WO2009104901A2

WO2009104901A2 - Composition comprising an extract of anemarrhena asphodeloides bunge for preventing and treating arthritic diseases

Info

Publication number: WO2009104901A2
Application number: PCT/KR2009/000776
Authority: WO
Inventors: Dong-Suk Park; Myung Chul Yoo; Do-Young Choi; Hyung In Yang; Yong-Hyeon Baek; Jeong-Eun Huh; Kyoung Soo Kim; Yong-Baik Cho; In Ho Jung; Jong Hyun Hur; Jae Dong Lee
Original assignee: WHAN IN PHARMACEUTICAL Co; Kyung Hee University
Current assignee: WHAN IN PHARMACEUTICAL Co; Kyung Hee University
Priority date: 2008-02-19
Filing date: 2009-02-18
Publication date: 2009-08-27
Anticipated expiration: 2010-08-19
Also published as: KR20090089672A; WO2009104901A3

Abstract

The present invention is related to the inventive compositions comprising the extract of Anemarrhena asphodeloides BUNGE showing potent anti-inflammatory activity, therefore, it can be used as the effective and safe therapeutics or health food for treating and preventing arthritic disease.

Description

COMPOSITION COMPRISING AN EXTRACT OF ANEMARRHENA ASPHODELOIDES BUNGE FOR PREVENTING AND TREATING ARTHRITIC DISEASES

The present invention relates to a composition comprising an extract of Anemarrhena asphodeloides BUNGE for the prevention and treatment of arthritic disease.

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-α (TNF-α) 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 patients suffering with rheumatoid arthritis present the imbalance in immune system, which causes an overproduction of pro-inflammatory cytokines, e.g., TNF-α, 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. RA progression is associated with elevated levels of TNF-α and IL-1β produced by macrophages and dendrite cells, an imbalance of Th1/Th2 and over-production of antigen specific immunoglobulins. Especially, TNF-α and IL-1β directly induce the synthesis of proteolytic enzyme such as matrix metalloproteinase (MMPs) which can break down the extracellular matrix macromolecules. 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.

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-α antagonists which plays role in binding to its receptor or directly binding to the TNF-α 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-α blockage, etc.

Degenerative arthritis, one of representative osteo-joint diseases is chronic arthritis. It is difficult to treat the disease with conventionally available anti-inflammatory drugs in clinic. Moreover, the drugs give rise to systemic adverse response such as digestive disorder, gastro-intestinal disorder and renal function disorder and the adverse response of the drugs occurs more frequently as the age of patient increases, which causes lots of problems in case of long-term systemic treatment in older people. Therefore, the new drug development targeting anti-inflammatory effect, protecting and regenerating effect on cartilage has been urgently needed than previous systemic treatment therapy recently. The recent theory of drug development has been focused on joint tissue lyase inhibitor, free radical scavenger such as SOD, conservation therapy using by long-term treatment of joint tissue components such as chondroitin or glucosamine etc (Badger A. M. et al., J. Pharmacol. Exp. Ther., 290, pp587-593, 1999; Choi J.H. et al., Osteoarthritis Cartilage, 10(6), pp471-478, 2002).

Anemarrhena asphodeloides BUNGE belonged to Haemodoraceae, has been conventionally used to treat or prevent bacterial infection or fever and known to contain various saponins such as timosaponin A-I, timosaponin A-II, timosaponin A-III, timosaponin A-IV, timosaponin B-I, and timosaponin B-II, nicotinic acid, magniferin, isomangiferin etc (B.S. Chung et al., HyangYakDaeSaJeon, Youngrimsa, pp203-204, 1989).

However, there has been not reported or disclosed about the therapeutic effect of an extract of Anemarrhena asphodeloides BUNGE on the arthritic disease in any of above cited literatures, the disclosures of which are incorporated herein by reference.

Accordingly, the present inventors have confirmed that the extract of Anemarrhena asphodeloides BUNGE shows potent analgesic activity, anti-inflammatory activity and anti-rheumatic activity through various experiments, i.e., the analgesic activity proved by formalin analgesic test, radiation-induced tail flick analgesia test, and rat-paw pressure analgesia test; anti-inflammatory activity proved by croton-oil induced ear oedema test; and anti-rheumatic activity proved by MIA-induced arthritis animal model test, therefore, it can be used as the effective and safe therapeutics or health food for treating and preventing arthritic disease.

According to one aspect of the present invention, the present invention provides a composition comprising an extract of Anemarrhena asphodeloides BUNGE for the prevention and treatment of arthritic disease.

Accordingly, it is an object of the present invention to provide a pharmaceutical composition comprising an extract of Anemarrhena asphodeloides BUNGE as an active ingredient for preventing and treating of arthritic diseases.

The term "extract" disclosed herein includes; the crude extract soluble in water, C₁ to C₄ lower alcohol and the mixture thereof, preferably, the mixture of water and ethanol, more preferably, 30∼70% ethanol solution; the purified extract by fractionating the crude extract to collect butanol soluble extract containing abundant mangiferin, most preferably, the extract containing mangiferin, preferably, 4.0∼8.0 (w/w%) mangiferin as a standard component in total extract.

The term "arthritic disease" disclosed herein includes degenerative arthritis, rheumatic arthritis or Lupus arthritis, preferably, rheumatic arthritis.

The present invention also provided a use of an extract of Anemarrhena asphodeloides BUNGE for the preparation of therapeutic agent for the treatment and prevention of arthritic disease in mammal or human.

The present invention also provided a pharmaceutical composition comprising an extract of Anemarrhena asphodeloides BUNGE as an active ingredient and a pharmaceutically acceptable carrier thereof for treating and preventing arthritic disease.

It is an object of the present invention to provide a method of treating or preventing arthritic disease in human or mammal, wherein the method comprises administering a therapeutically effective amount of an extract of Anemarrhena asphodeloides BUNGE, as an effective ingredient, together with a pharmaceutically acceptable carrier thereof.

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, Anemarrhena asphodeloides BUNGE are dried, cut, crushed and added to 1 to 20-fold, preferably, approximately 5 to 10-fold volume of distilled water, C₁ to C₄ lower alcohols or the mixtures thereof, preferably the mixture of water and ethanol with approximately 1: 0.1 to 1: 10, more preferably, 1: 0.5 to 1:5 (v/v); the solution is performed to extraction at the temperature ranging from 10℃∼100℃, preferably, 60℃∼100℃, for the period ranging from 1 to 6 hours, preferably, 2 to 4 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 inventive crude extract of the present invention.

To obtain inventive purified extract of the present invention, the crude extract prepared by the above-described method is fractionated with equal volume of saturated butanol to collect inventive butanol soluble extract of the present invention.

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^rd Ed. pp6-7, 1998).

Accordingly, the present invention also provided a use of an extract of Anemarrhena asphodeloides BUNGE prepared by the above-described preparation method for the preparation of therapeutic agent for the treatment and prevention of arthritic disease in mammal or human.

The present invention also provided a pharmaceutical composition comprising an extract of Anemarrhena asphodeloides BUNGE prepared by the above-described preparation method as an active ingredient and a pharmaceutically acceptable carrier thereof for treating and preventing arthritic disease.

It is an object of the present invention to provide a method of treating or preventing arthritic disease in human or mammal, wherein the method comprises administering a therapeutically effective amount of an extract of Anemarrhena asphodeloides BUNGE prepared by the above-described preparation 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 analgesic activity, anti-inflammatory activity and anti-rheumatic activity through various experiments, i.e., the analgesic activity proved by formalin analgesic test, radiation-induced tail flick analgesia test, and rat-paw pressure analgesia test; anti-inflammatory activity proved by croton-oil induced ear oedema test; and anti-rheumatic activity proved by MIA-induced arthritis animal model test.

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 the other object of the present invention to provide a functional health food comprising an extract of Anemarrhena asphodeloides BUNGE as an active ingredient.

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 Anemarrhena asphodeloides BUNGE, 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 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 care 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 can be added to food or beverage for prevention and improvement of aimed disease. The amount of inventive extract 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 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 Anemarrhena asphodeloides BUNGE shows potent analgesic activity, anti-inflammatory activity and anti-rheumatic activity through various experiments, i.e., the analgesic activity proved by formalin analgesic test, radiation-induced tail flick analgesia test, and rat-paw pressure analgesia test; anti-inflammatory activity proved by croton-oil induced ear oedema test; and anti-rheumatic activity proved by MIA-induced arthritis animal model test, therefore, it can be used as the effective and safe therapeutics or health food for treating and preventing arthritic disease, therefore, it can be used as the effective and safe therapeutics or health food for treating and preventing 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 result of chromatogram analysis on the content of mangiferin, a standard component contained in the extract of Anemarrhena asphodeloides BUNGE.

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

Example 1. Preparation of the extract of Anemarrhena asphodeloides BUNGE(AA50E)

100 g of Anemarrhena asphodeloides BUNGE purchased from Kyunghee Medical Center in Korea, were dried, cut into small pieces and added to 0.7 L of 50% ethanol. The solution was refluxed for 4 hours with stirring at 80℃ and the residue was filtered. The filtrate was concentrated to the extent that the volume of solution reached to 0.1 L and the equal volume of saturated butanol was added thereto to perform fractionation. The butanol soluble fraction was collected, concentrated and dried to obtain 13 g of the extract of Anemarrhena asphodeloides BUNGE, which was used as a test sample (designated as 'AA50E' hereinafter).

As shown in Fig. 1, it has been confirmed that the AA50E extract contains 5.5% mangiferin (w/w%) through chromatogram analysis ( See , Fig. 1).

Example 2. Preparation of the extract of Anemarrhena asphodeloides BUNGE(AB50E)

100 g of Anemarrhena asphodeloides BUNGE purchased from Kyunghee Medical Center in Korea, were dried, cut into small pieces and added to 0.7 L of 50% ethanol. The solution was refluxed for 4 hours with stirring at 80℃ and the residue was filtered. The filtrate was concentrated and dried to obtain 40 g of the extract of Anemarrhena asphodeloides BUNGE, which was used as a test sample (designated as 'AB50E' hereinafter).

it has been confirmed that the AB50E extract contains 7.0% mangiferin (w/w%) through chromatogram analysis.

Experimental Example 1. Formalin analgesia test

In order to determine the analgesic activity of the inventive extract of the present invention, formalin-induced analgesia test was performed according to the method disclosed in the literature as follows (Frazli-Tabaei S et al., Behav. Pharmacol. , 16, pp613-619, 2005).

Male ICR mouse (Orient Bio, Japan) weighing from 20 to 25 g was acclimated for several days and each group consists of 8 mice. The test samples were administrated orally into the mice and 10 % formalin solution (v/v Sigma Co. USA) was subcutaneously administrated to the left posterior limb one hour after the administration. The licking frequency of the foot sole was observed at the 1^st phase (from initial time to 5 minutes after the administration) and 2^nd phase (from 15 minutes to 20 minutes after the administration) to record. The inhibition rate (%) was calculated by setting the inhibition rate in positive control group treated with Celecoxib, a conventionally used NSAID, to 100.

Table 1

Group	Concentration (mg/kg)	Inhibition rate(%)*
Group	Concentration (mg/kg)	1^st phase	2^nd phase
AA50E (Example 1)	400	88.2	73.9
AB50E (Example 2)	400	90.5	85.6
* Relative inhibition rate by setting the inhibition rate in positive control group treated with Celecoxib to 100

As shown in Table 1, it has been confirmed that both of AA50E and AB50E showed potent analgesic effect.

Experimental Example 2. MIA (Monosodium Iodoacetate) model

In order to confirm the analgesic activity of inventive extract prepared in Examples, MIA (monosodium iodoactetate)-induced arthritis animal model test was performed with the procedure described in the literature (James D. Pomonis et al., Pain, 114, pp339-346, 2005).

Male SD rat (Orientbio. Japan) weighing from 200 to 220 g, was acclimated for several days and MIA (Sigma, cat# I2512, USA) dissolved in PBS was injected into the glenoid cavity of left hind knee to induce arthritis. After a week of recovery, the subjects induced with arthritis were selected by using incapacitance tester apparatus (Linton, Stoelting Co., Wood Dale, IL) and the subjects were grouped to consist of 8 animals for each group. On the 8^th day after the induction, the test samples were orally administered at the routine time for every day and the measurement of resulting data was started at one week after the administration for 3 weeks, once a week. The data was measured using by incapacitance tester (Linton, Stoelting Co., Wood Dale, IL) and calculated according to following Math Method 1. The inhibition rate (%) was calculated by setting the inhibition rate in positive control group treated with Celecoxib, a conventionally used NSAID, to 100.

[Math Method 1]

% Weight of left hind ＝ (｛Weight of left hind/(Weight of left hind + Weight of right hind)｝×100)

Table 2

Group	Concentration (mg/kg)	Inhibition rate(%)*(at 3^rd week)
AA50E (Example 1)	400	89.1
AB50E (Example 2)	400	92.5
* Relative inhibition rate by setting the inhibition rate in positive control group treated with Celecoxib to 100

As shown in Table 2, it has been confirmed that both of AA50E and AB50E showed potent analgesic effect.

Experimental Example 3. Radiation-induced tail flick analgesia test

In order to confirm the analgesic effect of inventive extract prepared in Examples, radiation-induced tail flick analgesia test was performed with the procedure described in the literature (Shaw FZ et al., Brain Res., 911(2), pp105-115, 2001).

Male ICR mouse (Orientbio. Japan) weighing from 20 to 25 g was acclimated for several days and grouped to 8 animals for each group. Test samples were orally administrated thereto. One hour after the treatment, the medianus of tails was irradiated with infrared ray to determine the time until the avoidance response appeared. The inhibition rate (%) was calculated by setting the inhibition rate in positive control group treated with Celecoxib, a conventionally used NSAID, to 100.

Table 3

Group	Concentration (mg/kg)	Inhibition rate(%)*
AA50E (Example 1)	400	120.6
AB50E (Example 2)	400	135.1
* Relative inhibition rate by setting the inhibition rate in positive control group treated with Celecoxib to 100

As shown in Table 3, it has been confirmed that both of AA50E and AB50E showed potent analgesic effect.

Experimental Example 4. Paw pressure analgesia test

In order to confirm the analgesic effect of inventive extract prepared in Examples, rat paw pressure analgesia test was performed with the procedure described in the literature (Randall LO and Selitto JJ, Arch Int. Pharmacodyn., 111, pp409-419, 1957).

Male SD rat (Orientbio. Japan) weighing from 180 to 200 g was acclimated for several days and grouped to 8 animals for each group. Test samples were orally administrated thereto. One hour after the treatment, 2% carrageenan (Sigma Co., USA) was subcutaneously injected into the sinistral ramus posterior of the rat. 3 hours after the injection, the weight at the time that avoidance response appeared, was measured by using analgesic meter (Ugobasile, Italy). The inhibition rate (%) was calculated by setting the inhibition rate in positive control group treated with Celecoxib, a conventionally used NSAID, to 100.

Table 4

Group	Concentration (mg/kg)	Inhibition rate(%)*
AA50E (Example 1)	400	109.5
AB50E (Example 2)	400	115.3
* Relative inhibition rate by setting the inhibition rate in positive control group treated with Celecoxib to 100

As shown in Table 4, it has been confirmed that both of AA50E and AB50E showed potent analgesic effect.

Experimental Example 5. Test with croton oil induced ear oedema

Croton oil induces various skin inflammations such as rash, swelling, blister and so on.

In order to determine the anti-inflammatory activity of inventive extract, following test using croton oil-induced ear edema was performed according to method disclosed in the literature literature (Gabor M, Mouse ear inflammation models and their pharmacological applications, Published by Akademiai Kiado, Budapest, pp 24-28, 2000).

Male ICR mouse (Orientbio. Japan)) weighing from 20 to 25g was used as an experimental animal and each group consists of 6 mice. The test samples were administrated orally and after 1 hour, 2.5% croton oil dissolved in acetone was spread on the inner and outer surface of right ear to induce ear-edema. After 4 hours, the increased rate of ear thickness was calculated by comparing with that of left ear of dead mouse with ether using by thickness gauge according to velocity transformation technique (Patrick et al., Toxicol. Appl. Pharmacol., 81, pp476-490, 1985).

The inhibition rate (%) was calculated by setting the inhibition rate in positive control group treated with Celecoxib, a conventionally used NSAID, to 100.

Table 5

Group	Concentration (mg/kg)	Inhibition rate(%)*
AA50E (Example 1)	400	88.7
AB50E (Example 1)	400	95.1
* Relative inhibition rate by setting the inhibition rate in positive control group treated with Celecoxib to 100

As shown in Table 5, it has been confirmed that both of AA50E and AB50E showed potent anti-inflammatory effect.

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

AA50E 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

AB50E 500mg

Corn Starch 100mg

Lactose 100mg

Talc 10mg

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

Preparation of tablet

AA50E 200mg

Corn Starch 100mg

Lactose 100mg

Magnesium stearate optimum amount

Tablet preparation was prepared by mixing above components and entabletting.

Preparation of capsule

AB50E 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

AA50E 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

AB50E 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 0.2㎍

Vitamin C 10mg

Biotin 10㎍

Amide nicotinic acid 1.7mg

Folic acid 50㎍

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

AA50E 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 extract of Anemarrhena asphodeloides BUNGE 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 arthritic disease.

Claims

A pharmaceutical composition comprising an extract of Anemarrhena asphodeloides BUNGE as an active ingredient for the treatment and prevention of arthritic diseases.
The pharmaceutical composition of claim 1, wherein said extract is the crude extract soluble in water, C₁ to C₄ lower alcohol and the mixture thereof, or the purified extract by fractionating the crude extract to collect butanol soluble extract containing abundant mangiferin.
The pharmaceutical composition of claim 1, wherein said extract contains 4.0∼8.0 (w/w%) mangiferin as a standard component in total extract.
The pharmaceutical composition of claim 1, wherein said arthritic disease is degenerative arthritis, rheumatic arthritis or Lupus arthritis.
A use of an extract of Anemarrhena asphodeloides BUNGE for the preparation of therapeutic agent for the treatment and prevention of arthritic disease in mammal or human.
A method of treating or preventing arthritic disease in human or mammal, wherein the method comprises administering a therapeutically effective amount of an extract of Anemarrhena asphodeloides BUNGE, as an effective ingredient, together with a pharmaceutically acceptable carrier thereof.
A health care food comprising an extract of Anemarrhena asphodeloides BUNGE, together with a sitologically acceptable additive for the prevention and alleviation of arthritic diseases.
The health care food of claim 7, wherein said health care food is provided as powder, granule, tablet, capsule or beverage type.