WO2013012117A1

WO2013012117A1 - Pharmaceutical compositions for preventing or treating inflammatory diseases, comprising phytosterol compound

Info

Publication number: WO2013012117A1
Application number: PCT/KR2011/006095
Authority: WO
Inventors: Jeong Min Lee; Jung Ho Choi; Ji Na Choi; Yun Hyeok Choi; Ill Chan Noh
Original assignee: Sinil Pharmaceutical Co Ltd
Current assignee: Sinil Pharmaceutical Co Ltd
Priority date: 2011-07-20
Filing date: 2011-08-18
Publication date: 2013-01-24
Anticipated expiration: 2014-01-20
Also published as: KR20130011111A

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating inflammatory diseases, having a phytosterol compound or a pharmaceutically acceptable salt thereof as an active ingredient. Having therapeutic activity for inflammatory diseases, the phytosterol compound is also applicable to a quasi-drug composition, a health functional food composition, and a cosmetic composition, all of which are intended to prevent or improve inflammation. Also, a method is provided for treating an inflammatory disease by administering the pharmaceutical composition to a subject in need thereof. As a natural materials, the phytosterol compound can be used as a safe therapeutic relatively free of fungal infection or other side-effects, compared to synthetic medicines.

Description

PHARMACEUTICAL COMPOSITIONS FOR PREVENTING OR TREATING INFLAMMATORY DISEASES, COMPRISING PHYTOSTEROL COMPOUND

The present invention relates to a pharmaceutical composition for preventing or treating of an inflammatory disease, comprising a phytosterol compound or a pharmaceutically acceptable salt thereof as an active ingredient, a quasi-drug composition, a health functional food composition and a cosmetic composition for preventing or improving inflammation all comprising the phytosterol compound, and a method for treating an inflammatory disease, comprising administering the pharmaceutical composition to a subject suspected of having the inflammatory disease.

Inflammation is a biological response of tissue to harmful stimuli, such as tissue damage, external irritants, or pathogens, characterized by complex pathological events produced by the organic interaction between various inflammatory mediators within vessels and body fluids and various immune cells, including enzyme activation, the release of inflammatory mediators, cellular infiltration and fluid exudation, circulatory disturbance, tissue degradation, hypertrophy, etc. The process of inflammation is initiated by cells already present in all types of tissue, mainly resident macrophages. At the onset of an infection, macrophages gather at the site of the injury and attack the invaders. Then, plasma accumulates because of the increased blood flow to the injured site, causing increased heat, redness, swelling, pain, etc. However, prolonged or excessive inflammatory response are likely to progress to pathological phenomena (e.g., hypersensitive allergy, chronic inflammatory diseases), causing abnormalities associated with inflammation.

Non-steroidal anti-inflammatory drugs (NSAIDS), widely used to treat most inflammatory diseases, function to inhibit cyclooxygenase (COX), an enzyme involved in the biosynthesis of prostaglandin from arachidonic acid, and have anti-inflammatory effects. However, their long-term use is problematic because they cause significant adverse effects on the gastrointestine, the liver and the kidney (Rainsford KD., Subcell biochem., 42, pp3-27, 2007; Guruprasad P. Aithal., Rheumatology., 7, pp139-150, 2011; Praveen P. N. Rao et al., Pharmaceuticals., 3, pp1530-1549, 2010). Therefore, there has been a widespread need for novel drugs that have excellent anti-inflammatory effects without causing adverse effects after long-term use, which is the reason why material development following the validation of the effects of natural resources has been actively studied.

In the body, various biochemical events are involved in the onset of inflammation. In response to chemical stimuli, macrophages produce various cytokines and NO that play an important role in the inflammatory response. As a result of the inflammatory response, the supraphysiological concentrations of nitrogen oxide produced by iNOS (inducible nitric oxide synthase) play a major role in the pathobiology of various inflammatory diseases (Kobayashi Y. et al., J Leukoc Biol., 88, pp1157-62, 2010). LPS (lipopolysaccharide), an inflammatory response inducer, interacts with immune cells such as leukocytes and a large amount of nitrogen oxide is produced owing to the activation of iNOS isoform in monocytes/macrophages, contributing to the inflammatory response (Korhonen R. et al., Curr Drug Targets Inflamm Allergy., 4, pp471-79, 2005). LPS is an endotoxin located in the outer membrane of Gram-negative bacteria. The binding of LPS to TLR4 (toll-like receptor 4) initiates a signal transduction pathway that activates many cytokine genes such as IL-6 (interleukin-6) and chemokines, which culminates in inflammation (Dobrovolskaia MA, et al., J Immunol., 170, pp508-19, 2003; Ji Y, et al., Cell Physiol Biochem., 25, pp631-640, 2010). The inducible nitric oxide synthase (iNOS) expressed by the stimuli of cytokines such as interferon γ, TNF-α and the like produce a lot of NO for a long period of time. This oxidative stress is known to promote the activation of NFκB, a transcription factor of inflammatory response that complexes with IκB when inactivated. The activated NFκB translocates to the nucleus where it promotes the expression of the cytokine genes involved in the induction of inflammatory responses, such as iNOS, IL-1β and TNF-α. Inflammatory responses can be suppressed by inhibiting these factors (Karin M. et al., Cold Spring Harb Perspect Biol., 1, pp1-14, 2009). In the body, nitric oxide (NO) is produced from L-arginine, which is catalyzed by three major nitric oxide synthase isoforms (NOS), nNOS (neuronal NOS), eNOS (endothelial NOS) and iNOS (inducible NOS). nNOS and eNOS are regulated by Ca2+/calmodulin while iNOS is regulated at the transcription level by inflammatory stimuli such as interleukin, interferon, LPS, etc. The nitric oxide produced in a small amount by nNOS or eNOS accounts for normal physiological functions such as vasodilation, neurotransmission, disruption of pathogens, etc. while an excessive amount of nitric oxide generated by phagocytes armed with iNOS is involved in a variety of pathophysiological processes including inflammation and cancer and reacts with superoxides to produce peroxynitrite, which acts as a strong oxidant to damage cells. The nitric oxide is also known to activate NF-κB in the phagocytes activated by inflammatory stimuli to cause chronic diseases such as inflammation, cancer, arteriosclerosis, etc. (Gupta SC et al., Exp Biol Med., 236:658-671, 2011; Riehemann et al., FEBS Lett., 442:89-94, 1999; Stamleret al., Science, 258:1898-1902, 1992).

Various studies have shown that tumor necrosis factor-α(TNF-α), interleukin-6(IL-6) and interleukin-1β(IL-1β) are a kind of cytokine promoting the inflammatory responses(Punzi L. et al., Crit Rev Clin Lab Sci., 2002, 39(1):63-88), which are known to account for the onset of various inflammatory diseases including rheumatoid arthritis (Jang C. H. et al., Rheumatology, 2006, 45(6):703-710), fibromyalgia (Hernandez M. E. et. al., BMC Res. Notes., 2010, 3(1):156), and sjogren’s syndrome (Baturone R. et. al., Scand J Rheumatol., 2009, 38(5):386-389).

These research results indicate that if it has the function of inhibiting the production of nitric oxide (NO) or cytokines such as TNF-α, IL-6, and IL-1β, a material might be effectively used to prevent and treat various inflammatory diseases. In the context of the prevention and treatment of inflammatory diseases, there is the necessity for phytochemicals. Active research has been conducted on the isolation of physiologically active materials useful as medicines that prevent and treat inflammatory diseases and are from natural sources, particularly plant resources.

Trachelospermum asiaticum var. intermedium, also known as climbing bagbane, is an evergreen woody vine in the dogbane family, Apocyanaceae. The plant is found in the southern parts of Korea, such as on the seaside, bases of mountains, fields, wild lands, etc., and grows by means of creepers on rocks, walls, and other trees or plants. In ancient Korean publications of herbal medicines such as Tong-Ui-Bo-Gam, Hyang-Yak-Jib-Sung-Bang (Compendium of Prescriptions from the Countryside), and Kwangje-bikeup, Trachelospermum asiaticum var. intermedium is prescribed as a medicine to be used alone. However, these medicinal publications just mention Trachelospermum asiaticum var. intermedium in terms of morphological discrimination, medicinal effects and the methods of preparing a decoction. Nowhere are the medicinally active ingredients of the extract described in the publications. In addition, there are problems with Trachelospermum asiaticum var. intermedium that will have to be solved before the commercialization of its extract, not only because the content of index ingredients greatly varies depending on where it is grown and the time the plants are gathered but also because active fractions effective for anti-inflammatory analgesia, blood circulation and arthritis treatment cannot be reproducibly obtained. Representative among the ingredients of Trachelospermum asiaticum var. intermedium are tracheloside, arctiin, matairesinoside, arctigenin, and notracheloside. However, instructions pertaining to and disclosure of the anti-inflammatory activity of the phytosterol compounds isolated from Trachelospermum asiaticum var. intermedium have not been in previous documents.

Leading to the present invention, intensive and thorough research into phytochemicals therapeutic of inflammatory diseases, conducted by the present inventors, resulted in the finding that a phytosterol compound derived from Trachelospermum asiaticum var. intermedium has the activity of inhibiting the production of nitric oxide and the release of inflammatory cytokines, thus being useful for the prevention, treatment and improvement of inflammatory diseases.

It is therefore an object of the present invention to provide a pharmaceutical composition for preventing or treating of an inflammatory disease, comprising a phytosterol compound or a pharmaceutically acceptable salt thereof.

It is another object of the present invention to provide a quasi-drug composition for preventing or improving inflammation, comprising a phytosterol compound.

It is a further object of the present invention to provide a health functional food composition for preventing or improving inflammation, comprising a phytosterol compound.

It is still a further object of the present invention to provide a cosmetic composition for preventing or improving inflammation, comprising a phytosterol compound.

It is still another object of the present invention to provide a method for treating an inflammatory disease, comprising administering the pharmaceutical composition to a subject suspected of having the inflammatory disease.

Comprising as an active ingredient the phytosterol compound inhibitory of inflammatory cytokines and chemokines, the compositions of the present invention are useful for preventing, treating, and improving inflammatory diseases. As a natural material, the phytosterol compound can be used as a safe therapeutic relatively free of fungal infection or other side-effects, compared to synthetic medicines.

FIG. 1 is a diagram showing the separation of an extract of Trachelospermum asiaticum var. intermedium, fractions thereof, and a phytosterol compound from the fractions.

FIG. 2 is a ¹H NMR spectrum of the phytosterol compound isolated from Trachelospermum asiaticum var. intermedium.

FIG. 3 is a ¹³C NMR spectrum of the phytosterol compound isolated from Trachelospermum asiaticum var. intermedium.

FIG. 4 is a mass spectrum of the phytosterol compound isolated from Trachelospermum asiaticum var. intermedium.

FIG. 5 is a curve showing the data of an assay for the cytotoxicity of an extract of Trachelospermum asiaticum var. intermedium, a fraction thereof, and a phytosterol compound from the fraction.

FIG. 6 is a graph showing the data of an assay for the inhibitory activity of an extract of Trachelospermum asiaticum var. intermedium, a fraction thereof, and a phytosterol compound from the fraction against tumor necrosis factor-α (TNF-α).

FIG. 7 is a graph showing the data of an assay for the inhibitory activity of an extract of Trachelospermum asiaticum var. intermedium, a fraction thereof, and a phytosterol compound from the fraction against interleukin-1β (IL-1β).

FIG. 8 is a graph showing the data of an assay for the inhibitory activity of an extract of Trachelospermum asiaticum var. intermedium, a fraction thereof, and a phytosterol compound from the fraction against nitric oxide (NO).

FIG. 9 is a graph showing the data of an assay for the inhibitory activity of an extract of Trachelospermum asiaticum var. intermedium, a fraction thereof, and a phytosterol compound from the fraction against interleukin-6 (IL-6).

In accordance with an aspect thereof, the present invention provides a pharmaceutical composition for preventing or treating an inflammatory disease, comprising a compound represented by the following Chemical Formula 1 or a pharmaceutically acceptable salt thereof.

[Chemical Formula 1]

The compound represented by Chemical Formula 1 is a phytosterol compound commonly named beta-sitosterol-beta-D-glucoside. The compound of the present invention may be chemically synthesized, but is preferably isolated from a natural source such as Trachelospermum asiaticum var. intermedium, and more preferably from an extract of Trachelospermum asiaticum var. intermedium or a fraction thereof. The extract of Trachelospermum asiaticum var. intermedium may be obtained from various tissues including the roots, stems, leaves, flowers, flesh and peel of the fruit, and a plant tissue culture. In one embodiment of the present invention, a phytosterol compound was isolated from Trachelospermum asiaticum var. intermedium, and was identified as beta-sitosterol-beta-D-glucoside by analyzing its structure using molecular weight measurement, molecular formula estimation and nuclear magnetic resonance (NMR).

The pharmaceutically acceptable salt of the compound of Chemical Formula 1 may be an acid addition salt formed with a pharmaceutically acceptable free acid which may be an organic acid or an inorganic acid. Examples of the inorganic acid include chloric acid, bromic acid, sulfuric acid, sulfurous acid, and phosphoric acid. Among the organic acid useful in the present invention are citric acid, acetic acid, maleic acid, fumaric acid, gluconic acid, metal sulfonic acid, glycolic acid, succinic acid, tartaric acid, 4-toluenesulfonic acid, galacturonic acid, embonic acid, glutamic acid, and aspartic acid.

The addition salt of the present invention may be prepared per typical methods. For example, the compound of Chemical Formula 1 is dissolved in a water-miscible organic solvent, e.g., acetone, methanol, ethanol or acetonitrile, followed by adding an equivalent or excess amount of an organic acid or an aqueous inorganic acid solution to the solution. The addition salt thus formed can be obtained by precipitation or crystallization, or by evaporating the solvent or excess acid and drying or suction-filtering the precipitated salt.

Not only the compound of Chemical Formula 1 and pharmaceutically acceptable salts thereof, but also solvates, hydrates and stereoisomers prepared therefrom, if having the same effect, are within the scope of the present invention.

The compound of Chemical Formula 1 may be prepared using a method comprising (a) extracting Trachelospermum asiaticum var. intermedium with an extraction solvent, followed by filtrating and concentrating to obtain an extract of Trachelospermum asiaticum var. intermedium; (b) fractionating the extract of Trachelospermum asiaticum var. intermedium of step (a) with an organic solvent and performing column chromatography to obtain a fraction; and (c) performing high-performance liquid chromatography to isolate the compound from the fraction of step (b).

In step (a) of the method, Trachelospermum asiaticum var. intermedium is extracted with an extraction solvent, then filtrated and concentrated to obtain an extract of Trachelospermum asiaticum var. intermedium.

The term “Trachelospermum asiaticum var. intermedium,” known as climbing bagbane, as used herein, refers to climbing stems and branches of Trachelospermum asiaticum or its closely related plants (Apocyanaceae). In Korean ancient medicinal publications, an extract from the herb is described as functionally acting on the heart, the liver and the kidney to treat diseases caused by the wind and dampness, and to help the flow of the life-energy through the meridian system and is prescribed for splenosis, limb convulsion, lumbago, arthralgia, tonsillitis, and boils. Arctiin, arctigenin, tracheloside, matairesinoside, and cymarose, found thus far as physiologically active ingredients in Trachelospermum asiaticum var. intermedium, are known to have vasodilative and anti-hypertensive activity. In the present invention, aerial parts, roots and seeds of Trachelospermum asiaticum var. intermedium, whether cultivated or commercially available, may be used without limitation.

So long as it is typically used in the art, any extraction solvent may be used in the present invention. Preferably, water, alcohols of 1 to 6 carbon atoms, or a combination thereof may be used as the extraction solvent. Preferred is ethanol, methanol or butanol. The extraction solvent may be added in an amount 2- to 20-fold greater than the amount to be reduced, and preferably in an amount 4- to 10-fold greater, and more preferably in an amount 5-fold greater. Preferred examples of the extraction method include, but are not limited to, boiling extraction, hot-water extraction, cold precipitation, reflux condensation, and ultrasonication, with a greater preference for boiling extraction or cold precipitation. For extraction, the extraction solvent may be maintained preferably at a temperature of 20 to 100ºC and more preferably at room temperature, but the present invention is not limited thereto. The time of extraction may preferably take place over, but is not limited to, 5 to 8 days and more preferably for 7 days. The number of the extractions may be preferably one to five and more preferably three, but the present invention is not limited thereto. Under these extraction conditions, Trachelospermum asiaticum var. intermedium may be extracted with an extraction solvent, followed by filtration, concentration and lyophilization to afford an extract.

In step (b) of the method, the Trachelospermum asiaticum var. intermedium extract of step (a) is fractionated with an organic solvent and column chromatography is performed to obtain a fraction.

The organic solvent is a liquid organic compound capable of dissolving solids, gases and liquids and may be one typically used in the art. Preferably, methanol, chloroform, acetate, hexane, dichloromethane, acetone, acetonitrile, benzene or a combination thereof may be used as the organic solvent for creating the fractions.

In the column chromatography, the column is filled with particles as a stationary phase. If necessary, suitable fillers may be selected before several rounds of chromatography are conducted. Preferably, the filler suitable for use in isolation and purification may be selected from among silica gel, Sephadex, RP-18, polyamide, toyoperal, and XAD resin.

In step (c) of the method, the compound of interest is isolated from the fraction of step (b) performing HPLC.

HPLC is a chromatography technique which can separate a mixture of compounds at high speed typically using a column, different types of stationary phases, and a pump, and is used to finally isolate the compound of Chemical Formula 1.

The composition of the present invention is characterized by the ability to suppress the release of nitric oxide (NO), TNF-α, IL-6 or IL-1β. In one embodiment of the present invention, the compound of Chemical Formula 1 was found to have little toxicity as measured by a cytotoxicity assay and thus could be safely used in pharmaceutical compositions. In addition, the compound was identified to have an excellent ability to suppress the production and release of the inflammatory factors nitric oxide (NO), TNF-α, IL-6 and IL-1β, and thus is useful for the prevention or treatment of inflammatory diseases (FIGS. 6 to 9). Further, the compound of Chemical Formula 1 was found to have a therapeutic effect on arthritis at a level similar to that of the conventional medications as measured by an assay for anti-inflammatory activity in arthritis-induced mice. Previously, the animal test of the present inventors verified in Korean Patent No. 10-0847439 that an extract from Trachelospermum asiaticum var. intermedium is therapeutic and prophylactic for inflammatory diseases as well as being inhibitory of the release of inflammatory mediators, which indirectly indicates that the compound of Chemical Formula 1 found among the extract of Trachelospermum asiaticum var. intermedium is responsible for the prevention or treatment of inflammatory diseases. In this context, Korean Patent No. 10-0847439 is hereby incorporated by reference in its entirety.

The term “inflammatory disease,” as used herein, is a generic name of the diseases having inflammation as their main damaging factor. Examples of the inflammatory disease include, but are not limited to, edema, dermatitis, allergy, atopy, asthma, conjunctivitis, peridontitis, rhinitis, otitis media, laryngopharyngitis, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn’s disease, colitis, hemorrhoid, gout, ankylosing spondylitis, rheumatic fever, systemic lupus erythematosus, fibromyalgia, psoriatic arthritis, osteoarthritis, rheumatoid arthritis, periarthritis of shoulder, tendonitis, tenosynovitis, myositis, hepatitis, cystitis, nephritis, sjogren's syndrome or multiple sclerosis, with preference for rheumatoid arthritis or edema, the former being more strongly preferred.

As used herein, the term “prevention” is intended to refer to any action resulting in the suppression or delay of the onset of an inflammatory disease thanks to the administration of the pharmaceutical composition according to the present invention. The term “treatment” is intended to refer to any action resulting in improvements in symptoms of an inflammatory disease or the beneficial alteration of the inflammatory state thanks to the administration of the composition according to the present invention.

The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable vehicle. The pharmaceutical composition comprising a pharmaceutically acceptable vehicle may be in various oral or non-oral dosage forms. In this regard, the pharmaceutical composition of the present invention may be formulated in combination with a diluents or excipient such as a filler, a thickener, a binder, a wetting agent, a disintegrant, a surfactant, etc. Solid preparations intended for oral administration may be in the form of tablets, pills, powders, granules, capsules, and the like. In regards to these solid agents, the compound of the present invention is formulated in combination with at least one excipient such as starch, calcium carbonate, sucrose, lactose, or gelatin. In addition to a simple excipient, a lubricant such as magnesium stearate, talc, etc. may be used. Among liquid preparations intended for oral administration are suspensions, internal use solutions, emulsion, syrups, and the like. In addition to a simple diluent such as water or liquid paraffin, various excipients, such as wetting agents, sweeteners, aromatics, preservatives, and the like may be contained in the liquid preparations. Also, the pharmaceutical composition of the present invention may be in a parenteral dosage form such as sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilizates, suppositories, and the like. Injectable propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and esters such as ethyl oleate may be suitable for the non-aqueous solvents and suspensions. The basic materials of suppositories include Witepsol, macrogol, Tween 61, cacao butter, laurin butter, and glycerogelatin.

Further, the form of the dosage of the pharmaceutical composition of the present invention may be selected from the group consisting of a tablet, a pill, a powder, a granule, a capsule, a suspension, an internal use solution, an emulsion, a syrup, a sterile aqueous solution, a non-aqueous solution, a lyophilizate, and a suppository.

The pharmaceutical composition of the present invention is administered in a therapeutically or pharmaceutically effective amount. The term “therapeutically or pharmaceutically effective amount,” as used herein, is intended to refer to an amount of a pharmaceutical composition for treating a disease that is sufficient, at a reasonable benefit/risk ratio applicable to any medical treatment. The effective amount may vary depending on various factors including the severity of the disease being treated, the patient’s age and sex, drug activity, sensitivity to the drug, the time of administration, the route of administration, the rate of excretion, the period of time of treatment, the co-administration of drugs, and other parameters well known in the art.

The dose of the pharmaceutical composition of the present invention is not imparted with special limitations, but varies depending on various factors including in vivo absorption rate, patient’s age, sex, health state and diet, the time of administration, the route of administration, excretion rate, and the severity of disease. Typically, the extract from Trachelospermum asiaticum var. intermedium may be administered at a daily dose of from 0.1 to 100 mg/kg, preferably at a daily dose of from 30 to 80 mg/kg and more preferably at a dose of from 50 to 60 mg/kg. Preferably, the pharmaceutical composition of the present invention is formulated in consideration of the effective amount range. The unit dosage formulations thus obtained may be administered using a specialized regimen or at regular intervals of time in multiple doses according to the decision of an expert who is responsible for monitoring or observing the administration of the drug, or according to personal demands.

In accordance with another aspect thereof, the present invention provides a quasi-drug composition for preventing or improving inflammation, comprising the compound represented by Chemical Formula 1. As for the compound, its description is as given above. The quasi-drug composition may comprise a pharmaceutically acceptable salt of the compound. In greater detail, the composition of the present invention may be added to a quasi-drug composition to be used to prevent or improve inflammation.

As used herein, the term “quasi-drug” may be defined as 1) articles made from fiber, rubber, or similar materials used in humans or animals for the purpose of curing, alleviating, treating, or preventing diseases; 2) articles, other than instruments, machines or the like, which have a mild action on or have no direct influence on the human body; and, 3) articles, falling within the range of agents used to sterilize, kill insects and for similar purposes. All of the articles exclude those intended at the same time to be prescribed to diagnosing, curing, alleviating, treating or preventing diseases in humans or animals, and for pharmaceutically affecting the structure and function of humans or animals.

When used as a quasi-drug additive, the composition of the present invention may be suitably used as it is, or in combination with another quasi-drug or quasi-drug component using a typical method. Its amount in the mixture may be properly determined depending on the purpose of use.

Examples of the quasi-drug to which the composition of the present invention may be applied include, but are not limited to, disinfectants, shower foams, mouthwash, wet tissues, detergent soap, handwashing materials, a humidifier fillers, masks, ointments, and a filter coating.

In accordance with a further aspect thereof, the present invention provides a health functional food composition for the preventing or improving inflammation, comprising the compound represented by Chemical Formula 1. As for the compound, its description was given above. The health functional food composition may comprise a pharmaceutically acceptable salt of the compound. In greater detail, the composition of the present invention may be added to a health functional food composition to be used to prevent or improve inflammation.

When used as a health functional food additive, the composition of the present invention may be suitably used in unchanged form, or in a combination with another health functional food or health functional food component made using a typical method. Its amount in the mixture may be properly determined depending on the purpose of use. When used to prepare foods or drinks, typically, the composition of the present invention may be used in an amount of 15 parts by weight or less, based on 100 parts by weight of the total material, and preferably in an amount of 10 parts by weight or less. If the foods or drinks are designed for long-term ingestion to provide health control and sanitation, the amount may be further decreased, but no problems are caused when used in greater amounts because the active ingredient is not toxic to the body.

There is no particular limitation on the kind of health functional food to which the composition of the present invention can be added. Examples of such a health functional food include meats, sausages, breads, chocolates, candies, snacks, confectionery, pizzas, ramen noodles, other noodles, gums, dairy products such as ice-creams, various soups, beverages, teas, drinks, alcoholic beverages, and vitamin complexes. All typically accepted health functional foods may contain the active ingredient according to the present invention. Also, animal feeds fall within the scope of the present invention.

When formulated into a beverage, the health functional food composition of the present invention may further comprise various sweeteners, fragrances or natural carbohydrates. Examples of such natural carbohydrates include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. The amount of the natural carbohydrate may be approximately 0.01 ~ 0.04 grams based on 100 mL of the beverage composition of the present invention, and preferably approximately 0.02 ~ 0.03 grams. The sweeteners may be natural sweeteners such as thaumatin or a stevia extract, or synthetic sweeteners such as saccaharin or aspartame.

In addition, the health functional food composition of the present invention may comprise various nutrients, vitamins, minerals, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH modifiers, stabilizers, antiseptics, glycerin, alcohols, and carbonating agents used in carbonated beverages. Moreover, the composition of the present invention may contain the fruit flesh used to prepare natural fruit juices, fruit beverages and vegetable beverages.

In accordance with still a further aspect thereof, the present invention provides a cosmetic composition for the preventing or improving inflammation, comprising the compound represented by Chemical Formula 1. As for the compound, its description is as given above. The cosmetic composition may comprise a pharmaceutically acceptable salt of the compound. In greater detail, the composition of the present invention may be added to a cosmetic composition to be used to prevent or improve inflammation.

The cosmetic composition of the present invention may be formulated into a general emulsion or water-soluble form. Examples of the emulsion cosmetics include nutrition lotions, creams, essences and the like. A skin lotion is a kind of the water-soluble cosmetic forms. Examples of the suitable cosmetic forms include, but are not limited to, solutions, gels, solid or paste preparations, oil-in-water emulsions, suspensions, microemulsions, microcapsules, microgranules or ionic liposomes, non-ionic vesicle dispersions, creams, skins, lotions, powders, ointments, sprays, conceal sticks, etc. Also, it may be prepared into a foam form or an aerosol form having a quantity of compressed propellant.

In addition, the cosmetic composition may comprise lipids, organic solvents, dissolving agents, thickening agents, gelling agents, softeners, anti-oxidants, suspending agents, stabilizers, foaming agents, aromatics, surfactants, water, ionic or non-ionic emulsifiers, fillers, sequestering agents, chelating agents, preservatives, vitamins, UV blocking agents, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic activators, liposomes, and/or other general supplements used in the skin science field.

In accordance with still another aspect thereof, the present invention provides a method of treating an inflammatory disease, comprising administering the pharmaceutical composition to a subject suspected of having the inflammatory disease.

The term “subject suspected of having an inflammatory disease,” as used herein, means an animal including a human, which has been attacked with or is apt to be attacked by an inflammatory disease. An inflammatory disease may be cured by administering the pharmaceutical composition comprising the compound of the present invention or a pharmaceutically acceptable salt thereof to a subject suspected of having the inflammatory disease. As for the inflammatory disease, its description was given above.

The term “administration,” as used herein, is intended to refer to introduction of the pharmaceutical composition of the present invention into a subject suspected of having an inflammatory disease in a suitable manner. So long as it allows the compound of the present invention to reach a target tissue, any administration, whether oral or parenteral, may be taken.

The treatment method of the present invention includes administering a pharmaceutically effective amount of the pharmaceutical composition comprising the compound. It will be apparent to those skilled in the art that the suitable total daily dose may be determined by an attending physician within the scope of sound medical judgment. Also, the composition may be administered in a single dose or it may be spread out over multiple doses per day. For purposes of the present invention, however, the therapeutically effective dose for a certain patient depends on various factors including the kind and extent of the response sought to be achieved, the use of any other agents according to the intended use, patient’s age, weight, general health state, sex and diet, the time of administration, the route of administration, the rate of excretion of the composition, the duration of the treatment, other drugs mixed with or concurrently administered together with the composition, and other factors well known in the medical art.

A better understanding of the present invention may be obtained through the following examples which are set forth to illustrate, but are not to be construed as limiting the present invention.

EXAMPLE 1: Isolation of phytosterol compound from Trachelospermum asiaticum var. intermedium

Extraction and fractionation processes were used to isolate a phytosterol compound from Trachelospermum asiaticum var. intermedium. To achieve this, Trachelospermum asiaticum var. intermedium was boiled for 2 hours in 95% ethanol in a water bath to a concentration of 20:1 and the concentrate was fractioned with chloroform and water. Thereafter, seven fractions were obtained by silica gel column chromatography using a gradient of from 100:0 to 1:1 of methanol to chloroform as an eluent. Among them, the fifth fraction was subjected to high-performance liquid chromatography using 85% methanol as a mobile phase to separate an active compound (FIG. 1).

EXAMPLE 2: Analysis and Identification of the Phytosterol Compound

The compound eluted at 15 min upon the HPLC of Example 1 was structurally identified by the measurement of molecular weight, the estimation of molecular formula, and nuclear magnetic resonance (NMR) analysis (FIGS. 2 to 4).

As a result, the compound was identified as beta-sitosterol-beta-D-glucoside, which is a phytosterol compound represented by Chemical Formula 1, as disclosed in Yayli N. et al., Turk. J. Chem., 2003, 27:749-755

[Chemical Formula 1]

EXAMPLE 3: Assay for Cytotoxicity

The Trachelospermum asiaticum var. intermedium extract, its fractions, and the phytosterol compound isolated from the fractions, prepared in Example 1, were assayed for cytotoxicity using MTT. The mouse macrophage RAW 264.7 was purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA) and maintained at 37℃ in DMEM supplemented with 10% fetal bovine serum (FBS), 100 units/ml penicillin, 100 μg/ml streptomycin under a 5% CO₂ condition.

RAW 264.7 macrophages were treated with different concentrations (0, 1, 3, 10, 30, 100 μg/ml) of the samples and cultured for 24 hours and then incubated for 1 hour at 37℃ with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; 500 μg/ml). After removal of the culture medium, a dimethylsulfoxide (DMSO) solution was added to the cells to dissolve the formed formazan, followed by measuring absorbance at 570 nm. Measurements were compared to the control to calculate relative cell viability (% of control).

There were no significant differences in cell viability between all of the Trachelospermum asiaticum var. intermedium extract, its fractions and the phytosterol compound isolated from the fractions, and the control over the concentration range of from 0 to 100 μg/ml (FIG. 5). Thus, the Trachelospermum asiaticum var. intermedium extract, its fractions and the phytosterol compound isolated from the fractions were identified as having no cytotoxicity.

EXAMPLE 4: Inhibitory Effect on Inflammatory Cytokines and Chemokines

An examination was made of the inhibitory effect of the Trachelospermum asiaticum var. intermedium extract, its fractions and the phytosterol compound isolated from the fractions, prepared in Example 1, on inflammatory mediators. In this regard, first, the mouse macrophage RAW 264.7 was incubated with lipopolysaccharide (LPS) (0.2 μg/ml) in the presence of each sample (100 μg/ml) for 18 hours, followed by quantitatively analyzing the inflammatory cytokines and chemokines, such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), nitric oxide (NO), and interleukin-6 (IL-6), released into the medium, using an ELISA kit or an NO Griess reagent system (Promega). The production amounts of the inflammatory cytokines and chemokines from the groups treated with the samples were compared to those from the control treated with LPS only to calculate relative amounts (% of the control). All measurements were expressed in mean±SE (standard error). Data was analyzed for statistical significance between experimental groups using Student’s t-test.

TNF-α was produced in an amount 12.8% reduced by the Trachelospermum asiaticum var. intermedium extract, 20.8% reduced by the chloroform fraction and 23.3% reduced by the phytosterol compound, compared to the control (FIG. 6).

IL-1β was produced in an amount 23.9% reduced by the Trachelospermum asiaticum var. intermedium extract, 30.2% by the chloroform fraction, and 46.7% by the phytosterol compound, compared to the control (FIG. 7).

NO was produced in an amount 20.9% reduced by the Trachelospermum asiaticum var. intermedium extract, 45.2% by the chloroform fraction, and 72.8% by the phytosterol compound, compared to the control (FIG. 8).

IL-6 was produced in an amount 42.4% reduced by the Trachelospermum asiaticum var. intermedium extract, 50.2% by the chloroform fraction, and 54.1% by the phytosterol compound, compared to the control (FIG. 9).

These data suggest that the Trachelospermum asiaticum var. intermedium extract, the fraction, and the phytosterol compound according to the present invention inhibit the production and release of the inflammatory cytokines and chemokines TNF-α, IL-1β, NO and IL-6 and thus are useful for preventing or treating various inflammatory diseases.

EXAMPLE 5: Assay for Anti-Inflammatory Activity in Arthritis-Induced Mice

Bovine type II collagen and an adjuvant were formulated into an emulsion which was subcutaneously injected at a dose of 100 μg into the tail. Three weeks later, a booster was achieved by the intraperitoneal injection of 100 μg of bovine type II collagen to induce arthritis. Immediately after the induction, drugs were administered and their inhibitory activity against arthritis was measured. Suspensions of the Trachelospermum asiaticum var. intermedium extract, the fraction and the phytosterol compound were orally administered at different doses. Severity of edema was measured in the test mice and compared to that of a normal group (arthritis not induced), a control (administered with vehicle), and the mice administered with a reference drug (commercially available medicine).

The Trachelospermum asiaticum var. intermedium extract, the fraction and the phytosterol compound in accordance with the present invention were found to have reduced edema in a dose-dependent manner and to show similar inhibitory activity against arthritis compared to the reference drug. As a natural material, the compound of the present invention has the therapeutic function of suppressing and alleviating inflammation.

Below, a description is given of preparation examples using the Trachelospermum asiaticum var. intermedium extract, its fraction and the phytosterol compound isolated from the fraction.

PREPARATION EXAMPLE 1: Preparation of Pharmaceutical Formulations

1-1. Preparation of Powder

Compound of Example 1 10 mg

Lactose 1 g

These ingredients were mixed and loaded into an airtight sac to give a powder.

1-2. Preparation of Tablet

Compound of Example 1 10 mg

Corn starch 100 mg

Lactose 100 mg

Magnesium stearate 2 mg

These ingredients were admixed together and directly compressed into a tablet using a typical method.

1-3. Preparation of Capsule

Compound of Example 1 10 mg

Corn starch 100 mg

Lactose 100 mg

Magnesium stearate 2 mg

These ingredients were admixed together and the admixture was loaded into a gelatin capsule using a typical method.

1-4. Preparation of Pill

Compound of Example 1 10 mg

Lactose 1.5 g

Glycecrin 1 g

Xylitol 0.5 g

These ingredients were admixed together and formulated into a pill weighing 3 g using a typical method.

1-5. Preparation of Granule

Compound of Example 1 10 mg

Soybean extract 50 mg

Glucose 200 mg

Starch 600 mg

These ingredients were admixed together and 100 mg of 30% ethanol was added to the admixture. Drying at 60℃ gave granules which were then loaded into a sac.

PREPARATION EXAMPLE 2: Preparation of Food

Food containing the Trachelospermum asiaticum var. intermedium extract, its fractions and the phytosterol compound isolated from the fractions was prepared as follows.

2-1. Preparation of Wheat Flour-Based Food

To 100 grams of wheat flour was added 1 mg of the compound of Example 1 and the mixture was used to make breads, cakes, cookies, crackers and noodles for health improvement.

2-2. Preparation of Dairy Products

To 100 mg of milk was added 1 mg of the compound of Example 1 and the milk was used to prepare various dairy products such as butter and ice-cream.

2-3. Preparation of Zen Food

Unmilled rice, barley, glutinous rice, and unshelled adlay were pre-gelatinized using a typical method, dried and roasted before grinding into powder with a particle size of 60 mesh. Black soybean, black sesame and wild sesame were steamed according to a typical method, dried and roasted before grinding into powder with a particle size of 60 meshes.

The grains, the seeds, and the compound of Example 1 were formulated at the following ratios to yield a zen food.

Grains (unmilled rice 30 wt %, unshelled adlay 15 wt %, barley 20 wt %),

Seeds (wild sesame 7 wt %, black soybean 8 wt %, black sesame 7 wt %),

Compound of Example 1 (2 wt %),

Ganoderma lucidum (0.5 wt %),

Foxglove (0.5 wt %)

PREPARATION EXAMPLE 3: Preparation of Beverages

3-1. Preparation of Health Drink

Compound of Example 1 1 mg

Citric acid 1000 mg

Oligosaccharides 100 g

Japanese Apricot Liquid Extract 2 g

Taurine 1 g

Purified water added to form a total volume of 900 mL

These ingredients were homogeneously formulated according to a typical method and the formulation was heated at 85°C for about 1 hour with stirring, loaded into a 2 L sterilized bottle, subjected to pasteurization and stored in a refrigerator until used.

The composition was given as a preferred example suitable for use in beverages, but may be changed depending on regional and national factors, such as consumer classes, countries, etc.

3-2. Preparation of Vegetable Juice

To 1,000 mL of typical tomato or carrot juice was added 1 mg of the compound of Example 1 to give a health-improving vegetable juice.

3-3. Preparation of Fruit Juice

To 1,000 mL of typical apple or grape juice was added 1 mg of the compound of Example 1 to give a health-improving fruit juice.

PREPARATION EXAMPLE 4: Preparation of Cosmetics

The Trachelospermum asiaticum var. intermedium extract, its fractions and the phytosterol compound isolated from the fractions in accordance with the present invention may be used as an active ingredient of a cosmetic for the prevention or improvement of inflammation. In this context, emulsion-type cosmetics, such as nutrition lotion, cream, essence, etc., and water-soluble cosmetics, such as skin lotion were prepared.

4-1. Preparation of Emulsion-Type Cosmetics

Emulsion-type cosmetics were prepared from the composition given in Table 1 as follows.

1) A mixture of materials 1 ~ 9 was heated at 65 ~ 70°C.

2) Material 10 was added to the mixture of step 1).

3) A mixture of materials 11 ~ 13 was heated at 65 ~ 70°C to complete dissolution.

4) During the heating of step 3), the mixture of step 2) was slowly added, followed by emulsification at 8,000 rpm for 2 ~ 3 min.

5) Material 14 was dissolved in a small amount of water, added to the mixture of step 4), and emulsified for 2 min.

6) Materials 15 ~ 17 were weighed and added to the mixture of step 5) before additional emulsification for 30 sec.

7) The mixture of step 6) was deaerated and cooled to 25 ~ 35°C to produce emulsion-type cosmetics.

Table 1

Composition		Emulsion Type	1	Emulsion Type 2	Emulsion Type 3
1	Stearic acid	0.3	0.3	0.3
2	Stearyl alcohol	0.2	0.2	0.2
3	Glyceryl monostearate	1.2	1.2	1.2
4	Beeswax	0.4	0.4	0.4
5	Polyoxyethylene sorbitan monolauric acid ester	2.2	2.2	2.2
6	Methyl paraoxybenzoate	0.1	0.1	0.1
7	Propyl paraoxybenzoate	0.05	0.05	0.05
8	Cetylethylhexanoate	5	5	5
9	Triglyceride	2	2	2
10	Cyclomethicone	3	3	3
11	Distilled water	~100	~100	~100
12	Conc. glycerin	5	5	5
13	Triethanol amine	0.15	0.15	0.15
14	Polyacrylic acid	0.12	0.12	0.12
15	Pigment	0.001	0.001	0.001
16	Fragrant	0.1	0.1	0.1
17	Cpd. of Example 1	0.01	0.1	1

4-2. Preparation of Water-Soluble Type Cosmetics

Water-soluble-type cosmetics were prepared from the composition given in Table 2, as follows.

1) Materials 2 ~ 6 were dissolved in material 1 (purified water) using an agitation mixer.

2) Materials 8 ~ 11 were completely dissolved in material 7 (alcohol).

3) The solution of step 2) was slowly added to the mixture of step 1) to produce water-soluble type cosmetics.

Table 2

Composition		Water-Soluble Type 1	Water-Soluble Type 2	Water-Soluble Type 3
1	Purified water	~100	~100	~100
2	Conc. glycerin	3	3	3
3	1,3-Butylene glycol	2	2	2
4	EDTA-2Na	0.01	0.01	0.01
5	Pigment	0.0001	0.0002	0.0002
6	Cpd. of Example 1	0.1	1	1
7	Alcohol (95%)	8	8	8
8	Methyl paraoxybenzoate	0.1	0.1	0.1
9	Polyoxyethylene hydrogenated ester	0.3	0.3	0.3
10	Fragrant	0.15	0.15	0.15
11	Cyclomethicone	-	-	0.2

Although the preferred embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims.

Claims

A pharmaceutical composition for preventing or treating an inflammatory disease, comprising a compound represented by the following Chemical Formula 1, or a pharmaceutically acceptable salt thereof.

[Chemical Formula 1]
The pharmaceutical composition of claim 1, wherein the compound is isolated from Trachelospermum asiaticum var. intermedium.
The pharmaceutical composition of claim 1, wherein the compound is prepared using a method comprising:

(a) extracting Trachelospermum asiaticum var. intermedium with an extraction solvent, followed by filtrating and concentrating to obtain an extract of Trachelospermum asiaticum var. intermedium;

(b) fractionating the extract of Trachelospermum asiaticum var. intermedium of step (a) with an organic solvent and performing column chromatography to obtain a fraction; and

(c) performing high-performance liquid chromatography to isolate the compound from the fraction of step (b).
The pharmaceutical composition of claim 3, wherein the solvent of step (a) is selected from the group consisting of water, alcohols of 1 to 6 carbon atoms, and a combination thereof.
The pharmaceutical composition of claim 3, wherein the organic solvent of step (b) is selected from the group consisting of methanol, chloroform, acetate, hexane, dichloromethane, acetone, acetonitrile, benzene and a combination thereof.
The pharmaceutical composition of claim 1, being suppressive of release of nitric oxide (NO), TNF-α, IL-6 or IL-1β.
The pharmaceutical composition of claim 1, wherein the inflammatory disease is selected from the group consisting of edema, dermatitis, allergy, atopy, asthma, conjunctivitis, peridontitis, rhinitis, otitis media, laryngopharyngitis, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn’s disease, colitis, hemorrhoid, gout, ankylosing spondylitis, rheumatic fever, systemic lupus erythematosus, fibromyalgia, psoriatic arthritis, osteoarthritis, rheumatoid arthritis, periarthritis of shoulder, tendonitis, tenosynovitis, myositis, hepatitis, cystitis, nephritis, sjogren's syndrome and multiple sclerosis.
A quasi-drug composition for preventing or improving inflammation, comprising the compound represented by Chemical Formula 1 of claim 1.
A health functional food composition for preventing or improving inflammation, comprising the compound represented by Chemical Formula 1 of claim 1.
A cosmetic composition for preventing or improving inflammation, comprising the compound represented by Chemical Formula 1 of claim 1.
A method for treating an inflammatory disease, comprising administering the pharmaceutical composition of any one of claims 1 to 7 to a subject suspected of having the inflammatory disease.