KR20040054940A - Cinnamic aldehyde containing composition having suppressive effect on inducible nitric oxide synthase and pro-inflammatory cytokine - Google Patents

Abstract

PURPOSE: Provided is a cinnamic aldehyde-containing composition which has suppressive effect on inducible nitric oxide synthase and pro-inflammatory cytokine. Therefore, the composition is used for the treatment of inflammation caused by increase of nitric oxides and cytokines. CONSTITUTION: A composition having suppressive effect on inducible nitric oxide synthase and pro-inflammatory cytokine is characterized by containing, as an active ingredient, 0.001-20 wt.% of cinnamic aldehyde represented by the formula(1), based on the total weight of the composition.

Description

Cinnamic aldehyde containing composition having suppressive effect on inducible nitric oxide synthase and pro-inflammatory cytokine}

The present invention provides an active ingredient comprising cinnamic (or sinam, cinnamic, cinamil: later cinnamic) aldehyde as an active ingredient in order to reduce and inhibit the production of nitrogen monoxide (hereinafter referred to as NO) and cytokines that cause inflammation. The composition relates to the inhibition of inducible nitric oxide synthase, which is produced upon inflammation, and also to the generation of inflammatory cytokines such as tumor necrosis factor (TNF-α), interleukin 1-beta (IL-1β), interleukin 6 It relates to a composition for inhibiting. Therefore, the composition of the present invention, which uses cinnamic aldehyde as an active ingredient, can be used for the treatment of inflammatory conditions characterized by an increase in NO and cytokines.

The main phenomena that occur in the living body when inflammation occurs are blood flow changes in vascular diameter, increased vascular permeability, and white blood cell infiltration. At the heart of the inflammation-producing process are T lymphocyte clonal augmentations caused by infections in the body, lipopolysaccharides (hereinafter referred to as LPS) or external antigenic stimuli from invading bacteria, activation of natural immune-related cells such as macrophages, It is initiated by cytokines and inflammatory mediators secreted from them. Representative cytokines secreted by immune-related cells include tumor necrosis factor (hereinafter referred to as TNF-α), interleukin 1-beta (hereinafter referred to as IL-1β), interleukin 6 (hereinafter referred to as IL-6), and inflammation. NO, Prostaglandins, Leukotrines, etc. can be mentioned as a mediator.

Among these, NO, which is involved in vasodilation, is produced and acted by several cells and is recognized as an inflammation mediator (Vane et al. 1994). The production of NO is catalyzed by a group of enzymes called nitric oxide synthase (hereinafter referred to as NOS). NOS removes guanidino nitrogen from L-arginine to form NO, which are generally separated into two types: constitutive NOS (hereinafter referred to as cNOS) and inducible NOS (hereinafter referred to as iNOS) (US CDNA clone in patent 5,468,630).

NOS in the form of cNOS produces small amounts of NO and mediates several specific actions, including mainly muscle relaxation and neurotransmission. In contrast, iNOS produces large amounts of NO upon inflammation, which is thought to mediate the cytotoxic activity of macrophages.

NO is a potent vasodilator that increases blood flow and vascular permeability. Examples of NO inducers include IL-1β, LPS, IFN-γ, and TNF-α.

NO formation has been found to increase in autoimmune diseases (ie rheumatoid arthritis, systemic lupus erythematosus, ulcerative colitis), and some traditional inflammatory symptoms (erythema, itching and vascular leakage) are recovered by NOS inhibitors (Schmidt) et al. 1994, Nathan et al. 1994, Marletta 1994).

Recently, cartilage affected by human osteoarthritis (OA) has been observed to spontaneously release enough NO to cause cartilage damage (Amin et al. 1995). Oral tissues have been reported to express higher amounts of NO and iNOS than healthy people (Hirose M et al. 2001).

In addition, IL-1β, TNF-α, and IL-6 are cytokines secreted in the inflammatory areas of rheumatoid arthritis and periodontitis-related patients. Recently, IL-1β, TNF-α antagonists are effective in the treatment of these patients. (R. Assuma et al. 1998). However, these antagonists are difficult to use as recombinant proteins due to their low production rate, fast activity loss at room temperature and high price.

The cinnamic aldehyde having the structure of Chemical Formula 1 is pale yellow with physicochemical properties and is an oily liquid with a scent. In addition, strong fungicidal action against fungi, analgesic, anti-inflammatory, anti-platelet aggregation has been known to be excellent. Cinnamic aldehydes are grown in the Cassia (Cinnamomum cassia Blume.), Cinnamomum obtussifolium Nees., Cinnamomum burmanni Blume., Cinnamomum iners Reinw. Ex Blume., Southern Japan It is found in the bark of Lauraceae, such as Cinnamomum sieboldii Meisn., Cinnamomum zeylanicum Nees. Or Cinnamomum cassia Presl var.

Looking at the prior art using the dynamic aldehyde, US Patent No. 6,042,812 and domestic patent (Registration No. 327,836) used the aldehyde as a fragrance component of the composition, and also in the case of Japanese Patent (P2001-89385A) Anti-adhesion to plaques was used as an active ingredient of anti-cavities (caries), which is different from the use of Cinnamic aldehyde as an active ingredient that inhibits the production of NO and inflammatory cytokines. It can be said.

In view of the above, it is apparent that for the treatment of acute and chronic inflammation accompanied with NO production and cytokine production, there is a need for a method of using a composition containing a aldehyde with low side effects and easily extracted.

The present inventors conducted a study on the inhibition of NO and inflammation-related cytokine production through cinnamic aldehyde on the basis of the above-mentioned prior art. As a result, a cinnamic aldehyde and a composition comprising the same as an active ingredient are produced by NO and IL-1β, TNF-α, The present invention was found to be able to effectively inhibit IL-6 and to complete the present invention.

Accordingly, it is an object of the present invention to provide a composition for the treatment of acute and chronic inflammation accompanied by the production of NO and cytokine by using the active ingredient with little side effects and easily extractable cinnamic aldehyde.

Figure 1 is an electrophoresis picture showing the change of iNOS mRNA by cinnamic aldehyde,

Figure 2 is an electrophoresis picture showing the change of IL-1β mRNA by cinnamic aldehyde,

Figure 3 is an electrophoresis picture showing the change of TNF-α mRNA by cinnamic aldehyde,

Figure 4 is an electrophoresis picture showing the change of IL-6 mRNA by cinnamic aldehyde.

The present invention relates to a composition having an inhibitory effect on the production of nitrogen monoxide and inflammatory cytokines containing cinnamic aldehyde having the structure of Formula 1 as an active ingredient.

The composition of the present invention, by the effect of inhibiting the production of nitric oxide and inflammatory cytokines, autoimmune diseases including rheumatoid arthritis, acute and chronic infections by bacteria and fungi, upper respiratory infections including common cold, gingiva and It can be used for the prevention and treatment of oral and periodontal diseases including periodontitis, skin diseases involving erythema and edema, or acute and chronic dermatitis. The usable formulation is not particularly limited, but may be formulated, for example, as toothpaste, dietary film, dentifrice, spray, paste (or patch), ointment, lotion, gel.

Cinnamic aldehyde as an active ingredient in the composition of the present invention may use purified high-purity reagents, and also cassia, spring and summer cinnamon, Java cinnamon, Thai cinnamon, broiler, Ceylon cinnamon or broadleaf cultivated in southern Japan From the powder ground crushed cyanide, extracted with at least one solvent selected from the group consisting of purified water, methanol, ethanol, propanol, butanol, ethylene glycol, propylene glycol, 1,3-butylene glycol, ethyl acetate and acetone Can be.

The preferred content of the cinnamic aldehyde in the composition of the present invention is 0.001 to 20% by weight, more preferably 0.1 to 10% by weight based on the total weight of the composition. When the content of the cinnamic aldehyde is less than 0.001% by weight, the effect on the inhibition of inflammation cannot be expected, and when it exceeds 20% by weight, the stimulation caused by the fragrance is severe, and thus the range is preferable.

The composition of the present invention may further include a steroidal drug such as dexamethasone, cyclohexidine or triclosan for the purpose of inhibiting inflammation and enhancing the therapeutic effect.

In the following Examples and Experimental Examples, the inflammation inhibitory effect of cinnamic aldehyde was identified in cells. Based on this, cinnamic aldehyde was used as an active ingredient to prepare compositions for oral cavity and periodontal disease. However, the scope of the present invention is not limited by the following examples and experimental examples.

Example 1 Effective Single Component Extraction of Cinnamic Aldehyde

Cynamic aldehyde can be prepared by a general extraction method, described above.

Cinnamon bark was pulverized into 10 to 200 mesh size, 100 g of ethanol (1,500 ml) was added to 300 g of the ground cinnamon powder, and the mixture was cooled for 48 hours, filtered, and the filtered extract was concentrated under reduced pressure at 45 ° C. The concentrated solution was redissolved in 70% ethanol aqueous solution to obtain 13% concentration of the extract as a natural natural aldehyde extract.

In this case, one or two or more mixed solvents selected from the group consisting of purified water, methanol, ethanol, propanol, butanol, ethylene glycol, propylene glycol, 1,3-butylene glycol, ethyl acetate, and acetone may be used as the extraction solvent.

Experimental Example 1: Inhibition of NO Production by GRIESS Reaction

RAW 264.7 murine macrophages were incubated for 1 hour in the presence and absence of 1 μg / ml to 10 μg / ml of the cinnamic aldehyde monocomponent obtained in Example 1, followed by LPS (1 μg / ml) and NO was induced by addition of IFN-γ (100 units / ml) (Sigma Chemical Co., St. Louis, Mo.) (Stuehr et al. 1991). After 24 to 48 hours of incubation, a portion of the medium was taken to assess nitrite accumulation by the modified GRIESS reaction (Gilliam et al. 1993). The OD average value of each (n = 4) was obtained at a given time interval, and the inhibition rate relative to the stimulus values of LPS and IFN-γ was calculated according to Equation 1 below, and the results are shown in Table 1 below. Dexamethasone, a steroidal anti-inflammatory agent, was treated as 10 ⁻⁵ to 10 ⁻⁶ M as a control.

% NO inhibition = [(O.D value at LPS and IFN-γ stimulation-sample O.D value) / (O.D value at LPS and IFN-γ stimulation-O.D value without stimulation)] × 100

Inhibition Effect of NO Production by Grease Reaction of Cyanide Aldehyde (Number of Samples: N = 3) Sample Name Concentration (㎍ / ml) N 1 N 2 N 3 Average NO inhibition rate (%) Cinnamicaldehyde 10 0.188 0.158 0.140 0.162 97.78 One 0.456 0.462 0.464 0.461 24.03 CTL (control) 0.172 0.146 0.134 0.153 100.00 0.168 0.154 0.142 LPS One 0.570 0.538 0.562 0.558 0.00 0.554 0.538 0.586 Dexamethasone 10 ^-5 M 0.221 0.201 0.243 0.222 83.05 10 ^-5 M 0.286 0.294 0.301 0.294 65.27

Inhibition of NO production by the GRIESS reaction revealed that the aldehyde suppressed NO production close to the default value when intracellular stimulation was not concentration dependent.

Compared with the dexamethasone used as a comparative group, it was found that 10 ㎍ / ml of the dynamic aldehyde has a stronger NO inhibitory power.

Experimental Example 2: Effect of Cinamic Aldehyde on iNOS mRNA Expression

Based on Experimental Example 1, the present inventors attempted to determine whether the action of synaldehyde on NO production inhibition in the murine macrophage model is iNOS mRNA level before the production of an enzyme that causes nitrite accumulation. This evidence is based on the observation that immunosuppressants (cyclosporine) or hydrocortisone, etc., in macrophages inhibit iNOS expression by decreasing the transcription rate of iNOS proteins (Nathan et al. 1994, Vodovotz et al. 1993).

Therefore, to confirm whether the aldehyde inhibits iNOS mRNA expression induced by LPS, RNA of RAW 264.7 cells was isolated and analyzed by semiquantitative RT-PCR method. The sense and antisense oligonucleotides for iNOS are 5′-TGG TGG TGA CAA GCA CAT TT-3 ′ (No. 1926-1945) and 5′-GCA GCC TCT TGT CTT TGA CC-3 ′ (No. 2605-2624), respectively. Oligonucleotides of 743 bp size with the base sequence of) were used, and the sense and antisense oligo primers for β-actin were 5′-TGT TAC CAA CTG GGA CGA CA-3 ′ and 5′-TCT CAG CTG TGG TGG, respectively. A primer having a size of 410 bp having a nucleotide sequence of TGA AG-3 ′ was used.

Polymerase chain reaction was performed with DNA heat cycle (Perkin-Elmer 9600). Total RNA was extracted using TRI-reagent (MRC Inc., Cincinnati, OH manufacturer) and cDNA was prepared from the same amount (1-2 μg) of total RNA using M-MLV (Promega Co, Madison manufacturer) Afterwards, the same amount of cDNA was used to amplify mRNA. PCR amplification was performed in a 20 μl solution containing 1.75 mM MgCl ₂ , 20 pM primer, 200 μM dNTP, 1 U units of Taq polymerase (ELPIS BIOTECH). After amplification conditions, iNOS (30 cycles) and β-actin (25 cycles) were denatured at 94 ° C. for 5 minutes, followed by 25 to 30 cycles of 30 seconds at 94 ° C., 30 seconds at 58 ° C., and 30 seconds at 72 ° C., 10 min at 72 ° C. The product was then analyzed by electrophoresis on 1.2% agarose gel. In the quantitative analysis, the amount of β-actin was evaluated after quantification using a densitometer.

As shown in FIG. 1, it was found that cinnamic aldehyde inhibited iNOS mRNA expression in a concentration-dependent manner similar to the results of Table 1. This data represents two separate experiments for the same sample concentration.

Experimental Example 3: Effect of Cinamic Aldehyde on Induced Inflammatory Cytokines

Based on Examples 2 and 3, the present inventors examined the inhibitory effect of cinnamic aldehyde against IL-1β, TNF-α, and IL-6, which occur frequently in patients with acute or chronic inflammation related to rheumatism or oral cavity. These inducible inflammatory cytokines are directly involved in inflammation as well as the inducers of NO as described above, and are based on data suggesting that water-soluble antagonists of IL-1β and TNF-α cytokines may be helpful in the treatment of rheumatism. The test method is the same as in Experimental Example 2, the primers used are shown in Table 2.

Oligonucleotide Sequences Used to Amplify IL-1β, TNF-α, and IL-6 Primer type division Oligonucleotide sequence Size (bp) IL-1β sense 5'-TCT TTG AAG TTG ACG GAC CC-3 '(142-161) 486 Antisense 5'-AGG CCA CAG GTA TTT TGT CG-3 '(608-627) TNF-α sense 5'-AGT TCT ATG GCC CAG ACC CT-3 '(388-407) 484 Antisense 5'-CGG ACT CCG CAA AGT CTA AG-3 '(852-871) IL-6 sense 5'-CCG GAG AGG AGA CTT CAC AG-3 '(127-146) 500 Antisense 5'-TGG TCT TGG TCC TTA GCC AC-3 '(607-626)

2 shows the sensitivity of cinnamic aldehydes to IL-1β mRNA. As a result, cinnamic aldehyde showed an inhibition rate of about 67% at the concentration of 10㎍ / ml, 50% inhibition even at 5㎍ / ml.

Figure 3 is a semi-quantitative RT-PCR analysis of TNF-α mRNA, it can be seen that the cinnamic aldehyde completely inhibits the expression of TNF-α mRNA at a concentration of 10㎍ / ml does not show cytotoxicity . It is interesting to note that even in CTL (Control: no treatment), TNF-α mRNA is expressed as little as possible, but the synaldehyde inhibits below this basic value.

Figure 4 shows the results for IL-6 mRNA, it can be seen that the cinnamic aldehyde inhibits the expression of IL-6 mRNA in a concentration-dependent manner as in IL-1β mRNA.

On the basis of the above results, it was confirmed that the cinnamic aldehyde inhibits the formation of cytokines such as IL-1β, TNF-α, IL-6, which are frequently occurring in rheumatism, gingiva, and periodontal disease.

Examples 2 to 5: Preparation of Toothpaste Compositions

Toothpaste compositions of the present invention (Examples 2 to 5) were prepared based on the ingredients shown in Table 3 below.

The toothpaste composition of the present invention uses cinnamic aldehyde as the main active ingredient, and may further contain triclosan, an abrasive, a humectant, a binder, a foaming agent, a sweetening agent, a preservative, a pH adjuster, a flavoring agent, and the like.

Cinnamic aldehydes are used in a mixture of 0.001 to 20% by weight, preferably 0.1 to 10% by weight, for oral and periodontal disease inhibition. To further improve the effect, 0.001 to 10% by weight of triclosan, preferably 0.01 to 2% by weight is used. The abrasive may be calcium carbonate, calcium dihydrogen phosphate, precipitated silica, hydrous alumina, silica gel, insoluble sodium metaphosphate, zirconium silicate, and the like. Preferably from 35 to 55% by weight. The humectant may be glycerin, sorbitol liquid, amorphous sorbitol liquid, polyethylene glycol, or the like, and 20 to 60% by weight, preferably 20 to 50% by weight, may be used alone or in combination of two or more of the humectants. The binder is usually suitable for carrageenan, xanthan gum, carboxymethyl cellulose sodium, carboxyvinyl polymer, sodium alginate and the like. The amount of the binder used is 0.1 to 3.0% by weight, preferably 0.5 to 2.0% by weight. A foaming agent can use anionic surfactants, such as sodium lauryl sulfate and sodium lauroyl sarcosinate, and nonionic surfactants, such as a sorbitan fatty acid ester, polyoxyethylene hardened castor oil, and a polyoxyethylene polyoxypropylene copolymer. The foaming agent uses 0.5 to 5.0% by weight, preferably 0.5 to 2.5% by weight, either alone or in combination of anionic and nonionic surfactants. The sweetening agent may be sodium saccharin, aspartame, acesulfame, stevioside, licorice acid, and the like, and the sweetener is preferably used in combination of 0.05 or 0.5% by weight.

In addition, a fluorine compound may be used as a caries preventive agent, and metabisulfite sodium may be used to stabilize the drug. The pH adjuster is preferably 0.02 to 1.5% by weight of phosphoric acid, sodium phosphate, citric acid, sodium citrate, succinic acid, sodium succinate, tartaric acid, sodium stannate, or a mixture of two or more.

The constituents of the fragrance are aromatic substances used in conventional toothpaste and substances used in food flavoring, such as peppermint oil, menthol, spearmint oil, carbon, anise oil, anitol, eucalyptus oil, and clove oil. , Eugenol, winter green oils may be used, and 0.1 to 2.0% by weight of one or two or more of the above components is used, preferably 0.5 to 1.5% by weight.

Preparation of Toothpaste Composition (Unit: wt%) division Ingredient Name Example 2 3 4 5 abrasive Calcium carbonate 40.0 40.0 40.0 40.0 Humectant Sorbitol Liquid 25.0 25.0 25.0 25.0 Foam Alkyl Sodium Sulfate 2.0 2.0 2.0 2.0 Binder Carboxymethyl Cellulose 1.0 1.0 1.0 1.0 Sweetener Zaccarin Sodium 0.2 0.2 0.2 0.2 antiseptic Paraoxy Benzoate 0.1 0.1 0.1 0.1 Spices Combination spices 1.0 1.0 1.0 1.0 Triclosan 0 0 0.1 0.1 Active ingredient Cinamic Aldehyde One 5 One 5 Purified water Remainder Remainder Remainder Remainder

Examples 6-9: Preparation of Dietary Film Compositions

Next, the dietary films for inhibiting oral inflammation of the present invention (Examples 6 to 9) were prepared based on the ingredients shown in Table 4 below.

Preparation of Dietary Film Composition (Unit: Weight%) division Example 6 7 8 9 Cinnamicaldehyde 0.25 2.5 12.5 25 PVP 30 30 30 30 glycerin 5 5 5 5 Sorbitol 4 4 4 4 Fruit flavoring 5 5 5 5 water 105.75 103.5 93.5 81 ethanol 100 100 100 100

Examples 10 to 13: Preparation of a spray composition for inhibiting inflammation

Next, the spray of the present invention (Examples 10 to 13) was prepared based on the ingredients shown in Table 5.

Preparation of spray composition for inhibiting inflammation (unit: wt%) division Example 10 11 12 13 Cinnamicaldehyde 0.1 One 5 10 ethanol 60 60 60 60 glycerin 7 7 7 7 Sodium fluoride 0.02 0.02 0.02 0.02 Sodium citrate 0.4 0.4 0.4 0.4 Citric acid 0.005 0.005 0.005 0.005 Saccharin Sodium 0.011 0.011 0.011 0.011 F-127 One One One One Methylparaben 0.1 0.1 0.1 0.1 Protylparaben 0.05 0.05 0.05 0.05 Combination spices 0.12 0.12 0.12 0.12 Purified water Remainder Remainder Remainder Remainder

As described above, the composition of the present invention containing cinnamic aldehyde as an active ingredient has the effect of reducing and inhibiting the production of nitrogen monoxide and cytokines, which cause inflammation, and thus, various kinds of NO and cytokine induced by It can be used for the prevention and treatment of inflammatory diseases.

Claims (6)

A composition having an inhibitory effect on the production of nitrogen monoxide and an inflammatory cytokine containing the cinnamic aldehyde having the following structure as an active ingredient. The method according to claim 1, wherein the effect of inhibiting the production of nitric oxide and inflammatory cytokines is autoimmune diseases including rheumatoid arthritis, acute and chronic infections caused by bacteria and fungi, upper respiratory infections including common cold, gingiva and Composition for use in the prevention and treatment of oral and periodontal diseases including periodontitis, skin diseases involving erythema and edema, or acute and chronic dermatitis. The composition of claim 2 wherein the formulation of the composition is a toothpaste, dietary film, dentifrice, spray, paste (or patch), ointment, lotion, gel formulation. The method of claim 1, wherein the aldehyde is Cinnamomum cassia Blume., Cinnamomum obtussifolium Nees., Cinnamomum burmanni Blume., Cinnamomum iners Reinw.Ex Blume ), Purified water, methanol, ethanol, propanol, from ground powder of broiler (Cinnamomum sieboldii Meisn.), Cinnamomum zeylanicum Nees. Or Cinnamomum cassia Presl var. Butanol, ethylene glycol, propylene glycol, 1,3-butylene glycol, ethyl acetate and acetone is a composition characterized in that extracted with at least one solvent selected from the group consisting of. The composition of claim 1, wherein the content of the cinnamic aldehyde is 0.001 to 20% by weight based on the total weight of the composition. The composition of claim 1, further comprising a steroidal drug, cyclohexidine or triclosan.