KR20060022631A - Pharmaceutical composition for treating or preventing an inflammatory disease comprising lignan compounds - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for the treatment or prophylaxis of inflammatory diseases containing a lignan compound, and more specifically, a lignan compound or Myristica fragrans extract represented by the following general formula I as an active ingredient: It relates to a pharmaceutical composition for the treatment or prevention of inflammatory diseases containing.

[Formula I]

또는

이다.In the above, R ₁ and R ₂ is C _1-5 is an alkoxy or hydroxy group, R ₃ is

or

to be.

The lignan compound of Formula I exhibits an anti-inflammatory effect by inhibiting the production or expression of inflammatory mediators NO, iNOS, COX-2 and TNF-α. Therefore, the pharmaceutical composition of the present invention containing the lignan compound or myristica fragrance extract of the present invention as an active ingredient can be very useful for the treatment or prevention of inflammatory diseases.

Lignan compound, mystica fragrance, anti-inflammatory

Description

Pharmaceutical composition for treating or preventing an inflammatory disease comprising lignan compounds             

1 is a process chart for separating lignan compounds from Myristica fragrans .

2 is a ¹³ C-NMR spectrum of the lignan compound of the present invention.

3 is a ¹ H-NMR spectrum of the lignan compound of the present invention.

4 is a ¹ H- ¹ H COSY spectrum of the lignan compound of the present invention.

5 is a ¹ H- ¹³ C HMBC spectrum of the lignan compound of the present invention.

6 is an EI-Mass spectrum of the lignan compound of the present invention.

7 is a result of examining the cytotoxic effect of the lignan compound of the present invention.

8 shows the results of investigating the NO production inhibitory effect of the lignan compound of the present invention.

9 is a result of investigating the iNOS expression inhibitory effect of the lignan compound of the present invention.

A: Western blot analysis

B: Graph showing relative iNOS protein levels relative to control stimulated by LPS

10 shows the results of investigating the effect of inhibiting PGE ₂ production by the lignan compound (A) and curcumin (B) of the present invention.

11 is a result of examining the inhibitory effect of COX-2 expression of the lignan compound of the present invention.

A: Western blot analysis

B: Graph showing relative COX-2 protein levels based on control stimulated by LPS

Figure 12 shows the results of the LN-stimulated macrophages irradiated with the inhibitory effect of the TNF-α production of the lignan compound (A) and curcumin (B) of the present invention.

FIG. 13 shows the results of the inhibition of TNF-α production of the lignan compound (A) and indomethacin (B) of the present invention in human monocytes U937 cells stimulated with Propionibacterium acnes .

The present invention relates to a pharmaceutical composition for treating or preventing an inflammatory disease containing a lignan-based compound, and more particularly, an inflammatory agent containing a lignan-based compound or myristica fragrance extract represented by the following general formula (I) as an active ingredient: It relates to a pharmaceutical composition for treating or preventing a disease.

[Formula I]

또는

이다.In the above, R ₁ and R ₂ is C _1-5 is an alkoxy or hydroxy group, R ₃ is

or

to be.

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, inflammatory mediator secretion, 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 and 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 delivery, 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 prostaglandin production, thereby exhibiting anti-inflammatory effects (Meade EA et al ., J. Biol. Chem. , 268: 6610, 1993). However, these NSAID drugs suppress not only COX-2, which is temporarily expressed by inflammatory stimuli, but also COX-1, which plays an important role in maintaining the normal function of the gastrointestinal tract and renal platelets, thereby preventing serious side effects such as gastrointestinal bleeding and renal failure. There is a problem that results (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.

On the other hand, the lignan (lignan) is a collection of natural compounds in which n-phenyl propane bound at the β site of the n-propyl side chain, it is widely distributed in nature. Various physiological activities of lignans have been studied, including hypoglycemic activity, anticancer activity, anti-asthma effect and whitening action. For example, it has been reported that sesamine, episesamin, sesaminol, sesamolin, and episaminol isolated from sesame seeds have anti-inflammatory effects (Korea) Korean Patent Laid-Open Publication No. 1997-7001043), It has been disclosed that lignan compounds isolated from Magnoliae flos can be used as anti-asthma agonists (Korean Patent No. 0263439). In addition, macelignan is a representative lignan compound found in Myristica fragrans (Tuchinda P. et al ., Phytochemistry , 59: 169-173, 2002), and apoptosis Inducing caspase-3 enhancement (Park BY et al ., Biol. Pharm. Bull. , 27 (8): 1305-1307,2004) and antioxidant activity (Sadhu, SK et al ., Chem. Pharm Bull. , 51 (9): 595-598, 2003). However, the anti-inflammatory activity of the lignan compound, including the mace lignan has not been reported so far.

Thus, the present inventors completed the present invention by confirming that the lignan compound separated and purified from Myristica fragrance extract showed 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 lignan compounds isolated and purified from Myristica fragrance extract.

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

[Formula I]

또는

이다.In the above, R ₁ and R ₂ is C _1-5 is an alkoxy or hydroxy group, R ₃ is

or

to be.

Hereinafter, the present invention will be described in detail.

The present invention is characterized by providing a novel use of the lignan compound isolated and purified from Myristica fragrance extract.

The lignan compound according to the present invention is represented by the following general formula (I).

[Formula I]

또는

이다.In the above, R ₁ and R ₂ is C _1-5 is an alkoxy or hydroxy group, R ₃ is

or

to be.

인 하기의 화학식 1의 메이스리그난[(8R, 8'S)-7-(3,4-methylenedioxyphenyl)-7'-(4-hydroxy-3-methoxyphenyl)-8,8'-dimethylbutane)]일 수 있다. Preferred lignan compound of the present invention is wherein R ₁ is a methoxy group, R ₂ is a hydroxy group, R ₃ is

It may be a may ligignan [(8R, 8'S) -7- (3,4-methylenedioxyphenyl) -7 '-(4-hydroxy-3-methoxyphenyl) -8,8'-dimethylbutane) of the following formula (1).

[Formula 1]

The lignan compound according to the invention can 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.

The lignan compound of the present invention can be obtained from a plant or part of a plant using a method 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. In addition, the synthetic compounds or derivatives thereof corresponding to the lignan compound represented by the general formula (I) are generally commercially available materials, or may be chemically prepared using known synthetic methods.

The lignan compound of the present invention represented by the general formula (I) can be isolated and purified from Myristica fragnance Houtt. (Jung Yun Lee et al ., Kor. J. Pharmacogn . 21 (4): 270-273, 1990; Masao Hattori et al., Chem. Pharm. Bull. , 34 (9): 3885-3893, 1986; Masao Hattori et al., Chem Pharm. Bull ., 35 (2): 668-674 , 1987). Preferably, it can be separated and purified from nutmeg or ail. The nutmeg refers to a seed that is in the mature or fruit of Myritica fragrance. In addition, the lignan compound of the present invention can be separated and purified from the oil obtained by pressing nutmeg. It can also be isolated and purified from other nutmeg plants, Myristica argentea Warb (Filleur, F. et al ., Natural Product Letters , 16: 1-7, 2002). It can also be isolated and purified from Machilus thunbergii (Park BY et al ., Biol. Pharm. Bull. , 27 (8): 1305- 1307,2004) and Leucas aspera (Sadhu). , SK et al ., Chem. Pharm. Bull. , 51 (9): 595-598, 2003).

As an extraction solvent for the separation of the lignan compound of the present invention, water or an organic solvent of C1-C6 may be used. Preferably purified water, methanol (ethanol), ethanol (propanol), isopropanol (isopropanol), butanol (acetone), acetone (ether), benzene (benzene), chloroform (chloroform), Various solvents such as ethyl acetate, methylene chloride, hexane, cyclohexane and petroleum ether may be used alone or in combination. More preferably methanol or hexane can be used. Isolation and purification of the lignan compound of the present invention from the myritica fragrance extract is performed by column chromatography and high performance liquid chromatography (HPLC) filled with various synthetic resins such as silica gel or activated alumina. And the like can 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.

As such, the lignan compound of the present invention may be used in the form of a purely separated and 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 seeds, fruits or seedlings of Myristica fragrance as described above or the seeds 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 of Myristica fragrance, ie nutmeg extract.

The lignan compound 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, and 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. The lignan compound of the present invention was shown to inhibit the production of NO and the expression of iNOS involved in NO production in macrophages in a concentration-dependent manner (see FIGS . 8 and 9 ).

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. In addition to inhibiting the expression of COX-2, the lignan compound of the present invention has an effect of inhibiting the production of PGE ₂ (Prostaglandin E2), which is one of the PG species, in a concentration-dependent manner (see FIGS . 10 and 11 ).

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). The lignan compound of the present invention has an effect of inhibiting the production of TNF-α in macrophages and human monocytes (see FIGS . 12 and 13 ).

In addition, as a result of topical application of the lignan compound of the present invention to the ears of rats induced with edema with TPA (12- O- tetradecanoylphorbol-13-acetate) treatment, the rate of production of edema was suppressed in a concentration-dependent manner, and is currently used as a commercial anti-inflammatory agent. It showed higher edema inhibition rate than methacin (see Table 2 ). In addition, when a cream containing a lignan compound was prepared and then topically applied 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 mysteria fragrance extract (methanol and hexane crude extract) was applied topically to the ears of rats that caused edema by TPA treatment, it was confirmed that the edema production rate is suppressed in a concentration dependent manner (see Table 5 ).

These results indicate that the lignan compound of the present invention exhibits excellent anti-inflammatory action by inhibiting COX-2 as well as various factors that mediate the inflammatory response. It also indicates that the same anti-inflammatory effect can be obtained with the myristica fragrance extract itself. The anti-inflammatory activity of the lignan compound and the mysticica fragrance extract of the present invention represented by the general formula (I) is first identified in the present invention.

In view of the non-steroidal anti-inflammatory drugs currently used commercially exhibit the anti-inflammatory effect by inhibiting the activity of the COX-2 enzyme, it can be seen that the lignan compound of the present invention can be used as an anti-inflammatory agent having a superior effect than the conventional anti-inflammatory agent Can be.

Therefore, the present invention provides a pharmaceutical composition for the treatment or prevention of inflammatory diseases containing the lignan compound represented by the general formula (I), 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 mystica fragrance extract as an active ingredient. The preparation of the myristica fragrance 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 a general medicine, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc. which are commonly 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, lignan-based compounds or myristica fragrance extracts. It is prepared by mixing sucrose 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 lignan compound of Formula I or mystica fragrance extract is mixed in water with a stabilizer or buffer to prepare a solution or suspension, which is formulated into a unit dosage form 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 lignan compound or mysticica fragrance extract represented by the general formula (I) of the present invention is a single dose of 0.1-50 mg / kg, preferably 1-10 mg / kg, 1-3 times a day. May be administered. 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 conducting oral administration of the lignan compound of the present invention 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 lignan compound or myristica fragrance 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 Preferably 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 disease to which the pharmaceutical composition of the present invention can be applied refers to a disease accompanied by inflammation caused by various stimulating factors causing a series of inflammatory reactions such as NO, iNOS, COX-2, PGE ₂ , TNF-α, and the like. . 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, intestinal disease spondylitis, juvenile arthrosis, juvenile ankylosing spondylitis, reactive arthrosis, infectious arthritis, post-infectious arthritis, gonococcal arthritis, Tuberculosis arthritis, viral arthritis, fungal arthritis, syphilis arthritis, Lyme disease, arthritis associated with 'angiopathy syndrome', nodular polyarthritis, irritable vasculitis, rheumatoid granulomatosis, rheumatoid polymyalgia, joint cell arteritis, calcium crystals Calm arthropathy, pseudogout, non-joint rheumatoid arthritis, 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 and chronic 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 activity assays were repeated three times or more, and the results are expressed as the mean value standard deviation. In addition, statistical analysis was performed using Students's t- test, and p was determined to be statistically significant when <0.05 or less.

<Example 1>

Isolation and Purification of Lignan Compounds from Myristica Fragrance

<1-1> Isolation and Purification of Lignan Compound

400 ml of 75 vol% methanol was added to 100 g (dry weight) of dry ground nutmeg and left to stand at room temperature for two days. The solution was filtered using Whatman filter paper # 2. The process was repeated twice. The methanol filtrate was concentrated in vacuo and lyophilized to prepare a crude crude methanol extract (7 g). The crude methanol extract was partitioned into ethyl acetate, butanol and water in order to obtain an ethyl acetate fraction (4.2 g). The ethyl acetate fraction was eluted with a solvent in which hexane and ethyl acetate were mixed at a ratio of 10: 1 (v / v) using silica gel column chromatography (Merck Kieselgel 66; 70-230 mesh), and fraction III (1.0 g). Got. The solvent was completely removed by a vacuum rotary concentrator to prepare a crude extract of nutmeg. Then, fraction III was eluted with a solvent in which hexane and ethyl acetate were mixed at a ratio of 20: 1 (v / v) using silica gel column chromatography (Merck Kieselgel 66; 70-230 mesh), and fraction III-B ( 0.52 g) was obtained. Fraction III-B was eluted with 80% methanol using Rp-18 column chromatography (Merck LiChroprep; 25-40 μm) to obtain single substance fraction III-B-2 (0.5 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. The results are shown in FIGS . 2 and 3 , respectively. ¹³ C-NMR spectrum and the ^{1 H-NMR 1 H- 1 H} COSY spectrum and ¹ H- ¹³ to measure the correlation of the correlation of the ¹ H- ¹ H, based on the result of the spectrum related to the ¹ H- ¹³ C HMBC C The spectrum was measured. The results are shown in FIGS . 4 and 5 , respectively. ^{The results of 1} H-NMR, ¹³ C-NMR, ¹ H- ¹ H COZY, and ¹ H- ¹³ C HMBC were comprehensively analyzed and shown in Table 1 .

Position ¹³ C-NMR ¹ H-NMR ¹ H- ¹ H COSY ¹ H- ¹³ C HMBC One 135.4 2 109.2 6.72 brs C-7, C-6, C-4, C-3 3 147.3 4 145.1 5 107.9 6.79 d (7.8) 6.61 C-6, C-4, C-3, C-1 6 121.7 6.61 dd (7.8) 6.79 C-7, C-5, C-4, C-2, C-1 7                                              38.2                                              2.23 dd (13.2, 9.3) 2.66 dd (13.2, 4.8) 1.64, 2.66 1.64, 2.23 C-8, C-6, C-2, C-1 C-9, C-8, C-6, C-2, C-1 8 38.7 1.64 brs 0.75, 2.23, 2.66 C-7 9 16.0 0.75 d (6.3) 1.64 C-8, C-7 One' 132.4 2' 112.9 6.66 brs C-7 ', C-6', C-4 ', C-3' 3 ' 147.1 4' 144.4 5 ' 115.2 6.66 d (7.9) 6.53 C-6 ', C-4', C-3 ', C-1' 6 ' 121.0 6.53 d (7.9, 1.1) 6.66 C-7 ', C-5', C-4 ', C-2', C-1 ' 7 '                                              38.0                                              2.17 dd (13.2, 9.3) 2.66 dd (13.2, 4.8) 1.64, 2.66 1.64, 2.17 C-8 ', C-6', C-2 ', C-1' C-9 ', C-8', C-6 ', C-2', C-1 ' 8' 38.7 1.64 brs 0.75, 2.17, 2.66 C-7 ' 9 ' 16.1 0.75 d (6.3) 1.64 C-8 ', C-7' OMe 55.5 3.72 (s) O-CH ₂ -O 100.6 5.95 d (4.8) C-3, C-4

<1-3> mass spectrometry

The results of EI / MS measured for mass spectrometry of the separated single substance are shown in FIG. 6 . The compound had a molecular weight of 328 with [M] ⁺ being observed at m / z 328 in EI / MS, and the molecular formula was C ₂₀ H ₂₄ O ₄ .

Results of the above ¹ H-NMR, ¹³ C-NMR, ¹ H- ¹ H COZY, ¹ H- ¹³ C HMBC and EI / MS and previously published research reports (Woo, WS et al ., Phytochemistry , 26: 1542-1543, 1987) was identified by comparative analysis, it was confirmed that the isolated single material is maceignan (macelignan) represented by the following formula (1).

[Formula 1]

<Example 2>

Investigation of Cytotoxic Effects of Lignan Compounds of the Present Invention

<2-1> Culture of RAW264.7 Cell Line

Macrophages RAW264.7 cells were used to determine the effect of the mace lignan obtained in Example 1 on the production of inflammatory mediators. Macrophage RAW264.7 cells were purchased from the American Tissue Culture Collection (ATCC TIB TIB-71, Rockville, MD, USA). The cell line was supplemented with 10% FBS (fetal bovine serum, Gibco, USA), 100 U / ml penicillin G, and 100 μg / ml streptomycin, inactivated to heat with DMEM (Dulbecco's Modified Eagle's Medium, Gibco, USA) medium. And incubated in a 37 ° C. incubator containing 5% CO ₂ .

<2-2> Cytotoxicity Measurement

In order to investigate the effect of the mayis lignan of the present invention on the survival rate of RAW 264.7 cells, an analysis based on MTT reduction, which is converted to a purple formazan product by mitochondrial dehydrogenase, was performed (Hayon T. et al . , Leuk.Lymphoma. 44 (11): 1957-1962, 2003). 1 × 10 ⁶ cells / mL of RAW264.7 cells were inoculated in RPMI 1640 medium and 6 hours later, the maye lignan of the present invention was treated to 1-80 μM / mL. After 24 hours, cell viability was measured using the MTT assay.

As a result, as shown in FIG. 7 , the maye lignan of the present invention did not significantly affect the viability of RAW264.7 cells at a concentration of 1-20 μM. Based on these results, the subsequent inflammatory experiments used Macelignan at a concentration of 1-20 μM.

<Example 3>

Investigation of NO Inhibition Effect of the Lignan Compound of the Present Invention

<3-1> NO production inhibition

Macrophages stimulated by IFN- [gamma] or LPS express large amounts of iNOS and produce large amounts of NO, a mediator of the inflammatory response (Miyasaka and Hirata., Immunol. Today., 16: 128-130, 1995; Guzik et al. , J. Physiol.Pharmacol ., 54 (4): 469-487, 2003). Therefore, we examined the effect of the maye lignan of the present invention on NO production in RAW264.7 cells activated with LPS.

RAW264.7 cells were diluted to a concentration of 1 × 10 ⁶ cells / ml and then inoculated in RPMI 1640 medium. After 5 hours, the maye lignan of the present invention was added at each concentration of 1-20 μM and pretreated for 2 hours. Thereafter, LPS (10 μg / ml) was treated and incubated for 24 hours. The control group was treated only with LPS. The amount of NO produced was quantified by measuring the NO ₂ ⁻ reaction product of NO using a cell culture filtrate (Han et al ., Life Sci., 75 (6): 675-684, 2004). Greiss reagent (0.5% sulfanilyamide, 0.05% N- (1-naphthyl) ethylene diamine dihydrochloride / 2.5% H ₃ PO ₄ ) in the same volume as 100 μl of the filtrate was mixed in a 96-well tissue culture plate in the dark at room temperature. The reaction was carried out for 10 minutes. The absorbance of the sample was then measured at 550 nm using an ELISA microplate reader. The concentration of NO ₂ ⁻ was made using NaNO ₃ to create a standard curve, and the amount of NO produced was measured. All experiments were repeated three more times and each was quantified using student's t- test.

As a result, as shown in FIG. 8 , the production of NO was greatly increased by LPS treatment alone, but it was confirmed that this was suppressed in a concentration-dependent manner by the treatment of mayis lignan according to the present invention. In particular, even at low concentrations of 1 μM and 5 μM it was confirmed that the production of NO is excellent ( P <0.01). In addition, when 20 μM of mace lignan was treated, NO production was suppressed to the same extent as the untreated group.

<3-2> iNOS expression inhibition

Stimulating macrophages with LPS overexpresses iNOS and produces large amounts of NO. Therefore, in order to examine the relationship between the inhibition of NO production and the iNOS of the maye lignan of the present invention identified in Example <3-1>, the effect of the maye lignan on iNOS expression was measured.

The protein was quantified by the Bradford method after lysing the May 26 lignin and LPS-treated RAW 264.7 cells of the present invention. 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.) attached with Horse radish peroxidase (HRP) 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 Fig. 9 , the maye lignan of the present invention inhibited the expression of iNOS in macrophages in a concentration-dependent manner, it was confirmed that the inhibitory effect is remarkable from 5 μM ( P <0.01).

From the above results, it was confirmed that the maye lignan of the present invention not only inhibits the production of NO, which is an inflammation-inducing factor, but also inhibits the expression of iNOS.

<Example 4>

Investigation of the COX-2 Inhibitory Effect of the Lignan Compound of the Present Invention

<4-1> PEG ₂ Suppress generation

Just as iNOS is closely related to inflammatory responses, COX-2 is an essential enzyme for generating PGs that mediate inflammatory responses, and the expression and activity of iNOS and COX are known to be correlated (Surh et al. , Mutat.Res., 481: 243-268, 2001). Therefore, we examined how the maye lignan of the present invention affects the production of PGE ₂ in macrophages activated by LPS.

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. Then, mayis lignan of the present invention was added at a concentration of 1-20 μM, respectively, and pretreated for 2 hours. Negative controls were not treated with anything, and positive controls were treated with reported curcumin (isolated from Curcuma longa , Sigma) with PGE ₂ inhibitory activity. Thereafter, 1 μg / ml of LPS was treated and incubated for 18 hours. The amount of PGE ₂ produced in macrophages was quantified by ELISA using the method of the assay kit (R & D System Inc, Minneopolis, USA) (Chen et al ., Biochem. Pharmacol., 68: 1089-1100, 2002). .

As a result, as shown in FIG. 10 , PGE ₂ was largely generated by LPS treatment alone, but it was confirmed that this was suppressed in a concentration-dependent manner by the treatment of the maye lignan of the present invention. The inhibitory effect was also observed at 5 μM. The PGE ₂ production inhibitory effect of the maye lignan of the present invention was almost similar to that of curcumin treatment, showing the same tendency as the NO and iNOS inhibitory effect confirmed in Example 3 ( P <0.05).

<4-2> COX-2 expression inhibition

We investigated the expression of COX-2, which directly affects PGE ₂ production, using Weston blot analysis. Except for using the anti-COX-2 antibody (1: 2,000) (Calbiochem) as the primary antibody and the anti-chlorine IgG-HRP (1: 2,000) (Calbiochem) as the secondary antibody. 3-2> was carried out in the same manner as described.

As a result, as shown in FIG. 11 , it was confirmed that the maye lignan of the present invention suppressed the expression of COX-2 protein in a concentration-dependent manner. In particular, the amount of expression was significantly reduced at a concentration of 10-20 μM.

From the above results, it was confirmed that the maye lignan of the present invention not only inhibits the production of PEG ₂ , which is an inflammation-inducing factor, but also inhibits the expression of COX-2.

<Example 5>

Investigation of TNF-α Inhibitory Effect of the Lignan Compound of the Present Invention

TNF-α is an inflammatory cytokine that plays an important role in the inflammatory response. Therefore, the effect of the mace lignan of the present invention on the production of TNF-α was investigated.

<5-1> Inhibition of TNF-α Production in Macrophage Cell Lines

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. Then, the mace lignan of the present invention was added to each concentration of 1 ~ 20 μM and pretreated for 2 hours. None were treated with the negative control and curcumin (Sigma) was treated with the positive control (Araujo and Leon, Mem. Inst. Oswaldo. Cruz., 96 (5): 723-728, 2001; Chainani- Wu, J. Altern.Complement., 9 (1): 161-168, 2003). Thereafter, 1 μg / ml of LPS was treated and incubated for 18 hours. The amount of TNF-α in macrophages was quantified by ELISA using the method of the assay kit (R & D System Inc, Minneopolis, USA) (Chen et al., J. Dermatol. Sci. 29: 97-103, 2002).

As a result, as shown in Fig. 12 , the production of TNF-α showed a tendency to decrease significantly from the treatment of Mayse lignan 5 μM of the present invention ( P <0.05).

<5-2> Inhibition of TNF-α Production in Human Monocytes

The present inventors measured the amount of TNF-α production in human monocytic U937 cells activated with Propionibacterium acnes according to the same method as in Example <5-1>. However, the positive control group was treated with indomethacin (Sigma) (Walch and Morris, Endocrinology. 143 (9): 3276-3283, 2002).

As a result, as shown in FIG. 13 , it was confirmed that TNF-α production in human monocytes was reduced in a concentration-dependent manner by the maye lignan of the present invention ( P <0.01) .

From the above results, it was confirmed that the maye lignan of the present invention inhibits the production of TNF-α which induces and / or mediates the systemic reaction of acute inflammation and inflammation.

<Example 6>

Investigation of anti-inflammatory activity of the lignan compound of the present invention using an animal model

The anti-inflammatory activity of the lignan compound 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). In Example 1, the purified pure mace lignan and the nonsteroidal anti-inflammatory agent, indomethacin, were dissolved in acetone to be 20, 200, 2000 ㎍ / ear. Mayslignan 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.

Medication Rat number (horse) Dose (μg / ear) Edema Thickness (㎛) Edema inhibition rate (%) Control 20 0 248 ± 8 - Mace lignan of the present invention 20 20 157 ± 9 ^* 36.7 20 200 98 ± 6 ^* 60.5 20 2000 52 ± 4 ^* 79.0 Indomethacin 20 20 185 ± 5 ^* 25.0 20 200 108 ± 8 ^* 56.5 20 2000 64 ± 7 ^* 74.2

^* p <0.01

As shown in Table 2 , the maye lignan 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 mayis lignan and indomethacin of the present invention were 120.6 µg / ear and 162.9 µg / ear, respectively.

<Example 7>

Preparation of Lignan Compound-Containing Cream of the Present Invention and Investigation of its Anti-inflammatory Activity

<7-1> Preparation of Cream

Using the mace lignan of the present invention, creams having various compositions shown in Table 3 were prepared, respectively. 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 maye lignan 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%, respectively. Finally, perfume was added and the quantity was adjusted to purified water to prepare a cream.

One 2 3 4 Main Components A Maces League 5.0% Maces League 0.5% Maces League 0.05% Maces League 0.005% B Glycerin 2.0% Glycerin 2.0% Glycerin 2.0% Glycerin 2.0% Propylene Glycol 2.0% Propylene Glycol 2.0% Propylene Glycol 2.0% Propylene Glycol 2.0% Lauryl chloride sulfide 8.0% Lauryl chloride sulfide 8.0% Lauryl chloride sulfide 8.0% Lauryl chloride sulfide 8.0% Stearin 5.4% Stearin 5.4% Stearin 5.4% Stearin 5.4% Mineral oil 4.5% Mineral oil 4.5% Mineral oil 4.5% Mineral oil 4.5% C Fragrance 0.02% Fragrance 0.02% Fragrance 0.02% Fragrance 0.02% Cetyl alcohol 6.5% Cetyl alcohol 6.5% Cetyl alcohol 6.5% Cetyl alcohol 6.5% Purified water balance Purified water balance Purified water balance Purified water balance 0.1% preservative 0.1% preservative 0.1% preservative 0.1% preservative

<7-2> Anti-inflammatory activity investigation

The anti-inflammatory activity of the mace lignan-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 Mace lignan content 0.005% 0.05% 0.5% 5% Edema inhibition rate (%) 14.2 42.3 64.6 88.7

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

<Example 8>

Investigation of Anti-inflammatory Activity of Myristica Fragrance Extract

<8-1> Methanol Extract Preparation

400 g of 95 vol% methanol was added to 100 g (dry weight) of dry ground nutmeg and left to stand at room temperature for two days. The solution was filtered using Whatman filter paper # 2. The process was repeated twice. The methanol filtrate was concentrated in vacuo and lyophilized to prepare methanol crude extract (16.2 g).

<8-2> Hexane Extract Preparation

To 100 g (dry weight) of dry ground nutmeg, 400 ml of 100% by volume hexane was added and left at room temperature for two days. The solution was filtered using Whatman filter paper # 2. The process was repeated twice. The hexane filtrate was concentrated in vacuo and freeze-dried to prepare a hexane crude extract (37.0 g).

<8-3> Anti-inflammatory Activity of Myristica Fragrance Extract Using Animal Model

The anti-inflammatory activity of the myristica fragrance extract prepared in Examples <8-1> and <8-2> 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.

Medication Rat number (horse) Dose (μg / ear) Edema Thickness (㎛) Edema inhibition rate (%) Control 20 0 248 ± 8 - Methanol crude extract 20 50 198 ± 3 ^* 20.2 20 500 119 ± 8 ^* 52.0 20 5000 83 ± 10 ^* 66.5 Hexane crude extract 20 50 207 ± 4 ^* 16.5 20 500 146 ± 9 ^* 41.1 20 5000 106 ± 7 ^* 57.3

^* p <0.01

As shown in Table 5 , it was confirmed that both the methanol crude extract and hexane crude extract of myritica fragrance of the present invention inhibit the edema of rats induced by TPA in a concentration-dependent manner (statistical significance p <0.01).

<Manufacture 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 the lignan compound or myristica fragrance 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 for direct-acting agent (low) -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

A syrup containing maye lignan of the present invention or a pharmaceutically acceptable salt thereof as an active ingredient of 2% (W / V) was prepared by the following method: 2 g of acid addition salt of maye lignan of the present invention, 0.8 g of saccharin, and 25.4 g of sugar was 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 lignan compound or myristica fragrance extract of the present invention, 50 mg of lactose, 46.5 mg of starch, 1 mg of talc, and an appropriate amount of magnesium stearate and filling it into a hard gelatin capsule.

<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 mayis lignan hydrochloride, 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 Disease

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 pyrobacteria, the enzyme COX-2 (cyclooxygenase-2) increases simultaneously (Nam NT et al., Clin. Cancer Res. 10 (23): 8105-8113, 2004). When Helicobacter is infected, gastric mucosa proliferates to cancer cells, and COX-2 inhibitors have been shown to inhibit gastric mucosa growth and cancer cell growth. The group treated with the COX-2 inhibitor was found to have a superior effect of killing cancer tissues by the method of apoptosis (Nam NT et al., Clin. Cancer Res. 10 (23): 8105) . -8113, 2004). Therefore, the COX-2 inhibitory effect of the lignan compound of the present invention suggests that it has a sufficient therapeutic effect since 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. New capillaries invade joints and are characterized by cartilage damage (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). In this case, it is reported that inflammatory reactions occur first and second depending on the type, and thus play an important role in the progression of disease by destroying cartilage, and the formation of new blood vessels into joints 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 lignan compound or myristica fragrance extract of the present invention as an active ingredient is very effective in preventing and treating arthritis.

As described above, the lignan compound of the present invention has an effect of inhibiting the inflammatory response by inhibiting the production or expression of inflammatory mediators NO, iNOS, PGE ₂ , COX-2 and TNF-α. Therefore, the pharmaceutical composition containing the lignan compound or myristica fragrance extract of the present invention as an active ingredient can be very useful for the treatment or prevention of inflammatory diseases.

Claims (6)

A pharmaceutical composition for the treatment or prophylaxis of an inflammatory disease containing a lignan compound represented by the following general formula (I) or a pharmaceutically acceptable salt thereof as an active ingredient. [Formula I]

In the above, R ₁ and R ₂ C _1-5 is an alkoxy or hydroxy group, R ₃ is

or

to be. According to claim 1, wherein R ₁ is a methoxy group, R ₂ is a hydroxy group, R ₃ is

The composition characterized in that the. Pharmaceutical composition for the treatment or prevention of inflammatory diseases containing myristica fragrans ( Myristica fragrans ) extracted with water or an organic solvent of C1-C6 as an active ingredient. According to claim 1 or 3, wherein the inflammatory disease is 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, spondyloarthropathies, 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, syphilis arthritis, Lyme disease, arthritis associated with 'angiopathy syndrome', nodular polyarthritis, irritable vasculitis, rheumatoid granulomatosis, rheumatoid polymyalgia, joint cell arteritis, calcium crystals Deposition arthrosis, pseudogout, non-joint rheumatoid, bursitis, hay fever, epicondylitis (tennis elbow), nerves Neuropathic joint disease, hemarthrosic, henoch-kön purpura, hypertrophic osteoarthritis, multicardiac retinopathy, scoliosis, hemochromatosis, hemoglobinopathy, hyperlipoproteinemia, hypomagglobulin Hyperemia, familial Mediterranean fever, Behatt's disease, systemic lupus erythematosus, recursive fever, multiple sclerosis, sepsis, septic shock, acute respiratory distress syndrome, multiple organ failure, chronic obstructive pulmonary disease, rheumatoid arthritis (rheumatoid arthritis), acute lung injury (acute lung injury), bronchopulmonary dysplasia (broncho-pulmonary dysplasia) and inflammatory skin diseases, the composition characterized in that any one selected from the group consisting of. The lignan compound according to claim 1 or 3, wherein the lignan compound is nitric oxide (NO), inducible nitric oxide synthase (iNOS), prostaglandin E2 (PGE2), cyclooxygenase-2 (COX-2), and tumor necrosis (TNF-α). composition for inhibiting the production or expression of factor α). An anti-inflammatory agent containing a pharmaceutical composition for the treatment or prevention of inflammatory diseases according to claim 1.

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