WO2010079914A2

WO2010079914A2 - A composition comprising the compound isolated from the extract of rubiae radix for preventing and treating inflammatory diseases

Info

Publication number: WO2010079914A2
Application number: PCT/KR2009/007837
Authority: WO
Inventors: Young Ho Kim; Do Youn Jun; Hyun Ju Woo; Mi Hee Woo; Chae Ha Yang
Original assignee: Industry Academic Coorperation Foundation of Daegu Haany University
Current assignee: Industry Academic Coorperation Foundation of Daegu Haany University
Priority date: 2009-01-06
Filing date: 2009-12-28
Publication date: 2010-07-15
Anticipated expiration: 2011-07-06
Also published as: WO2010079914A3; KR20100081627A; KR101072663B1

Abstract

The present invention is related to the inventive compositions comprising the compounds isolated from Rubiae radix showing potent anti-inflammatory effect through various experiments, therefore, it can be used as the effective and safe therapeutics or health food for treating and preventing inflammatory diseases.

Description

A COMPOSITION COMPRISING THE COMPOUND ISOLATED FROM THE EXTRACT OF RUBIAE RADIX FOR PREVENTING AND TREATING INFLAMMATORY DISEASES

The present invention relates to a composition comprising the compound isolated from the extract of Rubiae radix for preventing and treating inflammatory disease.

Inflammation is a barrier response in the body against the various stimuli caused by the contamination with various contaminants such as bacteria, virus or tissue injury etc, which plays an initial protective role in order to limiting the damage to the damaged region or injured region. Mostly, those inflammatory response results in the removal of etiological factors using the components of innate immunnity, and the induction of specific acquired immunity etc (Hawiger J., Innate Immunity and inflammation: A Transcriptional Paradigm, Immunologic Research, 23, pp99-109, 2001).

Inflammatory response is associated with rubor, tumor, calor, dolor etc, which results in consecutive immune responses such as the increase of focal blood flow and the decrease of blood velocity according to blood vasodilation caused by the action of inflammatory mediator, cytokines etc at inflamed area; the increased efflux of plasma components according to increased blood permeability, the increased efflux of immunocyte according to increased adhesive ability of blood endothelial cell to the circulatory immunocyte and increased movement to infected region caused by chemotaxis etc (Gallo RL et al., Biology and Clinical Relevance of naturally occurring antimicrobial peptides, J. Allergy Clin. Immunol., 110, pp823-831, 2002; Graem EB et al., Acute Inflammation, American Journal of Pathology, 86(1), pp185-274, 1977).

Inflammatory response at the infected region initiates the macrophage response against outer germ and inflammatory mediators such as ROS (reactive oxygen species), RNS (Reactive nitrogen species), prostaglandins, leukotriene etc, as well as pro-inflammatory cytokines such as TNF-α, IL-6, IL-1β etc are reported to be involved in inflammatory responses (Renauld JC, New Insights into the role of cytokines in asthma, J. Clin. Pathol., 54, pp577-589, 2001; Blake GJ et al., Tumor necrosis factor-α, inflammatory biomarkers, and atherogenesis, Eur. Heart J., 23, pp345-347, 2002). The activation of NF-κB, a transcription factor inducing the gene expression of the inflammatory mediators, plays important role in those inflammatory involved response of macrophage. The genes involved in inflammation such as iNOS (inducible nitric oxide synthase), cyclooxygenase (COX-2), TNF-α, IL-6, IL-1βetc in macrophage cell are transcribed by NF-κB.

NO (nitric oxide), a free radical having short half-life, has been reported to be produced during the oxidation reaction of L-arginine with L-citrulline by the catalyzed action of NOS (nitric oxide synthase). It shows various immune-related function in vivo (Moncada S. et al., Nitric oxide: Physiology, Pathology and Phamarcology. Phamacol. Rev., 43, pp109-142, 1991), however the over-production of NO may show cell-toxicity, which causes to the damage of host cell and tissue as well as may be involved in various pathological syndromes such as atherosclerosis, carcinogenesis etc (Mordan L. J, et al., Inhibitors of endogeneous nitrogen oxide formation block the promotion of neoplastic transformation in C3H10T1/2 fibroblasts, Carcinogenesis, 14, pp1555-1559, 1993; Lirk P. et al., Inducible nitric oxide synthase, Time for reappraisal, Current. Drug. Targets. Inflamm. Allergy, 1, pp.89-108, 2002). There exist three kinds of NOS catalyzing NO reproduction, and nNOS (neuronal NOS, NOS1) and eNOS (endothelial NOS, NOS3) always express constitutively, while iNOS is an inducible one which is transcribed by only transcription factors, NF-κB activated by LPS (lipopolysaccharide), a bacterial endotoxin.

A transcription factor, NF-κB binding with IκB, an its inhibitor, exists as an inactivated form within macrophage whereas NF-κB becomes activated through the phosphorylation of IκB by the action of signaling pathway induced when bacterial LPS binds to Toll-like receptor on cell surface, which results in the release and removal of IκB by the action of proteosome’s dissolution process. In the result, NF-κB is moved to nucleus to induce the transcription of inflammation-related genes and NF-κB activation is reported to be induced by Akt signaling pathway (Hattori Y. et al, lipopolysaccharide activate Akt in vascular smooth muscle cells resulting in the induction of inducible nitric oxide synthase through nuclear factor-k B activation, Eur. J. Pharmacol., 481, pp.153-158, 2003); as well as the signaling pathways of ERK, c-jun- and p38-MAPK (Kim SH, et al., Selenium attenuates lipopolysaccharide-induced oxidative stress responses through modulation of p38 MAPK and NF-κB signaling pathways, Exp. Biol. Medicine, pp565-701, 2004; Robinson MJ et al, mitogen-activated protein kinase pathways, Cur. Opi. Cell Biol., 9, pp.180-186, 1997).

The control of gene expression at the stage of iNOS transcription in connection with inflammation response, is mostly important to determine the duration period and reproduced level of NO. Accordingly, the control mechanism of iNOS expression and enzymatic activity of iNOS has been used as a main target to study novel ant-inflammatory agent for improving or treating chronic inflammatory disease.

Rubiae Radix is dried root of Rubia cordifolia L, which is belonged to Rubiaceae and cultivated in Korea and China. The radix has been reported to contain various anthraquinones, i.e., alizarin, rubierythric acid, purpurin, xanthopurpurin, munjistin, pseudopurpurin etc.

However, there has been not reported or disclosed on the therapeutic effect of the compound isolated from the extract of Rubiae radix on inflammatory diseases in any of above cited literatures, the disclosures of which are incorporated herein by reference.

Accordingly, the present inventors have confirmed that the compound isolated from the extract of Rubiae radix shows potent anti-inflammatory effect through various experiments, i.e., the inhibitory effect on the NO and PGE2 reproduction and the reproduction of pro-inflammatory cytokines such as iNOS, COX-2, TNF-α, IL-6 and IL-1βwhich is induced by LPS treatment as well as the reducing effect on gene expression of p-IκBα, iNOS, and COX-2 protein using by RAW 264.7 cell line together with in vivo animal model test, therefore, it can be used as the effective and safe therapeutics or health food for treating and preventing inflammatory disease.

According to one aspect of the present invention, the present invention provides a composition comprising the compound isolated from the extract of Rubiae radix for the prevention and treatment of inflammatory disease.

Accordingly, it is an object of the present invention to provide a pharmaceutical composition comprising the compound isolated from the extract of Rubiae radix as an active ingredient for the treatment and prevention of inflammatory diseases.

The term “the compound isolated from the extract of Rubiae radix “disclosed herein comprise at least one compound(s) selected from the group of 2-carbomethoxy-2,3-epoxy-3-prenyl-1,4-naphthoquinone, 3,3’-bis (3,4-dihydro-4-hydroxy-6-methoxy-2H-1-benzofuran), epoxymollugin, soranjidiol, oleanolic acid, 1-acetoxy-3-methoxy-9,10-anthraquinone, alizarin-2-methyl ether, furonollugin and mollugin, especially, 2-carbomethoxy-2,3-epoxy-3-prenyl-1,4-naphthoquinone, which may isolated from the extract of Rubiae radix.

The term “treatment and prevention of inflammatory diseases” disclosed herein is performed by way of inhibiting the NO reproduction and the reproduction of pro-inflammatory cytokines such as iNOS, COX-2, TNF-α, IL-6 and IL-1β as well as reducing gene expression of p-IκBα, iNOS, and COX-2 protein.

The term “inflammatory diseases” disclosed herein comprises atopic dermatitis, joint arthritis, urethritis, cystitis, artherosclerosis, allergy disease, rhinitis, asthma, asthma, acute pain disease, chronic pain disease, periodontitis, gingivitis, inflammatory intestine disease, gout, myocardiac infarction, congestive heart failure, hypertension, angina pectoris, stomach ulcer, ischemic stroke, Down syndrome, multiple sclerosis, obesity, diabetes, dementia, depression, schizophrenia, tuberculosis, sleep disturbance, sepsis, burn or pancreatitis, preferably, atopic dermatitis, joint arthritis, urethritis, cystitis, or periodontitis, more preferably, urethritis, cystitis, or periodontitis, which is occurred by the overproduction of NO and the over-reproduction of pro-inflammatory cytokines such as iNOS, COX-2, TNF-α, IL-6 and IL-1β.

The inventive compounds of the present invention can be transformed into their pharmaceutically acceptable salt and solvates by the conventional method well known in the art. For the salts, acid-addition salt thereof formed by a pharmaceutically acceptable free acid thereof is useful and can be prepared by the conventional method. For example, after dissolving the compound in the excess amount of acid solution, the salts are precipitated by the water-miscible organic solvent such as methanol, ethanol, acetone or acetonitrile to prepare acid addition salt thereof and further the mixture of equivalent amount of compound and diluted acid with water or alcohol such as glycol monomethylether, can be heated and subsequently dried by evaporation or filtrated under reduced pressure to obtain dried salt form thereof.

As a free acid of above-described method, organic acid or inorganic acid can be used. For example, organic acid such as methansulfonic acid, p-toluensulfonic acid, acetic acid, trifluoroacetic acid, citric acid, maleic acid, succinic acid, oxalic acid, benzoic acid, lactic acid, glycolic acid, gluconic acid, galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid, ascorbic acid, carbonylic acid, vanillic acid, hydroiodic acid and the like, and inorganic acid such as hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, tartaric acid and the like can be used herein.

Further, the pharmaceutically acceptable metal salt form of inventive compounds may be prepared by using base. The alkali metal or alkali-earth metal salt thereof can be prepared by the conventional method, for example, after dissolving the compound in the excess amount of alkali metal hydroxide or alkali-earth metal hydroxide solution, the insoluble salts are filtered and remaining filtrate is subjected to evaporation and drying to obtain the metal salt thereof. As a metal salt of the present invention, sodium, potassium or calcium salt are pharmaceutically suitable and the corresponding silver salt can be prepared by reacting alkali metal salt or alkali-earth metal salt with suitable silver salt such as silver nitrate.

The pharmaceutically acceptable salt of the present invention comprise all the acidic or basic salt which may be present at the compounds, if it does not indicated specifically herein. For example, the pharmaceutically acceptable salt of the present invention comprise the salt of hydroxyl group such as the sodium, calcium and potassium salt thereof; the salt of amino group such as the hydrogen bromide salt, sulfuric acid salt, hydrogen sulfuric acid salt, phosphate salt, hydrogen phosphate salt, dihydrophosphate salt, acetate salt, succinate salt, citrate salt, tartarate salt, lactate salt, mandelate salt, methanesulfonate (mesylate) salt and p-toluenesulfonate (tosylate) salt etc, which can be prepared by the conventional method well known in theart.

The present invention also provided a use of the compound isolated from the extract of Rubiae radix for the preparation of therapeutic agent for the treatment and prevention of inflammatory disease in mammal or human.

It is an object of the present invention to provide a method of treating or preventing inflammatory disease in human or mammal, wherein the method comprises administering a therapeutically effective amount of the compound isolated from the extract of Rubiae radix, as an effective ingredient, together with a pharmaceutically acceptable carrier thereof.

The term “extract” disclosed herein comprises crude extract, polar solvent soluble extract and non-polar solvent soluble extract of the extract of Rubiae radix.

Specifically, the term “crude extract” disclosed herein comprises the extract prepared by extracting plant material with water, lower alcohols such as methanol, ethanol, or the mixtures thereof, preferably, methanol or 50-100% methanol.

The term “polar solvent soluble extract” disclosed herein can be prepared by extracting the above described crude extract with polar solvent, for example, water, lower alcohol such as methanol, ethanol, preferably, butanol and the like, or the mixtures thereof.

The term “non-polar solvent soluble extract” disclosed herein can be prepared by extracting the above described crude extract with non-polar solvent, for example, hexane, methylene chloride, ethyl acetate or chloroform, preferably, methylene chloride.

The inventive compounds of the present invention may be chemically synthesized by the methods well-known in the art or be isolated from the extract of Rubiae radix belonged to Rubiaceae family which will be explained as follows, which are merely exemplary and in no way limit the invention.

Hereinafter, the present invention is described in detail.

An inventive extract of the present invention can be prepared in detail by following procedures.

For example, Rubia radix is dried, cut, crushed, and added to 1 to 20-fold, preferably, approximately, 1 to 7-fold volume of distilled water, C₁to C₄ lower alcohols or the mixtures thereof, preferably, the mixture of water and methanol; the solution is treated with hot water at the temperature ranging from 10℃∼100℃, preferably, 50℃∼90℃, for the period ranging from 1 to 20 hours, preferably, 5 to 15 hours with the extraction method by the extraction with hot water, cold water, reflux extraction, or ultra-sonication extraction, preferably, extraction with hot water; the extract is collected with filtration, concentrated under reduced pressure and dried to obtain an crude extract of the present invention.

And then, the crude extract prepared by above step, is suspended in water, and then is mixed with non polar solvent such as hexane, chloroform, or ethyl to fractionate into water soluble fraction and non-polar solvent soluble fraction;; the non-polar solvent soluble layer is collected to obtain non-polar solvent soluble extract of the present invention.

And then, the non-polar solvent soluble extract prepared by above step, is performed to repeated purification process using by flash column chromatography, RP C18 column chromatography and Silica gel column chromatography with increasing the polarity of developing solvent system such as mixture solvent of hexane and chloroform to afford the inventive compounds isolated from the extract of Rubia radix, i.e., 2-carbomethoxy-2,3-epoxy-3-prenyl-1,4-naphthoquinone, 3,3’-bis (3,4-dihydro-4-hydroxy-6-methoxy-2H-1-benzofuran), epoxymollugin, soranjidiol, oleanolic acid, 1-acetoxy-3-methoxy-9,10-anthraquinone, alizarin-2-methyl ether, furonollugin and mollugin.

Also, the above-described procedures may be modified or subjected to further step to fractionate or isolate more potent fractions or compounds by conventional procedure well- known in the art, for example, the procedure disclosed in the literature (Harborne J. B. Phytochemical methods: A guide to modern techniques of plant analysis, 3^rdEd. pp6-7, 1998).

Accordingly, the present invention also provided a pharmaceutical composition comprising the compounds isolated from the extract of Rubia radix prepared by the above-described preparation method as an active ingredient and a pharmaceutically acceptable carrier thereof for treating and preventing inflammatory disease.

The present invention also provided a use of the compounds isolated from the extract of Rubia radix prepared by the above-described preparation method for the preparation of therapeutic agent for the treatment and prevention of inflammatory disease in mammal or human.

It is an object of the present invention to provide a method of treating or preventing inflammatory disease in human or mammal, wherein the method comprises administering a therapeutically effective amount of the compounds isolated from the extract of Rubia radix prepared by the above-described preparation method, as an effective ingredient, together with a pharmaceutically acceptable carrier thereof.

It is confirmed that the inventive compounds of the present invention prepared by above-described method shows potent anti-inflammatory effect through various experiments, i.e., the inhibitory effect on the NO and PGE2 reproduction and the reproduction of pro-inflammatory cytokines such as iNOS, COX-2, TNF-α, IL-6 and IL-1βwhich is induced by LPS treatment as well as the reducing effect on gene expression of p-IκBα, iNOS, and COX-2 protein using by RAW 264.7 cell line together with in vivo in animal model test.

The inventive composition for treating and preventing inflammatory diseases may comprises the above-described compound as 0.1 ~ 50% by weight based on the total weight of the composition.

The inventive composition may additionally comprise conventional carrier, adjuvants or diluents in accordance with a using method well known in the art. It is preferable that said carrier is used as appropriate substance according to the usage and application method, but it is not limited. Appropriate diluents are listed in the written text of Remington’s Pharmaceutical Science (Mack Publishing co, EastonPA).

Hereinafter, the following formulation methods and excipients are merely exemplary and in no way limit the invention.

The composition according to the present invention can be provided as a pharmaceutical composition containing pharmaceutically acceptable carriers, adjuvants or diluents, e.g., lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starches, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinyl pyrrolidone, water, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate and mineral oil. The formulations may additionally include fillers, anti-agglutinating agents, lubricating agents, wetting agents, flavoring agents, emulsifiers, preservatives and the like. The compositions of the invention may be formulated so as to provide quick, sustained or delayed release of the active ingredient after their administration to a patient by employing any of the procedures well known in the art.

For example, the compositions of the present invention can be dissolved in oils, propylene glycol or other solvents that are commonly used to produce an injection. Suitable examples of the carriers include physiological saline, polyethylene glycol, ethanol, vegetable oils, isopropyl myristate, etc., but are not limited to them. For topical administration, the extract of the present invention can be formulated in the form of ointments and creams.

Pharmaceutical formulations containing present composition may be prepared in any form, such as oral dosage form (powder, tablet, capsule, soft capsule, aqueous medicine, syrup, elixirs pill, powder, sachet, granule), or topical preparation (cream, ointment, lotion, gel, balm, patch, paste, spray solution, aerosol and the like), or injectable preparation (solution, suspension, emulsion).

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

The desirable dose of the inventive extract or compound varies depending on the condition and the weight of the subject, severity, drug form, route and period of administration, and may be chosen by those skilled in the art. However, in order to obtain desirable effects, it is generally recommended to administer at the amount ranging 0.1 to 1000 mg/kg, preferably, 1 to 100 mg/kg by weight/day of the inventive extract of the present invention. The dose may be administered in single or divided into several times per day. In terms of composition, the amount of inventive extract should be present between 0.01 to 50% by weight, preferably 0.5 to 40% by weight based on the total weight of the composition.

The pharmaceutical composition of present invention can be administered to a subject animal such as mammals (rat, mouse, domestic animals or human) via various routes. All modes of administration are contemplated, forexample, administration can be made orally, rectally or by intravenous, intra muscular, sub cutaneous, intra-cutaneous, intrathecal, epidural or intra-cerebroventricular injection.

Accordingly, it is the other object of the present invention to provide a functional health food comprising an extract of the compound isolated from the extract of Rubiae radix as an active ingredient for the improvement and prevention of inflammatory diseases.

The term “a functional health food” defined herein is “the functional food having enhanced functionality such as physical functionality or physiological functionality by adding the extract of the present invention to conventional food to prevent or improve aimed disease in human or mammal”.

It is the other object of the present invention to provide a health care food comprising compound isolated from the extract of Rubiae radix, together with a sitologically acceptable additive for the prevention and alleviation of aimed disease.

The term “a health care food” defined herein is “the food containing inventive extract or compounds of the present invention showing no specific intended effect but general intended effect in a small amount of quantity as a form of additive or in a whole amount of quantity as a form of capsule, pill, tablet etc.

The term “a sitologically acceptable additive” defined herein is “any substance the intended use which results or may reasonably be expected to result-directly or indirectly-in its becoming a component or otherwise affecting the characteristics of any food” for example, thickening agent, maturing agent, bleaching agent, sequesterants, humectant, anti-caking agent, clarifying agents, curing agent, emulsifier, stabilizer, thickner, bases and acid, foaming agents, nutrients, coloring agent, flavoring agent, sweetner, preservative agent, antioxidant, etc, which had been well-known in the art.

If a substance is added to a food for a specific purpose in that food, it is referred to as a direct additive and indirect food additives are those that become part of the food in trace amounts due to its packaging, storage or other handling.

Above described health foods can be contained in food, health beverage, dietary therapy etc, and may be used as a form of powder, granule, tablet, chewing tablet, capsule, beverage etc for preventing or improving aimed disease.

Also, inventive extract or compounds can be added to food or beverage for prevention and improvement of aimed disease. The amount of inventive extract or compounds in food or beverage as a functional health food or health care food may generally range from about 0.01 to 100 w/w % of total weight of food for functional health food composition. In particular, although the preferable amount of inventive extract of the present invention in the functional health food, health care food or special nutrient food may be varied in accordance to the intended purpose of each food, it is preferably used in general to use as a additive in the amount of inventive extract of the present invention ranging from about 0.01 to 5% in food such as noodles and the like, from 40 to 100% in health care food on the ratio of 100% of the food composition.

Providing that the health beverage composition of present invention contains inventive extract or compounds as an essential component in the indicated ratio, there is no particular limitation on the other liquid component, wherein the other component can be various deodorant or natural carbohydrate etc such as conventional beverage. Examples of aforementioned natural carbohydrate are monosaccharide such as glucose, fructose etc; disaccharide such as maltose, sucrose etc; conventional sugar such as dextrin, cyclodextrin; and sugar alcohol such as xylitol, and erythritol etc. As the other deodorant than aforementioned ones, natural deodorant such as taumatin, stevia extract such as levaudioside A, glycyrrhizin et al., and synthetic deodorant such as saccharin, aspartam et al., may be useful favorably. The amount of above described natural carbohydrate is generally ranges from about 1 to 20 g, preferably 5 to 12 g in the ratio of 100 ㎖ of present beverage composition.

The other components than aforementioned composition are various nutrients, a vitamin, a mineral or an electrolyte, synthetic flavoring agent, a coloring agent and improving agent in case of cheese, chocolate et al., pectic acid and the salt thereof, alginic acid and the salt thereof, organic acid, protective colloidal adhesive, pH controlling agent, stabilizer, a preservative, glycerin, alcohol, carbonizing agent used in carbonate beverage et al. The other component than aforementioned ones may be fruit juice for preparing natural fruit juice, fruit juice beverage and vegetable beverage, wherein the component can be used independently or in combination. The ratio of the components is not so important but is generally range from about 0 to 20 w/w % per 100 w/w % present composition. Examples of addable food comprising aforementioned extract therein are various food, beverage, gum, vitamin complex, health improving food and the like.

It will be apparent to those skilled in the art that various modifications and variations can be made in the compositions, use and preparations of the present invention without departing from the spirit or scope of the invention.

The present invention is more specifically explained by the following examples. However, it should be understood that the present invention is not limited to these examples in any manner.

The present invention comprising the compound isolated from the extract of Rubiae radix shows potent anti-inflammatory effect through various experiments, i.e., the inhibitory effect on the NO and PGE2 reproduction and the reproduction of pro-inflammatory cytokines such as iNOS, COX-2, TNF-α, IL-6 and IL-1βwhich is induced by LPS treatment as well as the reducing effect on gene expression of p-IκBα, iNOS, and COX-2 protein using by RAW 264.7 cell line together with in vivo animal model test, therefore, it can be used as the effective and safe therapeutics or health food for treating and preventing inflammatory disease.

The present invention is more specifically explained by the following figures and examples. However, it should be understood that the present invention is not limited to these examples in any manner.

The above and other objects, features and other advantages of the present invention will more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which;

Fig. 1 shows the cell cytotoxicity result of the compounds isolated from the extract of Rubiae radix (0, 6.25 and 12.5 microgram/ml);

Fig. 2 shows the decreasing effect on the level of NO reproduction of the compounds isolated from the extract of Rubiae radix (0, 6.25 and 12.5 microgram/ml);

Fig. 3 shows the decreasing effect on the level of NO reproduction of 2-carbomethoxy-2,3-epoxy-3-prenyl-1,4-naphthoquinone (0, 1, 2, and 4 microgram/ml);

Fig. 4 shows the cell cytotoxicity result of 2-carbomethoxy-2,3-epoxy-3-prenyl-1,4-naphthoquinone (0, 1, 2, and 4 microgram/ml);

Fig. 5 presents decreasing effect on the reproduced level of inflammation-related proteins (iNOS, COX-2) of 2-carbomethoxy-2,3-epoxy-3-prenyl-1,4-naphthoquinone;

Fig. 6 presents decreasing effect on the reproduced level of inflammation-related proteins (p-IkBα, IkBα) of 2-carbomethoxy-2,3-epoxy-3-prenyl-1,4-naphthoquinone;

Fig. 7 presents the inhibitory effect of 2-carbomethoxy-2,3-epoxy-3-prenyl-1,4-naphthoquinone on the mRNA expression of iNOS, COX-2, TNF-α, IL-6 and IL-1β(0, 1, 2, and 4 microgram/ml);

Fig. 8 presents the inhibitory effect of 2-carbomethoxy-2,3-epoxy-3-prenyl-1,4-naphthoquinone on the level of IL-6 in RAW264.7 cell induced by LPS treatment (0, 1, 2, and 4 microgram/ml);

Fig. 9 presents the inhibitory effect of 2-carbomethoxy-2,3-epoxy-3-prenyl-1,4-naphthoquinone on the level of TNF-α in RAW264.7 cell induced by LPS treatment (0, 1, 2, and 4 microgram/ml).

Fig. 10 represents the inhibitory effect of 2-carbomethoxy-2,3-epoxy-3-prenyl-1,4-naphthoquinone on the level of IL-1β in RAW264.7 cell induced by LPS treatment (0, 1, 2, and 4 microgram/ml);

Fig. 11 represents the inhibitory effect of 2-carbomethoxy-2,3-epoxy-3-prenyl-1,4-naphthoquinone on the level of reproduced level of prostaglandin E2 in RAW264.7 cell induced by LPS treatment (0, 1, 2, and 4 microgram/ml);

Fig. 12 represents the inhibitory effect of 2-carbomethoxy-2,3-epoxy-3-prenyl-1,4-naphthoquinone on the expressed level of NF-k B protein in nucleus as well as IkB protein blocking the shift of NF-kB to nucleus in RAW264.7 cell induced by LPS treatment (0, 1, 2, and 4 microgram/ml);

Fig. 13 represents the inhibitory effect of 2-carbomethoxy-2,3-epoxy-3-prenyl-1,4-naphthoquinone on the binding affinity of p65 in RAW264.7 cell induced by LPS treatment (0, 1, 2, and 4 microgram/ml);

Fig. 14 represents the inhibitory effect of 2-carbomethoxy-2,3-epoxy-3-prenyl-1,4-naphthoquinone on the PMA-induced ear edema of mouse in vivo.

The following Reference Example, Examples and Experimental Examples are intended to further illustrate the present invention without limiting its scope.

Example 1. Preparation of extract of Rubiae radix

1-1. crude extract of Rubiae radix

10 g of dried power of Rubiae radix purchased from Daegu Yangyeongsi Herb Medicine Market in Korea, were added to 40.5 L of 95% methanol and the solution was refluxed for 8 hours with stirring at 80℃. The residue was filtered and the extraction process was repeated four times. The filtrate was collected and concentrated to obtain 1,048.4 g of methanol soluble extract of Rubia radix.

1-2. methylene chloride soluble extract of Rubiae radix

1,048.4 g of methanol soluble extract of Rubiae radix was suspended in 2.2 L of distilled water and 2 L of methylene chloride was added thereto to fractionate into two fractions, i.e., water layer and methylene chloride soluble layer. The methylene chloride soluble layer was collected and concentrated with vaccuo to obtain 360 g of methylene chloride soluble extract of Rubiae radix.

Example 2. Preparation of the compounds isolated from the extract of Rubiae radix

360 g of methylene chloride soluble extract of Rubiae radix prepared in step 1-2, was performed to flash column chromatography [YMC Gel ODS-A, 60A, 22 mesh; column size (75cm x 9 cm); stationary phase (230-400 mesh silica gel); eluting solvent = 100% hexane] to obtain 360 g of purified methylene chloride soluble extract and then Silica gel column chromatography [stationary phase (720 g of 70-230 mesh silica gel); eluting solvent as a mobile phase = Hexane: methylene chloride(98:2 → 95:5→ 90:10 → 85:15 → 80:20 → 60:40 → 50:50) to methylene chloride:methanol(99:1 → 98:2 → 96:4] to afford 34 fractions, i.e., fr. RA-MC-1～ RA-MC-34 according to TLC pattern.

Among the 34 fractions, 6.2 g of the 15^thfraction (RA-MC-15) dissolved in 100% CH₃CN was performed to RPC18 column chromatography [columnsize (57cm x 3.5cm) ; filled with stationary phase (40-63 micrometer RPC18) to about 22cm; eluting solvent = started with CH₃CN:H₂O(3:7)] to afford 1.2 g of purified fraction (RA-MC-15-7) and then the fraction adsorbed with 0.72 g of Silica gel SiO₂open column chromatography [columnsize (53cm x 2.3cm); filled with stationary phase (less than 70 mesh silica gel) to about 15cm; eluting solvent = hexane:EtOAc (100:0 → 18:0.3)] to obtain 754 mg of 2-carbomethoxy-2,3-epoxy-3-prenyl-1,4-naphthoquinone (RA-MC-AA).

51 mg of 3,3’-bis (3,4-dihydro-4-hydroxy-6-methoxy-2H-1-benzofuran) (RA-MC-BB), 36.4 mg of epoxymollugin (RA-MC-CC), 30 mg of soranjidiol(RA-MC-DD)., 28mg of oleanolic acid(RA-MC-EE)., 31 mg of 1-acetoxy-3-methoxy-9,10-anthraquinone(RA-MC-FF), 42 mg of alizarin-2-methyl ether(RA-MC-GG), 76 mg of furonollugin(RA-MC-Ha). and 670 mg of mollugin(RA-MC-Hb) were isolated from the purified fractions (RA-MC) according to the similar methods to the above-described method respectively ( see Table 1)

Table 1

Plant	Name	Chemical name of Isolated compounds
Rubiae radix	RA-MC-AA	2-carbomethoxy-2,3-epoxy-3-prenyl-1,4-naphthoquinone
	RA-MC-BB	3,3’-bis(3,4-dihydro-4-hydroxy-6-methoxy-2H-1-benzofuran)
	RA-MC-CC	epoxymollugin
	RA-MC-DD	soranjidiol
	RA-MC-EE	oleanolic acid
	RA-MC-FF	1-acetoxy-3-methoxy-9, 10-anthraquinone
	RA-MC-GG	alizarin-2-methyl ether
	RA-MC-Ha	furonollugin
	RA-MC-Hb	mollugin

Reference Example 1. Preparation of Reagents

DMEM (Dubecco’s modified eagle medium) and FBS (Fetal bovine serum) were procured from Hyclone Co. Ltd. (Gaithersbug, MD, USA); LPS (Lipopolysaccaride), Griess reagent, PMA (Phorbol 12-myristate 13-acetate) and MTT (3-(4,5-dimthylthiazol-2-yl)-2,5-dipheny-tetrazolium bromide) were procured from Sigma Co. Ltd. (St. Louis, MO, USA); BCA Protein assay reagent was procured from Pierce Co. Ltd. (Rockford, IL, USA); ECL Western Blot reagent was procured from Amersham Co. Ltd. (Arlington Heights, IL, USA); anti-IκBα, and anti-p-IκBαmonoclonal antibody were procured from Cell Signaling Co. Ltd.; mAb (iNOS monoclonal antibody) was procured from BD science Co. Ltd.; COX-1, COX-2 and NF-kB p65 antibody was procured from Santa Cruz Biotechnology Co. Ltd.; and anti-mouse or anti-goat IgG horseradish peroxide (HRP)-conjugated antibody were procured from Amersham Co. Ltd. (Arlington Heights, IL, USA).

All the reagent used in the experiment were higher grade than analytical grade.

Reference Example 2. Cell Culture

RAW264.7 cell line, a mouse macrophage cell, (ATCC, No. TIB-71) was culture in DMEM medium (Dulbecco’s Modified eagle Medium) supplemented with 10% FBS and 100 microgram/ml of gentamycin at 37oC in humidified 5% CO₂incubatorand used in the experiment.

Experimental Example 1. Cell Cytotoxicity

To determine the cell cytotoxicity of the compounds prepared in Examples, MTT assay was performed according to the method disclosed in the literature (Jun DY et al., Apoptogenic activity of Zanthoxylum schinifolium toward human acute leukemia Jurkat T cells is associated with ER-stress-mediated caspase-8 activation that stimulates mitochondria-dependent or independent cascade, Carcinogenesis, 28, pp1303-1313, 2007).

RAW264.7 cell was inoculated into 96-well plate with adjusting cell concentration to 2 x 10⁵cells/well to incubate for 16 hours and various concentrations of the compounds prepared in Example 2 was added thereto. 0.1 microgram/ml of LPS was added thereto to incubate for 12 hours and 50 microliter of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] to react with each other for 4 hours. After formazan was formed, the solution was centrifuged for 15 min sat the speed of 2300 rpm to remove its supernatant and 150 microliter/well of DMSO was added thereto to dissolve the formazan. Resulting absorbance was determined at 540nm using by ELISA reader (Molecular Devices, Thermo Max, USA).

At the result, the group treated with 6.25 microgram/ml of test sample did not significantly affect on cell viability of RAW264.7 cell whereas the group treated with 12.5 microgram/ml of the test sample (2-carbomethoxy-2,3-epoxy-3-prenyl-1,4-naphthoquinone) showed more reduced cell survival rate by 23.2% compared with the control group, which confirms that the test sample (2-carbomethoxy-2,3-epoxy-3-prenyl-1,4-naphthoquinone) showed cell cytotoxicity against RAW264.7 cell as can be seen in Table 2 and Fig. 1.

Table 2

Name	Cell viability (%)
Name	6.25 microgram/ml	12.5 microgram/ml
RA-MC-AA	85	23
RA-MC-BB	93	112
RA-MC-CC	108	111
RA-MC-DD	112	110
RA-MC-EE	110	93
RA-MC-FF	106	110
RA-MC-GG	108	102
RA-MC-Ha	108	109
RA-MC-Hb	120	115
LPS sole treatment	100
No treatment of LPS	114

Experimental Example 2. Determination of the amount of NO reproduction

To determine the effect on the amount of NO reproduction of the compounds prepared in Examples, the assay for determine the amount of NO reproduction was performed according to the method disclosed in the literature (Wang S. et al., J. Ethnopharmacol.,114(3),pp458-462,2007).

RAW264.7 cell was inoculated into 96-well plate with adjusting cell concentration to 2 x 10⁵cells/well to incubate for 16 hours and various concentrations of the compounds prepared in Example 2 was added thereto. 0.1 microgram/ml of LPS was added thereto to incubate for 16 hours and 100 microliter of supernatant was added with 100 microliter of Griess reagent to react with each other for 15 mins at room temperature in shadow condition. The absorbance was determined at 540 nm using by ELISA reader (Molecular Devices, Thermo Max, USA). The amount of NO in culture medium was determined using by dose dependent standard curve of sodium nitrite.

At the result, the test sample (6.25 microgram/ml; 2-carbomethoxy-2,3-epoxy-3-prenyl-1,4-naphthoquinone; compound A) potently inhibited the NO reproduction by almost 99% of mouse macrophage RAW264.7 cell ( See Table 3 and Fig.2).

Additionally, we have studied whether the test sample treatment groups with 1, 2 or 4 microgram/ml, also show inhibiting effect on the NO reproduction of RAW264.7 cell caused by LPS treatment or not.

At the result, it has been confirmed that the test sample treatment groups with 1, 2 or 4 microgram/ml, also inhibited showed the NO reproduction of RAW264.7 cell caused by LPS treatment in a dose dependent manner by 58%, 82% and 99% respectively ( See Fig.3). As can be seen in Fig.3, the test sample (2-carbomethoxy-2,3-epoxy-3-prenyl-1,4-naphthoquinone) did not show any cell cytotoxicity under the same concentrations in MTT assy.

Table 3

Name	NO synthesis inhibition (%)
Name	6.25 microgram/ml	12.5 microgram/ml
RA-MC-AA	99	100
RA-MC-BB	8	10
RA-MC-CC	3	7
RA-MC-DD	6	4
RA-MC-EE	19	40
RA-MC-FF	7	5
RA-MC-GG	7	6
RA-MC-Ha	10	6
RA-MC-Hb	43	92
LPS sole treatment	0

Experimental Example 3. Western Blot Analysis

To determine the effect on the gene expression of p-IκBα, iNOS, and COX-2 protein using by RAW 264.7 cell line of the compounds prepared in Examples, Western blot analysis was performed according to the method disclosed in the literature (Heo et al., J. Immunol., 179(9), pp6305-6310, 2007).

4 x 10⁶cells of RAW264.7 cell prepared in Reference Example 2, was washed with PBS (ice-cold phosphate buffered saline: 2.7 mM KCl, 137 mM NaCl, 10 mM Na₂HPO₄, 2 mM KH₂PO₄, pH 7.4) three times, suspended in lysis buffer (137 mM NaCl, 15 mM EGTA, 1 mM sodium orthovanadate, 15 mM MgCl₂, 0.1% Triton X-100, 25 mM MOPS, 2.5 microgram/ml proteinase inhibitor E64, pH 7.2), crushed with ultra grinder, reacted for 30 mins at 4℃ and centrifuged for 20 mins to obtain supernatant.

The same amount of protein was isolated with 4-12% SDS-PAGE (Sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and the isolated protein was transferred to Immobilon membrane. The membrane was reacted with blocking buffer (TBS solution comprising 3% non-fat milk and 0.1% Tween 20) for 1 hour to prevent non-specific binding with antibody, and then reacted with the respective antibodies (anti-iNOS and anti-COX 2) for 1 or 2 hours. The product was washed with TBST solution comprising 0.1% Tween 20 three times for 10 mins per every washing step, reacted with the secondary antibodies (anti-mouse or anti-rabbit IgG horseradish peroxidase-conjugated antibody) for the period ranging from 90 mins to 2 hours, and reacted with ECL system to check the protein on X-ray film (AGFA, Belgium). The quantitative analysis on the amount of protein for each sample was determined at 562 nm using by Micro BCA protein assay kit.

Macrophage cell plays an important roles in the initiation and development of inflammation response and Toll-like receptor of the cell surface was stimulated with bacterial LPS resulting in activating NF-κB through cell signaling pathway and then increasing the gene expression of iNOS due to the action of activated NF-κB. At the result, the reproduction of NO was increased with the increased catalytic activity of iNOS within the cell from L-arginine (Haiqi He, et al., MolecularImmunology,43,p783,2006).

At the result, the gene expression of iNOS and COX-2 involved in NF-κB activation was decreased with the treatment of test sample (2-carbomethoxy-2,3-epoxy-3-prenyl-1,4-naphthoquinone in a dose dependent manner ( See Fig.5).

Additionally, the phosphorylaton of IκBα involved in NF-κB activation was sharply increased under same condition while it was remarkably reduced in a dose dependent manner under the presence with test sample (2-carbomethoxy-2,3-epoxy-3-prenyl-1,4-naphthoquinone) ( See Fig.6).

Experimental Example 4. Reverse Transcription Polymerase Chain Reaction (RT-PCR)

To determine whether the inhibitory effect of test samples on NO reproduction is caused by the inhibitory mechanism on the gene expression of iNOS, mRNA and pro-inflammatory cytokines or not, RT-PCR was performed according to the method disclosed in the literature (Lee et al., J. Ethnopharmacol., 97, pp561-566, 2005).

Various concentrations of test sample (2-carbomethoxy-2,3-epoxy-3-prenyl-1,4-naphthoquinone), i.e., 1-4 microgram/ml, were treated to RAW264.7 cell for 1 hour, and the cell was pre-treated with 0.1 microgram/ml of LPS for 4 hours to induce NO reproduction. 4 hours after the treatment of LPS, the cell was treated with Trypsin-EDTA and the total RNA of the recovered cell was isolated using by TRIzole (Invitrogen, Carlsbad, CA, USA). The 1^ststrand of cDNA was synthesized from 2 microgram of total RNA using by MMLV-reverse transcriptase (GibcoBRL). The sequences of the primer used in the experiment was shown in Table 4.

Table 4

Genes		Oligonucleotide Sequence	Size
iNOS	Sense	5-ATGTCCGAAGCAAACATCAC-3 (SEQ. I.D. 1)	449 bp
	Anti-sense	5-TAATGTCCAGGAAGTAGGTG-3(SEQ.I.D.2)
COX-2	Sense	5-CAGCAAATCCTTGCTGTTCC-3′ (SEQ. I.D. 3)	490 bp
	Anti-sense	5-TGGGCAAAGAATGCAAACATC-3(SEQ.I.D.4)
TNF-α	Sense	5-TACTGAACTTCGGGGTGATCGGTCC-3(SEQ.I.D.5)	295 bp
	Anti-sense	5-CAGCCTTGTCCCTTGAAGAGAACC-3(SEQ.I.D.6)
IL-6	Sense	5-GAAATGATGGATGCTTCCAAACTGG-3(SEQ.I.D.7)	414 bp
	Anti-sense	5-GGATATATTTTCTGACCACAGTGAGG-3(SEQ.I.D.8)
IL-1β	Sense	5-CAAGGAGAACCAAGCAAC-3(SEQ.I.D.9)	350 bp
	Anti-sense	5-GGGGAAGGCATTAGAAAC-3(SEQ.I.D.10)
GAPDH	Sense	5-CCACTGGCGTCTTCACCAC-3(SEQ.I.D.11)	349 bp
	Anti-sense	5-CCTGCTTCACCACCTTTTG-3(SEQ.I.D.12)

The PCR was performed using by thermal cycler apparatus (Bio-Rad, USA) according to following procedure. After denaturing at 98℃ for 2 minutes, the PCR is performed in the order of reaction for 10 sec at 98℃, 30 sec at 50℃, 60 sec at 72℃. The cycles were repeated 20 times and the last extension was performed at 72℃ for 5 minutes. The product produced by PCR was subjected to electrophoresis (5 V / cm) in 1.8% agarose gel and stained for 5 minutes with 0.5 ㎍/㎖(microgram/ml) of ethidium bromide (EtBr). The stained product was washed for 10 minutes with distilled water and the result was determined at UV wavelength (260 nm).

At the result, the expressed mRNA of iNOS is remarkably increased in RAW264.7 cell treated with LPS but those of iNOS and COX-2 are decreased in a dose dependent manner in the cell treated with LPS and test sample (2-carbomethoxy-2,3-epoxy-3-prenyl-1,4-naphthoquinone). The expressed mRNAs of TNF-α, IL-6 and IL-1βare remarkably increased in RAW264.7 cell treated with LPS whereas those mRNAs are decreased in a dose dependent manner in the cell treated with LPS and test sample (2-carbomethoxy-2,3-epoxy-3-prenyl-1,4-naphthoquinone) ( See Fig.7).

Accordingly, it has been confirmed that the inhibitory effect of test sample on NO reproduction caused by LPS treatment is caused by inhibiting NF-κB activation involved in the gene expression of iNOS and the inhibition of NF-κB activation is caused by blocking the phosphorylation of IκBα, a inhibitory factor of NF-κB.

The inhibitory effect of test sample (2-carbomethoxy-2,3-epoxy-3-prenyl-1,4-naphthoquinone) on NF-κB activation is proved by the decreased level of TNF-α, IL-6 and IL-1β, pro-inflammatory factors induced by NF-κB activation.

In summary, the compounds isolated from the extract of Rubia radix, especially, 2-carbomethoxy-2,3-epoxy-3-prenyl-1,4-naphthoquinone decreased the phosphorylation of IκB, inhibited NF-κB activation, which results in inhibiting the gene transcription of TNF-α, IL-6 and IL-1β, pro-inflammatory factors induced by NF-κB activation and decreasing the level of TNF-α, IL-6 and IL-1β, and showed potent ant-inflammatory effect in the end.

Experimental Example 5. The determination of reproduced level of pro-inflammatory cytokines using by ELISA method

To determine the reproduced level of pro-inflammatory cytokines using by ELISA method, ELISA method was performed according to the method disclosed in the literature (Chun SC et al., Evid Based Complement Alternat Med, 4, 3, pp327-333, 2007).

After 3 x 10⁶RAW264.7 cell was pre-treated with 2-carbomethoxy-2,3-epoxy-3- prenyl-1,4-naphthoquinone in the concentration of 1-4 microgram/ml for 1 hour, the cell was treated with 0.1 microgram/ml of LPS to incubate for 4 hours and the effect of collected incubated supernatant on the reproduced level of IL-6 (Pierceendogen, Rockford, IL, USA, EM2IL6), TNF-α (Pierce endogen, Rockford, IL, USA, EMTNFA) and IL-1β (Pierce endogen, Rockford, IL, USA, EMIL1B) was determined by ELISA kit. Both of 50 microliter of biotinylat

ed antibody and various concentrations of 2-carbomethoxy-2,3-epoxy-3-prenyl-1

,4-naphthoquinone, i.e., 0,1,2 or 4 microgram/ml, were added to 96 well plate pre-coated with ant-mouse TNF-α, IL-6 and IL-1β and the plates were reacted for 2 hours at room temperature. The plate was washed with washing buffer three times and 100 microliter of streptavidin-horseradish peroxidase (HRP) solution was added thereto. The plate was reacted for 30 mins, washed three times and 100 microliter of 3,3’,5,5’-tetramethylbenzidine (TMB) substrate solution was added thereto to react for 30 mins in shadow. 100 microliter of stopping solution was added thereto and the absorbance was determined by plate reader (Titertek Multiskan Automatic ELISA microplate reader, Model MCC/340, Huntsville, AL, USA) at 450 nm.

At the result, various concentrations of 2-carbomethoxy-2,3-epoxy-3-prenyl-1,4-naphthoquinone, i.e., 0, 1, 2 or 4 microgram/ml decreased the level of IL-6, TNF-α and IL-1β induced by LPS treatment in a dose dependent manner (Figs. 8-10).

Experimental Example 6. The determination of reproduced level of prostaglandin E2 (PGE₂)

To determine the reproduced level of prostaglandin E2, following experiment was performed by the procedure according to the method disclosed in the literature (Chun SC et al., Evid Based Complement Alternat. Med, 4, 3, pp327-333, 2007).

After 3x 10⁶RAW264.7 cell was pre-treated with 2-carbomethoxy-2,3-epoxy-3-prenyl-1,4-naphthoquinone in the concentration of 1-4 microgram/ml for 1 hour, the cell was treated with 0.1 microgram/ml of LPS to incubate for 4 hours and the effect of collected incubated supernatant on the reproduced level of prostaglandin E2 was determined by ELISA kit (R&D Systems, Minneapolis, MN, USA, KG3004B). The cultures incubate was added to 96 well plate pre-coated with goat ant-mouse IgG antibody, and the plates were added with HRP-labeled PGE2 and anti-PGE2 monoclonal antibody to incubate for 18 hours at 4℃. The plate was washed with washing buffer three times and 100 microliter of streptavidin-horseradish peroxidase (HRP) solution was added. The plate was reacted for 30 mins, washed with washing buffer five times and 200 microliter of substrate solution was added thereto to react for 1 hour at 37℃. The absorbance was determined by plate reader (Titertek Multiskan Automatic ELISA microplate reader, Model MCC/340, Huntsville, AL, USA) at 405 nm.

At the result, 1, and 2 microgram/ml of 2-carbomethoxy-2,3-epoxy-3-prenyl-1,4-naphthoquinone, inhibited the level of NO reproduction in RAW264.7 cell induced by LPS treatment by 58% and 82% respectively. The expressed level of COX-2 (Cyclooxygenase 2) catalyzing the reproduction of PGE2 was remarkably inhibited by the test sample treatment, which is confirmed by Western blot analysis and RT-PCR. Accordingly, 1,2 and 4 microgram/ml of test sample inhibited the reproduction of PGE2 in RAW264.7 cell induced by LPS treatment by approximately 62, 85 and 100% respectively (Fig. 11).

Experimental Example 7. Extraction of cytoplasm and nucleus protein & EMSA (Electrophoretic Mobility Shift Assay)

To determine the effect of test sample on the shift from cytoplasm to nucleus as well as the binding ability to target DNA of NF-kB, Western blot analysis and EMSA were performed by the procedure according to the method disclosed in the literature (Ling et al., Blood, 92, 4, pp1334-1341, 1998).

To extract nucleus protein, after 5 x 10⁶RAW264.7 cell was pre-treated with 2-carbomethoxy-2,3-epoxy-3-prenyl-1,4-naphthoquinone in the concentration of 1-4 microgram/ml for 2 hour, the cell was treated with 0.1 microgram/ml of LPS to incubate for 2 hours. The cell was washed with cold PBS twice at 4℃ and the collected cells were suspended in buffer A (10mM HEPES, pH 7.2, 10mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1mM DTT, 1 mM PMSF, 2.5 microgram/ml of E-64) to react for 15 mins at 4℃. Nonidet P-40 (Sigma Co. Ltd., St. Louis, MO, USA, I3021) was added to the cell in the concentration of 0.63% to afford cell lysis and the cell lysis was centrifuged for 30 sec with the speed of 14000 rpm to afford the supernatant as cytoplasm protein solution. The pellet was suspended in buffer B (20 mM HEPES, pH 7.9, 25% glycerol, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM PMSF, 2.5 microgram/ml of E-64) to react for 20 mins at 4℃ and centrifuged at the speed of 14000 rpm for 10 mins to afford nucleus protein solution. In order to investigating the binding affinity against the target DNA of transcription factor (NFkB) included in nucleus protein, about 5 microgram of the nucleus protein was mixed with double-stranded NF-kB-target oligonucleotide having NF-kB binding moiety (5’-AGTTGAGGGGACTTTCCCAGGC-3’). The terminus of oligonucleotide was radio-labelled with[γ-³²P] ATP using by T4 polynucleotide kinase. To determine the reaction specificity between NF-kB and target oligonucleotide, non-labeled abundant oligonucleotide was added thereto by 10 and 30 fold to react and the supershift was confirmed by adding 1 microgram of anti-p65 antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA, sc-8008X). The DNA-protein complex was performed to electrophoresis on 4% polyacrylamide gel using by 0.5X TBE and then the gel was dried with gel drying apparatus (Bio-Rad laboratories Inc.,model 583) to expose onto X-ray film at -70℃.

At the result, various concentrations of 2-carbomethoxy-2,3-epoxy-3-prenyl-1,4-naphthoquinone, remarkably inhibited the expressed level of NF-k B protein in nucleus as well as IkB protein blocking the shift of NF-kB to nucleus in a dose dependent manner ( See Fig.12).

At the result of EMSA assay using the nucleus protein, various concentrations of 2-carbomethoxy-2,3-epoxy-3-prenyl-1,4-naphthoquinone, remarkably reduced the sharply increased binding affinity of p65 caused by LPS treatment ( See Fig.13). It has been confirmed that it is p65 specific binding by confirming the supershift band using the test group and anti-p65 antibody which are competitively treated with abundant non-labelled oligonucleotide with [γ-³²P] ATP.

Experimental Example 9. PMA induced ear edema (in vivo assay)

To determine the anti-inflammatory effect of test sample on the PMA-induced ear edema of mouse in vivo, following PMA induced ear oedema was performed by the procedure according to the method disclosed in the literature (Khan MN et al., Agric. Food Chem. 55, 17, pp6984-pp6988, 2007).

6-weeks aged male ICR mouse (Daehan Biolonk Co. Korea) weighing from 26 to 28g was used as an experimental animal. The animals were randomly divided into 5 groups and each group consists of 5 mice. 10 microliter of the test samples dissolved in ethanol was spread onto the ear and after 1 hour, PMA (Sigma St. Louis, MO; 0.4 microgram/10 microliter of ethanol) was spread on the inner surface of right ear to induce ear-edema. After 6 hours, the increased rate of ear thickness was calculated by comparing with that of left ear of anesthetized mouse by ether inhalation for 30 sec using digital thickness gauge (Mitutoyo Co., Kawasaki-shi, JAPAN).

The value of edema was calculated by the following math formulae 1.

MathFigure 1

At the result, it has been confirmed that the ear thickness of test group treated with 20, 40 and 60 microgram of 2-carbomethoxy-2,3-epoxy-3-prenyl-1,4-naphthoquinone was decreased by approximately 36-53% compared with that of PMA treatment, which confirms that IC₅₀of 2-carbomethoxy-2,3-epoxy-3-prenyl-1,4-naphthoquinone is 50 microgram.

In summary, it has been confirmed that the compounds isolated from Rubia radix shows potent anti-inflammatory effect through various experiments, therefore, it can be used as the effective and safe therapeutics or health food for treating and preventing inflammatory disease.

Experimental Example 10. Acute toxicity test of oral administration in rat

The acute toxicity test was performed by administrating inventive compounds to 6-weeks aged SPF Sprague-Dawley rats.

250 mg/kg, 500 mg/kg, 1000 mg/kg, 5000 mg/kg of inventive compounds was orally administrated to each group consisting of 2 rats and the symptoms of rats were observed for 14 days. After administrating the extract, all the clinical changes i.e., mortality, clinical signs, body weight changes was observed and blood test such as haematological test and hematological biochemistry test was performed. The abnormal changes of abdominal organ and thoracic organ were observed after autopsy.

There did not show any changes in mortality, clinical signs, body weight changes and gross findings in any group or either gender. Furthermore, there showed any toxicity in test group treated with 5000 mg/kg of inventive compounds.

Accordingly, it has been confirmed that the inventive compounds prepared in the present invention was potent and safe substance showing LD₅₀(more than 5000 mg/kg) in oral administration.

Hereinafter, the formulating methods and kinds of excipients will be described, but the present invention is not limited to them. The representative preparation examples were described as follows.

Preparation of injection

2-carbomethoxy-2,3-epoxy-3-prenyl-1,4-naphthoquinone 100mg

Sodim methabifulfite 3.0mg

Methyl paraben 0.8mg

Propyl paraben 0.1mg

Distilled water for injection optimum amount

Injection preparation was prepared by dissolving active component, controlling pH to about 7.5 and then filling all the components in 2 ml ample and sterilizing by conventional injection preparation method.

Preparation of powder

2-carbomethoxy-2,3-epoxy-3-prenyl-1,4-naphthoquinone 500mg

Corn Starch 100mg

Lactose 100mg

Talc 10mg

Powder preparation was prepared by mixing above components and filling sealed package.

Preparation of tablet

2-carbomethoxy-2,3-epoxy-3-prenyl-1,4-naphthoquinone 200mg

Corn Starch 100mg

Lactose 100mg

Magnesium stearate optimum amount

Tablet preparation was prepared by mixing above components and entabletting.

Preparation of capsule

2-carbomethoxy-2,3-epoxy-3-prenyl-1,4-naphthoquinone 100mg

Lactose 50mg

Corn starch 50mg

Talc 2mg

Magnesium stearate optimum amount

Tablet preparation was prepared by mixing above components and filling gelatin capsule by conventional gelatin preparation method.

Preparation of liquid

2-carbomethoxy-2,3-epoxy-3-prenyl-1,4-naphthoquinone 1000mg

Sugar 20g

Polysaccharide 20g

Lemon flavor 20g

Liquid preparation was prepared by dissolving active component, and then filling all the components in 1000 ㎖ ample and sterilizing by conventional liquid preparation method.

Preparation of health care food

2-carbomethoxy-2,3-epoxy-3-prenyl-1,4-naphthoquinone 1000mg

Vitamin mixture optimum amount

Vitamin A acetate 70㎍

Vitamin E 1.0mg

Vitamin B₁ 0.13mg

Vitamin B₂ 0.15mg

Vitamin B6 0.5mg

Vitamin B12 0.2mg

Vitamin C 10mg

Biotin 10mg

Amide nicotinic acid 1.7mg

Folic acid 50mg

Calcium pantothenic acid 0.5mg

Mineral mixture optimum amount

Ferrous sulfate 1.75mg

Zinc oxide 0.82mg

Magnesium carbonate 25.3mg

Monopotassium phosphate 15mg

Dicalcium phosphate 55mg

Potassium citrate 90mg

Calcium carbonate 100mg

Magnesium chloride 24.8mg

The above mentioned vitamin and mineral mixture may be varied in may ways. Such variations are not to be regarded as a departure from the spirit and scope of the present invention.

Preparation of health beverage

2-carbomethoxy-2,3-epoxy-3-prenyl-1,4-naphthoquinone 1000mg

Citric acid 1000mg

Oligosaccharide 100g

Apricot concentration 2g

Taurine 1g

Distilled water 900 ㎖

Health beverage preparation was prepared by dissolving active component, mixing, stirred at 85℃ for 1 hour, filtered and then filling all the components in 1000 ㎖ ample and sterilizing by conventional health beverage preparation method.

The invention being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the present invention, and all such modifications as would be obvious to one skilled in the art are intended to be included within the scope of the following claims.

As described in the present invention, the inventive compositions comprising the compounds isolated from Rubiae radix shows potent anti-inflammatory effect through various experiments, therefore, it can be used as the effective and safe therapeutics or health food for treating and preventing inflammatory disease.

SEQ.I.D.1 5-ATGTCCGAAGCAAACATCAC-3

SEQ.I.D.2 5-TAATGTCCAGGAAGTAGGTG-3

SEQ.I.D.3 5-CAGCAAATCCTTGCTGTTCC-3

SEQ.I.D.4 5-TGGGCAAAGAATGCAAACATC-3

SEQ.I.D.5 5-TACTGAACTTCGGGGTGATCGGTCC-3

SEQ.I.D.6 5-CAGCCTTGTCCCTTGAAGAGAACC-3

SEQ.I.D.7 5-GAAATGATGGATGCTTCCAAACTGG-3

SEQ.I.D.8 5-GGATATATTTTCTGACCACAGTGAGG-3

SEQ.I.D.9 5-CAAGGAGAACCAAGCAAC-3

SEQ.I.D.10 5-GGGGAAGGCATTAGAAAC-3

SEQ.I.D.11 5-CCACTGGCGTCTTCACCAC-3

SEQ.I.D.12 5-CCTGCTTCACCACCTTTTG-3

Claims

A pharmaceutical composition comprising at least one compound(s) selected from the group of 2-carbomethoxy-2,3-epoxy-3-prenyl-1,4-naphthoquinone, 3,3’-bis (3,4-dihydro-4-hydroxy-6-methoxy-2H-1-benzofuran), epoxymollugin, soranjidiol, oleanolic acid, 1-acetoxy-3-methoxy-9,10-anthraquinone, alizarin-2-methyl ether, furonollugin and mollugin, which may be isolated from the extract of Rubia radix as an active ingredient for the treatment and prevention of inflammatory diseases.
The pharmaceutical composition of claim 1, wherein said compound is 2-carbomethoxy-2,3-epoxy-3-prenyl-1,4-naphthoquinone.
The pharmaceutical composition of claim 1, wherein said extract is crude extract, polar solvent soluble extract or non-polar solvent soluble extract of Rubia radix.
The pharmaceutical composition of claim 3, wherein said crude extract is the extract prepared by extracting plant material with water, lower alcohols such as methanol, ethanol, or the mixtures thereof.
The pharmaceutical composition of claim 3, wherein said non-polar solvent soluble extract is prepared by extracting the crude extract as set forth in claim 4 with non-polar solvent, for example, hexane, methylene chloride, ethyl acetate or chloroform.
The pharmaceutical composition of claim 1, wherein said inflammatory disease is atopic dermatitis, joint arthritis, urethritis, cystitis, artherosclerosis, allergy disease, rhinitis, asthma, asthma, acute pain disease, chronic pain disease, periodontitis, gingivitis, inflammatory intestine disease, gout, myocardiac infarction, congestive heart failure, hypertension, angina pectoris, stomach ulcer, ischemic stroke, Down syndrome, multiple sclerosis, obesity, diabetes, dementia, depression, schizophrenia, tuberculosis, sleep disturbance, sepsis, burn or pancreatitis, which is occurred by the overproduction of NO and the over-reproduction of pro-inflammatory cytokines such as iNOS, COX-2, TNF-α, IL-6 and IL-1β.
A use of an a use of at least one compound(s) selected from the group of 2-carbomethoxy-2,3-epoxy-3-prenyl-1,4-naphthoquinone, 3,3’-bis (3,4-dihydro-4-hydroxy-6-methoxy-2H-1-benzofuran), epoxymollugin, soranjidiol, oleanolic acid, 1-acetoxy-3-methoxy-9,10-anthraquinone, alizarin-2-methyl ether, furonollugin and mollugin, especially, 2-carbomethoxy-2,3-epoxy-3-prenyl-1,4-naphthoquinone, which may be isolated from the extract of Rubia radix for the preparation of therapeutic agent for the treatment and prevention of inflammatory disease in mammal or human.
A method of treating or preventing inflammatory disease in human or mammal, wherein the method comprises administering a therapeutically effective amount of at least one compound(s) selected from the group of 2-carbomethoxy-2,3-epoxy-3-prenyl-1,4-naphthoquinone, 3,3 -bis (3,4-dihydro-4-hydroxy-6-methoxy-2H-1-benzofuran), epoxymollugin, soranjidiol, oleanolic acid, 1-acetoxy-3-methoxy-9,10-anthraquinone, alizarin-2-methyl ether, furonollugin and mollugin, especially, 2-carbomethoxy-2,3-epoxy-3-prenyl-1,4-naphthoquinone, which may be isolated from the extract of Rubia radix, as an effective ingredient, together with a pharmaceutically acceptable carrier thereof.
A functional health food comprising at least one compound(s) selected from the group of 2-carbomethoxy-2,3-epoxy-3-prenyl-1,4-naphthoquinone, 3,3’-bis (3,4-dihydro-4-hydroxy-6-methoxy-2H-1-benzofuran), epoxymollugin, soranjidiol, oleanolic acid, 1-acetoxy-3-methoxy-9,10-anthraquinone, alizarin-2-methyl ether, furonollugin and mollugin, which may be isolated from the extract of Rubia radix as an active ingredient for the improvement and prevention of inflammatory diseases.
A health care food comprising at least one compound(s) selected from the group of 2-carbomethoxy-2,3-epoxy-3-prenyl-1,4-naphthoquinone, 3,3’-bis (3,4-dihydro-4-hydroxy-6-methoxy-2H-1-benzofuran), epoxymollugin, soranjidiol, oleanolic acid, 1-acetoxy-3-methoxy-9,10-anthraquinone, alizarin-2-methyl ether, furonollugin and mollugin, which may be isolated from the extract of Rubia radix, together with a sitologically acceptable additive for the prevention and alleviation of inflammatory disease.
The health care food of claim 10, wherein said health care food is provided as powder, granule, tablet, capsule or beverage type.