KR20100121343A - A composition for preventing or treating anti-inflammatory disease or infectious disease comprising extracts isolated from ligustrum obtusifolium s. et z - Google Patents

Abstract

PURPOSE: A composition containing Ligustrum obtusifolium S. et Z. is provided to prevent and treat inflammatory disease or viral disease. CONSTITUTION: A composition for preventing or treating inflammatory disease or virus infectious disease contains Ligustrum obtusifolium S. et Z. extract. The virus is rhinovirus or HIV-1. The Ligustrum obtusifolium S. et Z. extract is ethanol soluble extract from leaves of Ligustrum obtusifolium S. et Z. The extract contains (4S,5E,6S)-4-[2-[2-(3,4-dihydroxyphenyl)ethoxy]-2-oxoethyl]-5-ethylidene-6-[[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)-2-tetrahydropyrane]oxy]-4H-pyrane-3-carboxylic acid and methyl aster, carboxylic acid, and methyl ester.

Description

A composition for the prevention or treatment of inflammatory diseases or infectious diseases, including extracts of the leaves of the leaves of the leaves of the leaves. A COMPOSITION FOR PREVENTING OR TREATING ANTI-INFLAMMATORY DISEASE OR INFECTIOUS DISEASE COMPRISING EXTRACTS ISOLATED FROM

The present invention relates to a composition for the prophylaxis or treatment of inflammatory diseases or viral infectious diseases, including the extract of Ligustrum obtusifolium S. et Z. , more specifically, the extract or fractions thereof The present invention relates to a composition for preventing or treating the disease, in particular, a composition capable of preventing or treating the disease by inhibiting cell adhesion activity between ICAM-1 and LFA-1, and a composition for health food comprising the extract as an active ingredient. .

Inflammation is a body response caused by injury, infection, or an immune response following the introduction of a foreign substance, which involves pain, swelling, or the like of a diseased tissue. Although the ability to initiate an inflammatory response is essential for survival, the ability to control inflammation is also necessary for health. Inflammatory diseases are characterized by abnormal activation of the immune system in tissues or organs, which can cause dysfunction and / or disease in tissues or organs. These inflammatory diseases are the main causes of morbidity and death, which are all organs and tissues such as blood vessels, heart, brain, nerves, joints, skin, lungs, eyes, gastrointestinal tract, kidneys, thyroid, adrenal glands, pancreas, liver and muscles. This is because it can invade where possible. The treatment of such inflammatory diseases has been intensively received from the pharmaceutical industry since the past, but as a result of research and development, but somewhat developed in the management and therapeutic aspects, there is no fundamental treatment for cure or inflammation.

ICAM-1 (Intercellular adhesion molecule-1; CD54) is an adhesion receptor belonging to the Ig-superfamily of vascular endothelial cells, some lymphocytes and monocytes. are expressed at low density in monocytes (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), it is a molecule known to increase the expression of ICAM-1 in multiple cell types (Hubbard and Rothlein, Free Radic. Biol. Med. , 28: 1379-1386, 2000).

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

In order for white blood cells to infiltrate inflamed sites, leukocytes must adhere to vascular endothelial cells by rotating the vascular endothelial cells surface. 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. In addition, ICAM-1 plays an important role in the adhesion of platelets to vascular endothelial cells (Thomas and DeGraba, Neurology 49: 15-19, 1997).

On the other hand, ICAM-1 is not only used for inflammatory diseases but also for infectious diseases such as viruses.

Plays an important role. Several known viruses have 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), for example human immunodeficiency virus type-1 (HIV-1) Statin compounds, which are infected by the binding between ICAM-1 and LFA-1 and are known as inhibitors of binding of ICAM-1 and LFA-1, have been reported to inhibit the proliferation of HIV-1 virus (Giguere and Tremblay, J. Virology 78: 12062-12065, 2004).

Currently, the development of ICAM-1 and LFA-1 mediated cell adhesion inhibitors has been mainly targeted to their neutralizing antibodies, and only a few kinds of inhibitors have been reported from natural products. As an example, Abbott has reported ICAM-1 expression inhibitors as synthetics (Stewart et al., J. Med. Chem., 44: 988-1002, 2001), Novatis and Roche ( Roche developed an antagonist of LFA-1, a synthetic compound based on the epitope of ICAM-1 (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, seco-limonids-based materials (Trichilia rubra, IC50; 10-25 nM), cucurbitacins, Conobea scoparioides, IC50; 0.18-1.36 mM) and adzanthromycins (adxanthromycins) from natural products , Streptomyces sp.Na-148, IC50; 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), with only a few inhibitors known to be.

Accordingly, the present inventors have made diligent efforts to develop an agent capable of effectively treating inflammatory diseases or viral infection diseases by inhibiting ICAM-1 and LFA-1 mediated cell adhesion. It was confirmed that can effectively prevent or treat the present invention was completed.

An object of the present invention is to provide a composition for the prevention or treatment of inflammatory diseases or viral infectious diseases, including the extract of Ligustrum obtusifolium S. et Z.

Another object of the present invention is to provide a composition for health food comprising the extract of Ligustrum obtusifolium S. et Z.

Another object of the present invention to provide a composition for the prevention or treatment of inflammatory diseases or viral infectious diseases represented by the formula (1).

As one embodiment, the present invention relates to a composition for the prevention or treatment of inflammatory diseases or viral infectious diseases, including extracts of Ligustrum obtusifolium S. et Z.

In the present invention, the term 'leaving leaf tree ( Ligustrum obtusifolium S. et Z.'), belonging to the ash family, also called deciduous wood (落葉 低 木). The leaves of the larch tree are opposite and the flowers are white, and hang at the end of the branches in May-June. The fruits are nucleus-shaped, round, ripened in black in October, distributed in valleys in the sub-Hwangdo, and also in Japan. The fruit of the larch tree is called Rupaceae and has long been tonic, hemostatic effect, and used for the treatment of hemostasis and blood stool, but the specific anti-inflammatory action or anti-viral action is not known until now. Preferably the present invention provides a composition for the prevention or treatment of inflammatory diseases or viral infectious diseases using the extract of the leaves of the leaves. The extract used in the present invention may be an extract of any part of the larch oak tree, preferably an extract separated from the leaves of the oak tree, and the extract is an active ingredient (4S, 5E, 6S) -4 -[2- [2- (3,4-dihydroxyphenyl) ethoxy] -2-oxoethyl] -5-ethylidene-6-[[(2S, 3R, 4S, 5S, 6R) -3, 4,5-trihydroxy-6- (hydroxymethyl) -2-tetrahydropyranyl] oxy] -4H-pyran-3-carboxylic acid, methyl ester.

In addition, the extract is extractable using an extraction solvent commonly used in the art, preferably ethanol extract. In a preferred embodiment of the present invention it was prepared by extracting the leaves of the larch tree with ethanol and concentrated under reduced pressure to dissolve the concentrated active fraction in ethanol. Preferably the ethanol extract of the present invention may be an extract extracted with 80% to 100% ethanol.

In addition, the extract of the leaves of the present invention preferably inhibits the binding of ICAM-1 and LFA-1, thereby providing a composition for preventing or treating inflammatory diseases or viral infection diseases caused by the factors.

As used herein, the term 'ICAM-1 (Intercellular adhesion molecule-1, CD54)', a glycoprotein having a molecular weight of 90-120 kD, includes five immunoglobulin domains, and includes vascular endothelial cells and lymphocytes. Adhesion receptors are distributed in various cell types such as epithelial cells. ICAM-1 increases expression when stimulated by cytokines such as IL-1, TNF-α or γ-interferon, and LFA in β2 integrins distributed in lymphocytes, neutrophils, monocytes, etc. It acts as a ligand of -1 (lymphocyte function-associated antigen-1). In addition, it enhances antigen recognition and cytotoxicity with LFA-1, and regulates the migration of white blood cells or the attachment of white blood cells to epithelial cells and fibroblasts.

In the present invention, the term 'LFA-1 (Leukocyte function associated antigen-1, leukocyte function associated antibody-1; CD11a / CD18)' is a ligand of ICAM-1, and mainly β2 integrins distributed in lymphocytes, neutrophils, monocytes, etc. Integrin refers to a molecule belonging to β2-integrin expressed on white blood cells.

In addition, cell adhesion is very closely associated with atherosclerosis, arthritis, osteoporosis, bacterial infections, cancer metastasis, and refractory 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 in the blood vessel wall, and transform into foam cells that store cholesterol introduced into the blood. Progression of fibrosis leads to irreversible degeneration of narrowing 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.

For this reason, ICAM-1 and LFA-1 mediated cell adhesion inhibitors are of great value 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).

That is, ICAM-1 and LFA-1 may act as very important factors in inflammatory diseases or viral infectious diseases. First, as ICAM-1 can be seen in monoclonal antibodies and animal models of various disease models, the combination of ICAM-1 and LFA-1 is associated with psoriasis, atopy, asthma, glomerulonephritis and arthritis. It is closely related to autoimmune diseases, and it has been found that antigen-specific signals such as ICAM-1 / LFA-1 are essential for general immune responses (Corniejo et al. L, Adv. Pharmacol. 39: 99). -141, 1997; Patel et al., Circulation 97: 75-8, 1998; Nishikawa et al., J. Exp. Med. 177: 667-77, 1993; Kobayashi et al., Cell Immunol. 164: 295-305 Nagase et al., Am. J. Respir. Crit.Care Med. 152: 81-86, 1995). Therefore, many studies are being conducted to inhibit the binding of CAM-1 and LFA-1, and therapeutic compositions such as monoclonal antibodies are being actively studied, but the oak extract is an inflammatory disease as described above. There is no known fact that inflammatory diseases can be effectively treated by inhibiting the binding of ICAM-1 and LFA-1.

In addition, ICAM-1 is known to play an important role in viral infection, and the cold virus rhinovirus (rhino virus) has been reported to use ICAM-1 as a major receptor in the infection pathway (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). Therefore, the present inventors are contemplated that the blocking of the pathway of ICAM-1 / LFA-1 may be useful not only for the treatment of inflammatory diseases but also for the infectious diseases of the virus that uses ICAM-1 as every individual in the path of infection. As a result of administering the Larch extract as a composition, it was confirmed that the virus proliferation is significantly suppressed.

The extract of the present invention provides a composition for preventing or treating an inflammatory disease or a viral infectious disease. In the present invention, 'inflammatory' generally refers to localized protection of body tissues against host invasion due to foreign substances or harmful stimuli. As a result of reactions, the cause of this inflammation may be infectious causes such as bacteria, viruses and parasites, physical causes such as burns or radiation, or chemicals such as toxins, drugs or industrial reagents, or allergic and autoimmune reactions. It may be an immune response, or an abnormal condition associated with oxidative stress, preferably the inflammatory disease is meant to be caused by ICAM-1 / LFA-1 mediated cell adhesion. Examples of inflammatory diseases of the present invention include autoimmune diseases associated with 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, arteriosclerosis, 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, Crohn's disease, autoimmune pancreatitis, periodontal Diseases, 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, adhesion formation, Chronic inflammatory diseases such as chronic hepatitis and tuberculosis, multicuring and chronic prostatitis, and the like, and preferably, but not limited to, asthma, psoriasis, atopic dermatitis and transplant rejection (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; Poston et al, Am J Pathol. 140: p665-673, 1992; Macchioni et al., J. Reumatol. 21 (10): p1860-1864, 1994; Mason et al., Arthritis Rheum. 36: p519-527, 1993; Kling et al., Clin.Invest . 71: p299-304, 1993; US Registered Patent No. 5,629,162; Korean Patent Application No. 10-2007-7006370).

According to a preferred embodiment of the present invention, Ligustrum obtusifolium S. et Z. extract of the present invention effectively inhibits the adhesion and infiltration of blood cells such as monocytes, neutrophils and lymphocytes to the vascular endothelial cells, ICAM It was confirmed that it is effective in inflammatory diseases caused by -1 / LFA-1 mediated cell adhesion (Example 3).

As used herein, the term 'infection' refers to the growth of pathogenic microorganisms in human tissues, body tissues, body fluids, surfaces, and proliferation, and may be classified into various types according to infection paths and infectious conditions. In particular, the "infectious disease" in the present invention is a disease caused by ICAM-1 / LFA-1 mediated cell adhesion, and includes a viral infectious disease that is infected using ICAM-1, preferably a rhinovirus. (rhinovirus) and HIV-1 infection.

'Rhinovirus' is an RNA virus belonging to the family Picornaviridae, which is isolated from the nasal mucosa and pharynx of a cold patient. Renoviruses include human renovirus and bovine renovirus, of which human renovirus is pathogenic to humans and chimpanzees. There are more than about 100 types of serology, especially cold syndrome (acute upper respiratory tract infection) caused by rhinoviruses. Immunity due to rhinovirus infection continues for 2-16 weeks regardless of serotype, and resistance to infection is assumed not to be due to serum antibodies but to secreted antibodies. Epidemiologically, rhinoviruses are the most common cause of colds and are isolated in 15-40% of adults with cold-like diseases. The overall infection rate by rhinoviruses is high in infants and infants and decreases with age. In particular, the mechanism of action of rhinoviruses infects cells by attaching them to specific cellular receptors. The main group of receptors has recently been known as intracellular adhesion molecule (ICAM-I). Inflammatory cells, including lymphocytes, mast cells and eosinophils, are lightly infiltrated.

'HIV-1 (Human immunodeficiency virus-1)' is a human immunodeficiency virus belonging to the retroviral family (Retroviridae), a pathogen of AIDS (AIDS) and has not been accurately identified at present. HIV is known to be transmitted to body fluids such as blood transfusions, semen, vaginal secretions, and breast milk, but mainly infects immune cells such as helper T cells (CD4 + T cells), macrophages, and dendritic cells. HIV infection significantly reduces the number of CD4 + T cells. Three mechanisms exist: the destruction of cells due to life cycle progression following the first virus infection, the increased probability of apoptosis of second infected T cells, and finally CD8 cells. It induces toxic lymphocytes to destroy infected CD4 + T cells. When the number of CD4 + T cells falls below the level at which immunity can be maintained, humoral immunity is completely lost and the infected are exposed to opportunistic infections.

In a preferred embodiment of the present invention, it was confirmed that the extract of Ligustrum obtusifolium S. et Z. can inhibit the proliferation of viral infectious diseases, in particular, rhinovirus (Example 4).

In another aspect, the present invention relates to a composition for health food comprising the extract of Ligustrum obtusifolium S. et Z.

As used herein, the term 'health food' refers to a food prepared and processed by a method of extracting, concentrating, refining, and mixing a specific ingredient as a raw material or contained in a food ingredient for the purpose of health supplement, It refers to foods that are designed and processed to sufficiently exert bioregulatory functions such as biological defense, biorhythm control, disease prevention and recovery by the above ingredients to the living body, and the composition for health foods prevents diseases and prevents diseases. It can perform a function related to the recovery.

When using the extract of the leaves of the present invention as a health food, the extract can be added as it is or used in conjunction with other food or food ingredients, it can be selected as appropriate and used appropriately. In addition, the amount of the extract to be added may be appropriately determined depending on the purpose of use (prevention, health or therapeutic purpose).

In addition, there is no particular limitation on the kind of health foods in which the extract of the present invention can be used. Examples of foods that can be added include meat, sausages, breads, chocolates, candy, snacks, confections, pizzas, ramen noodles, dairy products including other noodles, gums, ice creams, various soups, beverages, teas, drinks, alcoholic beverages and Vitamin complexes and the like, it can be prepared by adding the extract according to the present invention as a main ingredient juice, tea, jelly and juice.

In addition, the mycelium extract of the present invention may be prepared by mixing known additives with other appropriate auxiliary ingredients that may be contained in the health functional food according to the choice of those skilled in the art.

As another aspect, the present invention relates to a composition for preventing or treating inflammatory diseases or viral diseases represented by the following formula (1).

The IUPAC name of the compound represented by Formula 1 is (4S, 5E, 6S) -4- [2- [2- (3,4-dihydroxyphenyl) ethoxy] -2-oxoethyl] -5-ethylidene -6-[[(2S, 3R, 4S, 5S, 6R) -3,4,5-trihydroxy-6- (hydroxymethyl) -2-tetrahydropyranyl] oxy] -4H-pyran-3 -Carboxylic acid, methyl ester ((4S, 5E, 6S) -4- [2- [2- (3,4-dihydroxyphenyl) ethoxy] -2-oxoethyl] -5-ethylidene-6-[[(2S, 3R, 4S, 5S, 6R) -3,4,5-trihydroxy-6- (hydroxymethyl) -2-tetrahydropyranyl] oxy] -4H-pyran-3-carboxylic acid, methyl ester) and oleuropein Collectively referred to as molecular weight 540.51, molecular formula C ₂₅ H ₃₂ O ₁₃ .

According to a preferred embodiment of the present invention, ICAM-1 and LFA-1 mediated cell adhesion inhibitory activity comprising the larch leaf extract and the compound according to Formula 1 extracted therefrom as an active ingredient, cell adhesion inhibitory activity And it was confirmed that the virus proliferation inhibitory effect (Examples 2, 3 and 4).

The compound of Formula 1 may be used in the form of a pharmaceutically acceptable salt, and includes all salts, hydrates, and solvent compounds prepared by conventional methods. Extraction and separation and purification method of the compound from the leaves of the leaves according to the invention is as follows.

First, the leaves of the larch tree, preferably the leaves of the tree was extracted with ethanol and concentrated under reduced pressure, and the concentrated active fraction was dissolved in ethanol and subjected to chromatography to concentrate the cell adhesion inhibitory activity fraction. After separation by high performance liquid chromatography, preferably, the elution solvent used in the high performance liquid chromatography may be methanol. The compound of formula 1 was obtained as a compound of the present invention eluted by the above method, and the ICAM-1 / LFA-1 mediated cell adhesion inhibitory activity assay and virus proliferation inhibitory activity were analyzed.

The leaves of the leaves of the present invention or the compounds of the formula (1) extracted from the ICAM-1 and LFA-1 mediated cell adhesion inhibitory activity of blood cells such as monocytes, neutrophils and lymphocytes adhere to and invade vascular endothelial cells By inhibiting it can be usefully used as a medicament for the prevention or treatment of inflammatory diseases or viral infectious diseases. In addition, the polyphenol-based compound of the present invention was used by extracting, separating and purifying from the leaves of larch, but can be obtained by any conventional method, and commercially available reagents can be used.

The 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 a suitable collection.

The form of the pharmaceutically acceptable salt of the compound of the present invention may be used in the form of a pharmaceutically acceptable salt, and acid addition salts formed by free acid are useful. The compound of Formula 1 may form a pharmaceutically acceptable acid addition salt according to a conventional method in the art. Organic acids and inorganic acids may be used as the free acid, and citric acid, acetic acid, lactic acid, tartaric cid, umarin acid, maleic acid, fumaric acid, formic acid, and propionic acid may be used as the organic acid. , Oxalic acid, trifluoroacetic acid, benzoic acid, gluconic acid, methanesulfonic acid, glycolic acid, succinic acid, 4-toluenesulfonic acid, galluturonic acid, embonic acid, glutamic acid or aspartic acid, and the like can be used.

In addition, the composition comprising the leaves of the leaves of the larch tree of the present invention or the compound of formula 1 may additionally include suitable carriers, excipients and diluents commonly used in the preparation of pharmaceutical compositions.

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 may include various excipients, such as wetting agents, sweeteners, fragrances or preservatives, in addition to water and liquid paraffin, which are commonly used as simple suspensions, solvents, emulsions or syrups. have. 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 present invention vary depending on the condition and weight of the patient, the severity 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 1 to 100 mg / day per kg of adult patient weight, preferably 5 to 20 mg / day It may be administered several times a day, preferably two to three times a day at regular time intervals according to the judgment of a doctor or a pharmacist. In addition, the administration may be administered once a day, may be administered several times divided.

The pharmaceutical compositions of the present invention can be administered to mammals such as mice, mice, livestock, humans, and the like by various routes. All modes of administration can be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection. In addition, the pharmaceutical composition of the present invention may be administered parenterally, and parenteral administration may be by subcutaneous injection, intravenous injection, intramuscular injection, or intrathoracic injection. To formulate into a parenteral formulation, the compound of Formula 1 is mixed in water with a stabilizer or buffer to prepare a solution or suspension, which is formulated in unit dosage forms of ampoules or vials.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the examples.

In the case of using the extract of Ligustrum obtusifolium S. et Z. of the present invention or the compound of Formula 1, blood cells such as monocytes, neutrophils and lymphocytes are inhibited by inhibiting ICAM-1 and LFA-1 mediated cell adhesion. Since it is effective in reducing adhesion and penetration to vascular endothelial cells, it can be effectively used as a composition for the prevention or treatment of inflammatory diseases and viral infectious diseases infected using ICAM-1.

Example 1 Isolation and Purification of Antiviral Inhibitors

In order to search for compounds capable of inhibiting antiviral, the present inventors have isolated and purified ICAM-1 and LFA-1 mediated cell adhesion inhibitory active compounds from the leaves of the bark by using extraction and chromatography. After extracting 150 g of the leaves of the larch leaf tree sold by the Korea Research Institute of Bioscience and Biotechnology for 72 hours with 100% ethanol and concentrating under reduced pressure, the concentrated active fraction was dissolved in ethanol and then Sephadex LH-20 (sephadex LH20) Chromatography was performed to concentrate ICAM-1 and LFA-1 mediated cell adhesion inhibitory activity fractions. The solvent was concentrated with a reduced pressure dryer and finally separated by high performance liquid chromatography (YMC Jsphere ODS H-80 (250 × 20 mm) while flowing 100% methanol at 6 ml / min as elution solvent. Was separated into a column to obtain 98 mg of the pure compound of formula 1. Detection of the active substance was carried out at UV 280 nm, ICAM-1 and LFA-1 mediated cell adhesion inhibitory substance eluted at 10 minutes.

Example 2: Physicochemical Characterization of Active Substances

The present inventors used ESI-MS (electrospray ionization mass spectrometry, Fisons VG Quattro 400 mass spectrometer, USA) to determine the molecular weight of the active material that inhibits cell adhesion. Nuclear magnetic resonance method was used to investigate the structure of the active material. In NMR experiments, each sample was dissolved in DMSO, and hydrogen and carbon nuclear magnetic resonance spectra were measured in a 5 mm NMR tube. The peaks of the solvents were measured based on peaks of tetramethylsilane (TMS) or internal standards. As a result of analyzing the property, molecular weight, molecular formula and mass of the compound, it was confirmed that the compound of the present invention had the following physical and chemical properties. The appearance of the active substance appears as a film in glass containers. The mass spectrometry (MH) ⁺ of the compound was measured at 539.5 m / z, and (M + Na) ⁺ was measured at 563.4 m / z (FIG. 2). The molecular formula of the active material was estimated to be C ₂₅ H ₃₂ O ₁₃ , and the actual molecular weight was found to be 540.51. The structure of the active material by analyzing the EIMS spectrum (FIG. 3), the hydrogen nuclear magnetic resonance spectrum (FIG. 4), and the carbon nuclear magnetic resonance spectrum (FIG. 5) measured by dissolving the active substance in DMSO d- 6 while referring to the physicochemical characteristics. As a result, the active compound was consistent with Oleuropein (Damtoft et al 1992, Phytochemistry 31 (12) 4197-4201) (Fig. 2).

Example 3: Assay for Cell Adhesion Inhibitory Activity of the Compound

Cells used to measure cell adhesion activity were HL-60 cells (ATCC; CCL-240), 10% FBS (v / v), 60.0 mg / L kanamycin sulfate, 2.0 g / L NaHCO ₃ Using the prepared RPMI-1640 as a culture medium, cells were cultured under conditions of 37 ° C and 5% CO ₂ . HL-60 cells aggregate with ICAM-1 / LFA-1 mediated cell adhesion when stimulated with PMA (phorbol-1,2-myristate-1,3-acetate). Using this property, HL-60 cells were dispensed into 1 × 10 ⁶ cells / ml (200 μl / well) in 96 well flatted-bottom plates, respectively, and the samples to be tested for inhibitory activity were pretreated for 30 minutes. In order to measure the concentration-dependent inhibitory activity, the samples were diluted in steps and treated to cells, and anti-ICAM-1 monoclonal antibody was used as a positive control. PMA was added to each well at a concentration of 25 nM, and after 48 hours of incubation, the aggregation of cells was observed under a microscope (Nikon; ECLIPSE TS100), and the state was photographed (Nikon; COOLPIX 995).

The ethanol extracts of the leaves of L. japonica were found to inhibit the adhesion of soluble intercellular adhesion molecule-1, sICAM-1, and human monocyte line THP-1 cells, at concentrations of 50 and 25 [mu] g / ml. The active material isolated from the fraction inhibited adhesion with HL-60 cells at a concentration of 25 μg / ml (FIG. 1).

Example 4: Evaluation of virus proliferation inhibitory ability

In order to measure the virus proliferation inhibitory ability against the compound 1 obtained in Example 1, a rhinovirus was used as a target, and the rhinovirus was infected with HeLa cells and grown and cultured. HeLa cells were cultured in a 37 ° C. incubator using MEM medium containing 10% of fetal bovine serum, and culture medium containing 20 mM MgCl ₂ in 1% serum was used for virus growth and virus growth inhibition. In order to measure the virus proliferation inhibitory ability against the rhinovirus of Compound 1 obtained in Example 1, HeLa cells were incubated for 24 hours in 2 × 10 ⁴ cells in each well of 96 well plates. After 24 hours, the culture supernatant of each well was removed and the rhinovirus infection solution titrated with TCID ₅₀ was added to each well, and each well and each plant extract and compound were brought to a final concentration of 100, 10, 1, 0.1 μg / ml. Was administered. The virus growth inhibition capacity of each extract was measured after 48 hours by the SRB assay (Choi et al., 2009). 100 μl of 70% acetone was added to each well, and then left at −20 ° C. for 1 hour and washed several times with distilled water. After drying at room temperature, 100 μl of a 0.4% (w / v) SRB (sulforhodamine B) solution dissolved in 1% (v / v) acetic acid was added and stained for 30 minutes. SRB stains that did not bind to cells were washed several times with 1% (v / v) acetic acid and then dried again. 100 μl of 10 mM Tris solution (pH 10.5) was added to each well to sufficiently dissolve the dye bound to the cells, and the absorbance was measured at 562 nm. Each treatment group was labeled as virus-free group (A), extract-only group (B), virus-only group (C), and virus and extract-treated group (D). Comparative cell growth rate (%) (Equation 1) and the plant growth inhibitory activity against the virus (%) (Equation 2) was calculated. The statistics on the results were calculated from the mean and standard deviation of three experimental results under the same conditions. Cytotoxicity evaluation of each sample was compared with the cells of the well without the extract at the concentration of 100, 10, 1, 0.1 ㎍ / ㎖ of the antiviral agent of Compound 1 isolated in Example 1.

Inhibition of Viral Proliferation of Compound 1 Extracted from the Leaves of the Leaves of Lepidoptera (HRV3 Virus in HeLa Cell Line) Test material CC ₅₀ ^a IC ₅₀ ^b TI ^C Active compound 1 > 100 90.59 ± 2.26 > 1.10 Ribavirin > 100 92.87 ± 1.05 > 1.07

^a sample concentration (/ mL) that reduces HeLa cells by 50%

^b cell lesions 50% reduction in sample concentration (CPE) (/ mL)

^c antiviral activity = CC ₅₀ / IC ₅₀

In order to measure the virus growth inhibitory ability by Compound 1 extracted from the leaves of the leaves of the leaves of the leaves of the ribinovirus infected with HeLa cells, during the growth of the sample was measured by the concentration of the virus growth inhibitory effect Ribavirin (Ribavirin) The extract showed higher virus growth inhibition.

Since the composition of the present invention is not an artificially synthesized compound, side effects can be minimized, and a pharmaceutical composition for preventing or treating inflammatory diseases or viral infectious diseases associated with ICAM-1 / LFA-1 mediated cell adhesion, health It can be usefully used as a composition for food.

1 is a photograph of HL-60 cell aggregation inhibition by ICAM-1 / LFA-1 of compound 1. FIG.

((A) cells not treated with PMA, (B) treated with PMA at a concentration of 25 nM, and 48 hours later cells, (C) treated with samples (25 μg / ml) one hour before, and treated with PMA at a concentration of 25 nM) One 48 hours later)

Figure 2 is a diagram showing the structure of the active material to inhibit the cell adhesion represented by the formula (1) according to the present invention.

3 is a diagram showing an EIMS spectrum of the active compound represented by Formula 1 according to the present invention.

4 is a hydrogen nuclear magnetic resonance spectrum (DMSO, 300 MHz) of the compound represented by Formula 1 according to the present invention.

5 is a carbon nuclear magnetic resonance spectrum (DMSO, 75 MHz) of the compound represented by Formula 1 according to the present invention.

6 is a DEPT spectrum (DMSO, 75 MHz) which is a compound represented by Chemical Formula 1 according to the present invention.

Claims (14)

A composition for the prevention or treatment of inflammatory diseases or viral infectious diseases, including the extract of Ligustrum obtusifolium S. et Z. The method of claim 1, wherein the inflammatory disease is systemic lupus erythematosus, scleroderma, ulcerative colitis, Crohn's disease, atopic dermatitis, psoriasis, anaphylaxis, dermatitis, diabetic retinopathy, retinitis, macular degeneration, uveitis, conjunctivitis, arthritis , Rheumatoid arthritis, ankylosing spondylitis, osteoarthritis, osteoporosis, allergies, diabetes, diabetic nephropathy, nephritis, nephritis, Sjogren's syndrome, Crohn's disease, autoimmune pancreatitis, periodontal disease, asthma, graft-versus-host disease, chronic pelvic inflammatory disease, uterus A composition for preventing or treating at least one inflammatory disease selected from the group consisting of endometritis, rhinitis, transplant rejection and chronic prostatitis. According to claim 1, wherein the virus is rhinovirus or HIV-1 composition for the prevention or treatment of viral infectious diseases. According to claim 1, wherein the extract is a prophylactic or therapeutic composition, characterized in that to prevent or treat viral infectious diseases by exhibiting cell adhesion inhibitory activity between ICAM-1 and LFA-1. The method of claim 1, wherein the extract is a prophylactic or therapeutic composition, characterized in that extracted from the leaves of the leaves. The prophylactic or therapeutic composition of claim 1, wherein the extract is an ethanol soluble extract. The method of claim 1, wherein the extract is (4S, 5E, 6S) -4- [2- [2- (3,4-dihydroxyphenyl) ethoxy] -2-oxoethyl] -5-ethylidene- 6-[[(2S, 3R, 4S, 5S, 6R) -3,4,5-trihydroxy-6- (hydroxymethyl) -2-tetrahydropyranyl] oxy] -4H-pyran-3- Carboxylic acid, a prophylactic or therapeutic composition which is an extract containing methyl ester as an active ingredient. Ligustrum obtusifolium S. et Z. A composition for health food comprising the extract. A compound for preventing or treating an inflammatory disease or a viral disease, comprising a compound represented by Formula 1 or a pharmaceutically acceptable salt thereof. [Formula 1]

The method of claim 9, wherein the inflammatory disease is systemic lupus erythematosus, scleroderma, ulcerative colitis, Crohn's disease, atopic dermatitis, psoriasis, anaphylaxis, dermatitis, diabetic retinopathy, retinitis, macular degeneration, uveitis, conjunctivitis, arthritis , Rheumatoid arthritis, ankylosing spondylitis, osteoarthritis, osteoporosis, allergies, diabetes, diabetic nephropathy, nephritis, nephritis, Sjogren's syndrome, Crohn's disease, autoimmune pancreatitis, periodontal disease, asthma, graft-versus-host disease, chronic pelvic inflammatory disease, uterus A composition for preventing or treating at least one inflammatory disease selected from the group consisting of endometritis, rhinitis, graft rejection and chronic prostatitis. The composition of claim 9, wherein the virus is rhinovirus or HIV-1. The method of claim 9, wherein the composition exhibits ICAM-1 and LFA-1 mediated cell adhesion inhibitory activity to prevent or treat a viral infectious disease, characterized in that the composition for preventing or treating. The prophylactic or therapeutic composition of claim 9, wherein the composition comprises an acid addition salt of the compound of Formula 1. 11. The prophylactic or therapeutic composition of claim 9, wherein the composition further comprises a pharmaceutically acceptable carrier.

Images