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Title:
FOMITOPSIS PINICOLA EXTRACT AND IT’S USE IN THE TREATMENT OF DIABETES
Document Type and Number:
WIPO Patent Application WO/2007/075069
Kind Code:
A1
Abstract:
Disclosed herein are Fomitopsis pinicola extracts and use thereof. Fruit body extracts and cultured mycelial extracts of Fomitopsis pinicola effectively inhibit the ROS production induced by diabetes mellitus, thereby finding various applications for the development of functional foods for preventing ROS-caused tissue injury.

Inventors:
OH, Seung-Hee (# Ssangyong Apt, Yongheung-dong Buk-g, Pohang-si Gyeongsangbuk-do 791-767, 103-1102, KR)
KIM, Soon-Dong (609-1 Jung-dong, Suseong-gu, Daegu 706-838, KR)
LEE, Sang-Il (49-11 Jincheon-dong, Dalseo-gu, Daegu 704-834, KR)
LEE, Hyun-Goo (# Yangjimaeul, 36 Sunae-dong Bundang-g, Seongnam-si Gyeonggi-do 463-838, 208-1101, KR)
Application Number:
KR2006/005877
Publication Date:
July 05, 2007
Filing Date:
December 29, 2006
Export Citation:
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Assignee:
EUGENE BIO.FARM CO.LTD (#204, c/o Advanced-Agribusiness Incubator of Korea National Agricultural, College 11-1 Donghwa-ri, Bongdam-eu, Hwaseong-si Gyeonggi-do 445-893, KR)
OH, Seung-Hee (# Ssangyong Apt, Yongheung-dong Buk-g, Pohang-si Gyeongsangbuk-do 791-767, 103-1102, KR)
KIM, Soon-Dong (609-1 Jung-dong, Suseong-gu, Daegu 706-838, KR)
LEE, Sang-Il (49-11 Jincheon-dong, Dalseo-gu, Daegu 704-834, KR)
LEE, Hyun-Goo (# Yangjimaeul, 36 Sunae-dong Bundang-g, Seongnam-si Gyeonggi-do 463-838, 208-1101, KR)
International Classes:
A61K36/07
Attorney, Agent or Firm:
YOU ME PATENT AND LAW FIRM (Seolim Bldg.Poonglim Building, 649-10 Yoksam-don, Kangnam-gu Seoul 135-080, KR)
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Claims:

Claims

[1] A composition, effective for the inhibition of diabetes mellitus-induced reactive oxygen species production, comprising a fruit body extract or cultured mycelial extract of Fomitopsis pinicola.

[2] The composition according to claim 1, wherein the fruit body extract is a hot- water extract or an alkali extract.

[3] The composition according to claim 2, wherein the hot-water extract is prepared by cutting Fomitopsis pinicola fruit bodies into fine pieces, pulverizing the pieces, heating the pulverized pieces in water, concentrating the aqueous solution, precipitating the solution with ethanol, and freeze-drying the precipitate, in order.

[4] The composition according to claim 3, wherein the pulverized pieces of the fruit bodies are heated at 100°C for 24 hours and the aqueous solution is concentrated at 40°C to one tenth of the initial volume.

[5] The composition according to claim 2, wherein the alkali extract is prepared by cutting Fomitopsis pinicola fruit bodies into fine pieces, pulverizing the pieces, swelling the pulverized pieces in IN KOH, homogenizing the pieces, filtrating the homogenate, neutralizing the filtrate, washing the neutralized filtrate with distilled water, and drying the washed filtrate at 60°C, in order.

[6] The composition according to claim 5, wherein the pulverized pieces of the fruit bodies are mixed with IN KOH in a ratio of 1:1 (w/v), left to swell for 1 hour, homogenized, filtered through a 100 mesh sieve, and neutralized with cone. HCl.

[7] The composition according to claim 1, wherein the cultured mycelial extract is prepared by seed- and sub-culturing mycelia of Fomitopsis pinicola, inoculating the mycelia in a sterile and cold potato medium, neutralizing the culture, precipitating with ethanol, and dialyzing the precipitate against distilled water.

[8] The composition according to claim 7, wherein the mycelia are inoculated in the potato medium to an amount of 2% (v/v), and the culture is neutralized with sodium hydrogen carbonate (NaHCO ) to a pH of 6.5.

Description:

Description FOMITOPSIS PINICOLA EXTRACT HAVING INHIBITORY

ACTIVITY AGAINST REACTIVE OXYGEN SPECIES PRODUCTION INDUCED BY DIABETES MELLITUS AND THE

USE THEREOF

Technical Field

[1] The present invention relates to a Fomitopsis pinicola extract and the use thereof.

More particularly, the present invention relates to the fruit body extracts and cultured mycelial extract of Fomitopsis pinicola, which effectively inhibit the reactive oxygen species production induced by diabetes mellitus. Background Art

[2] In the condition of hyperglycemia, which is one of the classic symptoms of diabetes mellitus, glucose enters either glycolysis or the polyol pathway. The latter case occurs when hexokinase, which is involved in anaerobic glycolysis, is saturated so that excess glucose is reduced to sorbitol via aldose reductase and then metabolized to fructose via sorbitol dehydrogenase. An abundance of intracellular sorbitol generates osmotic stress due to its low permeability through the cell membrane, thus injuring tissue cells. In addition, excessive glucose and excessively generated fructose may be associated with proteins to form advanced glycation end products (AGEs), which are thought to be major factors in aging and age related chronic diseases. They are also believed to play a causative role in the vascular complications of diabetes mellitus.

[3] ROSs, including superoxide radical (O " ), hydroxyl radical (HO ), hydrogen peroxide (H O ) and singlet oxygen ( O ), form as a natural byproduct of the normal metabolism of oxygen. However, during times of physical, chemical and environmental stress, ROS levels can increase dramatically, which can result in significant damage to cell structures. That is, ROSs cause non-selective irreversible destructive damage to cell constituents, such as lipids, proteins, sugar, and DNA, and have been found to be involved in the occurrence of various diseases, such as cancer, apoplexy, Parkinson's disease, Alzheimer's disease, aging, heart failure, ischemia, arteriosclerosis, skin disease, inflammation, rheumatoid arthritis, autoimmune diseases, etc.

[4] For example, when accumulated in the cell membrane, lipid peroxides, which may be formed through the reaction of ROSs, such as free radicals, with phospholipids of cell membranes, have adverse effects on the fluidity and functionality of cell membranes, and thus on cellular functions, resulting in local defects of tissues, such as

deformed cell structure. Therefore, the accumulation of lipid peroxides is now known to be a factor causative of various diseases, including cerebral apoplexy, myocardial infarction, hyperlipidemia, acute inflammation, rheumatoid arthritis, alcoholic hepatitis, etc. To date, the synthetic antioxidants BHT (butylated hydroxytoluene) and BHA (butylated hydroxyanisole) have been used, for the most case, to inhibit lipid peroxidation. However, these synthetic antioxidants, although showing fine inhibitory activity against lipid peroxidation, are not safe to apply to the body over the long term.

[5] Free radicals, ROSs, and peroxides are formed during the normal metabolism of oxygen in cells. Cells are normally able to defend themselves against ROS damage through the use of enzymes such as superoxide dismutase (SOD), catalase and glutathione peroxidase. Small molecule antioxidants such as vitamin E, vitamin C, glutathione, ubiquinone, and uric acid also play important roles as cellular antioxidants against oxidative stress. When any abnormality occurs in, or ROSs are predominant over, these cellular defense systems, oxidative stress brings about the destruction of cells. Therefore, it is well known that Antioxidants capable of scavenging free radicals and ROSs can be developed as therapeutics for various oxidative-stress implicated diseases, or as antiaging agents.

[6] Particularly, intensive attention has recently been paid to natural antioxidants and to the defense mechanism thereof against oxidative damage, leading to extensive studies into the development of ROS scavengers for use in the treatment of diseases based on oxidative damage.

[7] Recently, tissue injury caused by diabetes mellitus has been reported to be based on an excess of ROSs. Therefore, there is a need for natural materials that effectively scavenge the ROSs generated by diabetes mellitus. Disclosure of Invention Technical Problem

[8] Leading to the present invention, intensive and thorough research on a natural material involved in scavenging ROSs, conducted by the present inventors, resulted in the finding that extracts from the fruit body and cultured mycella of Fomitopsis pinicola inhibit the activity of xanthine oxidase (XOD), a ROS generating enzyme and decrease the level of lipid peroxides and alanine-aminotransferase (ALT) in blood, while enhance the activity of the reactive oxygen species scavengers such as glutathione peroxidase (GPX) and superoxide dismutase (SOD), and increase the level of the antioxidant such as glutathione. In addition, one of the materials which bring about these inhibitory effects was analyzed to be 'β- 1 ,3-glucano-β- 1 ,6-heterogalactomannan-protein complex'.

[9] It is therefore an object of the present invention to provide a fruit body extract or

cultured mycelial extract of Fomitopsis pinicola which has inhibitory activity against the ROS production induced by diabetes mellitus.

[10] It is another object of the present invention to provide a composition based on the fruit body extract or cultured mycelial extract of Fomitopsis pinicola, which effectively inhibits the ROS production induced by diabetes mellitus. Technical Solution

[11] In order to accomplish the above objects, there is provided a composition, effective for the inhibition of diabetes mellitus-induced reactive oxygen species production, comprising a fruit body extract or cultured mycelial extract of Fomitopsis pinicola.

[12] Preferably, the fruit body extract is a hot- water extract or an alkali extract.

[13] In an embodiment, the hot- water extract may be prepared by cutting Fomitopsis pinicola fruit bodies into fine pieces pulverizing the pieces, heating the pulverized pieces in water, concentrating the aqueous solution, precipitating the solution with ethanol, and freeze-drying the precipitate, in order. In one modification of this embodimemt, the pulverized pieces of the fruit bodies are heated at 100°C for 24 hours and the aqueous solution is concentrated at 40°C to one tenth of the initial volume.

[14] In another embodiment, alkali extract may be prepared by cutting Fomitopsis pinicola fruit bodies into fine pieces pulverizing the pieces, swelling the pulverized pieces in IN KOH, homogenizing the pieces, filtrating the homogenate, neutralizing the filtrate, washing the neutralized filtrate with distilled water, and drying the washed filtrate at 60°C, in order. In one modification of this embodiment, the pulverized pieces of the fruit bodies are mixed with IN KOH in a ratio of 1 : 1 (w/v), left to swell for 1 hour, homogenized, filtered through a 100 mesh sieve, and neutralized with cone. HCl.

[15] In another embodiment, the cultured mycelial extract is prepared by seed- and sub- culturing mycelia of Fomitopsis pinicola, inoculating the mycelia in a sterile and cold potato medium, neutralizing the culture, precipitating with ethanol, and dialyzing the precipitate against distilled water. In one modification of this embodiment, the mycelia are inoculated in the potato medium to an amount of 2% (v/v), and the culture is neutralized with sodium hydrogen carbonate (NaHCO ) to a pH of 6.5. Advantageous Effects

[16] Fruit body extracts and cultured mycelial extracts of Fomitopsis pinicola according to the present invention effectively inhibit the ROS production induced by diabetes mellitus, thereby finding various applications for the development of functional foods for preventing ROS-caused tissue injury. Best Mode for Carrying Out the Invention

[17] In accordance with an aspect thereof, the present invention is directed to an extract from the fruit body or cultured mycelia of Fomitopsis pinicola, and a composition

comprising the extract as an active ingredient for inhibiting the ROS production caused by diabetes mellitus.

[18] In the present invention, the extract from the fruit body of Fomitopsis pinicola may be prepared with hot water or alkali.

[19] A hot- water extract from the fruit body of Fomitopsis pinicola can be obtained as follows. The fruit body is finely cut, pulverized, immersed in water, and heated, followed by concentration and precipitation with ethanol. The precipitate is then freeze-dried. Water is preferably used in an amount of about 30 times the weight of the pulverized fruit body, but is not limited thereto. Heating is preferably conducted at 100°C for about 24 hrs, but is not limited thereto. As for the concentration, it is preferable that the supernatant be evaporated at 40°C until the final volume is reduced to one tenth of the initial volume.

[20] An alkaline extract from the fruit body of Fomitopsis pinicola can be obtained as follows. The fruit body is finely cut, pulverized and left to swell for 1 hr in IN KOH, followed by homogenization using a homogenizer. The homogenate is filtered through a 100 mesh sieve, neutralized with cone. HCl, washed with distilled water, and then dried at 60°C. For the swelling, it is preferable that IN KOH be used in a ratio of approximately 1:1 (w/v). This alkaline extraction, called HAS (homogenization after alkali swelling) method, is exceptionally improved in yield over conventional ones.

[21] Extraction from the cultured mycelia of Fomitopsis pinicola starts with seed culturing the mycelia of Fomitopsis pinicola. After sub-culturing for enrichment, Fomitopsis pinicola is inoculated into a sterile and cold potato medium and cultured. The resulting culture is neutralized and precipitated with ethanol. The precipitate is dialyzed against distilled water to yield a desired extract. The neutralization is preferably conducted to a pH of 6.5 with sodium hydrogen carbonate (NaHCO ), but is not limited thereto.

[22] The extract from Fomitopsis pinicola according to the present invention is assayed for inhibitory activity against diabetes mellitus-caused ROS production as follows. First, the extract is orally administered to rats in which diabetes mellitus has been induced with STZ (streptozotocin). After being raised, each rat is sacrificed to excise the liver, which is then homogenized in ice. The homogenate is analyzed for the activity of enzymes having influence on ROS production and toxification. In addition, the levels of lipid peroxides, glutathione and ALT in the blood are measured to determine whether the extract of the present invention can inhibit the ROS production caused by diabetes mellitus.

[23] Furthermore, in order to identify the material which brings about the inhibitory effect, the Fomitopsis pinicola extract is analyzed for ingredients and the molecular weights thereof. In this regard, the Fomitopsis pinicola extract is fractioned and

purified through DEAE-cellulose ion exchange resin and a Sepharose CL-4B gel column, followed by methylation analysis using gas chromatography.

[24] A better understanding of the present invention may be obtained in light of the following examples which are set forth to illustrate, but are not to be construed to limit the present invention. Mode for the Invention

[25] EXAMPLES

[26] EXAMPLE 1 : Preparation of the Fruit Body and Mycelial Extract of Fomitopsis pinicola

[27]

[28] EXAMPLE 1-1 : Preparation of the Fruit Body of Fomitopsis pinicola with Hot

Water

[29]

[30] 90 Grams of the fruit body of Fomitopsis pinicola, purchased from Jeseng farm, located in Pohang City, Korea, were finely sectioned to a mean size of 5x5 mm, pulverized, added to 2.5 liters of water, and heated at 100°C for 24 hrs. The resulting solution was concentrated at 40°C to a volume of 250 mL, followed by precipitation with ethanol to produce an alcohol-insoluble material. The precipitate, insoluble in alcohol, was freeze-dried to give a hot- water extract of the fruit body of Fomitopsis pinicola.

[31]

[32] EXAMPLE 1-2: Preparation of the Mycelial Extract of Fomitopsis pinicola with

Alkali

[33]

[34] 90 Grams of the fruit body of Fomitopsis pinicola, purchased from Jeseng farm, located in Pohang City, Korea, were finely sectioned to a mean size of 5x5 mm, pulverized, swelled for 1 hr in IN KOH (1:1, w/v), and homogenized using a ho- mogenizer. Following filtration through a 100 mesh sieve, the filtrate was neutralized with cone. HCl, washed with distilled water and dried at 60°C to give an alkali extract of the fruit body of Fomitopsis pinicola.

[35]

[36] EXAMPLE 1-3: Preparation of Extract from Cultured Mycelia of Fomitopsis pinicola

[37]

[38] Seed mycelia of Fomitopsis pinicola, obtained from Jeseng farm, located in Pohang

City, Korea, were inoculated on a YM agar plate (yeast extract: 0.5%(w/v), peptone: 0.5%(w/v), malt extract: 0.2%(w/v), glucose: 1.0%(v/v), agar: 2.0%(w/v), pH 6.5) and

sub-cultured at 30°C every 15 days. The mycelia were seed cultured in YM broth using a rotary agitator (150 rpm).

[39] After culturing for 10 days, the mycelia were inoculated in a sterilized (120°C, 30 min), cold potato medium (water: 16L, potato powder: 300g, glucose: 150g, peptone: O.lg) to an amount of 2%(v/v), and cultured at 30°C for 10 days in a rotary agitator operating at 150 rpm with sterile air (10cm 3 /min) provided thereto. Then, neutralization with sodium hydrogen carbonate (NaHCO ) to a pH of 6.5 preceded precipitation with 10 volumes of ethanol.

[40] The precipitate was dialyzed against distilled water for 48 hrs using a membrane with a molecular weight cutoff of 3,000 to obtain a cultured mycelial extract of Fomitopsis pinicola.

[41] [42] EXAMPLE 2: Inhibition Effect of Fomitopsis pinicola Extract on ROS Production [43] [44] EXAMPLE 2-1: Experimental Animal and Method [45] [46] SD rats, each having body weight of 200+5g, were divided into 5 groups, which were respectively set as a normal control group (NC), a diabetes mellitus control group (DM), in which diabetes mellitus was induced by STZ, a diabetes mellitus-induced, hot- water extract-administered group (DM-WE) which was administered with 1% of the hot-water extract after treatment with STZ, a diabetes mellitus-induced, alkali extract- administered group (DM-AE) which was administered with 1% of the alkali extract after treatment with STZ, and a diabetes mellitus-induced, cultured mycelial extract- administered group (DM-CM) which was administered with 1% of the cultured mycelial extract after treatment with STZ. Each of the group has 7 rats and they were reared for four weeks according to the dietary schedules of Table 1, below. In order to produce ROSs in hepatic tissues, STZ (55mg/kg) was intramuscularly injected. Animals which had a blood sugar level of 300 mg/dL, measured 48 hrs after injection with STZ, were regarded as having ROSs induced in the hepatic tissues thereof. Blood sugar levels were measured using a bio-sensor and a kit. [47] Table 1

Basic Dietary Composition for Animal Test (g/kg)

[48] AIN-mineral mix(g/kg): Calcium lactate 620.0, sodium chloride 74.0, potassium phosphate dibasic 220.0, potassium sulfate 52.0, magnesium oxide 23.0, manganous carbonate 3.3, ferric citrate 6.0, zinc carbonate 1.0, copper carbonate 0.2, potassium iodide 0.01, sodium selenite 0.01 and potassium chromium sulfate 0.5 were mixed to form a total weight of l,000g and finely powdered

[49] 2) AIN- vitamin mix (mg/kg): thiamin hydrochloride 600, riboflavin 600, pyridoxine hydrochloride 700, nicotinic acid 3,000, calcium D-pantothenate 1,600, folic acid 200, D-biotin 20, vitamin B 12 2.5, vitamin A 400,000 IU, vitamin D3 100,000 IU, vitamin E 7,500 IU and vitamin K 75 were mixed to form a total weight of 1,000 g and finely powdered.

[50] In addition, while being fed four weeks according to a different dietary schedule, the experimental animals were observed for weight gain, dietary intake, and feed efficiency ratio (see: Table 2). The DM group was remarkably decreased in weight gain and feed efficiency ratio compared with the NC group. The DM-AE group, although unable to keep pace with the NC group, was found to significantly increase in weight gain and feed efficiency ratio compared to the DM group. Considerable increases of diet intake were observed in the DM, DM-WE, and DM-CM groups compared to the NC group, whereas the dietary intake of the DM-AE group was recovered to a level comparable to that of the NC group. As for feed efficiency ratio, it was considerably decreased with all experimental groups compared to the NC group,

but all of the groups administered with Fomitopsis pinicola extract were observed to have a feed efficiency ratio superior to that of the DM group.

[51] Table 2

Effect of 4- Week Administration with Fomitopsis pinicola Extracts on Weight Gain, Diet Intake and Feed Efficiency Ratio of Diabetes Mellitus-Induced Rats

[52] NC: normal control, DM: diabetes mellitus control, DM-WE: 1% of Fomitopsis pinicola fruit body hot-water extract was administered after treatment with STZ, DM- AE: 1% of Fomitopsis pinicola fruit body alkali extract was administered after treatment with STZ, DM-CM: 1% of the cultured mycelial extract of Fomitopsis pinicola was administered after treatment with STZ

[53] feed efficiency ratio: weight gain/dietary intake [54] After being fed with the experimental diets for 4 weeks, the animals were starved for 16 hrs with only water fed thereto. They were etherized and subjected to laparotomy along the ventral median line to expose the ventral aorta, from which blood was then sampled and analyzed for sugar level. The liver was also excised and homogenized in a homogenizer to obtain a postmitochondrial fraction, which was analyzed for the activity and level of xanthine oxidase (XOD), glutathione peroxidase (GPX), superoxide dismutase (SOD), glutathione, lipid peroxide, and blood alanine aminotransferase (ALT).

[55] [56] EXAMPLE 2-2: Inhibitory Effect of Fomitopsis pinicola Extracts on Diabetes Mellitus-Induced ROS Production

[57] [58] EXAMPLE 2-2-1: Activity change of xanthine oxidase (XOD) in hepatic tissue [59] Livers, excised from the experimental animals which had been fed with diets different from those of Example 2-1 for 4 weeks, were assayed for the activity of

XOD, which catalyzes the oxidation of hypoxanthine to xanthine and the oxidation of xanthine further to uric acid with the concomitant production of hydrogen peroxide. Total XOD activity was measured according to the Stripe method, in which the uric acid, oxidized from xanthine in the presence of the enzyme and NAD+ at 30°C for 10 min in 0.1 M PBS (pH 7.4) is measured at 292 nm. Type 0 activity of XOD, obtained when NAD+ was absent, was measured. XOD activity was expressed as nmoles of the uric acid produced from the substrate for 1 min by 1 mg of the hepatic protein. Type 0 activity of XOD was observed to increase by approximately 68% for the DM group, compared to the NC group, and to decrease by approximately 32% for the DM-AE group, compared to the DM group. It was also observed that the DM-WE group and the DM-CM group both tended to decrease in type 0 activity (see Table 3).

[60] Table 3

Effect of 4- Week Administration with Fomitopsis pinicola Extracts on Hepatic XOD Activity of Diabetes Mellitus-Induced Rats

[61] υ NC: normal control, DM: diabetes mellitus control, DM-WE: 1% of Fomitopsis pinicola fruit body hot-water extract was administered after treatment with STZ, DM- AE: 1% of Fomitopsis pinicola fruit body alkali extract was administered after treatment with STZ, DM-CM: 1% of the cultured mycelial extract of Fomitopsis pinicola was administered after treatment with STZ

[62]

[63] EXAMPLE 2-2-2: Activity change of superoxide dismutase (SOD) and glutathione peroxidase (GPX) in hepatic tissue

[64] [65] The scavenging enzymes superoxide dismutase (SOD) and glutathione peroxidase (GPX) within the liver of the experimental animals, which had been fed diets different from the experimental diet, were assayed for activity. SOD activity was analyzed according to the Martin method by which the extent to which the oxidation of hematoxylin in 5OmM PBS (pH 7.5) in the presence of 0.1 mM EDTA was inhibited

was measured at 560nm.

[66] GPX activity was determined by the method of Paglia, using NADPH-coupled reduction of GSSG, catalysed by glutathione reductase. H2O2 was used as a substrate. Oxidation of NADPH was conducted at 340 nm. One unit of enzyme activity is defined as the oxidation of 1 nmole NADPH/min/mg protein in a solution of 5 mM EDTA in 50 mM PBS (pH 7.0) at 25°C. The DM group was remarkably decreased in SOD and GPX activity compared to the NC group. The Fomitopsis pinicola extract- administered groups were increased in SOD and GPX activity compared to the DM group, but this difference was not significant (see Table 4).

[67] Table 4

Effect of 4- Week Administration with Fomitopsis pinicola Extracts on Hepatic SOD and GPX Activity of Diabetes Mellitus-Induced Rats

[68] υ NC: normal control, DM: diabetes mellitus control, DM-WE: 1% of Fomitopsis pinicola fruit body hot-water extract was administered after treatment with STZ, DM- AE: 1% of Fomitopsis pinicola fruit body alkali extract was administered after treatment with STZ, DM-CM: 1% of the cultured mycelial extract of Fomitopsis pinicola was administered after treatment with STZ

[69] [70] EXAMPLE 2-2-3: Change in hepatic glutathione and lipid peroxide level and blood ALT level

[71] [72] The hepatic glutathione and hydrogen peroxide level and blood ALT level of the experimental animals, which had been fed diets different from the experiment diet for 4 weeks, were assayed. Hepatic glutathione level was analyzed by the method of Ellman, and hepatic lipid peroxide level by the method of Ohkawa. Blood ALT activity was determined using a commercially available kit (Asan Pharmaceuticals, Korea). Enzyme activity was expressed as karmen units per 1 mL of blood. The DM group was observed to decrease in hepatic glutathione level by approximately 22% compared to the NC group. The hepatic glutathione level was measured to be ap-

proximately 21% and 13% larger in the DM-WE and DM-AE groups respectively, than in the DM group. DM-CM group had little difference from the DM group.

[73] Although the hepatic lipid peroxide level of the DM group increased by approximately 146% compared to that of the NC group, the hepatic lipid peroxide level of the DM-AE group was reduced by approximately 36% compared to that of the DM group. However, the hot-water extract and the cultured mycelial extract did not significantly reduce the lipid peroxide level which was increased by STZ administration. Blood ALT activity was measured to increase 2.97 times for the DM group compared to the NC group. A significant decrease in blood ALT activity was found in the DM- AE group and the DM-CM group. Also, the DM-WE group tended to decrease in blood ALT activity (see Table 5).

[74] Table 5

Effect of 4- Week Administration with Fomitopsis pinicola Extracts on Hepatic Glutathione and Lipid Peroxide and Blood ALT Activity of Diabetes Mellitus-Induced Rats

[75] υ NC: normal control, DM: diabetes mellitus control, DM-WE: 1% of Fomitopsis pinicola fruit body hot-water extract was administered after treatment with STZ, DM- AE: 1% of Fomitopsis pinicola fruit body alkali extract was administered after treatment with STZ, DM-CM: 1% of the cultured mycelial extract of Fomitopsis pinicola was administered after treatment with STZ

[76] [77] EXAMPLE 3: Qualitative Assay for Fomitopsis pinicola Extracts [78] [79] Fomitopsis pinicola extracts of the present invention were identified as inhibitors of ROS production in diabetes mellitus-induced rats, as evident in Example 2. In order to examine the principle of inhibiting diabetes mellitus-induced ROS production, the Fomitopsis pinicola extracts were qualitatively analyzed.

[80] The Fomitopsis pinicola fruit hot- water extract, the Fomitopsis pinicola fruit body alkali extract, and the cultured mycelial extract of Fomitopsis pinicola, prepared as described above, were fractioned and purified through DEAE-Cellulose (Cl " ) ion

exchange resin and Sepharose CL-4B gel according to a well-known method (see, Lee Shin young, Kang Tae soo, Structure Analysis of Antitumoral Exo-polysaccharide (BWS) obtained from submerged cultivation of Ganoderma lucidum mycelium, The Korean J. Mycology, 27: 76-81, (1999)), followed by methylation analysis using gas chromatography. For the measurement of molecular weight, dextran, having MWs of 2,000,000, 500,000, and 300,000 (Sigma), was used. Absorbance at 280 nm was utilized to detect the protein composition of the fractions. Six-carbon sugars were analyzed according to the anthrone method (see, Spiro RG, Analysis of sugars found in glycoprotein in Method in Enzymology, Academic Press, New York 8: 4-10, (1966)).

[81] Polysaccharides obtained from the Fomitopsis pinicola fruit body hot- water extract, alkali extract and cultured mycelial extract were subjected to affinity chromatography to determine their configurations. No saccharides were detected in the absorption region, indicating that the polysaccharides were of β structure. Methylation analysis gave detailed information, indicating that, as shown in Table 6, 2,3,4,6-tetramethyl glucose and 2,4,6-trimethyl glucose were detected, evincing the presence of β- 1,3-glucan, and that β-l,6-heterogalactomannan was also present. Taken together, the data demonstrated that one main component of the Fomitopsis pinicola extract was identified as β-l,3-glucano-β-l,6-heterogalactomannan, in which β-l,3-glucan is linked to β-l,6-heterogalactomannan. This complex polysaccharide was found to be a proteoglycan, ranging in molecular weight from 300,000 to 500,000 as measured by gel filtration.

[82] Table 6

Structure Analysis of Polysaccharides from Fomitopsis pinicola Fruit Body Extracts and Cultured Mycelial Extract

[83]

Industrial Applicability [84] Fruit body extracts and cultured mycelial extracts of Fomitopsis pinicola according to the present invention effectively inhibit the ROS production induced by diabetes mellitus, thereby finding various applications for the development of functional foods for preventing ROS-caused tissue injury.

[85] The present invention has been described in an illustrative manner, and it is to be understood that the terminology used is intended to be in the nature of description rather than of limitation. Many modifications and variations of the present invention are possible in light of the above teachings. Therefore, it is to be understood that within the scope of the appended claims, the invention may be practiced otherwise than as specifically described.