WO2010143825A2

WO2010143825A2 - Anti-arthritic agent using cyathula officinalis

Info

Publication number: WO2010143825A2
Application number: PCT/KR2010/003190
Authority: WO
Inventors: Hyun Pyo Kim; Yong Soo Kwon
Original assignee: University-Industry Cooperation Foundation Kangwon National University
Priority date: 2009-06-09
Filing date: 2010-05-20
Publication date: 2010-12-16
Also published as: KR101084942B1; KR20100132172A; WO2010143825A3

Abstract

The present invention is related to the inventive compositions comprising an extract of Cyathula officinalis or sesquiterpene isolated therefrom, show 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 a disease in a subject mediated by MMP-13, preferably, arthritic disease.

Description

ANTI-ARTHRITIC AGENT USING CYATHULA OFFICINALIS

The present invention relates to a composition comprising an extract of Cyathula officinalis or sesquiterpene isolated therefrom for the prevention and treatment of a disease in a subject mediated by MMP-13, preferably, arthritic disease and the use thereof.

Arthritis is an autoimmune disease characterized by their symptoms such as pain, swelling and stiffness in the joints. The two major forms of arthritis in mammals are inflammatory arthritis such as rheumatoid arthritis (RA), and osteoarthritis (OA), a progressive, degenerative loss of cartilage often secondary to mechanical stress, aging, dysplastic conditions and/or injury. The symptoms of arthritis generally relates to arthrosis of spine, e.g., hallux rigidus, arthrosis psoriaticum, or rheumatic arthritis.

Osteoarthritis manifests similar symptoms to a rheumatoid arthritis (RA). In particular, although osteoarthritis begins as a degeneration of articular cartilage, RA begins as an inflammation in synovium. In osteoarthritis, as cartilage deteriorates, a reactive synovitis often develops later on. Conversely, as rheumatoid arthritis erodes cartilage, the secondary osteoarthritis changes the bone and cartilage development. At the final stages of both osteoarthritis and rheumatoid arthritis, the joints suffering with both diseases appear the same phenomena with each other.

Osteoarthritis usually represents a pain which become worsen with exercise or a simple X-ray radiation showing clearly a thinned cartilage. Commonly affected joints are knees, hips, spine, finger, base of thumb or big toe etc. The disease is involved in the destruction of articular cartilage by MMPs (matrix metaloproteinases) which mainly works for cartilage depletion, which is characterized by degenerative changes in the articular cartilage and caused by the over-production of inflammatory cytokines, e.g., interleukin-1 (IL-1), tumor necrosis factor-a (TNF-a) etc, causing to extremely severe pain in joints, tendons, muscles and ligament (Fernandes J.C., The role of cytokines in osteoarthritis pathophysiology, 39, pp237-246, 2002). Rheumatoid arthritis (RA) is a common autoimmune disease characterized by the swelling, deformation and destruction of joint, which culminates in severe physical disability. Rheumatic diseases include the diseases occurring at muscles, tendons, joints, bones or sinus, which are generally characterized by an inflammation and/or degeneration. The patient ssuffering with rheumatoid arthritis present the imbalance in immune system, which causes an overproduction of pro-inflammatory cytokines, e.g., TNF-a, IL-1 etc, and a lack of anti-inflammatory cytokines, e.g., IL-10, IL-1, etc. RA is characterized by synovial inflammation, which progresses to a cartilage destruction, bone erosion and subsequent joint deformity. During the inflammation process, polymorphonuclear cells, macrophages and lymphocytes are released from the joint. Activated T-lymphocytes produce cytotoxins and pro-inflammatory cytokines, while macrophages stimulate the release of the prostaglandins and cytotoxins. Vasoactive substances such as histamine, kinins and prostaglandins, are released at the site of inflammation and they cause to an edema, erythema and pain at the region of the inflamed joints.

The main pathology of the affected synovial tissue is a hyperplasia and the sub-intimal infiltration of T and B lymphocytes. Synovial tissue hyperplasia forms in pannus tissue, which irreversibly destroys the cartilage and bone in the affected joint. There have been reported that various mediators, for example, pro-inflammatory cytokines such as IL-1 etc, eicosanoids such as prostaglandins, leukotrienes, and main enzymes such as cyclooxygenase-1 or 5-lipoxygenase etc, are closely involved in the progression of the arthritic disease.

Especially, TNF-a and IL-1b directly induce the synthesis of proteolytic enzyme such as matrix metalloproteinase (MMPs). The over-expression of MMP occurs by the action of various mediators and the bio-synthesized MMP break down the extracellular matrix macromolecules (ECM), which get worsen the arthritic disease. MMP-13 (mammalian collagenase), one of MMPs, has been known to play key roles in the progression of arthritis.

ECM, the component of cartilage, can be classified into proteoglycan and glycoprotein, which are synthesized at chondrocyte in joint cartilage and fibroblast-like synoviocyte (FLS) in synovial membrane, and released to extracellular space. They also synthesize and release MMP for their turn-over process. Namely, they synthesized not only ECM but also MMP in order to maintaining their balance. Under normal condition, the tissue inhibitors of metalloproteinases (TIMPs) normally bind to MMPs with the ratio of 1:1. The imbalanced ratio of TIMPs to MMPs which is generally caused by the up-regulation of MMPs, results in the continued matrix destruction in RA by way of the attack of higher level of MMPs.

The first aid drugs for treating arthritis includes the drug for alleviating pain and inflammation, which is classified into non-steroidal anti-inflammatory drugs (NSAIDs), e.g., aspirin, ibuprofen, naproxen, methotrexate, etc. Secondary aid drugs include corticosteroids, slow acting antirheumatic drugs (SAARDs) or disease modifying drugs (DMs), e.g., penicilamine, cyclophosphamide, gold salts, azethioprine, levamisole, etc. The first groups of biological-response modifiers (BRMs) approved by FDA for treatment of RA are TNF-a antagonists which plays role in binding to its receptor or directly binding to the TNF-a protein. However, the use of DMARDs has been impeded by various disadvantages, for example, the potential of its long-term side effects and toxicity, high cost, hypersensitivity to the medications and infections due to TNF-a blockage, etc.

Cyathula officinalis Kuhn belonged to Amaranthaceae, has been conventionally used to treat or prevent dysmenorrhea or dysfunctional voiding in China and known to contain 4-[(1-ethoxy-2-hydroxy)ethyl]phenol, 2,3-isopropylidene cyasterone, 24-hydroxycyasterone, 2,3-isopropylidene isocya sterone, betavulgarin, daidzin, cyasterone, sengosterone etc (Zhou et al., Chemical study on Cyathula officinalis Kuan, Journal of Asian natural Products Research, 7, pp245-252, 2005).

There have been reported on the cytotoxic activity on MDA-MB-231 of several glycosides isolated from Cyathula officinalis, for example, 3-O-[alpha-L-rhamnopyranosyl-n-butyl-beta-D-glucopyranosiduronate]]-28-O-beta-D-glucopy ranosyloleanol ic acid et al (Zhou et al., Glycosides from Roots of Cyathula officinalis Kuan, Journal of integrative Biology, 47(3), pp368-374, 2005) and the anti-tumor activity using by Lewis pulmonary carcinoma cells of the fructan polysaccharides isolated from Cyathula officinalis (Chen et al., Structural elucidation and antitumor activity of a fructan from Cyathula officinalis Kuan, Carbohydrate Research, 338, pp1235-1241, 2003).

However, there has been not reported or disclosed about the therapeutic effect of an extract of Cyathula officinalis or sesquiterpene isolated therefrom for the prevention and treatment of a disease in a subject mediated by MMP-13, preferably, arthritic disease in any of above cited literatures, the disclosures of which are incorporated herein by reference.

Accordingly, the present inventors have tried to find the pharmacological activity of an extract of Cyathula officinalis or sesquiterpene isolated therefrom and confirmed that they show potent anti-inflammatory effect through various experiments, i.e., in vitro tests, for example, the inhibitory effect on the over-expression of MMP-13, and the production of proteoglycan and collagen degradation products as well as in vivo tests, for example, anti-inflammatory effect on carrageenan-induced rat paw-edema model and anti-rheumatic effect on the rheumatic arthritis animal model and degenerative arthritis animal model, therefore, it can be used as the effective and safe therapeutics or health food for treating and preventing a disease in a subject mediated by MMP-13, preferably, arthritic disease.

According to one aspect of the present invention, the present invention provides a composition comprising an extract of Cyathula officinalis or sesquiterpene isolated therefrom for the prevention and treatment of a disease in a subject mediated by MMP-13, preferably, arthritic disease and the use thereof.

Accordingly, it is an object of the present invention to provide a pharmaceutical composition comprising an extract of Cyathula officinalis or sesquiterpene isolated therefrom as an active ingredient for the prevention and treatment of a disease in a subject mediated by MMP-13, preferably, arthritic disease.

The present invention also provided a pharmaceutical composition comprising an extract of Cyathula officinalis or sesquiterpene isolated therefrom as an active ingredient and a pharmaceutically acceptable carrier thereof for treating and preventing a disease in a subject mediated by MMP-13, preferably, arthritic disease.

The term “extract” disclosed herein includes a crude extract, a polar solvent soluble extract and non-polar solvent soluble extract of Cyathula officinalis.

The term “crude extract” disclosed herein can be prepared by the steps consisting of: extracting the root or herb of plant material with extracting solvent, for example, water, C1-C4 lower alcohols such as methanol, ethanol and the like, hexane, chloroform, methylene chloride, ethylacetate, glycerin, butylene glycol or the mixtures thereof, preferably, water, ethanol, chloroform or the mixture thereof.

The term “polar solvent soluble extract and non-polar solvent soluble extract extract” disclosed herein can be prepared by fractionating the crude extract into two fractions, i.e., (a) non-polar solvent soluble extract which is soluble in non-polar solvent, for example, hexane, chloroform, methylene chloride, ethyl acetate, glycerin or butylene glycol, preferably hexane or methylene chloride to obtain non-polar solvent soluble extract and (b) polar solvent soluble extract which is soluble in polar solvent, for example, water, C1-C4 lower alcohol such as methanol, ethanol, butanol or the mixtures thereof, preferably, water or butanol.

The term “sesquiterpene isolated Cyathula officinalis”disclosed herein includes 5alpha-H-10-beta-selina-4(14), 7(11)-dien-8-one of Chemical formulae (I) and atractylenoide I of Chemical formulae(II).

ChemistryFigure 1

ChemistryFigure 2

The term “a disease in a subject mediated by MMP-13” disclosed herein includes acute and chronic inflammatory disease, degenerative arthritis, rheumatic arthritis, degenerative cartilage loss caused by traumatic joint injury, spondyloarthropathy, ankylosing spondylitis, Lupus arthritis, Neuronal demyelinating Polyradiculoneurpathy, periodontal membrane disease, diabetic retinopathy, Proliferative vitreous retinopathy, immature retinopathy, keratoconus, or Sjogren’s syndrome, preferably, acute and chronic inflammatory disease, degenerative arthritis, rheumatic arthritis, degenerative cartilage loss caused by traumatic joint injury, spondyloarthropathy, ankylosing spondylitis, or Lupus arthritis, more preferably, arthritis, or rheumatic arthritis.

The present invention also provided a use of an extract of Cyathula officinalis or sesquiterpene isolated therefrom for the preparation of medicament for the treatment and prevention of a disease in a subject mediated by MMP-13 in mammal or human.

It is the other object of the present invention to provide a method of inhibiting activity of MMP-13 in a subject, comprising administering to the subject a therapeutically effective amount of an extract of Cyathula officinalis or sesquiterpene isolated therefrom, as an effective ingredient, together with a pharmaceutically acceptable carrier thereof.

It is the other object of the present invention to provide a method of treating a disease in a subject mediated by MMP-13, wherein the method comprises administering to the subject a therapeutically effective amount of an extract of Cyathula officinalis or sesquiterpene isolated therefrom, as an effective ingredient, together with a pharmaceutically acceptable carrier thereof.

Hereinafter, the present invention is described in detail.

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

For example, Cyathula officinalis are dried, cut, crushed, mixed together and added to 1 to 1000-fold, preferably, approximately 10 to 500-fold volume of water, C1-C4 lower alcohols such as methanol, ethanol and the like, hexane, chloroform, methylene chloride, ethylacetate, glycerin, butylene glycol or the mixtures thereof, preferably, water, ethanol, chloroform or the mixture thereof; the solution is subjected to extraction according to we1l-known extraction method in the art such as hot water extraction, Enfleurage extraction, reflux extraction, or ultra-sonication extraction, preferably, extraction with Enfleurage extraction at the temperature ranging from 4℃~40℃, preferably, 10℃~33℃, for the period ranging from 1 to 24 hours, preferably, 2 to 4 hours; the extract is collected with filtration, concentrated under reduced pressure and dried to obtain an inventive crude extract of the present invention.

At then, the crude extract prepared by above step, is suspended in 0.1 to 1000-fold, preferably, approximately 1 to 500-fold volume (v/w) of water, and then is mixed with non polar solvent such as hexane, chloroform, methylene chloride, ethyl acetate, glycerin or butylene glycol, preferably hexane or methylene chloride to fractionate into two fractions, i.e., (a) non-polar solvent soluble extract which is soluble in non-polar solvent, for example, hexane, chloroform, methylene chloride, ethyl acetate, glycerin or butylene glycol, preferably hexane or methylene chloride to obtain non-polar solvent soluble extract and (b) polar solvent soluble extract which is soluble in polar solvent, for example, water, C1-C4 lower alcohol such as methanol, ethanol, butanol or the mixtures thereof, preferably, water or butanol to afford the inventive non-polar solvent soluble extract and polar solvent soluble extract of the present invention.

At then, the non-polar solvent soluble extract prepared by above step, is performed to repeated purification processes using by flash column chromatography, ODS column chromatography and/or Silica gel column chromatography with increasing the polarity of developing solvent system such as mixture solvent of hexane and ethylacetate to afford the inventive compounds isolated from the extract of Cyathula officinalis, i.e., 5alpha-H-10-beta-selina-4(14), 7(11)-dien-8-one (compound I) and atractylenoide I (compound II).

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, it is an object of the present invention to provide a pharmaceutical composition comprising an extract of Cyathula officinalis or sesquiterpene isolated therefrom prepared by the above-described method as an active in gredient for the prevention and treatment of a disease in a subject mediated by MMP-13, preferably, arthritic disease.

It is an object of the present invention to provide a use of an extract of Cyathula officinalis or sesquiterpene isolated therefrom prepared by the above-described method for the preparation of medicament for the treatment and prevention of a disease in a subject mediated by MMP-13 in mammal or human.

It is the other object of the present invention to provide a method of inhibiting activity of MMP-13 in a subject, comprising administering to the subject a therapeutically effective amount of an extract of Cyathula officinalis or sesquiterpene isolated therefrom prepared by the above-described method, as an effective ingredient, together with a pharmaceutically acceptable carrier thereof.

It is the other object of the present invention to provide a method of treating a disease in a subject mediated by MMP-13, wherein the method comprises administering to the subject a therapeutically effective amount of an extract of Cyathula officinalis or sesquiterpene isolated therefrom prepared by the above-described method, as an effective ingredient, together with a pharmaceutically acceptable carrier thereof.

It is confirmed that the inventive extract of the present invention prepared by above-described method shows potent anti-inflammatory effect through various experiments, i.e., in vitro tests, for example, the inhibitory effect on the over-expression of MMP-13, and the reproduction of proteoglycan and collagen degradation products as well as in vivo tests, for example, anti-inflammatory effect on carrageenan-induced rat paw-edema model and anti-rheumatic effect on the rheumatic arthritis animal model and degenerative arthritis animal model, therefore, it can be used as the effective and safe therapeutics or health food for treating and preventing a disease in a subject mediated by MMP-13, preferably, arthritic disease.

The inventive composition for treating and preventing arthritic diseases may comprises the above-described extract 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, Easton PA).

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 composition 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, for example, administration can be made orally, rectally or by intravenous, intramuscular, subcutaneous, intra-cutaneous, intrathecal, epidural or intra-cerebroventricular injection.

Accordingly, it is another object of the present invention to provide a health functional food comprising an extract of Cyathula officinalis or sesquiterpene isolated therefrom as an active ingredient for the prevention and improvement of a disease in a subject mediated by MMP-13, preferably, arthritic disease.

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 an extract of Cyathula officinalis or sesquiterpene isolated therefrom, together with a sitologically acceptable additive for the prevention and alleviation of a disease in a subject mediated by MMP-13, preferably, arthritic disease.

The term “a health care food” defined herein is the food containing inventive extract 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, anticaking 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 compound can be added to food or beverage for prevention and improvement of aimed disease. The amount of inventive extract or compound 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 or compound 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 or compound 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 compound 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 an extract of Cyathula officinalis or sesquiterpene isolated therefrom show potent anti-inflammatory effect through various experiments, i.e., in vitro tests, for example, the inhibitory effect on the over-expression of MMP-13, and the reproduction of proteoglycan and collagen degradation products as well as in vivo tests, for example, anti-inflammatory effect on carrageenan-induced rat paw-edema model and anti-rheumatic effect on the rheumatic arthritis animal model and degenerative arthritis animal model, therefore, it can be used as the effective and safe therapeutics or health food for treating and preventing a disease in a subject mediated by MMP-13, preferably, arthritic 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 inhibition of test samples on MMP-13 expression (1: CO-F-BuOH (50 microgram/ml), 2: CO-F-hexane (50 microgram/ml), 3: CO-F-DCM (50 microgram/ml), 4: water fraction (50 microgram/ml), Co-1: 5alpha-H-10-beta-selina-4(14), 7(11)-dien-8-one, Co-2: atractylenoide I);

Fig. 2 represents the histological examined result (a: control, b: iodoacetate-treated, c: CO-EtOH (300 mg/kg/day), d: CO-F-DCM (100 mg/kg/day).

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

Comparative Example 1. Preparation of root extract of Achyranthes japonica NAKAI.

100 g of Achyranthes japonica NAKAI purchased from Kyungdong herbal market in Korea, was dried, cut into small pieces and added to 1 L of 100% ethanol. The solution was performed to Enfleurage extraction for 3 hours with stirring at 20℃ repeatedly and the residue was filtered. The filtrate was concentrated and dried to obtain 5.4 g of root extract of Achyranthes japonica NAKAI to use as a comparative test sample (designatedas‘AJ extract’hereinafter).

Example 1. Preparation of crude extract of Cyathula officinalis .

100 g of Cyathula officinalis purchased from Kyungdong herbal market in Korea, was dried, cut into small pieces and added to 1 L of various extraction solvents as shown in Table 1. Each solution was performed to Enfleurage extraction for 3 hours with stirring at 20℃ repeatedly and the residue was filtered. The filtrate was concentrated and dried to obtain various kinds of extracts of Cyathula officinalis according to various extraction solvents, which were used as test samples.

Table 1

Name	Extraction Solvent	Yield (%, w/w)
CO-W	Distilled water	15.3
CO-5-EtOH	5% EtOH	10.7
CO-10-EtOH	10% EtOH	11.4
CO-25-EtOH	25% EtOH	13.8
CO-50-EtOH	50% EtOH	25.1
CO-75-EtOH	75% EtOH	22.0
CO-EtOH	100% EtOH	20.9
CO-MeOH	100% MeOH	21.9
CO-hexane	Hexane	2.2
CO-DCM	Dichloromethane	3.5
CO-CF	Chloroform	4.1
CO-Ethylacetate	Ethylacetate	7.2

Example 2. Preparation of purified extract of Cyathula officinalis .

100 g of 100% ethanol extract of Cyathula officinalis prepared in Example 1 was added to 0.2 L of distilled water and the solution was performed to successive fractionation with 0.5 L of hexane, methylene chloride, and butanol repeatedly to afford hexane soluble fraction (5.4 g), and methylene chloride soluble fraction (9.2 g) as non-polar solvent soluble extracts as well as butanol soluble fraction (21.7 g) and water soluble fraction (36.2 g) as polar solvent soluble extracts, respectively as shown in Table 2, which were used as test samples.

Table 2

Name	Fractionation solvent	Yield (%, w/w)
CO-F-hexane	Hexane	5.4
CO-F-DCM	Methylene chloride	9.2
CO-F-BuOH	butanol	21.7

Example 3. Preparation of sesquiterpene compounds from Cyathula officinalis .

6 kg of dried root of Cyathula officinalis was performed to hot-water extraction with 18 L of 95% ethanol to obtain 1.2 kg of ethanol extract of Cyathula officinalis.

The ethanol extract was performed to fractionation using by 4 L of chloroform and 2 L of distilled water to obtain 10 g of chloroform soluble fraction.

The chloroform soluble extract was performed to open column chromatography [200 g of Silica gel, 70-230 mesh silica gel; glass column size (50 cm x 5 cm); eluting solvent as a mobile phase = chloroform: methanol (15:1)] to afford 5 fractions, i.e., fractions I~V according to TLC pattern and activity-guided separation technique.

Among the 5 fractions, fraction II was further performed to open column chromatography [200 g of Silica gel, 70-230 mesh silica gel; glass column size (40 cm x 3 cm); eluting solvent as a mobile phase = hexane: ethylacetate (4:1)] to afford 6 fractions, i.e., sub-fractions II-1~II-6 according to TLC pattern and activity-guided separation technique.

Among the 6 fractions, sub-fraction II-2 was further performed to open column chromatography [200 g of Silica gel, 70-230 mesh silica gel; glass column size (40 cm x 3 cm); eluting solvent as a mobile phase = hexane: ethylacetate (9:1)] to afford 4 fractions, i.e., sub-fractions II-2-1~II-2-4 according to TLC pattern and activity-guided separation technique.

Among the 4 fractions, sub-fraction II-2-2 and II-2-3 were further performed ODS column chromatography [column size (20 cm x 3 cm) ] using by 80% methanol and 100% chloroform as eluting solvents to afford 10 mg of 5alpha-H-10-beta-selina-4(14), 7(11)-dien-8-one (compound I) and 17 mg of atractylenoide I (compound II), which are identified by comparing the following physichemical data with those in the literature (Endo et al., Chem.Pharm.Bull., 27, pp.2594-2958, 1979; Pachaly et al., Planta Med., 55, pp.59-61, 19 89).

Compound I

¹H-NMR (600 MHz, CDCl₃)δ:0.77(3H, s), 1.82(3H, s), 1.99(3H, s), 4.61(1H, s), 4.86(1H, s); ¹³C-NMR (150 MHz, CDCl₃)δ: 17.33(C-15), 22.32(C-13), 23.14(C-2), 23.26(C-12), 29.19(C-6), 36.78(C-1orC-3), 38.20(C-10), 41.25(C-1orC-3), 47.06(C-5), 57.61(C-9), 106.95(C-14), 131.55(C-7), 142.74(C-11), 149.08(C-4), 203.83(C-8)

Compound II

¹H-NMR (600 MHz, CDCl₃)δ:0.93 (3H, s, CH₃), 1.89(3H, d, J=1.2Hz, CH₃), 4.62(1H, s, C=CH₂), 4.90(1H, s, C=CH₂), 5.59(1H, s, C=CH); ¹³C-NMR (150 MHz, CDCl₃)δ: 8.46(C-13), 18.57(C-14), 22.66(C-6), 23.00(C-2), 36.18(C-3), 38.13(C-10), 39.10(C-1), 48.41(C-5), 107.48(C-15), 119.16(C-9), 120.45(C-11), 147.99(C-4), 148.09(C-8), 148.34(C-7), 171.38(C-12)

Reference Example 1. Preparation of cartilage cell and cartilage tissue

Rabbit articular cartilage slice was incubated in complete DMEM medium for 14 days according to the literature (Baek et al., Biol. Pharm. Bull., 29, pp1423-1430, 2006). The slices were added to 48-well plates and treated with 10 ng/ml of IL-1alpha to incubate for 25 days. At then, various concentrations of test samples was treated therewith to incubate and the supernatant of the medium was collected at 3^rd, 7^th, 14^thand 28^thdays after the incubation to use in following experiment.

Experimental Example 1. Inhibition effect on MMP-13 expression

In order to determine the inhibitory effect of the inventive extract and compounds prepared in Examples on MMP-13 expression, following experiment was performed according the procedure disclosed in the literature (Pei et al., Osteoarthritis Cartilage, 14, pp749-758, 2006).

Human chondrosarcoma cell line (SW1353, ATCC) was incubated in DMEM medium supplemented with 10% FBS, penicillin/streptomycin and glutamine. In order to confirm the MMP-12 expression released to the medium, 10 ng/ml of IL-1beta was added to the medium and 24 hours after the incubation, the supernatant was collected. The amount of MMP in the supernatant was determined by using Western blot analysis as follows:

Trichloroacetic acid (Sigma Co.) was added to the supernatant of cell culture to be 15% of final concentration and the solution was centrifuged for 10 mins at the speed of 12,000 rpm. The supernatant was discarded and 1ml of acetone was added to the medium. The medium was further centrifuged for 10 mins at the speed of 12,000 rpm with vortexing and the supernatant was discarded and left alone at room temperature for more than 30 mins. The medium was added with 20 microliter of 1x sample buffer, vortexed, heated for 3 mins and performed to electrophoresis using by 10% gel. The gel was transferred and reacted on PVDF for 1 hour. The solution was reacted with diluted anti-MMP-13 antibody (Sigma, M4052) with 5% skim milk to 1:5000 for 1 hour and then reacted with diluted anti-rabbit antibody (Cell signalling) with 5% skim milk to 1:2000 for 1 hour. ECL solution (Amersham) was added thereto and exposed to X-ray film.

Various test samples comprising the extract of Achyranthes japonica NAKAI prepared in Comparative Example 1 and the inventive extract and compounds prepared in examples were treated thereto and the amount of MMP-13 was determined. The result was quantitatively analyzed by scanning the each blot using apparatus (HP laser jet 3051) and shown in Table 3 and Fig. 1.

Table 3

Name	Conc.(microgram/ml)	Inhibition ratio (%)	Name	Conc.(microgram/ml)	Inhibition ratio (%)
CO-W	200	10.3	CO-F-hexane	50	94.4
CO-5-EtOH	200	15.5	CO-F-DCM	50	82.2
CO-10-EtOH	200	22.3	CO-F-BuOH	50	32.8
CO-25-EtOH	200	42.9	Co-hexane	50	98.1
CO-50-EtOH	200	60.1	CO-DCM	50	96.7
CO-75-EtOH	200	72.4	CO-CF	50	95.5
CO-EtOH	200	98.0	Co-Ethylacetate	50	97.4
CO-EtOH	50	15.6	AJ extract	200	-
CO-MeOH	200	97.7	Compound I	20	72.2
CO-MeOH	50	12.3	Compound II	20	52.3
The result showed the mean value of test results (3x)

At the result, the crude extract, non-polar solvent soluble extract and polar solvent soluble extract prepared in Examples showed potent inhibition effect on the MMP-13 reproduction whereas the 100% ethanol extract of Achyranthes japonica NAKAI did not show any inhibition effect on the MMP-13 reproduction. Moreover, it has been confirmed that the active ingredients of the extract of Cyathula officinalis, are 5alpha-H-10-beta-selina-4(14), 7(11)-dien-8-one (compound I) and atractylenoide I (compound II) as can be seen in Table 3, which result verifies that the inventive extract and the inventive compounds isolated from the extract of Cyathula officinalis inhibited the dissociation of ECM at the joint therefore, they can be useful as a anti-rheumatic agent.

Experimental Example 2. Determination of proteoglycan and collagen degradation product

In order to determine the inhibitory effect of the inventive extract and compounds prepared in Examples on the amount of collagen degradation product and proteoglycan resulting from the inhibition of MMP-13 expression, following experiment was performed.

The degradation of cartilage glycosaminoglycan was determined by quantitatively analyzing the amount of glycosaminoglycan at 535 nm using by 1,9-dimethylene blue and the test result was calculated according to following math formulae 1 and shown in Table 4.

In order to determine the dissociation rate of cartilage collagen, type II collagen degradation product in the culture supernatant at 21^stdays after incubation as disclosed in Reference Example, was determined using by sircol collagen type II cleavage ELISA assay and the test result was calculated according to following math formulae 1 and shown in Table 4. Prednisolone (50 microM) was used as a positive control.

MathFigure 1

Table 4

Test sample	Conc.	Dissociation rate of glycosaminoglycan (%)	Dissociation rate of collagen (%)
Control	-	22.7 ± 4.5	30.5 ± 5.1
IL-1-treated	-	100.0 ± 3.1	100.0 ± 4.4
Prednisolone	50 micromole	38.1 ± 4.8*	47.1 ± 3.6*
CO-EtOH	1 microgram/ml	89.9 ± 7.1	92.6 ± 3.7
	5 microgram/ml	77.5 ± 4.1*	82.4 ± 5.3
	20 microgram/ml	45.5 ± 3.7*	55.9 ± 3.4*
CO-F-DCM	1 microgram/ml	63.6 ± 3.2*	67.8 ± 2.9*
CO-F-DCM	5 microgram/ml	44.9 ± 2.4*	51.7 ± 3.1*
CO-CF	1 microgram/ml	68.2 ± 4.4	69.0 ± 2.1*
CO-CF	5 microgram/ml	50.4 ± 3.7*	49.2 ± 3.7*
Compound I	1 micromole	82.3 ± 4.7*	88.2 ± 5.7*
Compound I	10 micromole	50.4 ± 5.2*	55.1 ± 2.6*
The evaluation of significance was calculated by performing student t-test (n= 3) for each test group to IL-1 treated group and expressed as *p<0.01.

At the result, the groups treated with 100% EtOH extract, methylenechloride soluble fraction, chloroform soluble fraction and compound I significantly inhibited the dissociation of ECM. Therefore, it has confirmed that they have inhibiting effect on the dissociation of joint cartilage as can be seen in Table 4.

Experimental Example 3. Anti-inflammatory activity in acute inflammation animal model ( in vivo )

In order to determine the anti-inflammatory activity of the inventive extract and compounds prepared in Examples, carrageenan-induced rat paw edema test as an in vivo acute inflammation animal model was performed by using ICR mouse (20-22 g, Orient-Bio Co.) according to the procedure disclosed in the literature (Winter et al., Proc. Soc. Exp. Biol. Med. 111. pp544-547, 1962).

The inventive extract and compounds prepared in Examples were orally administrated into the ICR mouse (20-22 g, Orient-Bio Co.) and 1 hour after the administration, 1% λ-carrageenan (CGN) solution was injected to the right rat paw. 5 hours after the injection, the paw edema was determined according to water-displacement method and the test result was shown in Table 5. Prednisolone (Sigma, 5 mg/kg) was used as a positive control.

Table 5

Test sample	Dose (mg/kg)	Increased vol. of right rat paw (ml)	Inhibition rate (%)
CGN-treated	-	0.153 ± 0.022	-
Prednisolone	5	0.083 ± 0.012	45.8
CO-EtOH	10	0.144 ± 0.038	5.9
	100	0.135 ± 0.027	11.8
	400	0.102 ± 0.018*	33.3
CO-F-DCM	100	0.124 ± 0.032	19.0
CO-F-DCM	400	0.098 ± 0.020*	35.9
CO-CF	100	0.118 ± 0.028	22.9
CO-CF	400	0.100 ± 0.019*	34.6
Compound I	10	0.130 ± 0.037	15.0
Compound I	100	0.106 ± 0.017*	30.7
The evaluation of significance was calculated by performing student t-test (n= 3) for each test group to CGN- treated group and expressed as *p<0.01.

At the result, the groups treated with 100% EtOH extract, methylenechloride soluble fraction, chloroform soluble fraction and compound I showed significantly potent anti-inflammatory activity in acute inflammation animal model as can be seen in Table 5.

Experimental Example 4. Inhibition effect on Rheumatic arthritis in chronic inflammation animal model ( in vivo )

In order to determine the anti-inflammatory activity of the inventive extract and compounds prepared in Examples, following test as an in vivo chronic inflammation animal model was performed by using female SD rat (200-220 g, Orient-Bio Co.) of which right paw was injected with heat-treated dry Mycobacterium butyricum according to the procedure disclosed in the literature (Kim et al., Planta Med. 65. Pp465-467, 1999).

The inventive extract and compounds prepared in Examples were orally administrated into the female SD rat (200-220 g, Orient-Bio Co.) daily. The degree of edema at 16^thday (late phase of secondary inflammation) and that of the left paw which had treated without any adjuvant and regarded as arthritic inflammation were determined and the inhibition rate was calculated. The test result was shown in Table 6 and Prednisolone (Sigma, 5 mg/kg) was used as a positive control.

Table 6

Test sample	Dose (mg/kg)	Increased vol. of left rat paw at 16^thday(ml)	Inhibition rate (%)
Adjuvant-treated	-	0.177 ± 0.028	-
Prednisolone	5	0.052 ± 0.010*	70.6
CO-EtOH	100	0.167 ± 0.042	5.6
CO-EtOH	400	0.103 ± 0.025*	41.8
CO-F-DCM	100	0.143 ± 0.026	19.2
CO-F-DCM	400	0.090 ± 0.024*	49.2
CO-CF	100	0.157 ± 0.038	11.3
CO-CF	400	0.129 ± 0.045	27.1
Compound I	10	0.158 ± 0.042	10.7
Compound I	100	0.114 ± 0.026*	35.6
The evaluation of significance was calculated by performing student t-test (n= 3) for each test group to adjuvant- treated group and expressed as *p<0.01.

At the result, the groups treated with 100% EtOH extract, methylenechloride soluble fraction, and compound I significantly inhibited the rheumatic arthritis in chronic inflammation animal model as can be seen in Table 6.

Experimental Example 5. Inhibition effect on degenerative arthritis in degenerative arthritis animal model ( in vivo )

In order to determine the inhibitory effect of the inventive extract and compounds prepared in Examples on degenerative arthritis, following test as an in vivo animal model was performed according to the procedure disclosed in the literature(Guingamp et al., Arthritis Rheum. 40. pp.1670-1679, 1997).

30 microliter of iodoacetic acid was intra-articularly injected to the glenoid cavity of right leg of female SD rat (200-220 g, Orient-Bio Co.) and the inventive extract and compounds prepared in Examples were orally administrated into the SD rat daily (5 days/week).

4 weeks after the injection of iodoacetic acid, the rats were sacrificed and the knee joint was excised. The sliced cartilage tissue was fixed according to histological method, stained with safranin O staining dye and the tissue was observed by microscope. The test result was shown in Fig.2.

At the result, the groups treated with 100% EtOH extract (300 mg/kg/day), and methylenechloride soluble fraction (100 mg/kg/day) significantly inhibited the degenerative arthritis and cartilage destruction in animal model as can be seen in Fig. 2.

Experimental Example 6. Acute toxicity test of oral administration in rat

The acute toxicity test was performed by administrating inventive extract and compound to ICR mice.

100% EtOH extract (CO-EtOH, up to 2 g/kg), methylenechloride soluble fraction (CO-F-DCM, up to 1 g/kg), chloroform soluble fraction (CO-CF, up to 1 g/kg), and Compound I (5alpha-H-10-beta-selina-4(14), 7(11)-dien-8-one, up to 400 mg/kg) were dissolved in 0.1% CMC, and diluted to twice (2x). The inventive extract and compound were orally administrated to the mice and the symptoms of mice were observed for 3 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. Accordingly, it has been confirmed that the inventive extract prepared in the present invention was potent and safe substance 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 powder

CO-EtOH 20 mg

Corn Starch 100 mg

Lactose 100 mg

Talc 10 mg

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

Preparation of tablet

CO-F-DCM 10 mg

Corn Starch 100 mg

Lactose 100 mg

Magnesium stearate optimum amount

Tablet preparation was prepared by mixing above components and entabletting.

Preparation of capsule

CO-CF 10 mg

Crystalline cellulose 3 mg

Lactose 14.8 mg

Magnesium stearate 0.2 mg

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

Preparation of injection

Compound I (5alpha-H-10-beta-selina-4(14), 7(11)-dien-8-one) 10 mg

Mannitol 180 mg

Na₂HPO₄.12H₂O 0.1 mg

Distilled water for injection 2974 mg

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 liquid

compound II (atractylenoide I) 20 mg

isomerized Sugar 10 g

Mannitol 5 g

Distilled water for injection optimum amount

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

CO-EtOH 1000 mg

Vitamin mixture optimum amount

Vitamin A acetate 70 mg

Vitamin E 1.0 mg

Vitamin B₁ 0.13 mg

Vitamin B₂ 0.15 mg

Vitamin B6 0.5 mg

Vitamin B12 0.2 mg

Vitamin C 10 mg

Biotin 10 mg

Amide nicotinic acid 1.7 mg

Folic acid 50 mg

Calcium pantothenic acid 0.5 mg

Mineral mixture optimum amount

Ferrous sulfate 1.75 mg

Zinc oxide 0.82 mg

Magnesium carbonate 25.3 mg

Monopotassium phosphate 15 mg

Dicalcium phosphate 55 mg

Potassium citrate 90 mg

Calcium carbonate 100 mg

Magnesium chloride 24.8 mg

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

CO-F-DCM 1000 mg

Citric acid 1000 mg

Oligosaccharide 100 g

Apricot concentration 2 g

Taurine 1 g

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 an extract of Cyathula officinalis or sesquiterpene isolated therefrom, show 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 a disease in a subject mediated by MMP-13, preferably, arthritic disease.

Claims

A pharmaceutical composition comprising an extract of Cyathula officinalis or sesquiterpene isolated therefrom as an active ingredient for the prevention and treatment of a disease in a subject mediated by MMP-13.
The pharmaceutical composition of claim 1, wherein said extract is a crude extract, a polar solvent soluble extract or non-polar solvent soluble extract of Cyathula officinalis.
The pharmaceutical composition of claim 2, wherein said crude extract is prepared by the steps consisting of: extracting the root or herb of plant material with extracting solvent, for example, water, C1-C4 lower alcohols such as methanol, ethanol and the like, hexane, chloroform, methylene chloride, ethylacetate, glycerin, butylene glycol or the mixtures thereof.
The pharmaceutical composition of claim 2, wherein said non-polar solvent soluble extract is soluble in non-polar solvent selected from the group of hexane, chloroform, methylene chloride, ethyl acetate, glycerin or butylene glycol.
The pharmaceutical composition of claim 2, wherein said polar solvent soluble extract is soluble in polar solvent selected from water, C1-C4 lower alcohol such as methanol, ethanol, butanol and the like, or the mixtures thereof.
The pharmaceutical composition of claim 1, wherein said sesquiterpene isolated from Cyathula officinalis is 5 alpha-H-10-beta-selina-4(14), 7(11)-dien-8-one or atractylenoide I.
The pharmaceutical composition of claim 1, wherein said disease in a subject mediated by MMP-13 is acute and chronic inflammatory disease, degenerative arthritis, rheumatic arthritis, degenerative cartilage loss caused by traumatic joint injury, spondyloarthropathy, ankylosing spondylitis, Lupus arthritis, Neuronal demyelinating Polyradiculoneurpathy, periodontal membrane disease, diabetic retinopathy, Proliferative vitreous retinopathy, immature retinopathy, keratoconus, or Sjogren’s syndrome.
A use of an extract of Cyathula officinalis or sesquiterpene isolated therefrom for the preparation of medicament for the treatment and prevention of a disease in a subject mediated by MMP-13 in mammal or human.
A method of inhibiting activity of MMP-13 in a subject, comprising administering to the subject a therapeutically effective amount of an extract of Cyathula officinalis or sesquiterpene isolated therefrom, as an effective ingredient, together with a pharmaceutically acceptable carrier thereof.
A method of treating a disease in a subject mediated by MMP-13, wherein the method comprises administering to the subject a therapeutically effective amount of an extract of Cyathula officinalis or sesquiterpene isolated the refrom, as an effective ingredient, together with a pharmaceutically acceptable carrier thereof.
A health care food comprising an extract of Cyathula officinalis or sesquiterpene isolated therefrom, together with a sitologically acceptable additive for the prevention and alleviation of a disease in a subject mediated by MMP-13, preferably, arthritic disease.
The health care food of claim 11, wherein said health care food is provided as powder, granule, tablet, capsule or beverage type.