Description

NEW ACYCLIC TRITERPENOIDS COMPOUND, AND PHARMACEUTICAL COMPOSITION COMPRISING ALPINIA KATSUMADAI EXTRACT OR ACYCLIC TRITERPENOIDS COMPOUNDS ISOLATED FROM THE

SAME Technical Field

[1] The present invention relates to a novel acyclic triterpenoid compound and a pharmaceutical composition comprising an Alpinia katsumadai extract or acyclic triterpenoid compound isolated therefrom.

[2]

Background Art

[3] 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, /. Immunol. 137: 1270-1274, 1986]. When various cells are treated with LPS (lipopolysarharide) and inflammatory cytokines such as IL-I, TNF-α, and IFN-γ, ICAM-I expression is rapidly augmented in multiple cell types, and adheres to immune cells including monocytes or lymphocytes in blood, to allow cell migration to inflammatory sites [Hubbard and Rothlein, Free Radic. Biol. Med. 28: 1379-1386, 2000]. Accordingly, ICAM-I expression plays a crucial role in inflammatory cell recruitment into inflammatory sites for amplification of inflammation.

[4] ICAM-I is a major ligand for leukocyte function associated antigen- 1 (LFA-I;

CDl la/CD 18) which is a molecule belonging to β2-integrins expressed on leukocytes. 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 so as to bind with ICAM-I [Dustin and Springer, Nature 341: 619-624, 1989, Rothlein and Springer, /. Exp. Med. 163: 1132-1149, 1986].

[5] Cell adhesion is strongly associated with chronic inflammatory diseases including arthritis, osteoporosis, microbial infection, vascular stenosis, cancer metastasis, an- giogenesis, psoriasis, asthma, allergy, lupus, and Crohn's disease [R>ston et al., Am. J. Pathol. 140: p665-673, 1992; Maαchioni 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].

[6] 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 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].

[7]

[8] Vascular stenosis progresses to chronic inflammation by arteriosclerosis due to excessive cholesterol in blood and vascular endothelial damage due to hypertension, toxin or pathogen, and monocytes infiltrate into vessel walls by cell adhesion molecules expressed on endothelial cells, and differentiate into macrophages in vessel walls, 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 or LFA-I can be valuable therapeutic agents for vascular stenosis-related diseases, which may progress to inflammation [Oppenheimer-marks and Lipsky, Clin. Immunol. Immunopαthol. 19: p203-210, 1996].

[9] The fact that ICAM- 1 is involved in inflammation progress has been clarified by animal testing using monoclonal antibody against ICAM-I. It has been suggested that the inhibition of ICAM- 1/LFA-l adhesion using monoclonal antibody against ICAM-I or LFA- 1 is useful for treating autoimmune diseases such as psoriasis, atopic dermatitis, glomerulonephritis, and arthritis. Further, it has been reported that in addition to antigen- specific signals transduced through T-cell receptors, antigen- nonspecific signals such as ICAM- 1/LFA- 1 are essential for T-cell activation in immune response [Cornejo et al., Adv. phαrmαcol. 39: 99-141, 1997, Patel et al., Circulation 97: 75-8, 1998, Mshikawa et al., J. Exp. Med. Ill: 667-77, 1993, Kobayashi et al., Cell Immunol. 164: 295-305, 1995, Nagase et al. Am. J. Respir. Cut. Care Med. 152: 81-86, 1995].

[10] 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. Sd. (U.S.A.) 86: 4907-4911 , 1989] . It was also reported that the interaction of ICAM- 1 with LFA- 1 is involved in the infection of human immunodeficiency virus type-1 (HIV-I), and the inhibitors of LF A-I /IC AM-I interaction, statins, are able to inhibit HIV-I replication [Giguere and Tremblay, J. Virology 78: 12062-12065, 2004]. Accordingly, the inhibitors of LFA-I HC AM-I interaction can be used as a therapeutic agent for inflammatory diseases and as an antiviral agent against rhino virus and HIV-I.

[11] 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., /. Med. Chem. 44: 988-1002, 2001], and a synthetic LFA-I antagonist generated by the transfer of the ICAM- 1 immunoregulatory epitope to a small molecule was suggested by Nwartis and Roche [Welzenbach et al., / Biol. Chem. Ill: 10590-10598, 2002, Gadek et al., Science 295: 1086-1089, 2002]. However, there has been no report so far on its in vivo activity in animal models.

[12] As an inhibitor of ICAM- 1/LFA-l mediated cell adhesion, seco-limonids ( Trichilia rubra, IC ; 10~25 nM), cucurbitadns ( Conobea scoparioides, IC ; 0.18-1.36 mM),

50 50 adxanthromydns ( Streptomyces sp. Na- 148, IC ; 1.5-6.5 mM) or the like are reported [Musza et al., Tetrahedron 50: 11369-11378, 1994, Musza et al., J. Net. Prod. 57: 1498-1502, 1994, Nakano et al., /. Antibiotics 53: 12-18. 2000].

[13] On the other hand, Alpinia katsumadai is a seed of Alpinia katsumadai belonging to thefamily Zingiberaceae, containing essential oil svch as d-4-Terpinyl acetate and d- α-Terpineol, and is produced chiefly in Guangxi and Guangdong provinces of China. Alpinia katsumadai has a spicy taste, warm energy, and affects the spleen and stomach. Further, since it has a warm and aromatic nature, it warms up the spleen and stomach, dispels chill, and vitalizes stagnant blood. Alpinia katsumadai is used for treating abdominal cold syndrome, emesis, weak appetite, diarrhea, chest pain, and vomiting It has been known that Alpinia katsumadai has pharmacological actions of antibacterial effect, intestinal stimulating effect, intestinal suppressing effect, anti-wrinkle effect, oral hygiene-promoting effect, and hormone-like action.

[14] As such, various pharmacological activities of Alpinia katsumadai have been reported. However, there is still no report about its inhibitory activity on the ICAM- 1/LFA-l mediated cell adhesion.

[15]

Disclosure of Invention Technical Problem

[16] Accordingly, the present inventors have conducted studies on the inhibitors of

ICAM-I /LF A-I mediated cell adhesion which is derived from natural sources. They found that an Alpinia katsumadai extract or acyclic triterpenoid compound isolated therefrom inhibits the ICAM- 1/LFA-l mediated cell adhesion, thereby completing the present invention.

[17]

Technical Solution

[18] The present invention provides a novel acyclic triterpenoid compound.

[19] Further, the present invention provides a pharmaceutical composition for preventing or treating diseases caused by ICAM- 1/LFA-l mediated cell adhesion, comprising an

Alpinia katsumadai extract or acyclic triterpenoid compound isolated therefrom. [20]

Brief Description of the Drawings [21] Rg. 1 illustrates the H-ISMR spectrum of acyclic triterpenoid compound of formula

1 according to the present invention; [22] Hg. 2 illustrates the C-ISMR spectrum of acyclic triterpenoid compound of Formula

1 according to the present invention; [23] Rg. 3 illustrates the HMBC spectrum of acyclic triterpenoid compound of Formula 1 according to the present invention; [24] Rg. 4 illustrates the FAB-Mass spectrum of acyclic triterpenoid compound of

Formula 1 according to the present invention; [25] Rg, 5 is a graph showing inhibitory activity of the Alpinia katsumadai ethanol extract of the present invention on adhesion of soluble intercellular adhesion molecule -

1 (sICAM-1) to human monocyte cell line (THP-I) expressing LFA-I; [26] Rg. 6 is a graph showing inhibitory activity of the Alpinia katsumadai chloroform fraction of the present invention on adhesion of soluble intercellular adhesion molecule- 1 (sICAM-1) to human monocyte cell line (THP-I) expressing LFA-I; and [27] Rg, 7 is a graph showing inhibitory activities of acyclic triterpenoid compounds of

Formulae 1 and 2 of the present invention on adhesion of soluble intercellular adhesion molecule- 1 (sICAM-1) to human monocyte cell line (THP-I) expressing LFA-I. [28]

Best Mode for Carrying Out the Invention

[29] The present invention provides an εcyclic triterpenoid compound represented by the following Ibrmula 1.

[30] [31] [Ibrmula 1]

[33] [34] The compound of Ibrmula 1 is characterized in that it is extracted, isolated, and purified from Alpinia katsumadai.

[35] Further, the present invention provides a pharmaceutical composition for preventing or treating diseases caused by ICAM- 1/LFA-l mediated cell adhesion, comprising an Alpinia katsumadai extract or acyclic triterpenoid compounds of Formulae 1 and 2 isolated from the Alpinia katsumadai extract.

[36] [37] [Formula 2]

[39] [40] Hereinafter, the present invention will be described in detail. [41] The acyclic triterpenoid compounds of Formulae 1 and 2 of the present invention 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. As the salt, acid addition salts produced with pharmaceutically acceptable free adds are preferred. As the free adds, organic adds and inorganic adds may be used. Examples of the inorganic adds may include hydrochloric add, bromic add, sulfuric add, and phosphoric add, and examples of the organic adds may include dtric add, acetic add, lactic add, maleic add, fumaric add, gluconic add, methanesulfonic add, glyconb add, surinic add, 4-toluenesulfonic add, trifluoroacetic add, galacturonic add, embonic add, glutamic add, and aspartic add.

[42] The acyclic triterpenoid compounds of Formulae 1 and 2 of the present invention may be extracted/isolated from a natural herb, for example, Alpinia katsumadai, and prepared by synthetic methods or purchased from commerdally available sources.

[43] The method for extracting, isolating and purifying the acyclic triterpenoid compound

from Alpinia katsumadai according to the present invention is as follows.

[44] Specifically, Alpinia katsumadai powder is mixed with about 2 to 20-fold, preferably, 3 to 5-fold volume of polar solvent, for example, water, C to C lower alcohol such as methanol, ethanol, butanol, or the mixtures thereof at a ratio of 1:0.1 to 1: 10, and is extracted at a temperature ranging from 20 to 100 ⁰ C, preferably room temperature, for a period ranging from about 12 hrs to 7 days, preferably 3 to 7 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 reduced pressure. The filtrate is concentrated at 20 to 100 ⁰ C, preferably room temperature under reduced pressure using a rotary vacuum evaporator to obtain an Alpinia katsumadai crude extract.

[45] The Alpinia katsumadai crude extract is 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 is added to the water- soluble fraction to obtain chloroform-soluble fraction and water-soluble fraction. The Alpinia katsumadai chloroform fraction is subjected to silica gel column chromatography with a mixture solvent of chloroform and methanol. At this time, chloroform and methanol are preferably mixed in a ratio of 100-0:0-100 (v/v). Inhibitory activities of the separated fractions on ICAM- 1/LFA-l mediated cell adhesion are measured, and fractions exhibiting high inhibitory activity are subjected to reverse-phase column chromatography using 60%, 70%, 80%, 90%, and 100% methanol as an eluting solvent. Among the final fractions, fractions exhibiting high inhibitory activity on ICAM- 1/LFA-l mediated cell adhesion were separated by HPLC using 90% methanol at a flow rate of 6 ml/min to obtain final compounds 1 and 2.

[46] The final compound of Ibrmula 1 is a novel acyclic triterpenoid compound, and designated as

2,3,5,22,23-pentahydroxy-2,6,10,15,19,23-hexamethyl-6,10, 14,18-tetracosatetraene. The final compound of Formula 2 is designated as 2,3,22,23-tertrahydroxy-2,6,10,15,19,23-hexamethyl-6,10,14,1 8-tetracosatetraene.

[47] The Alpinia katsumadai ethanol extract according to the present invention inhibits the sICAM-1 and THP-I cell adhesion by 68% and 54% at concentrations of 50 and 25 βglwλ, respectively. The Alpinia katsumadai chloroform fraction inhibits the sICAM-1 and THP-I cell adhesion by 85% and 75% or more at concentrations of 50 and 25 μg/ml, respectively. The acyclic triterpenoid compounds of Formulae 1 and 2 exhibit IC values of 6.98 μg/πύ and 7.59 μg/ml, respectively. Accordingly, the Alpinia

katsumadai ethanol extract, the Alpinia katsumadai chloroform fraction, and the acyclic triterpenoid compounds of Ibrmulae 1 and 2 isolated therefrom inhibit ICAM- 1 and LFA- 1 mediated cell adhesion to effectively inhibit the adhesion and infiltration of blood cells svch as monocytes, neutrophils and lymphocytes to vascular endothelial cells, thereby being used for preventing or treating diseases caused by ICAM-I /LFA-I mediated cell adhesion.

[48] The diseases caused by ICAM- 1/LF A-I mediated cell adhesion include inflammatory and infectious diseases. 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; IgE-mediated hypersensitivity (type I) sich 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, Crohn's disease, 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 (Marlin, et al., Cell 51:813-819 ,1987; Dustin, et al., J. Immunol. 137:245-254, 1986; Dustin, et al., Immunol. Today 9:213-215, 1988; Ibston et al., Am. J. Pathol. 140 : p665-673, 1992; Macchioni et al., /. Reumatol. 21(10): pl860-1864, 1994; Mason et al., Arthritis Rheum. 36: p519-527, 1993; Kling et al., Clin. Invest. 71: p299-304, 1993; US patent N). 5,629,162; Korean Patent Application N). 10-2007-7006370). Further, in the present invention, infectious diseases include diseases caused by infection of rhinovirus or HIV- 1 using ICAM- 1.

[49] The composition of the present invention may include one or more known active in-

gredients having inhibitory activity on ICAM- 1/LFA-l mediated cell adhesion, in addition to the Alpinia katsumadai extract or acyclic triterpenoid compound isolated therefrom.

[50] The composition of the present invention comprises the Alpinia katsumadai extract or acyclic triterpenoid oompound isolated therefrom in an amount of 0.0001 to 10% by weight, preferably 0.001 to 1% by weight, based on the total weight of the composition.

[51] R>r administration, the composition of the present invention can be provided as a pharmaceutical composition containing pharmaceutically acceptable carriers, excipients or diluents, e.g, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starches, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate and mineral oil.

[52] 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, and an aerosol, an external preparation, suppository, or a sterilized injectable solution. Specifically, svch preparations may be prepared using diluents or exάpients ordinarily employed, such as filler, extender, binder, wetting agent, disintegrating agent, and 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 Alpinia katsumadai extract or the compound of Ibrmula 1 or 2 with at least one excipient such as starch, calcium carbonate, sucrose, lactose, and gelatin. Further, in addition to the exripients, 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, and a syrup, and various extipients such as wetting agent, sweetener, flavor, and preservative may be contained, in addition 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 glyool, polyethylene glycol, plant oil such as olive oil, injectable ester such as ethyloleate or the like may be used. As a suppository base, witepsol, macrogpl, tween 61, cacao butter, lauric butter, glycerogelatin or the like may be used.

[53] The composition of the present invention may be administered by oral or parenteral

route (e.g, intravenous, subcutaneous, intraperitoneal, or topical administration) depending on its purpose. An effective dosage of the present composition may be determined depending on the patient's health state and body 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 Alpinia katsumadai extract or compound of Ibrmula 1 or 2 isolated therefrom 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.

[54] The composition of the present invention may be used singly or in combination with surgical operation, hormone therapy, drug therapy and biological response regulators in order to prevent or treat diseases caused by ICAM- 1/LFA-l mediated cell adhesion.

[55]

Mode for the Invention

[56] Hereinafter, the preferred Examples are provided for better understanding However, these Examples are for illustrative purposes only, and the invention is not intended to be limited by these Examples.

[57]

[58] Example 1 : Preparation of Alpinia katsumadai extract and acyclic triterpenoid compound isolated therefrom

[59] 1. Preparation of Alpinia katsumadai ethanol extract

[60] Alpinia katsumadai was washed with water, dried in shade, and then pulverized using a mill. 3-fold volume of ethanol (based on the weight of dried Alpinia katsumadai) was added to 100 g of the pulverized Alpinia katsumadai, and extracted at room temperature for 7 days, followed by filtration. The filtrate was concentrated under redvced pressure to obtain 20 g of Alpinia katsumadai ethanol extract.

[61] 2. Preparation of Alpinia katsumadai chloroform fraction

[62] 20 g of the Alpinia katsumadai ethanol extract prepared in the above 1 was suspended in 500 ml of water, mixed with an equivalent amount of n-hexane (500 ml x 3), and then subjected to fractionation to obtain n-hexane-soluble fraction and water- soluble fraction. Chloroform (500 ml x 3) was added to the water-soluble fraction to obtain 7.35 g of chloroform-soluble fraction and water-soluble fraction.

[63] 3. Isolation and purification of acyclic triterpenoid compound from

Alpinia katsumadai chloroform fraction

[64] 7.35 g of the Alpinia katsumadai chloroform fraction obtained in the above 2 was concentrated under reduced pressure, and subjected to silica gel column chro-

matography using a step gradient solvent system oonsisting of chloroform:methanol = (100/0 ~ 0/100) to obtain active fractions. Inhibitory activities of the separated fractions on ICAM- 1/LFA-l mediated cell adhesion were measured, and fractions exhibiting high inhibitory activity were subjected to reverse-phase column chromatography (ODS gel) using 60%, 70%, 80%, 90%, and 100% methanol as an eluting solvent. Among the fractions, fractions exhibiting high inhibitory activity on ICAM- 1/LFA-l mediated cell adhesion were separated by high performance liquid chromatography (HPLC, YMC Jsphere ODS H-80 (250 x 20 mm)) using 90% methanol at a flow rate of 6 ml/min to obtain 20.3 mg of oompound 1 and 50.8 mg of compound 2. Detection of the active compounds was performed by UV at 210 nm, and the substances showing the inhibition of ICAM- 1/LFA-l mediated cell adhesion were eluted at 40 min and 25 min, respectively.

[65] 4. Structural analysis of active substance

[66] The results of measuring the completely isolated and purified compounds 1 and 2 are as follows, and as compared with the data published in literatures (Nshiyama Y, Moriyasu M, Ichimaru M, Tachibana Y, Kato A, Mathenge SG et al. Acyclic triterpenoids from Ekebergia capensis. Phytochemistry 1996; 42(3): 803-807), it was found that the oompound of Formula 1 was a novel acyclic triterpenoid compound, designated as

2,3,5,22,23-pentahydroxy-2,6,10,15,19,23-hexamethyl-6,10, 14,18-tetracosatetraene [Figs 1 to 4], and the compound of Formula 2 was found to be 2,3,22,23-tertrahydroxy-2,6,10,15,19,23-hexamethyl-6,10,14,1 8-tetra»satetraene.

[67] <Compound 1>

[69] 1) Appearance: yellow oil,

[70] 2) Molecular formula: C H O ,

30 54 5

[71] 3) Molecular weight: 494,

[72] 4) Electron impact mass spectrometry (7OeV): m/z(rel. int) = 517 [M+Na] ,

[73] 5) ¹ H-NVIR (500MHz, chloroform-d ): δ(ppm) 5.43 (IH, t, J= 5.8 Hz, H-7), 5.19

(IH, t, /= 5.3 Hz, H-18), 5.14 (2H, m, H-I l, H-14), 4.26 (IH, m, H-5), 3.62 (IH, m, H-3), 3.35 (IH, dd, /= 8.5, 1.5 Hz, H-22), 2.23, 2.13, 2.1, 2.09, 2.05, 2.02 (each 2H, m, H-20, H-8, H- 17, H-21, H-9, H-16), 1.63, 1.62, 1.61, 1.60 (eadi 3H, s, 26, 27, 28, 29-Me), 1.63 (2H, m, H-4), 1.59 (2H, m, H-13), 1.20, 1.19, 1.17, 1.15 (each 3H, s, 1,

24, 25, 30-Me), 1.41 (2H, m, H- 12),

[74] 6) ¹³ C-NVlR(500MHz, chloroform-d ): δ(ppm) 11.86, 16.10, 16.18, 16.20 (C-26, C- 27, C-28, C-29), 23,46, 24,00(C-I, C-24), 26.21 (C-21), 26.43, 26.57 (C-25, C-30), 26.57 (C- 17), 26.62 (C-8), 28.38 (C- 16), 29.84 (C- 12), 36.06 (C-4), 37.04 (C-20), 39.44 (C-9), 39.82 (C-13), 72.79, 73.26 (C-2, C-23), 78.46, 78.63, 78.90 (C-3, C-5, C- 22), 124.66, 124.87 (C-I l, C-14), 125.28, 126.57 (C-7, C-18), 134.87, 135.07, 135.24, 137.32 (C-6, C-IO, C-15, C- 19).

[75] [76] <Compound 2>

[78] [79] It was found that oompound 2 was thick, colorless oil, its molecular weight was [M+Na]+= m/z 501, and its molecular formula was determined as C H O by high-

30 54 4 resolution ESI-MS. As the result of measuring UV absorbance, its maximum absorbance was found to be detected at 210 nm. In NVlR analysis to determine the structure of oompound 2, 15 carbons corresponding to half of 30 carbons measured in mass spectrometry were observed in C-NVlR spectrum, which indicates that the oompound 2 has a symmetrical structure. In H-NVIR spectrum, four olefinic protons (65.13, 5.18) were observed, four methyl protons (δl.59, 1.61) were observed, and six methylene protons linked to olefin group (δl.99, 2.08, 2.01) were observed. Further, two methin protons (63.36) and four terminal methyl protons (δl.15, 1.19) linked to four methylene protons (δl.40, 1.58, 2.06 and 2.23) and hydroxyl group were observed.

[80] [81] Experimental Example 1 : Inhibitory activity on adhesion of soluble intercellular adhesion molecule-1 (sICAM-1) to human monocyte cell line (THP-I)

[82] The following experiment was performed to confirm inhibitory activities of the Alpinia katsumadai extract and acyclic triterpenoid oompounds isolated therefrom on adhesion of sICAM-1 to THP-I cell.

[83] Recombinant soluble intercellular adhesion molecule-1 (Recombinant sICAM-1; R& D Systems, Abingdon, UK) was diluted in IBS to 10 jWg/ml, and then 100 μJl thereof was added to a 96-well plate. The reaction was allowed to proceed at 4 ⁰ C for 12 hrs.

The plate was washed with FBS once, and 100 jλ of bovine serum albumin (BSA, 10 mg/ml) was added thereto, followed by blocking at room temperature for 1 to 2 hrs. THP-I cells (lxlθ ^? cells/ml) was added to BCECF-AM

(2'7'-bis(2-carboxyethyl)-5,6-carboxy-fluorescein acetoxymethyl ester, 5 μM), and subjected to reaction at 37 ⁰ C for 30 to 60 min. The THP-I cells were washed with PBS, and treated with LFA- 1/2 antibody (500 ng/ml), followed by reaction at 37 ⁰ C for 10 to 20 min. Then, each 100 ill of the THP-I cells and Alpinia katsumadai ethanol extract, Alpinia katsumadai chloroform fraction and compounds of Ibrmulae 1 and 2 (50 and 25 /zg/ml) prepared in Example 1 was added to each well coated with recombinant sICAM-1 , and subjected to reaction at 37 ⁰ C for 30 to 60 min. After the reaction, unbound THP-I cells were removed, and washed with PBS three times. The cells were solubilized with PBS (1% Triton X-100), and then fluorescence was measured (excitation 485 nm, emission 592 nm).

[84] The results are shown in Rgs. 5 to 7.

[85] As shown in Hg. 5, the Alpinia katsumadai ethanol extract inhibited the adhesion of sICAM-1 to THP-I cell by 68% and 54% in its concentration of 50 and 25 μg/ml, respectively.

[86] As shown in Hg. 6, the Alpinia katsumadai chloroform fraction inhibited the adhesion of sICAM-1 to THP-I cell by 85% and 75% or more at concentrations of 50 and 25 βglvcλ, respectively.

[87] As shown in Hg. 7, the acyclic triterpenoid compounds of Formulae 1 and 2 exhibited IC value of 6.98 μg/ml and 7.59 μg/ml, respectively.

50

[88] Accordingly, the Alpinia katsumadai extract and acjclic triterpenoid compounds isolated therefrom of the present invention were found to exhibit excellent inhibitory activities on the adhesion of sICAM- 1 to THP- 1 cell.

[89]

[90] Experimental Example 2 : Acute oral toxicity study in rats

[91] The following experiment was performed to analyze acute toxicity of the Alpinia katsumadai extract and acyclic triterpenoid compounds isolated therefrom of the present invention.

[92] The Alpinia katsumadai ethanol extract, Alpinia katsumadai chloroform fraction and compounds of Formulae 1 and 2 prepared in Example 1 were suspended in a 0.5% methylcellulose solution, and each suspension was orally administered once to five 6 week-old specific-pathogen-free (SPF) rats per group in a dosage of 1 g/kg After administration, death, clinical symptoms, and weight change in rats were observed,

hematological and biochemical tests of blood were performed, and any abnormal signs in the gastrointestinal organs of the chest and the abdomen were visually checked upon autopsy.

[93] The results showed that the test compounds of the present invention did not cause any specific clinical symptoms or death in rats. N) weight change was observed and no hematological or biochemical changes were observed in the blood and in autopsy. Therefore, the Alpinia katsumadai extract and acyclic triterpenoid compounds isolated therefrom 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 1 g/kg and its LD value is at

50 least 1 g/kg [94]

[95] Hereinbelow, Preparation Examples for the composition of the present invention will be described.

[96] Preparation Example 1 : Preparation of powder

[97] Alpinia katsumadai extract (or compound of Formula 1 or 2) 0.1 g

[98] Lactose 1.5 g

[99] Talc 0.5 g [100]

[101] The ingredients were mixed and filled in an airtight sac to prepare a powder agent. [102]

[103] Preparation Example 2 : Preparation of tablet

[104] Alpinia katsumadai extract (or compound of formula 1 or 2) 0.1 g

[105] Lactose 7.9 g

[106] Crystalline cellulose 1.5 g

[107] Magnesium Stearate 0.5 g [108]

[109] The ingredients were mixed and prepared into a tablet using a direct tabletting method. [110]

[111] Preparation Example 3 : Preparation of capsule

[112] Alpinia katsumadai extract (or compound of Formula 1 or 2) 0.1 g

[113] Corn starch 5 g

[114] Carboxy cellulose 4.9 g [115]

[116] The ingredients were mixed to prepare powder, and then the powder was filled into a

gelatin capsule according to a typical procedure to give a capsule agent. [117] [118] Preparation Example 4 : Preparation of injectable formulation

[119] Alpinia katsumadai extract (or compound of Formula 1 or 2) 0.1 g

[120] Sterile distilled water proper amount

[121] pH adjuster proper amount

[122]

[123] According to a typical procedure, an injectable formulation comprising the above ingredients was prepared into a (2 ml) ampule.

[124]

[125] Preparation Example 5 : Preparation of liquid formulation

[126] Alpinia katsumadai extract (or compound of formula 1 or 2) 0.1 g

[127] High fructose corn syrup 1O g

[128] Mannitol 5 g

[ 129] Purified water proper amount

[130]

[131] According to a typical procedure, each ingredient was solubilized in 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.

[132]

Industrial Applicability

[133] The Alpinia katsumadai extract or acyclic triterpenoid compound isolated therefrom of the present invention inhibits ICAM- 1/LF A-I mediated cell adhesion to effectively inhibit the adhesion and infiltration of blood cells svch as monocytes, neutrophils and lymphocytes to vascular endothelial cells, thereby being used for preventing or treating diseases caused by ICAM- 1/LFA-l mediated cell adhesion.

[134]