloroform was added, vortexed for 30 seconds, centrifuged at 11500 rpm for 15 minutes at 4 ° C., the supernatant was again transferred to a new 1.5 mL Eppendorf tube, 2-propanol was added, and the mixture was allowed to stand for 10 minutes. . Ten minutes later, it was centrifuged at 11500 rpm for 10 minutes at 4 ° C., the supernatant was carefully removed and the pellet was washed with 500 μL of 75% EtOH. The pellet was collected by centrifugation at 9000 rpm for 5 minutes at 4 ° C., and the washing solution was carefully discarded and allowed to air dry for 30 minutes. After 30 minutes, 180 μL of DEPC water (invitrogen), 20 μL of 10 × Buffer (Takara bio, Ohtsu, Japan), 2 μL of DNase1 were added and reacted at 37 ° C. for 1 hour. Thereafter, 40 μL of sodium acetate (Nippon Gene, Tokyo, 3M, pH 5.2) and 150 μL of phenol chloroform solution (invitrogen) were added and vortexed. After the tube containing the phenol chloroform sample solution was centrifuged at 15000 rpm for 5 minutes, the supernatant was transferred to a new 1.5 mL Eppendorf tube, and total RNA was extracted with 800 μL of ethanol. Thereafter, the mixture was centrifuged at 15000 rpm for 30 minutes at 4 ° C. to collect the total RNA. Finally, the supernatant was discarded, air-dried, dissolved in DNase-RNase-free water, and used for the test. GAPDH was used as a standard marker for mRNA expression level in this experiment.

  After measuring the total RNA amount of the sample with a biophotometer, each RNA sample was adjusted to 100 ng / μL. Reverse transcription reaction was performed using Takara Bio Prime Script RT regent kit. PCR conditions were heat denaturation 94 ° C .: 5 minutes, heat denaturation 94 ° C .: 0.5 minutes, annealing 55 ° C .: 0.5 minutes, and extension reaction 72 ° C .: 0.5 minutes and 30 cycles. Extension reaction 72 ° C .: 7 minutes and storage 4 ° C .: infinite. As primers used for PCR, iNOS: SEQ ID NO: 1 (forward), iNOS: SEQ ID NO: 2 (reverse), GAPDH: SEQ ID NO: 3 (forward), and GAPDH: SEQ ID NO: 4 (reverse) were used.

  The sample DNA obtained by the reverse transcription reaction was subjected to a semiquantitative RT-PCR reaction using the reaction solution shown in Table 1 below (conditions: see Table 1). Thereafter, iNOS mRNA expression was examined by 1 × TAE-2% agarose gel electrophoresis. The results are shown in FIG. In the figure, in the LPS / IFNγ column and R-scytonemin column, + indicates addition and-indicates no addition.

<6. Western blot and mRNA expression results>
As shown in FIG. 4 (a), LPS / IFNγ-induced iNOS and COX-2 protein expression was significantly higher than that of untreated cells (with LPS / IFNγ-induced treatment). Declined. Further, as shown in FIG. 4 (b), when the effect of reduced skitonene on iNOS mRNA expression was examined by a semiquantitative RT-PCR method, iNOS mRNA expression was significantly increased by treatment with reduced skitonene. Declined. Based on the above, it was speculated that reduced skitonene reduced iNOS mRNA expression and decreased iNOS protein expression level, thereby reducing NO production causing inflammatory reaction.

<7. Effect of reduced skitonemin on intracellular NO production signal>
The effect of reduced skitonemin on intracellular signal transduction associated with LPS / IFNγ-induced NO production was examined by Western blotting. MAP kinases (p38, SAPK / JNK, ERK), IκBα, and STAT1 activated by LPS / IFNγ-induced NO production signals were examined. Western blotting uses commercially available monoclonal antibodies corresponding to MAP kinase and the like, and <4. Western blotting> The method described in the column was followed. In this experiment, β-actin was used as a standard marker for protein expression level. The results are shown in FIGS. 5 (a) and 5 (b). In addition, each lane (0, 15, 30, 60) of FIG. 5 shows the processing time (minute) after adding LPS / IFNγ.

  As shown in FIGS. 5 (a) and 5 (b), reduced skitonene significantly suppressed the activation of MAP kinase, IκBα, and STAT1 in LPS / IFNγ-induced inflammatory signals. For example, as shown in FIG. 6, reduced skitonene inhibits activation of MAP kinase, Iκ-B, and STAT1, and suppresses downstream activation of AP-1 and NFκ-B nuclear translocation. It was speculated that iNOS expression was decreased and NO production was finally decreased.

Claims (1)

Anti-inflammatory agents against bacterial inflammation via inducible nitric oxide synthase, characterized by containing a reduced form Sukitonemi in as an active ingredient.