KR100964906B1 - Pharmaceutical composition for treating or preventing an inflammatory disease comprising licarin E - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for the treatment or prevention of inflammatory diseases containing a lignan-based compound, more specifically, Licarin E (arcarin E) or nutmeg (aril of nutmeg) extract represented by the formula (I) It relates to a pharmaceutical composition for the treatment or prevention of inflammatory diseases containing as an active ingredient.

Lycarin of Formula I exhibits an anti-inflammatory effect by inhibiting the production or expression of inflammatory mediators iNOS, COX-2, TNF-α and IκBα. Therefore, the pharmaceutical composition of the present invention containing the lycarinic tooth or nutmeg alfalfa extract of the present invention as an active ingredient can be very useful for the treatment or prevention of inflammatory diseases.

Licarin E, nut of nutmeg, anti-inflammatory

Description

Pharmaceutical composition for treating or preventing an inflammatory disease comprising licarin E}

The present invention relates to a pharmaceutical composition for the treatment or prophylaxis of an inflammatory disease containing Licarin E, more specifically, the extract of Licarin Lee or Nutmeg (aril of nutmeg) represented by the following formula (I): It relates to a pharmaceutical composition for the treatment or prevention of inflammatory diseases containing as an active ingredient.

(I)

An inflammatory response occurs when a tissue (cell) is damaged or infected by an external source of infection, and various inflammatory mediators and immune cells are involved in local blood vessels and body fluids, resulting in enzyme activation, secretion of inflammatory mediators, fluid infiltration, cell migration, and tissue destruction. It presents a series of complex physiological reactions, external symptoms such as erythema and edema fever pain. The results also show symptoms of acute inflammation, chronic inflammation such as granuloma inflammation, rheumatoid arthritis and osteoarthritis (Goodwin JS et. al ., J. Clin . Immunol . , 9: 295-314, 1989).

Cyclooxygenase (hereinafter referred to as 'COX') plays an important role among enzymes that have an important effect on blood coagulation and inflammation. COX produces two main products, prostaglandin and thromboxane. do. Prostaglandins are unsaturated fatty acids with a wide variety of physiological activities that act as local hormone or cellular function regulators in our bodies, including inflammation and pain transmission, vasodilation, temperature regulation, and gastric juice secretion (Marnett, LJ et. al ., J. Biol . Chem . , 274: 22903-22906, 1999). COX-1 plays an important role in maintaining homeostasis of cells by maintaining normal physiological responses such as gastrointestinal protection, renal blood flow control and platelet aggregation. On the other hand, in the process of inflammation-induced delivery by external stimulation, COX-2, an inducible isoenzyme, is temporarily expressed to release excessive prostaglandin in the place where inflammation occurs. Prostaglandins cause erythema, swelling and pain, which are the main symptoms of inflammation, and the activity of increasing the action of histamine, an endogenous inflammatory mediator, can inhibit the production of prostaglandins at inflammation sites.

Non-steroidal anti-inflammatory drugs (NSAIDs) such as aspirin, indomethacin, naproxen and ibuprofen, which are currently commercially available, have been shown to inhibit COX-2 enzyme activity. It inhibits the production of prostaglandins and has anti-inflammatory effects (Meade EA et. al ., J. Biol . Chem. , 268: 6610, 1993). However, these NSAID drugs inhibit COX-2, which is temporarily expressed by inflammatory stimuli, as well as COX-1, which plays an important role in maintaining the normal functioning of the gastrointestinal tract and renal platelets, leading to serious side effects such as gastrointestinal bleeding and renal failure. (Surh YJ et al ., Mutation Research 480-481: 243-268, 2001). Therefore, it is very important industrially to find substances that provide anti-inflammatory action while minimizing side effects from natural products.

Nutmeg is a fruit of Myristica fragrans , a perennial plant grown in the tropics, and has been used as a spice for a long time. Nutmeg aril (aril) is growth inhibitory activity of Helicobacter pylori bacteria (In Vivo, 17;. 541-4, 2003 ), activation of detoxification mechanisms between (Food Chem . Toxicol, 31;. 517-21, 1993 ), chemopreventive action of skin warts (Cancer Lett ., 56; 59-63, 1991).

Licarin is a representative lignan compound found in nutmeg agar and has never been reported for its anti-inflammatory activity.

Thus, the present inventors have completed the present invention by confirming that the lycarinic tooth isolated from nutmeg alfalfa extract shows excellent anti-inflammatory activity as a result of long-term search for a natural-derived compound having anti-inflammatory activity.

Accordingly, it is an object of the present invention to provide a novel use of lycarin thereof isolated from nutmeg alfalfa extract.

As described above, lycarinic of the present invention has the effect of inhibiting the inflammatory response by inhibiting the production or expression of inflammatory mediators iNOS, COX-2, TNF-α and IκBα. Therefore, the pharmaceutical composition containing lycarinic or nutmeg agar ethanol extract of the present invention as an active ingredient can be very useful for the treatment or prevention of inflammatory diseases.

In order to achieve the above object, the present invention provides a pharmaceutical composition for the treatment or prevention of inflammatory diseases containing lycarinic yi or pharmaceutically acceptable salts thereof represented by the following formula (I) as an active ingredient.

(I)

Hereinafter, the present invention will be described in detail.

The present invention is characterized by providing a novel use of lycarin thereof isolated from nutmeg alfalfa extract.

Licarin according to the present invention is represented by the following formula (I).

(I)

Lycarin according to the invention may be used in the form of salts, preferably pharmaceutically acceptable salts. As the salt, an acid addition salt formed by a pharmaceutically acceptable free acid is preferable. Organic acids and inorganic acids may be used as the free acid. The organic acid is not limited thereto, citric acid, acetic acid, lactic acid, tartaric acid, maleic acid, fumaric acid, formic acid, propionic acid, oxalic acid, trifluoroacetic acid, benzoic acid, gluconic acid, metasulfonic acid, glycolic acid, succinic acid, 4-toluenesulfonic acid, Glutamic acid and aspartic acid. In addition, the inorganic acid includes, but is not limited to, hydrochloric acid, bromic acid, sulfuric acid and phosphoric acid.

Licarinic of the present invention can be obtained from a plant or part of a plant using methods of extracting and separating conventional materials. Stems, roots or leaves are properly dehydrated and macerated or only dehydrated to obtain the desired extracts, and the desired extracts are purified using purification methods known to those skilled in the art to which this invention pertains. In addition, the synthetic compound represented by the formula (I) or the derivatives thereof are generally commercially available materials, or may be chemically prepared using known synthetic methods.

Lycarin of the present invention represented by the above formula (I) can be purified separately from nutmeg alfalfa. The nutmeg refers to the mature fruit or seeds of the fruit of Myristica fragrans . In addition, the lycarinic tooth of the present invention may be purified separately from the oil obtained by pressing the nutmeg seeds.

As an extraction solvent for separating lycarinic of the present invention, water or an organic solvent of C1-C6 may be used. Preferably, purified water, methanol, ethanol, propanol, isopropanol, butanol, acetone, acetone, ether, benzene, chloroform, Various solvents such as ethyl acetate, methylene chloride, hexane, cyclohexane and petroleum ether may be used alone or in combination. More preferably ethanol or hexane can be used. Separation and purification of the lycarinic ions of the present invention from nutmeg seed agar extracts include column chromatography and high-performance liquid chromatography (HPLC) filled with various synthetic resins such as silica gel or activated alumina. It may be used alone or in combination. However, the extraction and separation and purification method of the active ingredient is not necessarily limited to the above method.

Thus, the lycarinic of the present invention may be used in the form of a purely purified compound, and may also be used in the form of an extract containing the same. For example, it may be used in the form of an oil obtained by compressing the seed, fruit or alfalfa extract of Myristica fragrance as described above or the seed of Myristica fragrance. The extract can be obtained by extracting mysticica fragrance with water or an organic solvent of C1-C6 as described above. Preferably it may be a seed or alfalfa of myritica fragrance.

Lycarinic of the present invention has anti-inflammatory activity by inhibiting substances that mediate various inflammatory responses.

NO is a substance involved in neurotransmission, blood vessel migration, and cell-mediated immune responses. It is produced from L-arginine by nitric oxide synthase (NOS) (Nathan and Xie, 1994; Alderton et. al ., 2001). In particular, when macrophages are stimulated by IFN-γ or lipopolysaccaride (LPS), inducible nitric oxide synthase (iNOS) is expressed and a large amount of NO is produced by the iNOS. Lycarinic of the present invention has been shown to inhibit the expression of iNOS involved in the production of NO in macrophages in a concentration-dependent manner (see Figure 6 ).

COX-2 also produces inflammatory PG as a substance involved in the body's inflammatory response. COX-2 is induced by LPS, an endotoxin secreted by bacteria, and inflammatory cytokines, IL-1, TNF-α, IFN-γ, and the like. Lycarinic of the present invention has the effect of inhibiting the expression of COX-2 concentration-dependent (see Figure 7 ).

Tumor necrosis factor TNF-α (tumor necrosis factor α) is a major mediator of acute inflammatory responses to Gram-negative bacteria and other infectious microorganisms. Macrophages stimulated by LPS increase the synthesis of TNF-α. The biological action of TNF-α induces acute inflammation by acting on white blood cells and endothelial cells at low concentrations. Medium concentrations mediate the systemic response of inflammation and high concentrations cause death due to pathological abnormalities of sepsis shock. TNF-α also increases the synthesis of PG, producing heat, and inhibiting the expression of trombomodulin, resulting in vascular plugging (Abbas and Lichtman, "Cellular and Molecular Immunology" the fifth edition pp. 247-253, 2003). Lycarin of the present invention has the effect of inhibiting the production of TNF-α in macrophages and human monocytes (see Figure 8 ).

Expression of iNOS and COX-2 occurs through complex cell transfer processes, involving various enzymes (kinase) that transmit external signals into cells, among which nuclear factor-kappa B (NF-κB) is iNOS and COX. It has a major influence on the expression of -2. Stimulation of LPS or TNF-α phosphorylates and activates tyrosine or serine / threonine kinase, while inhibitor-kappa B (IκB), an inhibitory component of the NF-κB complex present in the cytoplasm, is IκB kinase NF-κB is activated by phosphorylation and activation and proteolysis. Lycarinic of the present invention has the effect of inhibiting the phosphorylation of IκBα which affects the production of NF-κB (see FIG. 9 ).

In addition, as a result of topical application of the lycarinic agent of the present invention to the ears of rats induced with edema with TPA (12- O- tetradecanoylphorbol-13-acetate), edema production rate was suppressed in a concentration-dependent manner, and indometha is currently used as a commercial anti-inflammatory agent. It showed higher edema inhibition rate than renal (see Table 2 ). In addition, after preparing a cream containing lycarinic tooth and topically applying it to the rat's ear, the edema production rate was greatly suppressed (see Table 4 ).

On the other hand, the present inventors confirmed that the result of topical application of the nutmeg agar ethanol extract to the ears of rats induced edema by TPA treatment, the edema production rate is suppressed in a concentration-dependent manner (see Table 5 ).

These results indicate that the lycarinic disaccharide of the present invention exhibits excellent anti-inflammatory action by inhibiting not only COX-2 but also various factors mediating the inflammatory response. It also indicates that the nutmeg apricot extract itself can achieve the same anti-inflammatory effect. The anti-inflammatory activity of the lycarinic tooth and nutmeg alfalfa extract of the present invention represented by the above formula (I) was first identified in the present invention.

Given that most of the non-steroidal anti-inflammatory drugs currently used commercially exhibit anti-inflammatory effects by inhibiting the activity of the COX-2 enzyme, it can be seen that the lycarine of the present invention can be used as an anti-inflammatory agent having a superior effect than the conventional anti-inflammatory agents. have.

Accordingly, the present invention provides a pharmaceutical composition for the treatment or prevention of inflammatory diseases, wherein lycarin represented by Formula I contains a pharmaceutically acceptable salt thereof as an active ingredient. In another aspect, the present invention provides a pharmaceutical composition for the treatment or prevention of inflammatory diseases containing nutmeg adenocarcinoma extract as an active ingredient. Preparation of the nutmeg alfalfa extract is as described above.

The composition of the present invention can be administered orally or parenterally during clinical administration and can be used in the form of a general pharmaceutical formulation. When formulated in the form of general pharmaceuticals, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents and surfactants are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules and the like, which solid preparations comprise at least one excipient such as starch, calcium carbonate, water It is prepared by mixing cross or lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium styrate talc are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, ointments, creams. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used.

In addition, the composition of the present invention can be administered parenterally, parenteral administration is by subcutaneous injection, intravenous injection, intramuscular injection or intrathoracic injection injection method. To formulate into a parenteral formulation, the lycarinic or nutmeg agar extract of Formula I is mixed in water with a stabilizer or buffer to prepare a solution or suspension, which is formulated in unit dosage forms of ampoules or vials. Dosage units may contain, for example, one, two, three or four times, or 1/2, 1/3 or 1/4 times the individual dosage. The individual dosage preferably contains an amount in which the effective drug is administered at one time, which usually corresponds to all, 1/2, 1/3 or 1/4 times the daily dose.

The effective dose of the lycarinic tooth or nutmeg alfalfa extract represented by the formula (I) of the present invention is a single dose of 0.1-50 mg / kg, preferably 1-10 mg / kg, administered 1-3 times a day. Can be. Dosage levels for a particular patient may vary depending on the patient's weight, age, sex, health condition, diet, time of administration, method of administration, rate of excretion, severity of the disease, and the like.

As a result of the toxicity test of orally administrating lycarinic tooth to rats, the 50% lethal dose (LD ₅₀ ) by oral toxicity test was found to be 2,000 mg / kg or more.

In particular, the pharmaceutical composition containing the lycarinic tooth or nutmeg alfalfa extract of the present invention may be formulated in the form of a medicament for skin application, i.e. ointment, cream, 0.001-10.0% by weight relative to the total weight of the formulation, preferably Can be suitably formulated according to the formulation in the range of 0.005-5.0 wt%. This is because when added at less than 0.005% by weight, the anti-inflammatory activity is lowered, and when added at an amount exceeding 10.0% by weight, only the added amount is large, and there is no significant difference in anti-inflammatory activity.

Inflammatory diseases to which the pharmaceutical composition of the present invention can be applied are diseases accompanied by inflammation caused by various stimulating factors causing a series of inflammatory reactions such as NO, iNOS, COX-2, PGE ₂ , TNF-α, IκBα, and the like. Say The inflammatory diseases include general inflammatory symptoms such as edema, inflammatory bowel disease, peritonitis, osteomyelitis, cellulitis, pancreatitis, traumatic shock, bronchial asthma, allergic rhinitis, cystic fibrosis, acute bronchitis, chronic bronchitis, acute bronchitis, chronic Bronchiolitis, osteoarthritis, gout, spondyloarthropathy, ankylosing spondylitis, lighter syndrome, psoriatic arthrosis, enteropathic spondylitis, juvenile arthrosis, juvenile ankylosing spondylitis, reactive arthrosis, infectious arthritis, post-infectious arthritis, gonococcal arthritis, Mycobacterium tuberculosis, viral arthritis, fungal arthritis, syphilis arthritis, Lyme disease, arthritis associated with 'angiopathy syndrome', nodular polyarthritis, irritable vasculitis, rheumatoid granulomatosis, rheumatoid polymyalgia, articular cell arteritis, calcium crystal deposition Arthrosis, pseudogout, non-joint rheumatism, bursitis, hay fever, Inflammation (tennis elbow), neuropathic joint disease (also known as 'charcot joint'), hemarthrosic, henoch-Scholine purpura, hypertrophic osteoarthritis, multicardiac retinopathy, scoliosis (scoliosis), hemochromatosis, hemochromatosis, hyperlipoproteinemia, hypomagglobulinemia, familial Mediterranean fever, Behcet's disease, systemic lupus erythematosus, recurrent fever, multiple sclerosis, sepsis, septic shock, acute respiration Difficulty syndrome, multiple organ failure, chronic obstructive pulmonary disease, rheumatoid arthritis, acute lung injury, and broncho-pulmonary dysplasia, It is not limited to this. It also includes inflammatory skin diseases such as acute eczema, contact dermatitis, atopic dermatitis, seborrheic dermatitis, deprived dermatitis, sun dermatitis and psoriasis.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples. In addition, in the following Examples and Experimental Examples,% not specified as% by weight indicates weight%. In addition, all activities were repeated three times or more, and the results were expressed as the mean value and the standard deviation. In addition, statistical analysis was performed using Students's t- test, and p was determined to be statistically significant when <0.01 or less.

< Example  1>

nutmeg From alfalfa Licarin  Isolation and Purification

<1-1> Licarin  Isolation and Purification

400 g of 95 vol.% Ethanol was added to 100 g (dry weight) of dried and ground nutmeg seed skin, and left at room temperature for two days. The solution was filtered using Whatman filter paper # 2. The process was repeated twice. The ethanol filtrate was concentrated in vacuo and lyophilized to prepare an ethanol crude extract (26.61 g) of nutmeg seedlings. The ethanol crude extract was partitioned into ethyl acetate, butanol, and water in order to obtain an ethyl acetate fraction (18.44 g). The ethyl acetate fractions were eluted with a solvent in which chloroform and ethyl acetate were mixed at a ratio of 9: 1 (v / v) using silica gel column chromatography (Merck Kieselgel 66; 70-230 mesh), and fraction II (3.04 g). Got. The solvent was completely removed by a vacuum rotary concentrator to prepare a crude extract of nutmeg seedlings. Then, fraction II was purified by silica gel column chromatography (Merck Kieselgel 66; 70-230 mesh) to chloroform, hexane, ethyl acetate and acetone in a ratio of 15: 10: 0.1: 0.1 (v / v / v / v). Elution was performed with the mixed solvent to obtain fraction II-B. Fraction III-B was eluted with 100% methanol using preparative HPLC to afford single substance fraction II-B-2 (0.016 g). Such a separation process chart is shown in FIG. 1 .

<1-2> structural analysis

In order to determine the structure of the isolated single material III-B-2, ¹ H-NMR spectrum and ¹³ C-NMR spectrum were measured at 600 MHz and 150 MHz (solvent: DMSO), respectively, and the DEPT spectrum was measured . 2, 3 and 4 , respectively. Table 1 shows the results of the ¹ H-NMR and ¹³ C-NMR.

<1-3> mass spectrometry

The results of FAB / MS measured for mass spectrometry of the isolated single substance are shown in FIG. 5 . This compound had a molecular weight of 324 as [M] ⁺ was observed at m / z 324 in FAB / MS, and the molecular formula was C ₂₀ H ₂₀ O ₄ .

Results obtained by comparing and analyzing the results of the above ¹ H-NMR, ¹³ C-NMR, DEPT and FAB / MS and the previously published research report (Harvey DJ, J. Chromatogr., 110: 91-102, 1975) , The isolated single substance was confirmed to be Licarin E represented by the following Chemical Formula 1.

[Formula 1]

< Example  2>

Of the present invention Lignan system  Compound iNOS  Inhibitory effect investigation

Stimulating macrophages with LPS overexpresses iNOS and produces large amounts of NO. Therefore, the effect of the lycarinic tooth of the present invention prepared in Example 1 on iNOS expression was measured.

After lysing RAW 264.7 cells treated with lycarinine and LPS of the present invention, proteins were quantified by the Bradford method. After 10 μg of protein was separated by 10% SDS-PAGE, the separated protein was transferred to a nitrocellulose membrane using a transfer solution (20% methanol, 25 mM Tris, 192 mM glycine, pH 8.3) (Hall, Methods Mol . Biol ., 261: 167-174, 2004). The SDS-polyacrylamide gel and nitrocellulose membrane were brought into close contact and then mounted in a mini-gel transfer kit. The sample was then loaded and transferred for 1 hour at 100V. Then, washed once with a tris buffered saline tween-20 (TBST) solution, the membrane was transferred to a dry filter paper and dried at room temperature. In order to eliminate non-specific reactions, TBST containing 5% non-fat skimmed milk was allowed to stand at 4 ° C. for at least 24 hours. Then, washed three times with TBST solution and the anti-iNOS antibody (1: 2,000) (Calbiochem Co.) was injected and reacted at room temperature for 1 hour. Again the membrane was washed with TBST solution three times for 10 minutes. Anti-rabbit IgG-HRP (1: 2,000) (Calbiochem Co., Ltd.) to which a horse radish peroxidase (HRP) was attached was injected and reacted on a shaker for 1 hour. Then, the membrane was washed three times again with TBST solution, soaked in ECL (enhanced chemiluminescence) solution and shaken for 1 minute. The ECL solution is prepared by mixing Solution A (including luminol and enhancer) and Solution B (including hydrogen peroxide) in the same amount and shaking well for 1 minute. The membrane was removed from the ECL solution to remove water, and then scanned with an X-ray film in the dark room.

As a result, as shown in Figure 6 , lycarinic of the present invention inhibited the expression of iNOS in macrophages, it was confirmed that the inhibitory effect is significant at 25 μM ( P <0.01).

< Example  3>

Of the present invention Licarin  dental COX -2 Inhibitory effect investigation

We investigated the expression of COX-2, which directly affects PGE ₂ production, using Western blot analysis. Example 2, except that anti-COX-2 antibody (1: 2,000) (Calbiochem) was used as the primary antibody and anti-chlorine IgG-HRP (1: 2,000) (Calbiochem) was used as the secondary antibody. It carried out by the same method as described in.

As a result, as shown in Figure 7 , the lycarinic of the present invention was confirmed that the concentration-dependent inhibition of the expression of COX-2 protein. In particular, the amount of expression was markedly reduced at a concentration of 25 μM.

< Example  4>

Of the present invention Licarin  dental TNF -α inhibitory effect investigation

First, 1 × 10 ⁶ cells / ml of RAW264.7 cells were seeded in 96-well tissue culture plates and left at room temperature for 5 hours. Thereafter, the lycarin of the present invention was added at a concentration of 10 μM, respectively, and pretreated for 2 hours. None were treated with the negative control, and troglitazone (Sigma) was treated with the positive control. Thereafter, 1 μg / ml of LPS was treated and incubated for 18 hours. The amount of TNF-α gene expression in macrophages was quantified using real time PCR (real time-PCR).

As a result, as shown in Fig. 8 , the production of TNF-α showed a tendency to decrease significantly when the lycarin of the present invention treated 10 μM ( P <0.01).

< Example  5>

Invention Licarin  Investigation of its IκBα phosphorylation inhibitory activity

We investigated the inhibition of phosphorylation activity of IκBα involved in the activation of NF-κB that promotes the expression of iNOS and COX-2 using Western blot analysis. Except for using anti-IκBα antibody (1: 2,000) (Calbiochem) as the primary antibody and anti-goat IgG-HRP (1: 2,000) (Calbiochem) as the secondary antibody, It was carried out in the same manner as the method.

As a result, as shown in Figure 9 , it was confirmed that lycarinic of the present invention inhibits the phosphorylation expression of IκBα protein in a concentration-dependent manner. In particular, the amount of expression was markedly reduced at a concentration of 25 μM.

< Example  6>

Of the present invention using an animal model Licarin  Investigation of its anti-inflammatory activity

The anti-inflammatory activity of lycarin its isolated and purified in Example 1 was confirmed through animal experiments. The anti-inflammatory activity was measured through edema inhibition experiments in rats. Experimental animals used Wistar rats (Dae Biolink, Korea) of 5 weeks old. The feed was fed standard pelleted rat feed (Cheil Jedang), and the feed and water were fed freely. Rats were bred under conditions of 12 hours of fluorescent lamp sunshine, 25 ± 2 ° C. and 60 ± 10% humidity. TPA (12- O- tetradecanoylphorbol-13-acetate; Sigma Co., Ltd.) as a base agent was dissolved in acetone at 200 µg / mL. Edema of the rat ears was induced by adding TPA solution locally at 10 μl / ear to the outside and inside of the ear, respectively (4 μg / ear). Intact methasin, a nonsteroidal anti-inflammatory agent, was purified in acetone to be 20, 200, and 2000 ㎍ / ear as a control material. Lycarin and indomethacin were applied topically to the rat's ears 30 minutes after TPA treatment. Acetone was applied topically to the control group. Rat ear thickness was measured using a caliper 8 hours after each material was treated. Increasing the ear thickness was compared with the non-treated group, and the inflammation inhibition effect was measured by calculating the edema inhibition rate. The results are shown in Table 2 below.

^* p <0.01

As shown in Table 2 , the lycarinic of the present invention was confirmed that the concentration-dependent inhibition of edema of rats induced by TPA (statistical significance p <0.01). The ID ₅₀ values of lycarinine and indomethacin of the present invention were 117.4 μg / ear and 160.5 μg / ear, respectively, showing a higher edema inhibitory effect than indomethacin, which is used as a conventional anti-inflammatory agent.

< Example  7>

Of the present invention Licarin  Preparation of this containing cream and investigation of its anti-inflammatory activity

<7-1> Preparation of Cream

Ricarin of the present invention was used to prepare a cream having a variety of compositions shown in Table 3 below. First, the materials indicated by B in Table 3 were dissolved at 75 to 80 ° C. In addition, cetyl alcohol and a preservative in the material represented by C in Table 3 was dissolved at the same temperature. The C materials were emulsified in the B materials. Thereafter, the lycarinine of the present invention represented by A in Table 3 was mixed at a concentration of 5.0, 0.5, 0.05, and 0.005%. Finally, perfume was added and the quantity was adjusted to purified water to prepare a cream.

<7-2> Anti-inflammatory activity investigation

The anti-inflammatory activity of the lycarine-containing cream prepared in Example <7-1> was measured through edema inhibition experiments in rats. Edema inhibition experiment was carried out in the same manner as in Example 6. The results are shown in Table 4 below.

sample One 2 3 4 Licarin Lee
content 0.005% 0.05% 0.5% 5% Edema inhibition rate (%) 12.4 43.1 61.7 85.6

As shown in the results of Table 4 , it can be seen that the lycarinic tooth-containing cream of the present invention suppresses edema of rats induced by TPA depending on the concentration of lycarinic tooth contained in the cream.

< Example  8>

nutmeg Amalgam  Investigation of Anti-inflammatory Activity of Ethanol Extracts

<8-1> Ethanol Extract Preparation

400 ml of 95 vol. The solution was filtered using Whatman filter paper # 2. The process was repeated twice. The ethanol filtrate was concentrated in vacuo and lyophilized to prepare crude ethanol extract (26.61 g).

<8-2> using animal model nutmeg Amalgam  Investigation of Anti-inflammatory Activity of Extracts

Anti-inflammatory activity of the nutmeg agar ethanol extract prepared in Example <8-1> was confirmed through animal experiments. The anti-inflammatory activity was measured by edema inhibition experiments on rats in the same manner as in Example 6. The results are shown in Table 5 below.

^* p <0.01

As shown in Table 5 , the crude ethanol extract of nutmeg adenocarcinoma of the present invention was confirmed to inhibit the edema of rats induced by TPA concentration-dependently (statistical significance p <0.01).

< Production Example  1>

Preparation of a medicament containing the pharmaceutical composition for the treatment or prevention of inflammatory diseases according to the present invention

<1-1> Preparation of Tablet

25 mg of lycarinic or nutmeg alfalfa extract of the present invention, 26 mg of lactose for excipients and 3.5 mg of avicel (microcrystalline cellulose), 1.5 mg of sodium starch glyconate as a disintegration aid, and L-HPC (low-) for binders hydrosyprophylcellulose) was added to a U-type mixer with 8 mg and mixed for 20 minutes. After mixing was completed, 1 mg of magnesium stearate was further added as a lubricant and mixed for 3 minutes. Tablets were prepared by tableting and film coating after a quantitative test and a humidity test.

<1-2> Preparation of Syrup

Syrup containing lycarinic tooth of the present invention or a pharmaceutically acceptable salt thereof as an active ingredient of 2% (W / V) was prepared by the following method: lycarinic acid and acid addition salt of the present invention 2g, saccharin 0.8g And 25.4 g of sugar were dissolved in 80 g of warm water. After cooling the solution, 8.0 g of glycerin, 0.04 g of flavor, 4.0 g of ethanol, 0.4 g of sorbic acid, and an appropriate amount of distilled water were mixed thereto. Water was added to the mixture to make 100 mL.

<1-3> Preparation of Capsule

A capsule was prepared by mixing 50 mg of lycarinic tooth or nutmeg alfalfa extract, 50 mg of lactose, 46.5 mg of starch, 1 mg of talc and 1 mg of magnesium stearate and filling it into hard gelatin capsules.

<1-4> Preparation of Injection Solution

Injectable solutions containing 10 mg of the active ingredient were prepared by the following method: 100 ml were prepared by dissolving 1 g of lycarine and hydrochloride of the present invention, 0.6 g of sodium chloride and 0.1 g of ascorbic acid in distilled water. The solution was bottled and sterilized by heating at 20 ° C. for 30 minutes.

< Application example  1>

Gastric inflammatory  Digestive diseases

Gastritis is associated with various external factors and irregular eating habits, but Helicobacter pylori has been identified as the main cause. Helicobacter pyrobacteria affect not only gastric ulcer and gastritis, but also gastric cancer. During the growth of Helicobacter pylori, an enzyme called cyclooxygenase-2 (COX-2) increases simultaneously (Nam NT et al. al ., Clin . Cancer Res . 10 (23): 8105-8113, 2004). Helicobacter infection proliferates gastric mucosa to cancer cells, and COX-2 inhibitors have been shown to inhibit gastric mucosa growth / proliferation to cancer cells and to inhibit the change from normal tissue to cancer tissue. The group treated with the COX-2 inhibitor was found to have a superior effect of killing cancerous tissues by apoptosis (Nam NT et. al ., Clin . Cancer Res . 10 (23): 8105-8113, 2004). Therefore, the RICARIN COX-2 inhibitory effect of the present invention suggests that it has a sufficient therapeutic effect because it helps treat gastritis, which can prevent gastric cancer early.

< Application example  2>

arthritis

Arthritis is caused by autoimmune abnormalities, but as the disease progresses, chronic inflammation in the synovial cavity between joints induces angiogenesis and cartilage is destroyed. There are many types of arthritis, including infectious arthritis, degenerative arthritis, rheumatoid arthritis, femoral head avascular necrosis, ankylosing spondylitis, and arthritis caused by congenital malformations, but chronic inflammation of the synovial cavity during the course of the disease, regardless of the cause, may cause angiogenesis. It is known to induce new capillaries that invade joints and damage cartilage (Kocb AE et. al ., Arth. Rheum ., 29: 471-479, 1986; Stupack DG et al ., J. Med . Biol . Rcs ., 32: 578-281, 1999; Koch AE, Arthritis Rheum ., 41: 951-962, 1998). At this time, it is reported that the inflammatory response occurs first and second depending on the type, destroys cartilage and plays an important role in the progression of the disease, and the formation of new blood vessels into the joint is an important pathological mechanism (Colville-Nash, PR). et al ., Ann . Rheum . Dis . , 51, 919-925, 1992; Eisenstein, R., Pharmacol . Ther . , 49: 1-19, 1991). The treatment of arthritis prioritizes to suppress pain and to suppress inflammatory phenomena as much as possible to slow down the rate of destruction of joints and muscles and to minimize loss of function. Therefore, the anti-inflammatory composition containing the lycarinic tooth or nutmeg alfalfa extract of the present invention as an active ingredient is very effective in preventing and treating the progression of arthritis.

Figure 1 is a process for separating Licarin E (Licarin E) from the nutmeg ark of nutmeg (Aril of nutmeg).

Figure 2 is a lycarinic ¹ H-NMR spectrum of the present invention.

Figure 3 is a lycarinine ¹³ C-NMR spectrum of the present invention.

4 is a DEK spectrum of rivarin of the present invention.

5 is a RICARIN FAB-Mass spectrum of the present invention.

Figure 6 is a graph showing the inhibitory effect of lycarinic iNOS production of the present invention.

Figure 7 is a graph showing the effect of inhibiting the expression of RICARIN of the present invention.

Figure 8 is a graph showing the inhibitory effect of lycarinic TNF-α expression of the present invention.

Figure 9 is a graph showing the effect of inhibiting IkappaB expression of lycarinin of the present invention.

Claims (5)

A pharmaceutical composition for the treatment or prophylaxis of inflammatory diseases comprising lycarinic or a pharmaceutically acceptable salt thereof represented by the following formula (I) as an active ingredient. (I)

delete The method of claim 1, wherein the inflammatory disease is inflammatory bowel disease, peritonitis, osteomyelitis, fever, pancreatitis, trauma-induced shock, bronchial asthma, allergic rhinitis, cystic fibrosis, acute bronchitis, chronic bronchitis, acute bronchiolitis, chronic bronchiolitis, osteoarthritis , Gout, spondyloarthropathy, ankylosing spondylitis, lighter syndrome, psoriatic arthrosis, enteropathic spondylitis, juvenile arthrosis, juvenile ankylosing spondylitis, reactive arthrosis, infectious arthritis, post-infectious arthritis, gonococcal arthritis, tuberculosis arthritis, Viral arthritis, fungal arthritis, syphilitic arthritis, Lyme disease, arthritis associated with 'angioarthritis syndrome', nodular polyarthritis, irritable vasculitis, leukemic granulomatosis, rheumatoid polymyalgia, articular cell arteritis, calcium crystalline arthritis arthritis, Pseudogout, non-articular rheumatoid, bursitis, hay salt, epicondylitis (tennis elbow), neuropathic joints Diseases (neuropathic joint disease), hemarthrosic, henoch-Jehn purpura, hypertrophic osteoarthritis, multicardiac retinopathy, scoliosis, hemochromatosis, hemoglobinopathy, hyperlipoproteinemia, hypomagglobulinemia, Familial Mediterranean fever, Behatt's disease, systemic lupus erythematosus, recurrent fever, multiple sclerosis, sepsis, septic shock, acute respiratory distress syndrome, multiple organ failure, chronic obstructive pulmonary disease, rheumatoid arthritis Arthritis, acute lung injury (acute lung injury), bronchial pulmonary dysplasia (broncho-pulmonary dysplasia) and inflammatory skin diseases, the composition characterized in that any one selected from the group consisting of. The method of claim 1, wherein the lycarinic di is nitric oxide (NO), inducible nitric oxide synthase (iNOS), Prostaglandin E2 (PGE2), cyclooxygenase-2 (COX-2), tumor necrosis factor α (TNF-α) and IκBα (Inhibitor kappa Bα) composition characterized by inhibiting the production or expression. Anti-inflammatory agent containing a pharmaceutical composition for the treatment or prevention of inflammatory diseases of claim 1.

Images