WO2007078157A1

WO2007078157A1 - Fomitopsis pinicola extract and a process for manufacturing the same

Info

Publication number: WO2007078157A1
Application number: PCT/KR2007/000055
Authority: WO
Inventors: Seung-Hee Oh; Soon-Dong Kim; Sang-Il Lee; Hyun-Goo Lee
Original assignee: Eugene Bio.Farm Co.Ltd
Priority date: 2006-01-05
Filing date: 2007-01-04
Publication date: 2007-07-12

Abstract

Disclosed herein are Fomitopsis pinicola extracts and use thereof. Fruit body extracts and cultured mycelial extracts of Fomitopsis pinicola effectively inhibit the renal and retinal aldose reductase activity and decrease blood levels of triglycerides, total cholesterol and LDL cholesterol, thereby finding various applications for the development of functional foods for preventing diabetes mellitus complications and suppressing diabetes mellitus-induced hyperlipidemia.

Description

FOMITOPSIS PINICOLA EXTRACT AND A PROCESS FOR MANUFACTURING THE SAME

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 are effectively preventive or suppressive of diseases attributable to the increased activity of aldose reductase, including diabetes complications, such as cataracts, renal failure, etc., and diabetes mellitus-induced hyperlipidemia. Background Art

[2] With the westernization of the diet, lifestyle-related diseases, such as obesity, hyperlipidemia, diabetes, etc., are occurring at an incidence rate that is increasing in recent years, which is now emerging as a serious social problem in Asia, including Korea. In particular, the incidence rate of diabetes in Korea has drastically increased in the last decade, ranking as the 4th cause of death of Koreans (see Annual Report on Fatality Statistics in 1999, 2002).

[3] Diabetes mellitus is generally classified into type 1 and type 2, which are dependent on and independent of insulin, respectively. Approximately 90 to 95% of diabetes mellitus is observed to be type 2. Aside from acute glucose level abnormalities, the main risks to health, when diabetes mellitus occurs due to hormonal unbalance, are the characteristic long-term complications. These include arteriosclerosis, cardiovascular diseases, nerve damage, and renal failure, leading to death. This is believed to be attributed to a serious change which is brought about in lipid metabolism to increase levels of triglyceride, total cholesterol, and LDL cholesterol and decrease the level of HDL in the blood.

[4] Upon the onset 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.

[5] To treat diabetes mellitus, dietary or exercise therapy is usually conducted along with drug therapy. All of these therapies function to maintain a normal blood glucose level with the aim of preventing or retarding diabetes complications. Although extensive studies and research have been conducted on diabetes complications and diabetes-induced hypercholesterolemia, no perfect cures have been established. Thus, intensive and keen attention is paid to functional foods or medications for preventing or suppressing the onset of diabetes complication or diabetes-induced hypercholesterolemia.

[6] Fomitopsis pinicola, a Korean autogenous mushroom, was thought to be extinct, but has recently been discovered again. It was renamed ' Jeseng' and registered in the National Seed Management Office, Korea (Application No.: 2003-498). Fomitopsis pinicola grows with attachment to a wood stem and is leathery to woody, and hoof- shaped or shelved. The upper surface is usually zoned, and has a wide range in color from dark brown through grey to black, with a red band near the margin. Many mushrooms have been extensively reported to contain not only various nutrients but also various types of physiological substances useful in the prevention and treatment of metabolic diseases, such as diabetes mellitus, hyperlipidemia and arteriosclerosis, and effective for anticancer activity and immunopotentiation.

[7] Fomitopsis pinicola is known to have inhibitory activity against tumors, and has recently been reported to reduce blood sugar levels (refer to Korean Pat. Laid-Open Publication No. 2005-60726). However, Fomitopsis pinicola has not been sufficiently studied for physiological activity. Further, physiologically active principles of Fomitopsis pinicola remain unidentified. Therefore, it is required to assay Fomitopsis pinicola for various physiological activities, develop a method for preparing extracts from the fruit body and cultured mycelia of Fomitopsis pinicola, and analyze constituents of the extracts. Disclosure of Invention Technical Problem

[8] Leading to the present invention, intensive and thorough research into a material of

Fomitopsis pinicola capable of preventing or suppressing diabetes complications or diabetes-induced hyperlipidemia, conducted by the present inventors, resulted in the finding that extracts from the fruit body and cultured mycelia of Fomitopsis pinicola inhibits the activity of aldose reductase in the eye lens and the kidneys and reduces the level of triglycerides, total cholesterol and LDL cholesterol in the blood. In addition, the principle which brings about these inhibitory effects was analyzed to be 'β-l,3-glucano-β-l,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 can prevent or suppress the diseases attributable to the increased activity of aldose reductase, including diabetes complications, such as cataracts and renal failure, and diabetes-induced hypercholesterolemia.

[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 is useful in the treatment and prevention of diseases attributable to the increased activity of aldose reductase, including diabetes complications, such as cataracts and renal failure, and diabetes-induced hypercholesterolemia. Technical Solution

[11] In order to accomplish the above objects, there is provided a composition, effective for the prevention or suppression of diseases attributable to the increased activity of aldose reductase or diabetes mellitus-induced hyperlipidemia, comprising a fruit body extract or cultured mycelial extract of Fomitopsis pinicola.

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

[13] 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.

[14] Preferably, 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.

[15] The 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.

[16] Preferably, 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.

[17] The cultured mycelial extract may be 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.

[18] Preferably, 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.

[19] The diseases attributable to the increased activity of aldose reductase include a diabetes mellitus complication.

[20] In a preferred embodiment, the diabetes mellitus complication is cataract or renal failure. Advantageous Effects

[21] Fruit body extracts and cultured mycelial extracts of Fomitopsis pinicola according to the present invention effectively inhibit the renal and retinal aldose reductase activity and decrease blood levels of triglycerides, total cholesterol and LDL cholesterol, thereby finding various applications for the development of functional foods for preventing diabetes mellitus complications and suppressing diabetes mellitus-induced hyperlipidemia. Brief Description of the Drawings

[22] FIG. 1 is a diagram showing the mechanism of the incidence of diabetes mellitus and complications thereof. Because aldose reductase is responsible for the reduction of glucose to sorbitol in the polyol pathway, the inhibition of the activity thereof prevents the incidence of diabetes mellitus complications.

[23] FIG. 2 is a graph in which the activity of retinal aldose reductase is plotted against the dose of the Fomitopsis pinicola fruit body hot- water extract.

[24] FIG. 3 is a graph showing kinetics of the retinal aldose reductase according to the dose of Fomitopsis pinicola fruit body hot- water extract. Best Mode for Carrying Out the Invention

[25] 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 preventing or suppressing the diseases attributable to the increased activity of aldose reductase, including diabetes complications, such as cataracts and renal failure, and diabetes-induced hypercholesterolemia.

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

[27] 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 to that amount. Heating is preferably conducted at 100°C for about 24 hrs, but is not limited thereto. As for the con- centration, it is preferable that the supernatant be evaporated at 40°C until the final volume is reduced to one tenth of the initial volume.

[28] 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 at 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 methods.

[29] 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.

[30] The term "diseases attributable to the increased activity of aldose reductase", as used herein, is used to mean a condition in which diabetes complications, such as cataracts or renal failure, are caused by the increased intracellular level of sorbitol, reduced from glucose by aldose reductase, which generates osmotic stress due to its low permeability through the cell membrane, thus injuring the eye lens, the retina, and the glomeruli of the kidneys (refer to FIG. 1).

[31] The extract from Fomitopsis pinicola according to the present invention was assayed for preventive or suppressive activity against diseases attributable to the increased activity of aldose reductase and diabetes-induced hyperlipidemia as follows. First, the extract was orally administered to rats in which diabetes mellitus had been induced with STZ (streptozotocin). After being raised, each rat was sacrificed to excise the eye lens and kidneys, which were then homogenized in ice. The homogenate was analyzed for the activity of aldose reductase, having an influence on the incidence of diabetes complications. In addition, blood samples were measured for levels of triglycerides, total cholesterol, HDL cholesterol, and LDL cholesterol to determine whether the extract of the present invention could inhibit diabetes-induced hyperlipidemia.

[32] Furthermore, in order to identify the principle which brought about the inhibitory effect, the Fomitopsis pinicola extract is analyzed for ingredients and the molecular weights thereof. In this regard, the Fomitopsis pinicola extract was fractioned and purified through DEAE-cellulose ion exchange resin and a Sepharose CL-4B gel column, followed by methylation analysis using gas chromatography. [33] 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

[34] EXAMPLES

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

[36]

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

Water

[38] 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 yield a hot- water extract of the fruit body of Fomitopsis pinicola.

[39]

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

Alkali

[41]

[42] 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.

[43]

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

[45]

[46] 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).

[47] After culturing for 10 days, the mycelia were inoculated in a sterilized (120°C, 30 min), cold potato medium (water: 16 L, potato powder: 300 g, glucose: 150 g, peptone: 0.1 g) 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 /min) provided thereto. Then, neutralization with sodium hydrogen carbonate (NaHCO ) to a pH of 6.5 was conducted, followed by precipitation with 10 volumes of ethanol.

[48] The precipitate was dialyzed with 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.

[49] [50] EXAMPLE 2: Preventive Effect of Fomitopsis pinicola Extract on Diabetes Complications

[51] [52] EXAMPLE 2-1: Experimental Animals and Method [53] [54] SD male rats, each having body weight of 200+5 g, were divided into 5 groups of 7, which were respectively set as a normal control group (NC), a diabetes mellitus control group (DM), in which diabetes mellitus was induced through 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 groups was reared for four weeks according to the dietary schedules of Table 1, below. In order to induce diabetes mellitus, STZ (streptozotocin 55 mg/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 diabetes mellitus induced therein. Blood sugar levels were measured using a bio-sensor and a kit.

[55] Table 1 Basic Dietary Composition for Animal Test (g/kg)

[56] 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 1,000 g and finely powdered

[57] 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.

[58] [59] In addition, while being fed four weeks according to a different dietary schedule, the experimental animals were observed for weight gain, food 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 in food intake were observed in the DM, DM-WE, and DM-CM groups compared to the NC group, whereas the food 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.

[60] 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

[61] 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] feed efficiency ratio: weight gain/dietary intake

[63] [64] 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 kidneys and the eye lens were also excised and homogenized on ice in a homogenizer to obtain a postmitochondrial fraction, which was analyzed for the activity and level of aldose reductase (AR).

[65] [66] EXAMPLE 2-2: Preventive Effect of Fomitopsis pinicola Extracts on Diabetes Complications

[67] [68] Diabetes complications, such as cataracts, peripheral neuritis, pancreatic injury, etc., are known to be induced upon the metabolism of glucose through the polyol pathway instead of through glycosis. The polyol pathway takes place when hexokinase, which serves as the first step enzyme in anaerobic glycolysis, is saturated. Excess glucose is reduced to sorbitol via aldose reductase, the first step enzyme in the polyol pathway. An abundance of intracellular sorbitol generates osmotic stress due to its low permeability through the cell membrane, so that an excessive amount of water is introduced into the cell, thus injuring the non-insulin dependent organs, such as eye lenses, peripheral nerves, and the kidneys.

[69] Therefore, data on the activity of aldose reductase in the eye lens and kidney samples prepared in Example 2-1 would give information about the preventive effect of the Fomitopsis pinicola extracts of the present invention on diabetes complications.

[70] The activity of aldose reductase was analyzed according to the method of Hayman and Kinoshita (1965), in which, while lithium sulfate, NADPH and DL-α-glycerol were reacted at 25°C in the presence of the enzyme in 50 mM PBS (pH 6.2), decreasing NADPH levels were measured at 340 nm.

[71]

[72] EXAMPLE 2-2-1 : Suppressive effect on activity of aldose reductase in eye lens

[73]

[74] While the hot- water extract from Fomitopsis pinicola fruit bodies was added into the vial and a postmitochondrial fraction obtained by centrifuging a homogenate of the rat eye lens was used as an enzyme source, the aldose reductase was monitored for activity. The results are given in FIGS. 2 and 3. As seen in the plots of FIGS. 2 and 3, the aldose reductase activity was decreased in a manner dependent on the concentration of the hot- water extract. At a concentration of 125 μl/ml of the extract, the aldose reductase activity was reduced by as much as about 50% compared to that of the control. In addition, the aldose reductase activity change with the addition of the Fomitopsis pinicola fruit body extract was analyzed in terms of kinetics. The enzyme was remarkably increased in Km without variation of Vmax.

[75]

[76] EXAMPLE 2-2-2: Suppressive effect on activity of aldose reductase in kidney

[77]

[78] Postmitochondrial fractions obtained by centrifuging the homogenates of the kidneys were analyzed for aldose reductase (AR) activity. The DM group, in which diabetes mellitus was induced by STZ, was increased approximately 47% in AR activity compared to the NC group. An approximately 25% reduction was observed in the AR activity of the DM-AE group compared to the DM group. It was also found that both the DM-WE group and the DM-CM group tended to decrease in AR activity.

[79] Table 3

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

[80] ^υNC: 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

[81] [82] EXAMPLE 3: Suppressive Effect of Fomitopsis pinicola Extract on Diabetes Mellitus-Induced Hyperlipidemia

[83] [84] The same experimental groups and diets as in Example 2 were conducted. Following the diet programs for 4 weeks, the five experimental groups were measured for blood levels of triglycerides, total cholesterol, LDL cholesterol and HDL cholesterol. For the measurement of blood levels of triglycerides, total cholesterol and HDL cholesterol, commercially available kits were used (AM 157S-K, AM 202-K, AM 203-K, Asanpharm Co., Korea). The level of LDL cholesterol in the blood was calculated according to the method of Fridwell et al. (1972).

[85] A significant increase in blood triglyceride level was observed in the DM group compared to the NC group. However, the blood triglyceride level of the DM-AE group recovered to the same level as that of the NC group. Both the DM-WE group and the DM-CM group were observed to have blood triglyceride levels comparable to that of the DM group (see: Table 4).

[86] Table 4

Effect of 4- Week Administration with Fomitopsis pinicola Extracts on Blood Triglyceride Level of Diabetes Mellitus-Induced Rats

[87] 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

[88] [89] The DM-AE group was decreased about 18% in total blood cholesterol level compared to the DM group. Both the DM-AE group and the DM-CM group tended to decrease in total blood cholesterol level, although there was no significance (see: Table 5).

[90] Table 5

Effect of 4- Week Administration with Fomitopsis pinicola Extracts on Total Blood Cholesterol Level of Diabetes Mellitus-Induced Rats

[91] ^υNC: 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

[92] [93] As for blood LDL cholesterol level, it increased remarkably, by about 95%, in the DM group, but decreased significantly in the DM-AE group, unlike the DM group. The DM-AE group was observed to have a blood LDL cholesterol level comparable to that of the NC group. Also, a significant decrease was found in the DM-CM group, compared to the DM group. The DM-WE group tended to decrease in blood LDL cholesterol level, although this was not significant (see: Table 6).

[94] Table 6

Effect of 4- Week Administration with Fomitopsis pinicola Extracts on Blood LDL Cholesterol Level of Diabetes Mellitus-Induced Rats

[95] ^υNC: 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

[96] [97] All of the experimental groups administered with the extracts of the present invention were observed to be increased in blood HDL cholesterol level, but with no significance (see: Table 7).

[98] Table 7

Effect of 4- Week Administration with Fomitopsis pinicola Extracts on Blood HDL Cholesterol Level of Diabetes Mellitus-Induced Rats

[99] ^υNC: 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

[100] Taken together, the data thus obtained in the examples demonstrate that the administration of the Fomitopsis pinicola extracts to diabetes mellitus-induced rats for four weeks reduces blood levels of triglycerides, total cholesterol and LDL cholesterol, with the best effect with the alkali extract from Fomitopsis pinicola fruit bodies.

[101] [102] EXAMPLE 4: Qualitative Assay for Fomitopsis pinicola Extracts

[103] [104] Fomitopsis pinicola extracts of the present invention were identified as preventors of diabetes mellitus complications as well as suppressors of diabetes mellitus-induced hyperlipidemia, as evident in Examples 2 and 3. In order to examine the principle of the effects, the Fomitopsis pinicola extracts were qualitatively analyzed.

[105] 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)).

[106] 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 8, 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.

[107] 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.

[108] Table 8

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

[109]

Industrial Applicability

[HO] Fruit body extracts and cultured mycelial extracts of Fomitopsis pinicola according to the present invention effectively inhibit the renal and retinal aldose reductase activity and decrease blood levels of triglycerides, total cholesterol and LDL cholesterol, thereby finding various applications for the development of functional foods for preventing diabetes mellitus complications and suppressing diabetes mellitus-induced hyperlipidemia.

[111] [112] 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.

Claims

[1] A composition, effective for the prevention or suppression of diseases attributable to the increased activity of aldose reductase or diabetes mellitus- induced hyperlipidemia, 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.

[9] The composition according to claim 1, wherein the diseases attributable to the increased activity of aldose reductase include a diabetes mellitus complication.

[10] The composition according to claim 9, wherein the diabetes mellitus complication is cataract or renal failure.