WO2005067940A1

WO2005067940A1 - Novel use of water soluble glucan oligomer isolated from saccharomyces cerevisiae is2 for prevention and treatment of avian flu

Info

Publication number: WO2005067940A1
Application number: PCT/KR2005/000168
Authority: WO
Inventors: Won Kook Moon; Dong Woo Kim; Bong Hyun Chung; Jeong Hoon Park
Original assignee: Natural F and P Corp
Current assignee: Natural F and P Corp
Priority date: 2004-01-19
Filing date: 2005-01-19
Publication date: 2005-07-28
Anticipated expiration: 2006-07-19
Also published as: KR100485056B1; CN1909914A

Abstract

The present invention relates to novel use of water soluble glucan oligomer having a M.W. ranging from 1.000 to 10.000 prepared by treating insoluble beta-glucan isolated the cell wall of yeast variant IS2 with commercially available beta-glucan hydrolyzing enzymes for the preparation of pharmaceutical composition to treat and prevent avian flu, in a mammal including human in need thereof, therefore, it can be used as the therapeutic or health care food for treating and preventing avian flu.

Description

Description NOVEL USE OF WATER SOLUBLE GLUCAN OLIGOMER ISOLATED FROM SACCHAROMYCES CEREVISIAE IS2 FOR PREVENTION AND TREATMENT OF AVIAN FLU Technical Field

[1] The present invention relates to novel use of water soltble glt an oligomer isolated from Saccharomyces cerevisiae IS2 for the preparation of pharmaceutical composition to treat and prevent avian flu, specifically H1N2 type avian influenza in a mammal including htman in need thereof. Background Art

[2] Various kinds of avian flu including nonpathogenic and highly pathogenic flu are caused by avian viral infection mediated by poultries such as chick, duck, turkey, wild avian and the like on farms and in live markets. Among them, highly pathogenic avian St influenza has been designated as the 1 group of legal communicable disease in Korea and List A infectious disease in OIE (Office International des Epizooties). The pathogen, i.e., avian influenza virus, a sort of zoonotic virus is classified into highly pathogenic, weakly pathogenic and non-pathogenic virus according to their pathogenecity and there are 135 kinds of viruses according to their serotypes including 15 HA proteins and 9 NA proteins. Among those serotypes, virulent avian virus transmitted to htman such as H5Nland H9N2 stbtype has been reported recently.

[3] Avian influenza virus is frequently transmitted to htman through intermediate host such as swine, chicken, etc, modified by genetic nutation in the host and the mutated virus is transmitted to htman through respiratory tract and other pathway resulting in pathogenic avian flu.

[4] However, there have not yet developed effective agents to prevent or treat avian flu till now since avian influenza virus has lots of serotypes and shows frequent nutation. Accordingly, most of affected countries by pandemic avian flu have used to adopt mass cully or slaughter policy and it has been reported that barrier quarantine such as strict off limits and disinfection is the best way to protect avian flu.

[5] Recently, it has been found that the stbtype of swine influenza virus isolated from Korean pig is H1N2 and the PA and PB gene isolated from 8 gene fragment of H1N2 isolated strain are derived from avian influenza virus (K. Jung and C. Chae., Archives of Virology, in press, 2004). Accordingly, it is confirmed that the H1N2 isolated strain has the part of avian influenza virus.

[6] Beta-glucan can be isolated from various resources such as yeast, microorganism, mushroom, grain and algae. It has been studied and applied as various types of product till now. In particular, beta-glucan derived from yeast cell wall has been studied and known well.

[7] Yeast, a microorganism classified into GRAS (Generally Recognized As Safe) in FDA, has been used in various field including food field and the inner cell membrane of yeast comprises beta 1, 3- and 1, 6-gluccano as a main ingredients, and a small amount of chitin and mannoprotein, however, outer cell membrane thereof comprises mannoprotein, a protein linked to mannan.

[8] Beta-glucan, a major component of yeast cell wall has been reported to stirmlate immune system and reinforce the preventing mechanism of infected host by virus. TNF-α secreted from macrophage and monocytes during inflammation and NO (inducible nitric oxide) induced therefrom are reported to be effective in preventing RNA virus including avian influenza virus (Akaike T et al, Immunology, 101. pp30α308, 200Q Seo S. H. et al, Journal of Virology, 76, ppl071-1076, 2002).

[9]

[10] However, there has been not reported or disclosed about the therapeutic and preventing effects on the avian flu of specific soltble glt an oligomer isolated from yeast variant strain IS2 (KCTC 0959BP) in any of above cited literatures, the disclosures of which are incorporated herein by reference.

[11] The inventors of the present invention have been endeavored to find pharmacologically potent beta-glucan from specific yeast variant strain from investigate and finally completed present invention by confirming that the soltble gltcan oligomer having less than 50,000 D of M. W. obtained by extracting the cell wall of yeast mutant IS2 shows potent anti-viral activity of swine influenza virus derived from avian influenza virus directly and indirectly.

[12] These and other objects of the present invention will become apparent from the detailed disclosure of the present invention provided hereinafter. Disclosure of Invention Technical Problem

[13] According to one aspect of the present invention, the present invention provides a use of soltble gltcan oligomer derived from yeast variant strain IS2 (KCTC 0959BP) for the preparation of pharmaceutical composition to treat and prevent avian flu, specifically H1N2 type avian influenza in a mammal including htman in need thereof.

[14] Also, the present invention provides the pharmaceutical composition comprising soltble gltcan oligomer prepared by the method of the present invention for treating and preventing avian flu, specifically H1N2 type avian. Technical Solution

[15] Accordingly, it is an object of the present invention to provide a use of soltble gltcan oligomer derived from yeast variant strain IS2 (KCTC 0959BP) for the preparation of pharmaceutical composition to treat and prevent avian flu, specifically H1N2 type avian influenza in a mammal including htman in need thereof.

[16] It is another object of the present invention to provide a pharmaceutical composition comprising soltble gltcan oligomer derived from yeast variant strain IS2 (K C T C 0959 B P ) for treating and preventing avian flu, specifically H 1 N2 type avian flu.

[17] The term "soltble gltcan oligomer"disclosed herein can be prepared by follows:

[18] The soltble gltcan oligimer of the present invention may be prepared by the procedure comprising the steps consisting of: (a) culturing yeast ( Saccharomyces cerevisiae) variant IS2 (KCTC 0959BP) in the culture broth for inoculation; (b) inoculating above yeast culture solution to culture broth, culturing and centrifuging to obtain yeast; (c) adding NaOH thereto to extract beta-gltcan from yeast cell wall; (d) reacting extracted beta-gltcan with hydrolyzing enzyme and then stbjecting to filtration to obtain soltble gltcan oligomer; and (e) finally drying with lyophilization to obtain the soltble gltcan oligomer of the present invention.

[19] Above described yeast variant IS2 is characterized by treating and preventing avian flu.

[20] The soltble gltcan oligomer prepared by above described procedure comprises gltcan oligomer having a molecular weight of less than 50,000, preferably, ranging from 1,000 to 10,000.

[21] It is the other object of the present invention to provide a pharmaceutical composition comprising soltble gltcan oligomer derived from the cell wall of yeast variant strain (KTCT 0959BP) obtained by above described procedure as an active ingredient in an effective amount to treat and prevent avian flu, specifically H1N2 type avian flu, together with a pharmaceutically acceptable carrier thereof.

[22] The inventive soltble gltcan oligomer may be prepared in accordance with the following preferred eπi>odiment. [23] For the present invention, above described soltble gltcan oligomercan be prepared by following procedure;

[24] 1^st step, the step culturing yeast IS2 (KCTC 0959BP) consisting that yeast IS2 (KCTC 0959BP) is cultured in liquid culture median comprising 0.5 — 10 w/v% glucose, 0.1-5 w/v% yeast extract, 0.1-10 w/v% pepton;

[25] 2 step, the step obtaining yeast from yeast culture median consisting that the yeast culture median prepared from the first stage in a amount ranging from 0.1 to 10% (v/v) is inoculated to primary liquid culture median comprising 0.5 — 10 w/v% glucose, 0.1-5 w/v% yeast extract, Oβl-2 w/v% ammonian sulfate, 0fl01-l w/v% potassian phosphate, and 0fl01-l w/v% magnesian sulfate in the pH ranging from 5.0 to 6β, cultured for the period ranging from 12 hours to 48 hours at the speed ranging from 100 to 400 rpm, in the ventilating gas amount ranging from 0.3 to 3 vvm, at the temperature ranging from 20 to 40 °C in growth media and then stbjected to cen- trifugation to obtain yeast;

[26] 3^r step, the step extracting wet beta-gltcan from the cell wall of the yeast consisting that 1 — 10 % sodian hydroxide solution is added to the yeast, dispersed, reacted for the period ranging from 30 rrinutes to 5 hours at the temperature ranging from 70 to 100 °C, stbjected to centrifugation to obtain dried cell mass (DCW) of yeast, of which process may be repeated at several times to pool, titrating the pH of the mass ranging from 4β to 5.0 using by strong acid such as hydrochloric acid and hydrogen sulfuric acid, dispersed again in sodian hydroxide solution, further reacting for 1 hour at 75 °C, stbjecting centrifugation to separate to sodian hydroxide solution and solid component; and finally washing and purifying the solid component to obtain wet beta- glucan;

[27] 4 step, the step obtaining liquid phase of glucan oligomer consisting that distilled water at the amount equivalent to 1 to 10 times the volane of the gltcan (v/v%) and beta-gltcan hydrolyzing enzyme at the amount equivalent to 1/20 to 1/5 times of the glucan (v/w%) are added thereto, reacting for the period ranging from 6 to 24 hours at the temperature ranging from 30 to 80 °C, recovering supernatant solution by centrifuging after quenching the reaction, filtering supernatant with ultra filtration membrane to obtain inventive soltble gltcan oligomer solution having a molecular weight of less than 50,000.

[28] 5 step, the step obtaining dried powder form of final soltble gltcan oligomer consisting that the oligomer prepared from 4 step is left alone for the period ranging from 12 hours to 48 hours at less than - 70 °C, and then stbject to lyophilzation to obtain the powder form of soltble gltcan oligomer of the present invention.

[29] It is the other object of the present invention to provide the soltble gltcan oligomer derived from yeast variant IS2 (KCTC 0959BP) prepared by the preparation as described above and It is the other object of the present invention to provide a pharmaceutical composition comprising soltble gltcan oligomer derived from the cell wall of yeast variant strain (KTCT 0959BP) obtained by above described procedure as an active ingredient in an effective amount to treat and prevent avian flu, specifically H1N2 type avian flu, together with a pharmaceutically acceptable carrier thereof.

[30] Above described avian flu comprises the flu caused by the infection of the avian influenza virus such as H1N2, H5N1, H9N2 stbtype influenza virus, preferably H1N2 stbtype virus.

[31] It is an object of the present invention to provide a use of a pharmaceutical composition comprising a soltble gltcan oligomer derived from yeast variant strain IS2 (KCTC 0959BP) for the preparation of therapeutic agent for treatment and prevention of avian flu in human or mammal in need thereof.

[32] It is an object of the present invention to provide a method of treating or preventing avian flu in a mammal comprising the step of administering to said mammal an effective amount of composition comprising a soltble gltcan oligomer derived from yeast variant strain IS2 (KCTC 0959BP), together with a pharmaceutically acceptable carrier thereof.

[33] The inventive composition may additionally comprise conventional carrier, adjuvants or diluents in accordance with a using method. It is preferable that said carrier is used as appropriate stbstance 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).

[34] Hereinafter, the following formulation methods and excipients are merely exemplary and in no way Unit the invention.

[35] 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 rtbber, alginate, gelatin, calcian phosphate, calcian silicate, cellulose, methyl cellulose, polyvinyl pyrrolidone, water, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesian stearate and mineral oil. The forrmlations may additionally include fillers, anti-agglutinating agents, rubricating agents, wetting agents, flavoring agents, ermlsifiers, preservatives and the like. The compositions of the invention may be forrmlated 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.

[36] For example, the composition of the present invention can be dissolved in oils, propylene glycol or other solvents which 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 United to them. For topical administration, the extract of the present invention can be forrmlated in the form of ointments and creams.

[37] Pharmaceutical forrmlations containing crude drug 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), suppository, or sterile injectable preparation (solution, suspension, emulsion).

[38] 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 coniήnation with other pharmaceutically active ingredients.

[39] The desirable dose of the inventive composition varies depending on the condition and the weight of the stbject, 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 0.01-500ιηg/kg, preferably, 0.1 to lOOmg/kg by weight/day of the inventive composition of the present invention. The dose may be administered in a single or multiple doses per day.

[40] The pharmaceutical composition for preventing and alleviating avian flu could contain about 0.1 to 50 w/w%, of the above soltble gltcan oligomer derived from yeast variant strain IS2 (KCTC 0959BP) of present invention based on the total weight of the composition.

[41] The pharmaceutical composition of present invention can be administered to a stbject animal comprising mammals for example, rat, mouse, domestic animals such as dog, cat, cow, pig and htman via various routes. All modes of administration are contemplated well-known in the art, for example, administration can be made orally, rectally or by intravenous, intramuscular, stbcutaneous, intracutaneous, intrathecal, epidural or intracerebroventricular injection, or feeding by coniήng with animal feed. [42] It is still another object of the present invention to provide a health care food comprising a composition essentially comprising a soltble gltcan oligomer derived from yeast variant strain IS2 (KCTC 0959BP), together with a sitologically acceptable additive for preventing and improving avian flu.

[43] The health care food for preventing and alleviating avian flu could contain about 0.01 to 80 w/w%, preferably 1 to 50 w/w% of the above soltble gltcan oligomer derived from yeast variant strain IS2 (KCTC 0959BP) of present invention based on the total weight of the composition.

[44] The present invention provides a composition of the health care food beverage for preventing and alleviating avian flu comprising a soltble gltcan oligomer derived from yeast variant strain IS2 (KCTC 0959BP).

[45] Above inventive oligomer composition can be added to food and beverage for the preventing and alleviating avian flu.

[46] To develop for health care food, examples of addable food comprising above oligomer composition of the present invention are e.g., various food, beverage, bread, cookies, jam, candy, gan, tea, yogurt, vitamin complex, health improving food and the like, and can be used as power, granule, tablet, chewing tablet, capsule or beverage etc.

[47] Inventive composition of the present invention has no toxicity and adverse effect, therefore, they can be used with safe.

[48] Above described composition therein can be added to food, additive or beverage, wherein, the amount of above described oligomer in food or beverage may generally range from about 0.01 to 80 w/w % of total weight of food for the health care food composition and Offi to 30 g, preferably 0.3 to 5 g in the ratio of 100mβ of the health beverage composition.

[49] Providing that the health beverage composition of present invention contains above described oligomer 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 rronosaccharide 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 taanatin, 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 M of present beverage composition.

[50] The other components than aforementioned composition are various nutrients, a vitaπin, 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.

[51] The inventive composition can be used as the mixing agent in the lactic acid bacteria-formilated beverage or paste and the like.

[52] Above-mentioned component can be used independently or in coniήnation.

[53] The present invention provides a health care food comprising about Oβl to 30 w/w % of the vitaπin, oligosaccharides and dietary ingredients besides the composition of the present invention.

[54] The ratio of the components is not so important but is generally ranging from about 0.01 to 30 w/w % per 100 w/w % present composition. Examples of addable food comprising aforementioned extract therein are various food, beverage, gan, vitamin complex, health improving food and the like.

[55] The inventive composition may additionally comprise one or more than one of organic acid, such as citric acid, fanaric acid, adipic acid, lactic acid, malic acid; phosphate, such as phosphate, sodian phosphate, potassian phosphate, acid py- rophosphate, polyphosphate; natural anti-oxidants, such as polyphenol, catechin, α- tocopherol, rosemary extract, vitamin C, licorice root extract, chitosan, tannic acid, phytic acid etc.

[56] 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. Advantageous Effects

[57] The soltble gltcan oligomer having less than 50,000 D of M.W. prepared by treating insoltble beta-gltcan isolated the cell wall of yeast variant IS2 with commercially available beta-gltcan hydrolyzing enzymes showed potent anti-viral activity of swine influenza virus derived from avian influenza virus directly and indirectly, therefore, it can be used as the therapeutics or health care food for treating and preventing avian flu. Brief Description of the Drawings

[58] 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; [59] Fig. 1 shows the effect of soltble gltcan oligomer on NO production by mouse lymphocytes after peritoneal injection; [60] Fig. 2 shows the effect of soltble gltcan oligomer on 11_^2 production in spleen cell when it peritoneally injected to mouse; [61] Hereinafter, the present invention is more specifically explained by the following examples. However, it should be understood that the present invention is not United to these examples in any manner. Mode for the Invention [62] The following Examples and Experimental Examples are intended to further illustrate the present invention without Uniting its scope. [63] Example 1. Culture of yeast variant IS2 and harvest

[64] Liquid median containing 10 g/1 of glucose, 6 g/1 yeast extract, 3 g/1 of ammonian sulfate ((NH ) SO ), 1.5 g/1 of potassian phosphate (K PO ), 0.5 g/1 of magnesian 2 2 4 sulfate (MgSO ^• 7H O) was used as primary median. 4 2

[65] Liquid YPD median (glucose 20 g/1, yeast extract 10 g/1, peptone 20 g/1) was used for inoculation and growth media containing 400g/l of glucose, 30 g/1 yeast extract, 40 g/1 of ammonian sulfate ((NH ) SO ), 15 g/1 of potassian phosphate (K PO ) and 5.7 4 2 4 2 4 g/1 of magnesian sulfate (MgSO ^• 7H O). 4 2

[66] After autoclaving the growth media, 100 mβ of cultured yeast variant IS2 (KCTC 0959BP) was seeded thereto, cultured at rotating speed of 300 rpm, at the ventilating gas amount of 1 vvm, at 30 °C pH 5.5 and finally 50-55 g/1 of dried cell mass (DCW) of yeast was obtained through batch culture.

[67] Example 2. Extraction of beta gltcan from yeast variant IS2

[68] 80 g of DCW of yeast prepared in above Example 1, was suspended in 1000 mβ of 4% sodian hydroxide (NaOH) solution and then inctbated at 95 °C for 1 hour. The inctbated suspension was centrifuged at the speed of 2000 rpm for 15 minutes to separate into NaOH solution part and solid part.

[69] The separated solid part was suspended again in 2000 β of 3 % sodian hydroxide solution, inctbated at 75 °C for 3 hours and then centrifuged at the speed of 2000 rpm for 15 rrinutes to separate into NaOH solution and solid part again. [70] The pooled solid part was adjusted to pH 4.5 with HC1, dispersed to the extent the final volume of 2000 M and inctbated at 75 °C for 1 hour again. The inctbated suspension was centrifuged at the speed of 2000 rpm for 15 minutes to separate into NaOH solution part and solid part.

[71] The solid part was washed 3 times with distilled water to obtain 160 g of wet beta gltcan from the cell wall of yeast variant. f72] Example 3. Preparation of soltble gltcan oligomer from beta-gltcan of yeast variant IS2

[73] 160 g of wet beta gltcan prepared from Example 2 was put in 1000 M of flask and 480 M of distilled water and beta b-gltcanase at the amount equivalent to 1/10 of the gltcan (v/w) were added thereto and inctbated at 40 °C for 15 hours.

[74] After stopping the reaction, the reaction mixture was centrifuged at 7000 rpm for 15 minutes to collect the supernatant. The collected supernatant was filtered and the un- reacted enzymes were removed using by ultra filtration membrane (Filtron Go., MWCO 10K) to obtain the solution containing gltcan oligomer having MW of less than 10,000 Dalton. After the solution had been left alone at -74 °C for overnight, the solution was lyophilized to produce 5.8 g of powder form of soltble gltcan oligomer.

[75] Experimental Example 1. Effect of soltble gltcan oligomer on the production of NO (Nitric Oxide) and TNF-α.

[76] The effect of the test group containing soltble gltcan oligomer prepared from Example 3 and control group on the production of NO (Nitric Oxide) and TNF-α secreted from alveolar macrophage caused by LPS (Upopolysacchande) treatment was determined by following experiment.

[77] Cell culture

[78] Healthy weanUng pigs were stbjected to autopsy and their pneumoenterus were delivered aseptically. The alveolar macrophage was extracted from the organ with PBS 2 and the macrophage was floated in cell culture flask with a size of 75 cm using by DMEM median containing 10 % FBS and 2x antibacterial-antifungal solution. To confirm whether the macrophage has been contaminated with 7 porcine virus, i.e., porcine parvovirus, porcine circovirus type 2, porcine circovirus type 1, porcine reproductive and respiratory syndrome virus, Japanese encephalitis virus, encephalomye- locarditis virus and pseudorabies virus or not, PCR (polymerase chain reaction) examination was performed by extracting RNAs from the part of the floated solution for 12 hrs, which is modified by the procedure disclosed in the literature (Jabrane et al., Can. Vet. J. 35, pp86-92, 1994). [79] Prior to the inoculation of alveolar macrophage into plates, the macrophage was washed twice with PBS solution and detached from the surface of the flask using Trypsin-EDTA. The concentration of detached alveolar macrophage was adjusted to 1x10 -10 /ml/well in each 24 well plates.

[80] Determination of the production of NO (Nitric Oxide) and TNF-α

[81] lOmg/ml of water soltble gltcan oligomer dissolved in DMEM median was inoculated into each well with a concentration of 5mg/ml. 24 hours after, H1N2 swine influenza virus was inoculated into each well. After the inoculation, the supernatant was collected every 12 hours from 12 hrs to 60 hrs after the inoculation to determine the production of NO (Nitric Oxide) and TNF-α. The concentration of NO and TNF-α were determined by using nitrate/nitrite colorimetric assay kit (Cayman Chemical Go.) and porcine TNF-α ELISA kit (Endogem Go.) respectively.

[82] As the result of determination of NO production, the test groups injected with soltble gltcan oligomer showed about 4 to 5 folds of increased NO production in alveolar macrophage compared with that of negative control ( See Fig. 1).

[83] As the result of determination of TNF-α production, the expression of TNF-α has been increased from 12 hrs to 48 hrs after the inoculation and sharply decreased at 60 hr after the inoculation in virus treatment group. The expressed amount of TNF-α in test group treated with water soltble gltcan oligomer was significantly higher than that of negative control for the period ranging from 24 to 60 hours after the inoculation ( See Fig. 2, P<0D5).

[84] Experimental Example 2. Anti-viral Effect of soltble gltcan oligomer on porcine influenza virus

[85] Indirect antiviral activity of test sample group and negative control group was determined by treating the supernatants of the culture median in alveolar macrophage with virus and inoculating into culture cell to observe the cytopathic effect caused by influenza virus. Direct antiviral activity of test sample group and negative control group was determined by treating sample with influenza virus directly.

[86] 2-1. The preparation of virus and cell line

[87] H1N2 isolation strain was used as a swine influenza virus and the TCID value was 5x 10⁷⁵/ml.

[88] NO obtained from the supernatant of alveolar macrophage prepared in Experimental Example 1-2 was used in determining antiviral activity and MDCK (Mardin-Darby Canine Kidney, ATCC) cell line derived from dog kidney was used in experiment. [89] 2-2. Indirect determination of antiviral activity

[90] Mono-layer cell was cultured and grown in 96 well plates. 5mg/ml of the gltcan oligomer (GO group) and gltcan LPS (GL group) treated supernatant were used as NO treatment groups. As a negative control group, DMEM culture median and gltcan/ LPS non-treated supernatant was used in the experiment. The viruses were inoculated 3 2 in various concentrations, i.e., 10 , 10 , 10 TCID /well. 24 hrs and 36 hrs after the 50 viral inoculation, the CPE caused by inoculated viruses was determined by inverted microscope and the results were classified into four grades, i.e., +++(strongest CPE), ++ (moderate CPE), + (rrild CPE), and - (no CPE) according to the method disclosed in the literature (Belaid et al., J. Meά. Virol, 66(2). pp229-34, 2002). Serial three wells were used as each group and the mean of resulting values was determined.

[91] As can be seen in Table 1, the supernatant collected from the alveolar macrophage culture median treated with both of gltcan oligomer and LPS showing most potent NO producing ability had most potent anti-viral effect on porcine influenza virus. 3 Especially, the most abundant virus treatment group (10 TCID ) showed higher 50 inhibiting activity by about 70% and 30% of CPE caused by porcine influenza virus compared with that of control group at 24 hours and 36 hours after the inoculation re- 2 1 spectively. Furthermore, the least abundant virus treatment group (10 and 10 TCID ) 50 showed most potent inhibiting activity by about 100% of CPE caused by porcine influenza virus compared with that of control group at 36 hours after the inoculation

[92] Table 1

[93] 2-3. direct determination of antiviral activity [94] To determine the direct antiviral activity of water-soltble oligomer, 5mg/ml of gltcan prepared in Example 3 and porcine influenza virus were nixed directly and treated into rronolayer cells as a test group (GN group). The viruses were inoculated in 3 2 various concentrations, i.e., 10 , 10 , 10 TCID /well. The group treated with only 50 gltcan oligomer (G group) was used to compare with other control groups. 24 hrs and 36 hrs after the viral inoculation, the CPE caused by inoculated viruses was determined by inverted microscope and the results were classified into four grades, i.e., +++(strongest CPE), ++ (moderate CPE), + (rrild CPE), and - (no CPE) according to the method disclosed in the literature (Belaid et al., J. Meά. Virol, 66(2 . pp229-34, 2002). Serial three wells were used as each group and the mean of resulting values was determined.

[95] As can be seen in Table 2, the group treated with only gltcan oligomer (G group) showed 100% antiviral activity regardless of the amount of virus compared with control group till 24 hours. 36 hours after the inoculation, the most abundant virus 3 treatment group (10 TCID ) showed 70% of antiviral activity and the least abundant 2 ⁵° 1 virus treatment group (10 and 10 TCID ) showed 100% of antiviral activity. 50

[96] Table 2

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

[98] Preparation of powder

[99] Dried powder of Example 3 50mg

[100] Lactose lOOmg

[101] Talc lOmg

[102] Powder preparation was prepared by nixing above components and filUng sealed package.

[103] Preparation of tablet [104] Dried powder of Example 3 50mg [105] Cbrn Starch lOOπg [106] Lactose lOOmg [107] Magnesian Stearate 2mg [108] Tablet preparation was prepared by nixing above components and entabletting. [109] Preparation of capsule [110] Dried powder of Example 3 50mg

[111] Cbrn starch lOOmg

[112] Lactose lOOπg

[113] Magnesian Stearate 2mg

[114] Tablet preparation was prepared by nixing above components and filling gelatin capsule by conventional gelatin preparation method.

[115] Preparation of injection

[116] Dried powder of Example 3 50mg

[117] Distilled water for inj ection optiman amount

[118] PH controller optimm amount

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

[120] Preparation of Uquid

[121] Dried powder of Example 3 0.1~80g

[122] Sugar 5~10g

[123] Gtric acid 0.05-0.3%

[124] Caramel 0.005~0ffi%

[125] Nitanin C 0.1-1%

[126] Distilled water 79-94%

[127] CO gas 0.5-0.82% 2

[128] Liquid preparation was prepared by dissolving active component, filling all the components and sterilizing by conventional liquid preparation method.

[129] Preparation of health care food

[ 1 0] Extract of Example 3 1 OOOmg

[131] Nitaπin mixture optimm amount

[132] Nitaπin A acetate 70mg

[133] Nitanin E lβπg

[134] Nitanin B 0.13mg

[135] Nitanin B 0.15πg 2

[136] Nitanin B6 lOπg

[137] Biotin lOπg

[ 138] Aπide nicotinic acid 1 mg

[139] Folic acid 50mg

[140] Calcian pantothenic acid 0.5mg [141] Mineral mixture optimm amount

[142] Ferrous sulfate 1.75mg

[143] Zinc oxide 0.82πg

[144] Magnesian carbonate 25.3mg

[145] Monopotassian phosphate 15mg

[146] Dicalcian phosphate 55mg

[147] Potassian citrate 90mg

[148] Calcian carbonate lOOmg

[149] Magnesian chloride 24.8mg

[150] The above-mentioned vitaπin and mineral mixture 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.

[151] Preparation of health beverage

[152] Dried powder of Example 3 1 OOOmg

[153] Gtric acid lOOOmg

[154] Oligosaccharide lOOg

[155] Apricot concentration 2g

[156] Taurine lg

[157] Distilled water 900m£

[158] Health beverage preparation was prepared by dissolving active component, nixing, stirred at 85°C for 1 hour, filtered and then filling all the components in 1000mβ ample and sterilizing by conventional health beverage preparation method.

[159] 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. Industrial Applicability

[160] As described in the detailed description of the present invention, the soltble gltcan oligomer having a M.W. ranging from lβOO to 10,000 prepared by treating insoltble beta-gltcan isolated the cell wall of yeast variant IS2 with commercially available beta-gltcan hydrolyzing enzymes showed potent anti-viral activity of swine influenza virus derived from avian influenza virus directly and indirectly, therefore, it can be used as the therapeutics or health care food for treating and preventing avian flu.

Claims

[ 1 ] 1.A use of water-soltble gltcan oligomer derived from yeast variant strain IS2 (KCTC 0959BP) for the preparation of pharmaceutical composition to treat and prevent avian flu, in a mammal including htman in need thereof.

[2] 2.The use according to claim 1, wherein said water soltble oligomer is prepared by the method comprising the steps consisting of: (a) culturing yeast ( Sac- charomyces cerevisiae) variant IS2 (KCTC 0959BP) in the culture broth for inoculation; (b) inoculating above yeast culture solution to culture broth, culturing and centrifuging to obtain yeast; (c) adding NaOH thereto to extract beta-gltcan from yeast cell wall; (d) reacting extracted beta-gltcan with hydrolyzing enzyme and then stbjecting to filtration to obtain soltble gltcan oligomer; and (e) finally drying with lyophilization to obtain the soltble gltcan oligomer having a molecular weight of less than 50,000.

[3] 3.The use according to claim 1, wherein said avian flu is caused by H1N2, H5N1, H9N2 stbtype influenza virus.

[4] 4.The use according to claim 3, wherein said avian flu is caused by H1N2 stbtype influenza virus.

[5] 5. A pharmaceutical composition comprising the soltble gltcan oligomer derived from the cell wall of yeast variant strain (KCTC 0959BP) obtained for treating and preventing avian flu as an active ingredient.

[6] 6.A health care food comprising a soltble gltcan oligomer derived from yeast variant strain IS2 (KCTC 0959BP) as set forth in claim 1, together with a sitologically acceptable additive for the prevention and improvement of avian flu.

1/1 [Fig. 1]

Nitric Oxϊde □virus only ^■ «3O + tf rus

12 hrs 24 hrs 36 hrs after 48 hrs 60 hrs iπocuration

1/1/1 [Fig.2]

TNF-G αvirusr only

hrs 24 hrs 36 hrs after 48 hrs 60 hrs inoculation