WO2009041787A2

WO2009041787A2 - Pharmaceutical composition comprising inhibitors of cell adhesion molecule isolated from piper nigrum for the prevention and treatment of inflammatory disease

Info

Publication number: WO2009041787A2
Application number: PCT/KR2008/005723
Authority: WO
Inventors: Mun-Chual Rho; Young-Kook Kim; Hyun-Sun Lee; Chang-Duk Jun; Koanhoi Kim; Seung-Woong Lee; Jung Ho Choi; Jae Jun Song
Original assignee: Korea Research Institute Of Bioscience And Biotechnology
Priority date: 2007-09-28
Filing date: 2008-09-26
Publication date: 2009-04-02
Also published as: KR20090032601A; KR100954866B1; WO2009041787A3

Abstract

The present invention relates to a Piper nigrum extract having prophylactic and therapeutic effects on inflammatory or infectious diseases, and to amide compounds isolated therefrom. In addition, the present invention relates to a pharmaceutical composition for preventing or treating inflammatory or infectious diseases, comprising the Piper nigrum extract or amide compound isolated therefrom as an active ingredient.

Description

[DESCRIPTION]

[invention Title]

PHARMACEUTICAL COMPOSITION COMPRISING INHIBITORS OF CELL ADHESION MOLECULE ISOLATED FROM PIPER NIGRUM FOR THE PREVENTION AND TREATMENT OF INFLAMMATORY DISEASE

[Technical Field]

The present invention relates to a Pipernigrum extract having prophylactic and therapeutic effects on inflammatory or infectious diseases, and to amide compounds isolated therefrom. In addition, the present invention relates to a pharmaceutical composition comprising a Piper nigrum extract or amide compounds isolated therefrom as an active ingredient, which specifically inhibits the interaction between intercellular adhesion molecule-1 (ICAM-I) and leukocyte function associated antigen-1

(LFA-I; CDlla/CD18) to prevent and treat inflammatory diseases including vascular stenosis, arthritis, asthma, osteoporosis, psoriasis, atopic dermatitis, inflammatory bowel disease, cancer metastasis and transplant rejection, or infectious diseases caused by rhinovirus or human immuno-deficiency virus type-1 (HIV-I) .

[Background Art] Intercellular adhesion molecule-1 (ICAM-I; CD54) is an adhesion receptor expressed on endothelial cells, and is a protein belonging to the Ig-superfamily . ICAM-I is expressed at low levels on vascular endothelium, some lymphocytes and monocytes [Springer, Nature 346: 425-434, 1990, Rothlein et al., J. Immunol. 137: 1270-1274, 1986]. When various cells are treated with LPS (lipopolysaccharide) or inflammatory cytokines such as IL-I, TNF-α and IFN-γ, ICAM-I, expression is rapidly augmented in multiple cell types [Hubbard and Rothlein, Free Radic. Biol. Med. 28: 1379-1386, 2000] .

ICAM-I is a major ligand for leukocyte function associated antigen-1 (LFA-I; CDlla/CD18) . LFA-I is in an inactive state on resting leukocytes, and becomes activated by T-cell receptor-mediated signaling or various stimulators such as phorbol ester [Dustin and Springer, Nature 341: 619-624, 1989, Rothlein and Springer, J. Exp. Med. 163: 1132-1149, 1986].

To invade inflammatory lesions, leukocytes must roll along and adhere to the surface of vascular endothelium. ICAM-I is mainly expressed on vascular endothelium of inflammatory lesions, and functions to promote adhesion and infiltration of blood cells such as monocytes, neutrophils, and lymphocytes to the blood vessel wall. ICAM-I is also believed to play an important role in adherence of platelets to the vascular endothelium [Thomas and DeGraba, Neurology 49: 15-19, 1997].

Cell adhesion is strongly associated with chronic inflammatory diseases including vascular stenosis, arthritis, osteoporosis, microbial infection, cancer metastasis and angiogenesis, psoriasis, asthma, allergy, lupus, and Crohn's disease [Poston et al., Am. J. Pathol. 140: pββ5-673, 1992; Macchioni et al., J. Rheumatol. 21(10): pl860-1864, 1994; Mason et al., Arthritis Rheum. 36: p519-527, 1993; Kling et al., Clin. Invest. 71: p299-304, 1993]. Vascular stenosis progresses to chronic inflammation by arteriosclerosis due to excessive cholesterol in the blood and vascular endothelial damage due to hypertension, or the presence of a toxin or pathogen. Inaddition, monocytes infiltrate into vessel walls by cell adhesion molecules expressed on endothelial cells, and differentiate into macrophages in vessel walls to be fat-laden macrophages so called foam cells, resulting in gradual development of fibrosis [Ross, Annu. Rev. Physiol. 57: p791-804, 1995., Christoper and Joseph, Cell 104: p503-516, 2001]. Since cell adhesion molecules play a crucial role in such inflammation and vascular stenosis, inhibitors blocking their expression in monocyte and endothelium have been developed.

It can be understood that inhibitors of ICAM-I and LFA-I mediated cell adhesion can be valuable therapeutic agents for arteriosclerosis and immune-mediated inflammatory diseases [Oppenheimer-marks and Lipsky, Clin. Immunol. Immunopathol . 79: p203-210, 1996] . In particular, the fact that ICAM-I is involved in inflammation progress has been clarified by animal testing using a monoclonal antibody against ICAM-I . It has been suggested that the inhibition of ICAM-I/LFA-I adhesion using a monoclonal antibodies against ICAM-I or LFA-I is useful for treating autoimmune diseases such as arteriosclerosis, asthma, glomerulonephritis, or arthritis. Further, it has been reported that in addition to antigen-specific signals transduced through T-cell receptors, antigen-nonspecific signals such as ICAM-I/LFA-I are essential for T-cell activation in immune response [Cornejo et al . , Adv. pharmacol . 39: 99-141, 1997, Patel et al., Circulation 97: 75-8, 1998, Nishikawa et al., J. Exp. Med. Ill: 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-I plays an important role not only in inflammatory diseases but also in viral infection. Rhinovirus, which is the cause of the common cold, was clarified to employ the ICAM-I receptor for infection [Greve et al . , Cell 56: 839-847, 1989; Staunton et al., Cell 56: 849-853, 1989: Tomassini et al., Proc. Natl. Acad. Sci. (U.S.A.) 86: 4907-4911, 1989]. It was also reported that the interaction of ICAM-I with LFA-I is involved in the infection of human immunodeficiency virus type-1 (HIV-I) , and the inhibitors of LFA-l/ICAM-1 interaction, statins, are able to inhibit HIV-I replication [Giguere and Tremblay, J. Virology 78: 12062-12065, 2004]. Accordingly, the inhibitors of LFA-I or ICAM-I activity can be used as a therapeutic agent for inflammatory diseases and as an antiviral agent against rhinovirus or HIV-I.

Recently, development of the inhibitors has been focused on neutralizing antibodies against ICAM-I or LFA-I, and some inhibitors from natural sources were reported. An ICAM-I expression inhibitor synthesized at Abbott Laboratories was reported [Stewart et al., J. Med. Chem. 44: 988-1002, 2001], and a synthetic LFA-I antagonist generated by the transfer of the ICAM-I iπtmunoregulatory epitope to a small molecule was suggested by Novartis and Roche [Welzenbach et al., J. Biol. Chem. 277: 10590-10598, 2002, Gadeket al., Science 295: 1086-1089, 2002]. However, there has been no report so far on its in vivo activity in animal models. As an inhibitor of ICAM-I/LFA-I mediated cell adhesion, seco-limonids {Trichilia rubra, IC₅₀; 10~25 nM) , cucurbitacins (Conobea scoparioides, IC₅₀; 0.18~1.3β mM) , adxanthromycins {Streptomyces sp. Na-148, IC₅₀; 1.5-6.5 mM) or the like are reported [Muszaetal., Tetrahedron 50: 11369-11378, 1994, Musza et al., J. Net. Prod. 57: 1498-1502, 1994, Nakano et al., J. Antibiotics 53: 12-18. 2000].

[Disclosure]

[Technical Problem]

It is an object of the present invention to provide a Piper nigrum extract having prophylactic and therapeutic effects on inflammatory or infectious diseases, which is extracted using a solvent selected from the group consisting of water, Ci to C₄ lower alcohol, and the mixtures thereof.

It is another object of the present invention to provide a Pipernigrum extract having prophylactic and therapeutic effects on inflammatory or infectious diseases, which is obtained by using an eluting solvent selected from the group consisting of hexane, chloroform, and ethylacetate.

It is still another object of the present invention to provide a pharmaceutical composition for the prevention and treatment of inflammatory or infectious diseases, comprising the Piper nigrum extracts or compounds of Formulae 1 to 4 as active ingredients .

[Technical Solution]

The present inventors have conducted studies on the inhibitors of ICAM-I/LFA-I mediated cell adhesion, which are derived from natural sources. They found that Piper nigrum extracts, fractions thereof, or amide compounds isolated therefrom have inhibitory effects on the ICAM-l/LFA-1 mediated cell adhesion, thereby completing the present invention.

[Advantageous Effects]

As described above, the Piper nigrum extracts or compounds of Formulae 1 to 4 of the present invention inhibit ICAM-l/LFA-1 mediated cell adhesion to effectively inhibit the adhesion and infiltration of blood cells such as monocytes, neutrophils and lymphocytes to vascular endothelial cells, thereby being used as a prophylactic and therapeutic agent against inflammatory and infectious diseases related ICAM-I. [Description of Drawings]

FIG. 1 is a graph showing inhibitory activity of the Piper nigrum ethanol extract on adhesion of soluble ICAM-I (sICAM-1) and THP-I; FIG. 2 is a graph showing inhibitory activity of the Piper nigrum chloroformandwater fractions on adhesion of soluble ICAM-I ( sICAM-I) and THP-I; and

FIG. 3 is a graph showing inhibitory activity of compounds of Formulae 1 to 4, which are isolated from the Piper nigrum chloroform fraction, on adhesion of soluble ICAM-I (sICAM-1) and THP-I.

[Best Mode]

To achieve the above objects, in accordance with one aspect, the present invention relates to a Piper nigrum extract having prophylactic and therapeutic effects on inflammatory or infectious diseases, which is extracted using a solvent selected from the group consisting of water, Ci to C₄ lower alcohol, and the mixtures thereof. In the present invention, a method for obtaining the Piper nigrum extract is as follows. Piper nigrum powder is mixed with about 2 to 20-fold, preferably, about 3 to 5-fold volume of polar solvent, for example, water, Ci to C₄ lower alcohol such as methanol, ethanol, and butanol, or the mixtures thereof at a mixing ratio of about 1:0.1 to 1:10 as eluting solvent, and is extracted at a temperature ranging from 20 to 100 °C, preferably room temperature, for a period ranging from about 12 hrs to 4 days, preferably 3 days, by hot water extraction, cold-immersion extraction, reflux cold extraction or sonication, preferably cold-immersion extraction once to 5 times, and then filtered under reducedpressure . The filtrate is concentrated at 20 to 100^°C, preferably room temperature under reduced pressure using a rotary vacuum evaporator to obtain a Piper nigrum crude extract soluble in water, lower alcohol or the mixtures thereof.

The Piper nigrum extract of the present invention, as in the following Example, inhibited the adhesion of sICAM-1 and human monocyte cell line (THP-I expressing LFA-I) by 50% at a concentration of 50 /ig/ml (FIG.1) . Thus, the Piper nigrum extract of the present invention can be suitably used for the prevention and treatment of inflammatory or infectious diseases.

The Piper nigrum extract of the present invention may be, as described above, prepared by extracting Piper nigrum with a solvent selected from the group consisting of water, Ci to C₄ lower alcohol, and the mixtures thereof, and additionally by fractionating it with an eluting solvent selected from the group consisting of hexane, chloroform, and ethylacetate. Preferably, the present invention relates to a chloroform fraction prepared by repeating a sequential extraction of the Piper nigrum extract with hexane and chloroform as a solvent. In the additional step of the present invention, the Piper nigrum crude extract is suspended in distilled water, and mixed with about 1 to 100-fold, preferably, about 1 to 5-fold volume of nonpolar solvent such as hexane, ethylacetate and chloroform, and then extracted once to 10 times, preferably twice to 5 times, so as to obtain a nonpolar solvent-soluble layer. A conventional fractionation process may be additionally performed (Harborne J. B. Phytochemical methods : A guide to modern techniques ofplant analysis, 3rd Ed. pβ-7, 1998) . In particular, the Piper nigrum crude extract is suspended in water, and extracted using an equivalent volume of n-hexane and chloroform as a solvent, so as to obtain each solvent-soluble Piper nigrum extract. More particularly, the Pipernigrum crude extract is suspended in water, mixed with an equivalent volume of n-hexane, and then subjected to fractionation to obtain a n-hexane-soluble fraction and a water-soluble fraction. Chloroform may be added to the water-soluble fraction to obtain a chloroform-soluble fraction and a water-soluble fraction.

In the present invention, examples of inflammatory diseases, as described above, include multi-organ autoimmune diseases such as systemic lupus erythematosus and scleroderma; inflammatory bowel disease such as ulcerative colitis and Crohn's disease; inflammatory diseases of the central nervous system such as Alzheimer's disease, multiple sclerosis, motor neuron disease, Parkinson's disease, and chronic fatigue syndrome; inflammatory disease related IgE-mediated hypersensitivity (type I) such as atopic dermatitis, psoriasis, anaphylaxis, and dermatitis; ocular diseases such as diabetic retinopathy, retinitis, macular degeneration, uveitis, and conjunctivitis; vascular diseases such as stroke, coronary artery disease, myocardial infarction, unstable angina, angitis, arteriosclerosis, vascular stenosis, Wegener's granulomatosis, Churg-Strauss syndrome, Henoch-Schonlein purpura, Kawasaki disease, and giant cell arteritis; chronic inflammatory diseases such as arthritis, rheumatoid arthritis, ankylosing spondylitis, osteoarthritis, osteoporosis, allergy, diabetes, diabetic nephropathy, acute childhood diabetes, Addison's disease, Goodpasture's syndrome, IgA nephropathy, nephritis, nephropathy, glomerulonephritis, Sjogren' s syndrome, autoimmune chronic pancreatitis, periodontal disease, chronic obstructive pulmonary disease, acute leukemia-mediated lung injury, asthma, graft-versus-host disease, chronic pelvic inflammatory disease, endometriosis, rhinitis, metastasis, transplant rejection, paralysis, encephalitis, meningitis, AIDS dementia, fibrosis, adhesion formation, chronic hepatitis, and tuberculosis, multiple sclerosis and chronic prostatitis, but are not limited thereto. Further, in the present invention, infectious diseases include diseases caused by infection of rhinovirus or HIV-I. The Piper nigrum chloroform fraction of the present invention, as in the following Example, inhibited the adhesion of sICAM-I and human monocyte cell line, THP-I by 70% or more at a concentration of 50 μg/ml (FIG. 2) . Thus, the Piper nigrum chloroform fraction of the present invention can be suitably used for the prevention and treatment of inflammatory or infectious diseases . In accordance with another aspect, the present invention provides a pharmaceutical composition for the prevention and treatment of inflammatory or infectious diseases, comprising the Piper nigrum extracts or compounds of Formulae 1 to 4 as an active ingredient .

The amide compounds of Formulae 1 to 4 may be prepared in the form of a pharmaceutically acceptable salt, and include salts, hydrates, and solvates prepared according to the conventional method in the related art. The method for extracting, isolating and purifying the amide compounds from Piper nigrum according to the present invention is as follows. The chloroform-soluble fraction is subjected to silica gel column chromatography using a mixture solvent of n-hexane and ethylacetate. At this time, n-hexane and ethylacetate are preferably mixed in a ratio of 50:1 to 0:100 (v/v) . Inhibitory activities of the separated active fractions on ICAM-I/LFA-I mediated cell adhesion are measured, and fractions exhibiting high inhibitory activity on ICAM-l/LFA-1 mediated cell adhesion are subjected to reverse-phase column chromatography using 50%, 60%, 70%, 80%, 90%, and 100% methanol as an eluting solvent. Among the final fractions, fractions exhibiting high inhibitory activity on ICAM-l/LFA-1 mediated cell adhesion are separated by HPLC (high performance liquid chromatography) using 75% methanol and acetonitrile to obtain the final compounds of Formulae 1 to 4. As in the following Example, the compound of Formula 1 of the present invention inhibited the adhesion of siCAM-I and THP-I cell by 60% or more at a concentration of 25 /ig/ml, the compound of Formula 2 by 40% or more, the compound of Formula 3 by 80% or more, and the compound of Formula 4 by 70% or more. As the concentration of the compound of the present invention increased, the adhesion of siCAM-I and human monocyte cell line THP-I was effectively inhibited (FIG. 3) . Thus, the compounds of Formulae 1 to 4 of the present invention can be suitably used for the prevention and treatment of inflammatory or infectious diseases.

The Piper nigrum extracts or the compounds of Formulae 1 to 4 of the present invention inhibit ICAM-I and LFA-I mediated cell adhesion to effectively inhibit the adhesion and infiltration of blood cells such as monocytes, neutrophils and lymphocytes to vascular endothelial cells, thereby being used for the prevention and treatment of inflammatory diseases. The amide compounds used in the present invention are those extracted, isolated, and purified from Piper nigrum. Also, the amide compounds used in the present invention may be prepared by all typical methods or purchased from commercially available sources .

The pharmaceutical composition for the prevention and treatment of inflammatory or infectious diseases of the present invention comprises the Piper nigrum extracts or the compounds of Formulae 1 to 4 in an amount of0.0001tolO% by weight, preferably

0.001 to 1% by weight, based on the total weight of the composition .

The composition comprising the Piper nigrum extract or the compounds of Formulae 1 to 4 of the present invention may further comprise suitable carriers, excipients and diluents typically used for the preparation of pharmaceutical compositions.

The composition of the present invention may be used in the form of pharmaceutically acceptable salts, and also may be used alone or in appropriate association, as well as in combination with, other pharmaceutically active compounds.

According to conventional methods, the composition of the present invention may be formulated into an oral preparation such as a powder, a granule, a tablet, a capsule, a suspension, an emulsion, a syrup, or an aerosol, an external preparation, suppository, or a sterilized injectable solution. Examples of the carriers, excipients, and diluents contained in the composition of the present inventionmay include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starches, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate andmineral oil. Such preparations may be prepared using diluents or excipients ordinarily employed, such as a filler, an extender, a binder, a wetting agent, a disintegrating agent, and a surfactant. Examples of the solid preparation for oral administration include a tablet, a pill, a powder, a granule, and a capsule, and the solid preparation may be prepared by mixing the extract or fraction with at least one excipient such as starch, calcium carbonate, sucrose, lactose, or gelatin. Further, in addition to the excipients, lubricants such as magnesium stearate and talc may be used. Examples of a liquid preparation for oral administration include a suspension, a liquid for internal use, an emulsion, or a syrup, and various excipients such as a wetting agent, a sweetener, aflavor, or apreservativemaybe contained, inaddition to general diluents such as water and liquid paraffin. Examples of the preparation for parenteral administration include an aseptic aqueous solution, a non-aqueous solvent, a suspension, an emulsion, a lyophilized agent, and suppository. As the non-aqueous solvent and suspension, propylene glycol, polyethylene glycol, plant oil such as olive oil, or injectable ester such as ethyloleate may be used. As a suppository base, witepsol, macrogol, tween 61, cacao butter, lauric butter, glycerogelatin or the like may be used.

An effective dosage of the present composition may be determined depending on the patient' s health state andbody weight, severity of the diseases, drug formulation, administration routes, and administration time, and may be suitably selected by those skilled in the art . However, for better efficacy, the composition of the present invention may be administered at a daily dosage of 0.0001 to 100 mg/kg, preferably 0.001 to 100 mg/kg once or several times.

The pharmaceutical composition of present invention may be administered to a subject animal such as mammals (e.g., rat, mouse, domestic animals or human) via various routes. All modes of administration are contemplated; for example, administration can be made orally, rectally or by intravenous, intramuscular, subcutaneous, epidural or intracerebroventricular injection.

[Mode for Invention]

The present invention will be described in more detail with reference to the following Examples, but is not limited thereto.

Example 1. Extraction, isolation and purification of amide compounds from Piper nigrum

1-1. Extraction from Piper nigrum

Piper nigrum was washed with water, dried in shade, and then pulverized using a Waring blender. 3-fold volumes of methanol

(based on the weight of dried Piper nigrum) was added to 5 kg of the pulverized Piper nigrum, and extracted at room temperature for 7 days, followed by filtration. The filtrate was concentrated under reduced pressure to obtain a crude extract.

To isolate/purify active ingredients from the crude extract, the Piper nigrum crude extract was suspended in water, mixed with an equivalent volume of n-hexane, and then subjected to fractionation to obtain n-hexane-soluble fraction and water-soluble fraction. Chloroform was added to the water-soluble fraction to obtain chloroform-soluble fraction and water-soluble fraction. In accordance with the procedure of the following Example 2 , inhibitory activity of the obtained fractions on intercellular adhesion was examined. The chloroform fraction exhibited excellent inhibitory activity on intercellular adhesion.

1-2. Isolation and purification of chloroform fraction

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

Among the active fractions, fractions exhibiting high inhibitory activity on intercellular adhesion were subjected to reverse-phase column chromatography (ODS gel) using 50%, 60%,

70%, 80%, 90%, and 100% methanol as an eluting solvent. Among the final fractions, 80% methanol fraction exhibiting highest inhibitory activity was separated by low pressure liquid chromatography (LPLC) using 80% methanol at a flow rate of 8 ml/min, and the active fractions were subjected to high performance liquid chromatography (HPLC, YMC Jsphere ODS H-80, 250 x 20 mm) using 80% methanol at a flow rate of 4 ml/min. Active substances 3 (11 mg) , 1 (42 mg) and 2 (5 mg) were eluted at 25 min, 45 min and 53 min, respectively. The 80% methanol fraction (1.2 g) obtained by the first column chromatography was subjected to reverse-phase column chromatography (24 g, ODS gel) using MeOH-H₂O (50:50 ~ 0:100, v/v) as a solvent. The active fraction (1.07 g) was subjected to LPLC using 80% acetonitrile (CH₃CN) at a flow rate of 6 ml/min. The resulting active fraction (700 mg) was subjected to high performance liquid chromatography using 70% acetonitrile at a flow rate of 6 ml/min, and active substance 4 (123 mg) was eluted at 43 min.

1-3. Structural analysis of active substance

Compound 1 was completely isolated and purified as colorless oil and its molecular weight was (M+Na) ⁺=m/z 366, and its molecular formula was determined as C₂₁H₂₉NO₃ by high-resolution FAB-MS. As the result of measuring UV absorbance, maximum absorbance was detected at 260 nm, suggesting the presence of dienamide in the structure of the compound. NMR spectroscopy was carried out to determine the structure of the compound. In ¹H-NMR spectra, one methylenedioxy proton (55.92, s) was observed and five olefinic protons were detected at δ6.0~7.0. In addition, the methylene protons linked to -N- were observed at 53.54 and 53.38, two methylene protons at 52.30 and 52.15, and fourteen methylene protons at 51.31-1.67. Based on the obtained data, Compound 1 is very similar to the structure of piperolein B containing piperidine. By comparison and analysis with published data

(Kiuchi, F., et al., Chem Pharm Bull, 36(7), 2452-2465, 1988),

Compound 1 was identified as piperolein B.

Compound 2 was completely isolated and purified as colorless crystalline powder and its molecular weight was (M+Na)⁺ = m/z 380, and its molecular formula was determined as C₂₂H₃₁NO₃ by high-resolution FAB-MS . As the result ofmeasuring UV absorbance, maximum absorbance was detected at 260 nm, and shoulder absorbance appeared at 295 to 305 nm, suggesting the presence of a conjugated dienamide in the structure of the compound. NMR spectroscopy was carried out to determine the structure of the compound. In ¹H-NMR spectra, one methylenedioxy proton (5.93, s) was observed and seven olefinic protons were detected at 55.7~7.3. In addition, four methylene protons were observed at δl.30~1.50, which is similar to NMR spectrum of Compound 1. The data obtained above are very similar to those for the structure of piperchabamide D, which has an isobuthyl group and an amide bond in its structure. By comparison and analysis with published data (Morikawa, T., et al., Planta Med, 70:152-159, 2004), Compound 2 was identified as piperchabamide D.

Compound 3 was completely isolated and purified as dark yellow crystalline powder and its molecular weight was (M-H)⁺ = m/z 222, and its molecular formula was determined as C₁₄H₂₅NO by high-resolution FAB-MS. As the result ofmeasuring UV absorbance, maximum absorbance was detected at 260 nm, and shoulder absorbance appeared at 295 to 305 nm, suggesting the presence of a conjugated dienamide in the structure of the compound. NMR spectroscopy was carried out to determine the structure of the compound. In ¹H-NMR spectra, four olefinic protons were observed at 55.7-7.3, and the proton of -NH- (br s) was observed at 55.67. In addition, each one of methylene protons was observed at 53.15 and 52.13, three methylene protons atδl.20~1.45, andmethine proton at 51.88. Three methyl protons were also observed at 50.87, 50.90, and 50.92. The above data suggest that Compound 3 is pellitorine having an isobutyl group, but not a methylenedioxybenzyl group. By comparison and analysis with published data (Park, I. K. , et al. , J Agric Food Chem, 50:1866-1870, 2002), Compound 3 was identified as pellitorine.

Compound 4 was completely isolated and purified as light colorless plates and its molecular weight was (M+Na)⁺ = m/z 362, and its molecular formula was determined as C₂₁H₂₅NO₃ by ESI-MS.

As the result of measuring UV absorbance, absorbance was detected at 211 nm and 268 nm. IR spectra were obtained using a FTS-80 spectrometer (Bio-Rad Digilab Division) , indicating the presence of carbonyl group (1660 cm^"1) . Compound 2 shows a molecular formula different from that of Compound 3, in that Compound 2 has two less hydrogens than Compound 3, suggesting that Compound 2 is a derivative thereof. In ¹³C-NMR spectra, Compound 2 is similar to Compound 3, except that two signals of C-4 (δ 32.4) and C-5

(δ 27.9) were detected. NMR spectroscopy was carried out to determine the structure of the compound. In ¹H-NMR spectra, six olefinic protons were detected at δ 6.31 (IH, d, J = 15.9 Hz,

H-2), 7.23 (IH, dd, J = 10.5, 15.0 Hz, H-3) , 6.21 (IH, dd, J =

10.8, 15.0 Hz, H-4), 6.07 (IH, dt, J = 15.3, 6.0 Hz, H-5) , 6.02

(IH, m, H-8) , and 6.27 (IH, d, J= 14.7 Hz, H-9), and the position of the olefinic protons was confirmed by ¹H-¹H COSY and 2D-NMR spectrum of HMBC. By comparison and analysis with published data

(ShojiN, et al. , J. Pharmaceu. Sci. ,75:1188-1189, 1986), Compound 4 was identified as dehydropipernonaline having a piperidine group.

Example 2. Inhibitory activity on adhesion of sICAM-1 and THP-I cell

Recombinant siCAM-I (R&D Systems, Abingdon, UK) was diluted in PBS to 10 μl/ml, and then 100 μi thereof was added to a 96-well plate. The reaction was allowed to proceed at 4 ^°C for 12 hrs. The plate was washed with PBS once, and 100 μi of BSA (10 mg/ml) was added thereto, followed by blocking at room temperature for 1 to 2 hrs. After addition of BCECF-AM (5 μM) , THP-I cells (IxIO⁷ cells/ml) were incubated at 37^°C for 30 to 60 min. The THP-I cells were washed with PBS, and treated with LFA-I/2 antibody (500 ng/ml) , followed by reaction at 37^°C for 10 to 20 min. Then, each 100 μi of the THP-I cells and sample was added to each well coated with recombinant sICAM-1, and subjected to reaction at 37^°C for 30 to 60 min. After the reaction, unbound THP-I cells were removed. The cells were washed with PBS three times, and solubilized with PBS (1% Triton X-IOO), and then fluorescence was measured (excitation 485 nm, emission 592 nm) . The Piper nigrum ethanol extract and chloroform fraction inhibited the adhesion of sICAM-I and THP-I cell by 50% and 70% or more at a concentration of 50 /ig/ml, respectively (FIGs. 1 and 2) .

The amide compounds represented by Formulae 1, 3 and 4 of the present invention were found to have IC₅₀ value of 13.4 /_g/ml,

13.5 jWg/ml and 6 μg/ml, respectively. The compound of Formula

1 inhibited the adhesion of siCAM-I and THP-I cell by 60% or more at a concentration of 25 ^g/ml, the compound of Formula 2 by 40% or more, the compound of Formula 3 by 80% or more, and the compound of Formula 4 by 70% or more. As the concentration of the compound of the present invention increased; the adhesion of siCAM-I and THP-I was effectively inhibited (Fig. 3) .

Example 3. Acute oral toxicity study in laboratory rats The Piper nigrum extract, fraction thereof or an amide compound, pipernonaline was suspended in a 0.5% methylcellulose solution, and each suspension was orally administered once to five 6 week-old specific-pathogen-free (SPF) SD rats per group in a dosage of 10 mg/kg. After administration, death, clinical symptoms, and weight change in rats were examined, hematological and biochemical tests of blood were performed, and any abnormal signs in the chest and the abdominal organs were visually checked upon autopsy.

The results showed that the test compounds of the present invention did not cause any specific clinical symptoms or death in rats. No weight change was observed and no hematological or biochemical changes were observed in the blood or in autopsy. Therefore, the compounds of the present invention are evaluated to be safe substances, since they do not cause any toxic changes in rats up to the level of 10 mg/kg and its LD₅₀ value is at least 10 mg/kg.

Example 4. Formulation

4-1. Preparation of powder

0.1 g of the Piper nigrum extract, fraction thereof or dehydropipernonaline, 1.5 g of lactose, 0.5 g of talc were mixed with each other, and filled in an airtight sac to prepare a powder agent .

4-2. Preparation of tablet

0.1 g of the Piper nigrum extract, fraction thereof or dehydropipernonaline, 7.9 g of lactose, 1.5 g of crystalline cellulose and 0.5 g of magnesium stearate were mixed with each other, and prepared into a tablet using a direct tabletting method. A total weight of each tablet is 500 mg, in which the content of active ingredient is 50 mg.

4-3. Preparation of capsule

0.1 g of the Piper nigrum extract, fraction thereof or dehydropipernonaline, 5 g of corn starch, and 4.9 g of carboxy cellulose were mixed to prepare powder, and then 500 mg of the powder was filled into a hard capsule to give a capsule agent.

4-4. Preparation of injectable formulation

0.1 g of the Piper nigrum extract, fraction thereof or dehydropipernonaline, a proper amount of sterile distilled water, and a proper amount of pH adjuster were prepared according to a typical procedure, and thus an inj ectable formulation comprising the above ingredients was prepared into a (2 ml) ampule.

4-5. Preparation of liquid formulation

According to a typical procedure, 0.1 g of the Piper nigrum extract, fraction thereof or dehydropipernonaline, 10 g of high fructose corn syrup, and 5 g of mannitol were solubilized in a proper amount of purified water. A proper amount of lemon flavor was added to the above ingredients, and mixed. Then, purified water was added to a volume of 100 ml, filled in a brown bottle, and sterilized to prepare a liquid formulation.

[industrial Applicability]

As described above, Piper nigrum extracts or compounds of

Formulae 1 to 4 of the present invention inhibit ICAM-I/LFA-I mediated cell adhesion to effectively inhibit the adhesion and infiltration of blood cells such as monocytes, neutrophils and lymphocytes to vascular endothelial cells, thereby being used as a prophylactic and therapeutic agent against inflammatory and infectious diseases caused by ICAM-I mediated cell adhesion.

Claims

[CLAIMS]

[Claim 1]

A Piper nigrum extract for preventing and treating inflammatory or infectious diseases, extracted using a solvent selected from the group consisting of water, Ci to C₄ lower alcohol, and the mixtures thereof.

[Claim 2]

The Piper nigrum extract according to claim 1, wherein the lower alcohol solvent is selected from the group consisting of methanol, ethanol, and butanol.

[Claim 3]

The Piper nigrum extract according to claim 1, wherein Piper nigrum is extracted using a solvent selected from the group consisting of water, Ci to C₄ lower alcohol, and themixtures thereof, and then fractionated using an eluting solvent selected from the group consisting of hexane, chloroform, and ethylacetate.

[Claim 4]

The Piper nigrum extract according to claim 3, wherein the Piper nigrum extract is a chloroform fraction that is repeatedly extractedby sequentially using hexane and chloroformas a solvent .

[Claim 5]

The Piper nigrum extract according to any one of claims 1 to 4, wherein the inflammatory disease is selected from the group consisting of multi-organ autoimmune diseases including systemic lupus erythematosus and scleroderma; inflammatory bowel diseases including ulcerative colitis and Crohn's disease; inflammatory disease related IgE-mediated hypersensitivity (type I) including atopic dermatitis, psoriasis, anaphylaxis, and dermatitis; ocular diseases including diabetic retinopathy, retinitis, macular degeneration, uveitis, and conjunctivitis; vascular diseases including stroke, coronary artery disease, myocardial infarction, unstable angina, angitis, vascular stenosis, Kawasaki disease, and giant cell arteritis; arthritis, rheumatoid arthritis, ankylosing spondylitis, osteoarthritis, osteoporosis, allergy, diabetes, diabetic nephropathy, nephropathy, nephritis,

Sjogren' s syndrome, autoimmune chronic pancreatitis, periodontal disease, asthma, graft-versus-host disease, chronic pelvic inflammatory disease, endometriosis, rhinitis, metastasis, transplant rejection, and chronic prostatitis.

[Claim 6]

The Piper nigrum extract according to any one of claims 1 to 4, wherein the infectious disease is a disease caused by rhinovirus or HIV-I.

[Claim 7]

A pharmaceutical composition for preventing or treating inflammatory or infectious diseases, comprising a compound of Formulae 1 to 4, or the Piper nigrum extract of any one of claims 1 to 4 as an active ingredient.

[Claim 8]

The pharmaceutical composition according to claim 7, further comprising a pharmaceutically acceptable carrier.

[Claim 9]

The pharmaceutical composition according to claim 7, wherein the inflammatory disease is selected from the group consisting of multi-organ autoimmune diseases including systemic lupus erythematosus and scleroderma; inflammatory bowel disease including ulcerative colitis and Crohn's disease; inflammatory disease related IgE-mediated hypersensitivity (type I) including atopic dermatitis, psoriasis, anaphylaxis, and dermatitis; ocular diseases including diabetic retinopathy, retinitis, macular degeneration, uveitis, and conjunctivitis; vascular diseases including stroke, coronary artery disease, myocardial infarction, unstable angina, angitis, vascular stenosis, Kawasaki disease, and giant cell arteritis; arthritis, rheumatoid arthritis, ankylosing spondylitis, osteoarthritis, osteoporosis, allergy, diabetes, diabetic nephropathy, nephropathy, nephritis, Sj ogren' s syndrome, autoimmune chronic pancreatitis, periodontal disease, asthma, graft-versus-host disease, chronic pelvic inflammatory disease, endometriosis, rhinitis, metastasis, transplant rejection, and chronic prostatitis.

[Claim 10] The pharmaceutical composition according to claim 7, wherein the infectious disease is a disease caused by rhinovirus or HIV-I .

[Claim 11]

The pharmaceutical composition according to claim 7, wherein the compounds of Formulae 1 to 4 are substances isolated from Piper nigrum.