KR20090032601A - Pharmaceutical composition comprising inhibitors of cell adhesion molecule isolated from piper nigrum for the prevention and treatment of inflammatory disease - Google Patents

Abstract

Provided is a pharmaceutical composition containing Piper nigrum extracts or amide compounds isolated from Piper nigrum, which have effects of inhibiting ICAM-1(Intercellular adhesion molecule-1) and LFA-1(Leukocyte function associated antigen-1)-mediated cell adhesion. A pharmaceutical composition for treating inflammation diseases comprises Piper nigrum extracts or amide compounds isolated from Piper nigrum. The Piper nigrum extracts are extracted by water, C1-C4 low alcohol or mixed solvent thereof. The low alcohol indicates methanol, ethanol or butanol. A solvent extract of the Piper nigrum is fractioned by an extraction solvent such as hexane, chloroform and ethylacetate. The Piper nigrum extracts contain compounds represented by the formula 1 to 4 or one of compounds represented by the formula 1 to 4.

Description

Pharmaceutical composition comprising inhibitors of cell adhesion molecule isolated from Piper nigrum for the prevention and treatment of inflammatory disease}

The present invention is effective for treating inflammatory diseases or infectious diseases ( piper) nigrum ) extracts and amide compounds derived therefrom. In addition, the present invention as an active ingredient extracted from the reef extract or edible extract, and the binding between intercellular adhesion factor-1 (ICAM-1) and leukocyte function-related antigen-1 (LFA-1; CD11a / CD18) Specifically inhibited inflammatory diseases such as vascular stenosis, arthritis, asthma, osteoporosis, psoriasis, atopy, inflammatory enteritis, cancer metastasis or transplant rejection, or diseases caused by rhinovirus or human immunodeficiency virus type 1 (HIV-1) It relates to a pharmaceutical composition that can be usefully used for the prevention and treatment of infectious diseases such as.

Intercellular adhesion molecule-1 (ICAM-1; CD54) is an adhesion receptor belonging to the Ig-superfamily of vascular endothelial cells, some lymphocytes and monocytes. expressed in monocytes at low density (Springer, Nature , 346: 425-434, 1990; Rothlein et al., J. Immunol ., 137: 1270-1274, 1986) IL-1, TNF-α or IFN-γ When treated with inflammatory cytokines or lipopolysaccharides (LPS), the expression of ICAM-1 is increased in multiple cell types (Hubbard and Rothlein, Free). Radic . Biol . Med ., 28: 1379-1386, 2000).

Leukocyte function associated antigen-1 (LFA-1; CD11a / CD18) is a ligand of ICAM-1 that is inactive in resting leukocytes but is mediated by T cell receptors or Activated by various stimulators such as phorbolester (Dustin and Springer, Nature , 341: 619-624, 1989; Rothlein and Springer, J. Exp . Med ., 163: 1132-1149, 1986).

On the other hand, in order for the white blood cells to infiltrate the inflammation site, the white blood cells must adhere to the vascular endothelial cells on the surface of the vascular endothelial cells. ICAM-1 is particularly expressed in vascular endothelial cells that have undergone inflammatory reactions, and promotes adhesion and penetration of blood cells such as monocytes, neutrophils, and lymphocytes to blood vessel walls. ICAM-1 also plays an important role in adhesion between platelets and vascular endothelial cells (Thomas and DeGraba, Neurology, 49: 15-19, 1997).

Cell adhesion is closely related to atherosclerosis, arthritis, osteoporosis, bacterial infections, cancer metastasis, and intractable chronic diseases such as angiogenesis, psoriasis, asthma, allergies, lupus or Crohn's disease (Poston et al., Am . J. Pathol ., 140: 665-673, 1992; Macchioni et al., J. Reumatol ., 21 (10): 1860-1864, 1994; Mason et al., Arthritis Rheum ., 36: 519-527 , 1993; Kling et al., Clin . Invest ., 71: 299-304, 1993). Among these, arteriosclerosis develops in the form of chronic inflammation when microscopic damage occurs to endothelial cells of blood vessel walls by hypertension, toxins or pathogens along with the mechanism of atherosclerosis due to excessive cholesterol concentration in the blood. In addition, monocytes in the blood penetrate into the blood vessel wall by cell adhesion factors expressed in endothelial cells, differentiate into macrophage from the blood vessel wall, and transform into foam cells that store cholesterol introduced into the blood. Progression of fibrosis leads to irreversible degeneration that narrows blood vessels (Ross, Annu . Rev. Physiol., 57: 791-804, 1995; Christoper and Joseph, Cell, 104: 503-516, 2001). Therefore, cell adhesion factors play an important role in the process of inflammatory reaction and atherosclerosis, and various attempts have been made to develop inhibitors that block the expression of these adhesion factors between monocytes and endothelial cells.

These results suggest that ICAM-1 and LFA-1 mediated cell adhesion inhibitors are valuable as potential therapeutics for atherosclerosis and immune-related inflammatory diseases (Oppenheimer-marks and Lipsky, Clin . Immunol . Immunopathol ., 79: 203-210, 1996). In particular, the fact that ICAM-1 is important in the inflammatory response has been revealed through animal experiments with monoclonal antibodies of ICAM-1 and various disease models. Inhibition of binding between ICAM-1 and LFA-1 using monoclonal antibodies that directly correspond to ICAM-1 or LFA-1 has been shown to be effective in autoimmune diseases such as atherosclerosis, asthma, glomerulonephritis or arthritis In addition to antigen-specific signals through the T cell receptor, antigen-specific signals, such as ICAM-1 and LFA-1, are required for activation of T cells in the response (Cornejo et al., Adv . Pharmacol ., 39: 99-141, 1997; Patel et al., Circulation, 97: 75-78, 1998; Nishikawa et al., J. Exp . Med ., 177: 667-77, 1993; Kobayashi et al., Cell Immunol ., 164: 295-305, 1995; Nagase et al. Am . J. Respir . Crit . Care Med ., 152: 81-86, 1995).

 ICAM-1 plays an important role in viral infections as well as inflammatory diseases. A cold virus, rhinovirus, has been demonstrated to infect using ICAM-1 as a receptor (Greve et al., Cell, 56: 839-847, 1989; Staunton et al., Cell, 56: 849-853 , 1989; Tomassini et al., Proc. Natl. Acad. Sci. (USA), 86: 4907-4911, 1989). In addition, human immunodeficiency virus type-1 (HIV-1) is also infected by the binding between ICAM-1 and LFA-1 and is known as a statin known as an inhibitor of binding of ICAM-1 and LFA-1. ) Compounds have been reported to inhibit the proliferation of HIV-1 virus (Giguere and Tremblay, J. Virology 78: 12062-12065, 2004). Thus, inhibitors of activity of ICAM-1 or LFA-1 can be used not only for the treatment of inflammatory diseases, but also as antiviral agents for rhinoviruses or HIV-1.

Currently, the development of ICAM-1 and LFA-1 mediated cell adhesion inhibitors has been targeted by their neutralizing antibodies, and only a few kinds of inhibitors have been reported from natural products. Abbott has reported ICAM-1 expression inhibitors as a synthetic product (Stewart et al., J. Med . Chem ., 44: 988-1002, 2001) . The antagonist of LFA-1 was developed from Novartis and Roche (Welzenbach et al., J. Biol . Chem ., 277: 10590-10598, 2002, Gadek et al., Science , 295). : 1086-1089, 2002), there are no reports of in vivo activity using disease animal models. On the other hand, from the natural products seco-limonids-based material ( Trichilia rubra , IC ₅₀ ; 10-25 nM), cucurbitacins, Conobea scoparioides , IC ₅₀ ; 0.18-1.36 mM), and axanthromycins ( Streptomyces sp. Na-148, IC ₅₀ ; 1.5-6.5 mM) have been reported to inhibit ICAM-1 and LFA-1 mediated cell adhesion. Musza et al, Tetrahedron, 50: 11369-11378, 1994; Musza et al, J. Net Prod, 57:. 1498-1502, 1994; Nakano et al, J. Antibiotics, 53:.... 12-18, 2000).

The present inventors have searched for ICAM-1 and LFA-1 mediated cell adhesion inhibitors from natural resources, and as a result, aquatic extract, fractions thereof, or amide compounds derived from them inhibit ICAM-1 and LFA-1 mediated cell adhesion. The present invention has been completed by elucidating that there is an effect.

It is an object of the present invention to provide a reef extract having a prophylactic and therapeutic effect of an inflammatory disease or an infectious disease, wherein the reef is extracted with a solvent selected from the group consisting of water, lower alcohols having 1 to 4 carbon atoms and mixed solvents thereof.

Another object of the present invention is to provide a reef extract having a prophylactic and therapeutic effect of an inflammatory disease or an infectious disease obtained by using an elution solvent selected from the group consisting of hexane, chloroform and ethyl acetate.

Another object of the present invention to provide a pharmaceutical composition for the prevention and treatment of inflammatory diseases or infectious diseases comprising the above-mentioned extract of the edible extract or the compound of formula 1 to 4 as an active ingredient.

In order to achieve the above object, the present invention, as one embodiment, the preventive and therapeutic effect of inflammatory diseases or infectious diseases extracted with a solvent selected from the group consisting of water, lower alcohols having 1 to 4 carbon atoms and mixed solvents thereof It relates to a reef extract having a.

In the present invention, a method for obtaining a lake leaf extract is as follows. The lower ground alcohol is a lower alcohol having 1 (C ₁ ) to 4 (C ₄ ) carbon atoms, such as water, methanol, ethanol and butanol, in volumes of about 2 to 20 times, preferably about 3 to 5 times the dry weight. Using a polar solvent or a mixed solvent having a mixing ratio of about 1: 0.1 to 1:10 as the elution solvent, the extraction temperature is 20 to 100 ℃, preferably at room temperature, the extraction period is about 12 hours to 4 days Preferably, the extraction is performed using an extraction method such as hot water extraction, cold extraction, reflux cooling extraction, or ultrasonic extraction for three days. Preferably, the extract is filtered 1 to 5 times by cold extraction and subjected to filtration under reduced pressure, and the filtrate is concentrated under reduced pressure at 20 to 100 ° C., preferably at room temperature using a vacuum rotary condenser, and soluble in water, lower alcohols, or a mixed solvent thereof. One crude crude extract can be obtained.

Reef extract of the present invention inhibited more than 50% of adhesion between sICAM-1 and THP-1 cells (expressing LFA-1), which is a human monocyte cell line, at a concentration of 50 μg / ml as shown in the following example (FIG. 1). . Therefore, it is suitable for the prevention and treatment of inflammatory diseases or infectious diseases.

The vinegar extract of the present invention is extracted from the vinegar with a solvent selected from the group consisting of water, a lower alcohol having 1 to 4 carbon atoms and a mixed solvent thereof as described above, and then it is selected from the group consisting of hexane, chloroform and ethyl acetate. Fractionation may be further included using an eluting solvent. Preferably, the present invention relates to a chloroform fraction obtained by sequentially and repeatedly extracting the above seaweed extract using hexane and chloroform as a solvent.

In a further step in the present invention, the crude reef extract is suspended in distilled water, and then, by adding a nonpolar solvent such as hexane, ethyl acetate or chloroform in a volume of about 1 to 100 times, preferably about 1 to 5 times the suspension, 1 It can be obtained by extracting and separating the nonpolar solvent soluble layer from times to 10 times, preferably from 2 times to 5 times. It is also possible to further carry out conventional fractionation processes (Harborne JB Phytochemical methods: A guide to modern techniques of plant analysis , 3rd Ed. p6-7, 1998). Specifically, after suspending the crude crude extract in water, the same amount of n-hexane and chloroform can be obtained by continuous extraction of each solvent using a solvent, and more specifically, the edible crude extract is extracted in water. After suspension, the same amount of n-hexane can be mixed and fractioned to obtain n-hexane soluble fraction and water soluble fraction, and chloroform can be added to the water soluble fraction to obtain chloroform soluble fraction and water soluble fraction. .

Inflammatory diseases in the present invention include autoimmune diseases related to multiple organs such as systemic lupus erythematosus and scleroderma; Inflammatory bowel disease, including ulcerative colitis and Crohn's disease; Central nervous system inflammatory diseases such as Alzheimer's disease, multiple sclerosis, motor neuron disease, Parkinson's disease, chronic fatigue syndrome; Inflammation including IgE mediated (type I) hypersensitivity such as atopic dermatitis, psoriasis, anaphylaxis and dermatitis; Eye diseases such as diabetic retinopathy, retinitis, macular disease, uveitis, conjunctivitis; Stroke, Coronary Artery Disease, Myocardial Infarction, Unstable Angina, Vasculitis, Atherosclerosis, Vascular Stenosis, Wegener's Granulomatosis, Churg-Strauss Syndrome Vasculitis, Henoch-Schonlein Vascular diseases such as purpura, Kawasaki disease, giant cell arteritis; Arthritis, rheumatoid arthritis, ankylosing spondylitis, osteoarthritis, osteoporosis, allergy, diabetes, diabetic nephropathy, acute pediatric diabetes, Addison's disease, Goodpasture syndrome, IgA nephritis, nephritis, nephritis, Sjogren's syndrome, autoimmune pancreatitis, periodontal disease, chronic Obstructive pulmonary disease, acute leukemia-mediated lung injury, asthma, graft-versus-host disease, chronic pelvic inflammatory disease, endometritis, rhinitis, tumor metastasis, transplant rejection, stroke, encephalitis, meningitis, AIDS dementia, fibrosis, adhesions, chronic hepatitis and Chronic inflammatory diseases such as tuberculosis, multicuring and chronic prostatitis, and the like. In addition, the infectious disease in the present invention includes an infectious disease caused by rhinovirus or HIV-1.

The chloroform fraction of the weed of the present invention inhibited the adhesion between sICAM-1 and THP-1 cells, a human monocyte cell line, by 70% or more at a concentration of 50 μg / ml as shown in the following example (FIG. 2). Therefore, it is suitable for the prevention and treatment of inflammatory diseases or infectious diseases.

As another aspect, the present invention is to provide a pharmaceutical composition for the prevention and treatment of inflammatory diseases or infectious diseases comprising the above-mentioned extract of the edible extract or the compound of Formula 1 to 4 as an active ingredient.

Amide-based compounds of Formulas 1 to 4 may be used in the form of a pharmaceutically acceptable salt, and include all salts, hydrates, and solvates prepared by conventional methods. Extraction, separation and purification of the amide compound from the vinegar according to the present invention is as follows.

The chloroform soluble fraction is separated by silica gel column chromatography using a mixed solvent of n-hexane and ethyl acetate. At this time, n-hexane and ethyl acetate is preferably used a solvent of 50: 1 to 0: 100 (v / v). ICAM-1 and LFA-1 mediated cell adhesion inhibitory activity of the isolated active fractions was measured, and the highest inhibitory fractions were collected and 50%, 60%, 70%, 80%, 90% and 100% methanol was collected. Separating by reverse phase column chromatography using as eluent. Thus obtained fractions with high ICAM-1 and LFA-1 mediated cell adhesion inhibitory activity were collected to separate the compounds of Formulas 1 to 4 by HPLC (high performance liquid chromatography) using 75% methanol and acetonitrile. Can be.

As in the following examples, the compound of formula 1 of the present invention inhibited adhesion of sICAM-1 and THP-1 cells at least 60% at 25 μg / ml, the compound of formula 2 was at least 40%, the compound of formula 3 Silver more than 80%, the compound of formula 4 inhibited more than 70%, the compound of the present invention effectively inhibited the adhesion between sICAM-1 and THP-1 cells of human monocyte cell line as the concentration (Fig. 3). Accordingly, the compounds of formulas 1 to 4 of the present invention are suitable for the prevention and treatment of inflammatory diseases or infectious diseases.

Echo extract of the present invention or compounds of Formulas 1 to 4 inhibits the adhesion and infiltration of blood cells such as monocytes, neutrophils and lymphocytes to vascular endothelial cells with inflammation by inhibiting ICAM-1 and LFA-1 mediated cell adhesion. It can be usefully used as a medicine for the prevention and treatment of diseases. In addition, the amide compound of the present invention was used by extraction, separation and purification from the vinegar, but can be obtained by all conventional methods, and commercially available reagents can be used.

Pharmaceutical compositions for the prevention and treatment of inflammatory diseases or infectious diseases of the present invention, the total weight of the composition of 0.001 to 10% by weight, preferably 0.001 to 1% by weight of the extract or the compound of formula 1 to 4 Include.

The composition comprising the topical extract of the present invention or the compound of Formulas 1 to 4 may additionally include suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.

Pharmaceutical dosage forms of the compositions of the present invention may be used in the form of their pharmaceutically acceptable salts, and may be used alone or in combination with other pharmaceutically active compounds, as well as in any suitable collection.

The compositions according to the invention can be used in the form of oral formulations, external preparations, suppositories, or sterile injectable solutions, such as powders, granules, tablets, capsules, suspensions, emulsions, syrups or aerosols, respectively, according to conventional methods. have. Carriers, excipients and diluents that may be included in the compositions of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents and surfactants are usually used. Solid form preparations for oral administration include tablets, pills, powders, granules and capsules, and the like form at least one excipient such as starch, calcium carbonate, sucrose in the extract or fraction. (sucrose), lactose (lactose), gelatin, etc. are mixed and prepared. In addition to simple excipients, glidants such as magnesium stearate or talc are also used. Liquid preparations for oral use include various excipients, such as wetting agents, sweeteners, fragrances or preservatives, in addition to water and liquid paraffin, which are common diluents commonly used in suspensions, solutions, emulsions or syrups. Can be. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations or suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, or injectable ester such as ethyl oleate may be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter or glycerogelatin may be used.

Preferred dosages of the compositions of the present invention vary depending on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, and may be appropriately selected by those skilled in the art. However, for the desired effect, the composition of the present invention is preferably administered at 0.0001 to 100 mg / kg, preferably at 0.001 to 100 mg / kg. Administration may be administered once a day or may be divided several times.

The pharmaceutical composition of the present invention can be administered to various mammals such as rats, mice, livestock, humans, and the like. All modes of administration can be expected, for example by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.

As described above, the reef extract of the present invention or the compound of Formulas 1 to 4 inhibits ICAM-1 and LFA-1 mediated cell adhesion so that blood cells such as monocytes, neutrophils and lymphocytes adhere to and invade vascular endothelial cells. Since it is effective in lowering the amount, it can be usefully used as a medicament for preventing and treating inflammatory diseases and infectious diseases infected using ICAM-1.

The present invention is explained in more detail based on the following examples, but the present invention is not limited thereto.

Example  One. From the lake Amide  Extraction, Separation and Purification of Compounds

1-1. From the lake  extraction

The poncho was washed with water, dried in the shade, and then powdered by Waring brand. To 5 kg of powdered poncho, methanol was added three times the weight of the dried poncho, extracted at room temperature for 7 days, and filtered. The filtrate was concentrated under reduced pressure to obtain a crude extract.

In order to isolate and purify the active substance from the crude extract, the crude crude extract was suspended in water, and then the same amount of n-hexane was mixed and fractionated to obtain an n-hexane soluble fraction and a water soluble fraction. Chloroform was added to the fractions to give chloroform soluble fraction and water soluble fraction. The obtained fractions were measured for intercellular adhesion inhibitory activity by the method of Example 2 below, showing the best intercellular adhesion inhibitory activity in the chloroform layer.

1-2. Chloroform layer  Separation and Purification

The chloroform layer obtained in Example 1-1 was concentrated under reduced pressure (157.7 g), and silica gel was obtained using a step gradient solvent system consisting of n-hexane: ethyl acetate = (50: 1 to 0: 100). The active fractions were separated using column chromatography.

Reversed-phase column chromatography (ODS) using 50%, 60%, 70%, 80%, 90%, and 100% methanol as the elution solvent with the highest fraction of intercellular adhesion inhibitory activity in the active fraction gel) to separate the active fractions. The 80% methanol eluted fraction having the highest inhibitory activity in the fraction was separated by low pressure liquid chromatography (LPLC) while flowing 80 ml methanol at 8 ml / min, and the active fraction was again 80. Elution was performed at 25, 45 and 53 minutes by high performance liquid chromatography (HPLC, YMC Jsphere ODS H-80, 250 x 20 mm), flowing at 4 ml / min using% methanol. To 3 (11 mg), 1 (42 mg) and 2 (5 mg). Next, 80% methanol eluted fraction (1.2 g) of the first column chromatography with high inhibitory activity was reversed-phase column chromatography (24 g) using MeOH-H ₂ O (50:50 ~ 0: 100, v / v) as a solvent. , ODS gel), and LPLC was carried out while flowing the active fraction (1.07 g) at 6 mL / min in 80% acetonitrile (CH ₃ CN) solvent conditions. The active fraction (700 mg) was purified by high-performance liquid chromatography at 6 mL / min under 70% acetonitrile solvent to obtain active substance 4 (123 mg) eluted at 43 minutes.

1-3. Structure Analysis of Active Substances

Compound 1, which is completely separated and purified, is a colorless oil. The molecular weight is measured (M + Na).⁺Was m / z 366, and the molecular formula C in high resolution FAB-MS₂₁H₂₉NO₃Was estimated. Ultraviolet absorbance of the compound showed a maximum absorption at 260 nm, and it was estimated that dienamide was present in the structure of the compound. During an NMR test to determine the structure of a compound,^OneOne methylenedioxy proton (δ5.92, s) was observed in the H-NMR spectrum, and five olefin protons were observed between δ 6.0 and 7.0. On the other hand, methylene protons binding to -N- were observed at δ 3.54 and δ 3.38, and two methylene protons and 14 methylene protons were observed at δ 2.30 and δ 2.15 and between δ 1.31 and 1.67. From the measurement results, since compound 1 is very similar to the structure of piperolein B including piperidine, the published literature (Kiuchi, F., et al.,Chem Pharm Bull , 36 (7), 2452-2465, 1988), the compound of formula 1 was found to be a piperrolein ratio.

Compound 2, which was completely isolated and purified, was a dark colorless crystalline powder. As a result of molecular weight measurement, (M + Na) ⁺ was m / z 380 and the molecular formula was estimated to be C ₂₂ H ₃₁ NO ₃ in high resolution FAB-MS. . Ultraviolet absorbance of the compound was found to be maximum absorption at 260 nm and shoulder absorption at 295-305 nm, suggesting the presence of conjugated dieneamide in the structure of the compound. During the NMR test to determine the structure of the compound, one methylenedioxy proton (5.93, s) was observed in the ¹ H-NMR spectrum, and 7 olefinic protons between δ5.7 and 7.3. This was observed. In addition, four methylene protons were observed between δ1.30 and 1.50, similar to the NMR spectrum of Compound 1. The measurement results have an isobutyl group and are very similar to the structure of piperchabamide D, which contains an amide bond in its structure, and thus published literature (Morikawa, T., et al. ., Planta Med , 70: 152-159, 2004), the compound of formula (2) was found to be piperbamide di.

Compound 3, which was completely isolated and purified, was a dark yellow crystalline powder. As a result of molecular weight measurement, (MH) ⁺ was m / z 222, and the molecular formula was estimated to be C ₁₄ H ₂₅ NO in high resolution FAB-MS. Ultraviolet absorbance of the compound was found to be maximum absorption at 260 nm and shoulder absorption at 295-305 nm, suggesting the presence of conjugated dieneamide in the structure of the compound. During the NMR test to determine the structure of the compound, four olefinic protons were observed between δ5.7 and 7.3 in the ¹ H-NMR spectrum, and protons (br s) of -NH- were identified at δ5.67. Was observed. On the other hand, one methylene proton, three methylene protons between δ1.20 and 1.45 and methine proton of δ1.88 were observed at δ3.15 and δ2.13, respectively. In addition, three methyl protons were observed at δ0.87, δ0.90 and δ0.92. From the measurement results, compound 5 was estimated to be pellitorine having an isobutyl group but no methylenedioxybenzyl group, and published documents (Park, IK, et al., J Agric) Food Chem , 50: 1866-1870, 2002), the compound of formula ( 3 ) was found to be pelintorin.

Compound 4 was a pale colorless plate, the molecular weight was measured (M + Na) ⁺ was m / z 362, the molecular formula was estimated to C ₂₁ H ₂₅ NO ₃ in ESI-MS. Was born is displayed absorption at 211 and 268 nm results of measuring the UV absorbance of the compound, ratio recording infrared spectrophotometer (Bio-Rad Digilab Division, FTS -80) As a result of measuring the IR absorption by using a carbonyl group (1660㎝ ^- The presence of ¹ ) was confirmed. Compound 2 may be deduced from the fact that it has a molecular formula of two hydrogens less than that of compound 3 . Compound 2 in the ¹³ C-NMR spectrum is similar to compound 3 except for two signals C-4 (δ 32.4) and C-5 (δ 27.9). During the NMR test to determine the structure of the compound, the signals of the six olefinic protons in the ¹ H-NMR spectrum were δ 6.31 (1H, d, J = 15.9 Hz, H-2), 7.23 ( 1H, dd, J = 10.5, 15.0 Hz, H-3), 6.21 (1H, dd, J = 10.8, 15.0 Hz, H-4), 6.07 (1H, dt, J = 15.3, 6.0 Hz, H-5 ), 6.02 (1H, m, H-8) and 6.27 (1H, d, J = 14.7 Hz, H-9), where the positions of the olepic protons were ¹ H- ¹ H COSY and 2D- of HMBC. It was confirmed correctly from the NMR spectrum. As compared with the above instrumental analysis spectrum and previously reported data (Shoji N, et al., J. Pharmaceu . Sci., 75 : 1188-1189, 1986), Formula 4 is a dihydropipeno having a piperidine group. It turned out to be dehydropipernonaline.

Example  2. sICAM -1 and THP -1 cell  Interstitial  Inhibitory activity

Recombinant sICAM-1 (R & D Systems, Abingdon, UK) was diluted to 10 μg / ml (in PBS), 100 μl was added to a 96-well plate and reacted at 4 ° C. for 12 hours. After washing once with PBS, 100 μl of 10 mg / ml BSA was added and blocked for 1 to 2 hours at room temperature. THP-1 cells (1 × 10 ⁷ cells / ml) were reacted at 37 ° C. for 30 to 60 minutes after BCECF-AM (5 μM) was added. After THP-1 cells were washed with PBS and treated with LFA-1 / 2 antibody (500 ng / ml) for 10-20 minutes at 37 ° C, THP-1 cells and samples were coated with recombinant sICAM-1. 100 μl of each well of the plate was added and reacted at 37 ° C. for 30 to 60 minutes. After the reaction, unbound THP-1 cells were removed and washed three times with PBS. Cells were solubilized with PBS (1% Triton X-100) and fluorescence (ex. 485, em. 592) was measured.

The ethanol extract and chloroform fraction of the reefs inhibited adhesion between sICAM-1 and THP-1 cells by 50 and 70% or more, respectively, at a concentration of 50 μg / ml (FIGS. 1 and 2).

The IC ₅₀ values of the amide compounds represented by Formulas 1, 3, and 4 of the present invention were 13.4 mg / ml, 13.5 mg / ml, and 6 mg / ml, respectively. At least 60% inhibited adhesion of sICAM-1 to THP-1 cells, at least 40% of the compound of formula 2, at least 80% of the compound of formula 3, at least 70% of the compound of formula 4, Compounds effectively inhibited adhesion of sICAM-1 and THP-1 cells with increasing concentration (FIG. 3).

Example  3. Oral administration to experimental mice Acute Toxicity  exam

The SPF rats of 6 weeks of age were divided into 5 rats per group, and the reef extract of the present invention and its fraction, or an amide compound represented by pipefenalin, were suspended in 0.5% methylcellulose solution. Single oral administration at a dose of mg / kg. After administration, mortality, clinical symptoms and changes in body weight were observed, hematological and hematological examinations were performed, and autopsy was performed to visually observe abdominal and thoracic organ abnormalities.

As a result, no significant clinical symptoms or dead animals were noted in the animals to which the sample of the present invention was administered, and no toxic changes were observed in weight changes, blood tests, blood biochemical tests, and autopsy findings. The compound tested did not show toxic changes up to 10 mg / kg in rats and the minimum lethal dose (LD ₅₀ ) was determined to be 10 mg / kg or more.

Example  4. Formulation

4-1. Powder  Produce

A powder was prepared by mixing a chlorophyll extract and its fractions or 0.1 g of dihydropipefenolaline, 1.5 g of lactose and 0.5 g of talc and filling into an airtight bag.

4-2. Manufacture of tablets

A tablet was prepared by a direct tableting method after mixing a reef extract and its fraction or 0.1 g of dihydropipefenolaline, 7.9 g of lactose, 1.5 g of crystalline cellulose and 0.5 g of magnesium stearate. The total amount of each tablet is 500 mg, of which the active ingredient content is 50 mg.

4-3. Manufacture of powder

A powder is prepared by thoroughly mixing a vinegar extract and its fractions or 0.1 g of dihydropipefenalin, 5 g of corn starch and 4.9 g of carboxy cellulose. 500 mg of powder was put in a hard capsule to prepare a capsule.

4-4. Preparation of Injectables

A chlorine extract and its fractions or 0.1 g of dihydropipefenanoline, a suitable amount of sterile distilled water for injection, and a pH adjusting agent amount were prepared in the above-described content of ingredients per ampoule (2 ml) according to a conventional preparation method of injectables.

4-5. Liquid  Produce

0.1 g of distillate extract and fractions thereof or dihydropipefenolaline, 10 g of isomerized sugar, 5 g of mannitol and a suitable amount of purified water were dissolved in each of the ingredients in purified water according to a conventional method for preparing a liquid solution, and then a suitable amount of lemon flavor was added. After mixing the components of the purified water was added to the whole purified water was added to adjust the total 100 ㎖ filled in a brown bottle and sterilized to prepare a liquid.

Figure 1 shows the degree of inhibitory activity of the ethanol extract of the reefs inhibit the adhesion between soluble ICAM-1 (sICAM-1) and THP-1 cells.

Figure 2 shows the degree of inhibitory activity of the chloroform fraction and water fraction of the reefs inhibit the adhesion between soluble ICAM-1 (sICAM-1) and THP-1 cells.

Figure 3 shows the degree of inhibitory activity of the compounds of Formulas 1 to 4 isolated from the chloroform fraction of chlorophyll inhibit the adhesion between soluble ICAM-1 (sICAM-1) and THP-1 cells.

Claims (11)

A reef extract for preventing and treating an inflammatory disease or an infectious disease, obtained by extracting a reef ( Piper nigrum ) with a solvent selected from the group consisting of water, a lower alcohol having 1 to 4 carbon atoms, and a mixed solvent thereof. The extract of claim 1, wherein the lower alcohol solvent is selected from the group consisting of methanol, ethanol and butanol. The method according to claim 1, wherein the piper nigrum is extracted with a solvent selected from the group consisting of water, a lower alcohol having 1 to 4 carbon atoms and a mixed solvent thereof, and then it is selected from the group consisting of hexane, chloroform and ethyl acetate. An extract obtained further comprising the step of fractionating with an eluting solvent of choice. The extract according to claim 3, which is a chloroform fraction obtained by successive repeated extractions using hexane and chloroform as solvents. The method of claim 1, wherein the inflammatory disease comprises: autoimmune diseases associated with multiple organs such as systemic lupus erythematosus and scleroderma; Inflammatory bowel disease, including ulcerative colitis and Crohn's disease; Inflammation including IgE mediated (type I) hypersensitivity such as atopic dermatitis, psoriasis, anaphylaxis and dermatitis; Eye diseases such as diabetic retinopathy, retinitis, macular disease, uveitis, conjunctivitis; Vascular diseases such as stroke, coronary artery disease, myocardial infarction, unstable angina, vasculitis, vascular stenosis, Kawasaki disease, giant cell arteritis; Arthritis, rheumatoid arthritis, ankylosing spondylitis, osteoarthritis, osteoporosis, allergies, diabetes, diabetic nephropathy, nephritis, nephritis, Sjogren's syndrome, autoimmune pancreatitis, periodontal disease, asthma, graft-versus-host disease, chronic pelvic inflammatory disease, endometritis, An extract which is a disease selected from the group consisting of rhinitis, tumor metastasis, transplant rejection and chronic prostatitis. The extract according to any one of claims 1 to 4, wherein the infectious disease is an infectious disease caused by rhinovirus or HIV-1. A pharmaceutical composition for the prophylaxis and treatment of inflammatory diseases or infectious diseases comprising the compound of formula 1 to 4 or the aquatic extract of any one of claims 1 to 4.

8. The composition of claim 7, further comprising a pharmaceutically acceptable carrier. 8. The method of claim 7, wherein the inflammatory disease comprises: autoimmune diseases related to multiple organs such as systemic lupus erythematosus and scleroderma; Inflammatory bowel disease, including ulcerative colitis and Crohn's disease; Inflammation including IgE mediated (type I) hypersensitivity such as atopic dermatitis, psoriasis, anaphylaxis and dermatitis; Eye diseases such as diabetic retinopathy, retinitis, macular disease, uveitis, conjunctivitis; Vascular diseases such as stroke, coronary artery disease, myocardial infarction, unstable angina, vasculitis, vascular stenosis, Kawasaki disease, giant cell arteritis; Arthritis, rheumatoid arthritis, ankylosing spondylitis, osteoarthritis, osteoporosis, allergies, diabetes, diabetic nephropathy, nephritis, nephritis, Sjogren's syndrome, autoimmune pancreatitis, periodontal disease, asthma, graft-versus-host disease, chronic pelvic inflammatory disease, endometritis, An extract which is a disease selected from the group consisting of rhinitis, tumor metastasis, transplant rejection and chronic prostatitis. 8. The composition of claim 7, wherein said infectious disease is an infectious disease caused by rhinovirus or HIV-1. 8. The composition of claim 7, wherein the compound of formulas 1 to 4 is a material isolated from Piper nigrum .

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