WO2007091827A1

WO2007091827A1 - Extract from submerged culture of fomitopsis pinicola mycelia and hypoglycemic composition comprising the same

Info

Publication number: WO2007091827A1
Application number: PCT/KR2007/000646
Authority: WO
Inventors: Seung-Hee Oh; Soon-Dong Kim; Sang-Il Lee; Hyun-Goo Lee
Original assignee: Eugene Bio.Farm Co.Ltd
Priority date: 2006-02-07
Filing date: 2007-02-07
Publication date: 2007-08-16

Abstract

Disclosed herein are an extract from a submerged culture of Fomitopsis pinicola in a citrus peel medium or a green tea medium, and an anti-diabetic composition comprising the same. With great suppressive effects on blood glucose level, the extract can be applied to various foods for patients with diabetes mellitus.

Description

EXTRACT FROM SUBMERGED CULTURE OF FOMITOPSIS PINICOLA MYCELIA AND HYPOGLYCEMIC COMPOSITION

COMPRISING THE SAME

Technical Field

[1] The present invention relates to an extract from a submerged culture of Fomitopsis pinicola mycelia in a medium containing an extract from citrus peel or green tea, and a composition for lowering blood sugar levels, comprising the same. More particularly, the present invention relates to an extract from the cultured mycelia of Fomitopsis pinicola, which is superior in hypoglycemic, hypolipidemic and antioxidation activity. Background Art

[2] With rapid changes in dietary habits and hypernutrition, attributable to economic growth, degenerative adult diseases, such as cancer, strokes, hypertension, diabetes, etc., are occurring at an increasing rate. In particular, the rate of incidence of diabetes in Korea has drastically increased, amounting to about 3% of the total population, and in particular to about 6.5% of adults 65 years old or older. Diabetes mellitus, although the accurate mechanism remains unclear to date, is known to be treatable with medicaments and diet for one's entire life because it causes a reduction in the body's ability to utilize glucose, and the excess of blood sugar incurs various abnormal immune functions and complications, which lead to cardiovascular, neurological and renal diseases.

[3] Diabetes mellitus is generally classified into insulin-dependent type and non insulin-dependent type. Cytokines have been implicated as immunological effector molecules that mediate autoimmune destruction of pancreatic beta cells, which is associated with insulin-dependent diabetes mellitus. As for non insulin-dependent diabetes mellitus, it is characterized by tissue- wide insulin resistance due to obesity, resulting in the overproduction of TNF-α and in an increase in macrophage activity. Approximately 90% or more of diabetes mellitus in Korea is observed to be non insulin-dependent type 2. Patients with diabetes mellitus feature an acute increase in blood glucose level and an abnormality in lipid metabolism. Aside from acute glucose level abnormalities, the main risks to the health of patients with non insulin-dependent diabetes mellitus 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] As mentioned above, current therapies for diabetes mellitus rely on a combination of drugs and diet. Long term administration of drugs, such as sulfonyl urea, biguanide and troglitazone, is reported to have negative effects on the human immune system with a high risk of concomitant side effects. Thus, diet control is very important in practice. According to diet therapy aiming to normalize glucose and lipid levels in blood, it is recommended to eat low-fat and low-glucose diets to restrain an increase in blood glucose and insulin level. Acarbose, a polysaccharide, and dietary fibers are representative of low-fat and low-glucose diets. Recent studies have reported that physiologically active substances contained in red ginseng, Chinese matrimony vine, buckwheat, sicklepod(Cassia tora L.), and/or mushrooms have hypoglycemic effects, triggering the onset of active research thereto. Generally, mushrooms, which are eukaryotes, are low in lipid content, but contain ample nutrients, such as proteins, carbohydrates, minerals, nucleic acids and vitamins, and characteristic tastes and flavors. Particularly, the vitamin precursor Ergosterol is highly abundant therein.

[5] From mushrooms, reportedly, materials responsible for various biological activities including anticancer activity, immunity enhancement, hypocholestemic and hypoglycemic action, preventive and curative effects on stroke and heart failure, infection resistance, etc., can be obtained. Almost all of the biologically active materials are found to be polysaccharides, of which, for example, β-l,3-glucan from Lentinus edodes and PS-K extracted from cultured mycelia of Coriolus versicolor are representative.

[6] In studies of mushrooms, nutrient analysis, artificial culture, and polysaccharide extraction have usually been adopted as main themes. As for artificial culture, it is reported to utilize a solid method or a liquid method. The former is low in yield because of requirement for extensive labor and high expense while the latter, although having high yield, needs intrastructural techniques. Lee et al. suggested optimal conditions for culturing mycelia of Lentinus edodes, including a temperature of 25°C, a pH of 4.0, a stirring speed of 300rpm, an inoculum amount of 10%, an aeration rate of 1.0 v/v/m (Lee, B. W., et al., 'Cultural characteristics and pilot scale fermentation for the submerged mycelial culture of Lentinus edodes' Kor. J. Appl. Microbial Biotechnol., 21, 609-614, (1993)). Fraser reported yeast extract and casein as very effective nutrients for culturing mycelia of Agaricus bisporus (Fraser, LM. 'The growth promptive effect of several amino acids on the common cultivated mushroom' Mushroom ScL, 3, 190-200, (1956)). Aside from carbon and nitrogen sources, the growth of mycelia is also highly influenced by other environmental factors including vitamins and metal ions.

[7] Since submerged mycelial culture has a low growth rate and is apt to be contaminated, it is very important to determine medium ingredients in consideration of abilities to shorten culture periods, produce mycelia of uniform quality on a mass scale, be economically advantageous, easily extract useful materials, and develop mycelia and media as well as biologically active materials into foods. Thus far, submerged mycelial culture has been usually conducted in synthetic media, and although ginseng slices and brewery yeast extract were utilized, natural media have to be studied further.

[8] One of the most popular methods of extracting polysaccharides from mushroom comprises slicing fruit bodies, adding the slices to distilled water, heating at 85-110°C for 205 hrs, adding ethanol to form a precipitate, and dialyzing the precipitate. In another extraction method, a fermented solution is concentrated for 24 hrs in an ul- trafilter with a cutoff set at 100,000 and the concentrate is added to two volumes of acetone and left at 4°C for 24 hrs to obtain ex vivo crude polysaccharides from Phellinus ribis. It is known that polysaccharides can be obtained from Bacillus polymyxa KS-I by diluting a fermented solution with distilled water, adding NaOH to a concentration of l%(w/v) to separate the cells, centrifuging the solution, neutralizing the supernatant, and adding ethanol to form a precipitate. Lee, et al. extracted polysaccharides from Coriolus versicolor using various solvents including hot water(97°C), cold water(4°C), methanol(70°C), ethanol(70°C), acetone(50°C), hexane(50°C), 0.1N NaOH(60°C), and 0.1N HC1(6O°C), reaching the conclusion that polysaccharides can be obtained at a high yield from 10.4 to 18.4% when using hot water, cold water, 0.1N HCl and 0.1N NaOH.

[9] Fomitopsis pinicola, a Korean autogenous mushroom, is known to contain antibacterial steroids and functional, unknown proteoglycans and has been empirically shown to have antidiabetic effects. Korean Pat. Laid-Open Publication No. 2005-60726 discloses a hypoglycemic agent comprising an extract of Fomitopsis pinic ola cultured in a potato medium and a preparation method thereof. Recently, the present inventors have discovered various pharmaceutical and therapeutic functions of extracts from Fomitopsis pinicola, including the prevention of diabetes complications and diabetes-induced hypercholesterolemia(Korean Pat. Appl'n No. 2006-1430), the suppression of pancreatic cell injury(Korean Pat. Appl'n No. 2006-1431), and the suppression of diabetes-induced reactive oxygen species(Korean Pat. Appl'n No. 2005-133990). In our previous studies, the principle which brings about these inhibitory effects was also analyzed to be β- l,3-glucano-β-l,6-heterogalactomannan-protein complex, ranging in molecular weight from 300,000 to 500,000, in which β-l,3-glucan is linked to β- 1 ,6-heterogalactomannan.

[10] However, conventional methods are insufficient to culture Fomitopsis pinicola because synthetic media produce problems of being unsanitary and decreasing in production yield. Now, available natural media are under extensive study.

[11] The present inventors pay attention to green tea and citrus peel as ingredients of compositions for use in natural media. Green tea, containing free amino acids, caffeine, vitamin C, and polyphenols in small amounts, is known to have various physiological activities, including antioxidation, anticancer activity, anti-inflammation, hypocholes- terolemic action and hypertension prevention. Rich in the vitamin A precursor β- carotene, flavonoids, and terpenes, citrus peel is known to show effective antioxidation, anti-aging, cardiovascular disease preventive, immunopotentiation, and anticancer activity.

[12] The present inventors employed extracts from green tea and citrus peel as ingredients of natural media for culturing mycelia of Fomitopsis pinicola, which can overcome the unsanitary problem of synthetic media and which themselves can be utilized as drinks, and succeeded in establishing optimal conditions in the natural media (Korean Pat. Appl'n No. 2005-133989). In addition, a method for preparing a hot- water extract and an alkali extract from fruit bodies of Fomitopsis pinicola and an extract from cultured mycelia of Fomitopsis pinicola, which all are rich in polysaccharides, has been reported (Korean Pat. Appl'n No. 2006-1430).

[13] Therefore, extracts from fruit bodies of Fomitopsis pinicola, from submerged cultures in natural media, and from submerged cultures in synthetic media and their preparation methods have to be studied further to elicit the best hypoglycemic effects therefrom.

Disclosure of Invention Technical Problem

[14] Leading to the present invention, intensive and thorough research into culture conditions for obtaining biologically active materials of Fomitopsis pinicola and their applications, conducted by the present inventors, resulted in the finding that when Fomitopsis pinicola is cultured in a natural medium containing green tea or citrus peel, an extract from the mycelia thereof exhibits highly useful anti-diabetic effects.

[15] It is therefore an object of the present invention to provide an extract from the cultured mycelia of Fomitopsis pinicola, cultured in a green tea or citrus peel medium, which shows highly anti-diabetic activity.

[16] It is another object of the present invention to provide a hypoglycemic composition comprising the mycelial culture extract from Fomitopsis pinicola.

[17]

Technical Solution

[18] In order to accomplish the above objects, there is provided a composition, effective for lowering the blood sugar level in especially, patients with a diabetes mellitus, comprising an extract from Fomitopsis pinicola mycelia which is cultured in a natural medium containing green tea or citrus peel.

[19] The green tea or citrus peel medium used in the present invention can be prepared according to the method disclosed in Korean Pat. Appl'n No. 2005-133989, which is incorporated by reference herein. Green tea powder is added to distilled water, and heated for 2-3 hrs in the presence of a cooling tube. The filtered solution is mixed in an amount of 2.0%(v/v) to give a green tea medium. Likewise, a citrus peel medium can be prepared by adding distilled water to citrus peel powder, heating it for 2-3 hrs in the presence of a cooling tube, and mixing the filtered solution in an amount of 2.0%(v/v).

[20] The said hypoglycemic composition showing anti-diabetic effect comprises the extract from a submerged mycelial culture of Fomitopsis pinicola alone, but preferably in combination with a hot- water extract from fruit bodies of Fomitopsis pinicola and optionally further with the culture medium.

Advantageous Effects

[21] the extracts from submerged cultures of Fomitopsis pinicola in media containing green tea or citrus peel extracts are superior in anti-diabetic activity. With great hypoglycemic activity, the extracts of the present invention find various applications in the functional food industry. Best Mode for Carrying Out the Invention

[22] In accordance with an aspect, the present invention pertains to an anti-diabetic extract from a submerged mycelial culture of Fomitopsis pinicola cultured in a green tea or citrus peel medium.

[23] In accordance with another aspect, the present invention pertains to a hypoglycemic composition comprising the extract from a submerged mycelial culture of Fomitopsis pinicola.

[24] The hypoglycemic composition comprises the extract from a submerged mycelial culture of Fomitopsis pinicola alone, but preferably in combination with a hot- water extract from fruit bodies of Fomitopsis pinicola and optionally further with the culture medium.

[25] The green tea or citrus peel medium used in the present invention can be prepared according to the method disclosed in Korean Pat. Appl'n No. 2005-133989, which is incorporated by reference herein. Green tea powder is added to distilled water, and heated for 2-3 hrs in the presence of a cooling tube. The filtered solution is mixed in an amount of 2.0%(v/v) to give a green tea medium. Likewise, a citrus peel medium can be prepared by adding distilled water to citrus peel powder, heating it for 2-3 hrs in the presence of a cooling tube, and mixing the filtered solution in an amount of 2.0%(v/v).

[26] As for the hot- water extract from fruit bodies of Fomitopsis pinicola, it can be obtained by finely powdering the fruit bodies, adding distilled water to the powder, and heating the solution for 24 hrs in an extraction bath equipped with a cooling tube to give a concentrate.

[27] A potato medium is used to obtain an extract from cultured mycelia of Fomitopsis pinicola. First, the mycelia is inoculated in a YM medium and sub-cultured. Then, it is again inoculated in a sterile potato medium (potato hot-water extract 10~20%(v/v), sugar l~3%(w/v), corn starch powder 0.1~0.5%(w/v) and edible water 76.5~88.9%(v/v)) and cultured. The extract can be obtained by concentrating the culture medium(Korean Pat. Publication No. 2005-60726).

[28] These extracts from Fomitopsis pinicola were assayed for hypoglycemic and hypolipidemic activity as follows. First, feedstuff containing the extracts was fed for 4 weeks to rats in which diabetes mellitus had been induced with STZ(streptozotocin). Blood samples were taken from the tail vein of each rat to analyze blood sugar levels and from the ventral aorta to analyze blood lipid levels. In addition, each rat was sacrificed to excise the liver and kidneys. The weights of these organs were compared between the rats and the activity of xanthine oxidase in the liver was measured.

[29] 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

[30] EXAMPLES

[31]

[32] EXAMPLE 1 : Culturing of Mycelia of Fomitopsis pinicola in Green Tea Medium and Citrus Peel Medium

[33]

[34] EXAMPLE 1 - 1 : Separation of Mycelia of Fomitopsis pinicola

[35]

[36] Mycelia were separated from fruit bodies of 1 -year-old Fomitopsis pinicola, purchased from Jeseng Farm, located in Pohang City, Korea. The fruit bodies were cut to a size of 2x2x2mm, washed with 70%(v/v) ethanol and transferred in an amount of 5.0%(w/v) to a medium containing potato 2.0%(w/v), sugar 1.0%(w/v) and peptone 0.6%(w/v), followed by incubation for 10 days with shaking at 150rpm. The mycelia 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), and agar 2.0%(w/v), pH 6.5) and incubated at 30°C for 5-6 days before sub-culturing every 15 days.

[37]

[38] EXAMPLE 1-2: Preparation of Green Tea Medium and Citrus Peel Medium and Extract from Submerged Culture of Fomitopsis pinicola Therein

[39]

[40] A green tea extract was prepared with hot water as described above. Green tea, purchased from AmorePacific, Korea, was pulverized and filtered through a 100 mesh sieve. To lOOg of the green tea powder was added 2.5L of distilled water which was then heated for 2 hrs in an extraction bath equipped with a cooling tube. The solution was filtered through Miracloth (BioChem Co. U.S.A.) and the filtrate was used as a green tea extract in the present invention. Citrus peel, purchased from an herbal medicine market in Daegu, Korea, dried, pulverized, and passed through a 100 mesh sieve. To lOOg of the citrus peel powder was added 2.5L of distilled water which was then heated for 2 hrs in an extraction bath equipped with a cooling tube. The solution was filtered through Miracloth(BioChem Co. U.S.A.) and the filtrate was used as a citrus peel extract in the present invention.

[41] The green tea extract was mixed with necessary ingredients to prepare a green tea medium(the green tea extract 2%(v/v), glucose 5%(v/v), yeast extract 0.5%(w/v), peptone 0.5%(w/v) and malt extract 0.3%(w/v), pH 5.0). Likewise, a citrus peel medium was prepared using the citrus peel extract 2%(v/v) instead of the green tea extract 2%(v/v).

[42] The mycelia of Fomitopsis pinicola, obtained in Example 1 - 1 , were inoculated in the green tea medium and the citrus peel medium, and incubated at 30°C for 12 days with shaking at 150rpm. After being neutralized to pH 7.0 with NaHCO , the cultures were homogenized and concentrated at 40°C to 1/20 of their initial volume. These concentrates were used as extracts from the cultured mycelia of Fomitopsis pinicola.

[43]

[44] EXAMPLE 2: Hot- Water Extract from Fruit Body of Fomitopsis pinicola and

Mycelial Extract from Submerged Culture of Fomitopsis pinicola

[45]

[46] EXAMPLE 2-1 : Preparation of Hot- Water Extract from Fruit Body of Fomitopsis pinicola

[47]

[48] Fruit bodies of 1 -year-old Fomitopsis pinicola, purchased from Jeseng Farm, located in Pohang City, Korea, were cut to a size of 2x2x2mm and added to 4L of distilled water, followed by extraction for 24 hrs in a bath equipped with a cooling tube. The extract was concentrated to 1/4 of the initial volume to give a solution(3.75g/100mL).

[49]

[50] EXAMPLE 2-2: Preparation of Extract from Submerged Culture in Potato Medium

[51] [52] Mycelia of Fomitopsis pinicola, purchased from Jeseng Farm, located in Pohang

City, Korea, were inoculated in a YM medium(glucose l%(v/v), yeast extract 0.5%(w/v), peptone 0.5%(w/v) and malt yeast 0.3%(w/v), pH 6.5) and subcultured at 30°C for 10 days. This culture was inoculated in an amount of 5%(v/v) in a potato medium[(potato hot-water extract 15%(v/v), as a dry mass 4%(w/v)), sugar 2%(w/v), corn starch 0.3%(w/v), pH 6.5], which had been sterilized at 121°C for 1 hr and cooled, followed by incubation at 30°C for 12 days with shaking at 150rpm. After being neutralized to pH 7.0 with NaHCO , the culture was homogenized and concentrated at 40°C to 1/20 of its initial volume.

[53]

[54] EXAMPLE 3: Anti-Diabetic Effects of Extracts from Fomitopsis pinicola

[55]

[56] EXAMPLE 3-1 : Experimental Animals and Method

[57]

[58] SD male rats, each having body weight of 225+5.2g, were divided into 7 groups of

10. For environmental adaptation, they were pre-reared at 23+2°C and RH 60+5% for one week with general feedstuff(Purina Co., Seoul, Korea) in a 12:12 hr light-dark cycle before being fed the experimental diet.

[59] The seven groups are indicated using the following abbreviations.

[60] 1) NC: normal control group

[61] 2) DM: diabetes mellitus control group

[62] 3) DM-PD: diabetes mellitus-induced group administered with 2% of the concentrate(l/20) from a submerged culture of Fomitopsis pinicola in the potato(PD) medium

[63] 4) DM-CP: diabetes mellitus-induced group administered with 2% of the concentrate(l/20) from a submerged culture of Fomitopsis pinicola in the citrus peel(CP) medium

[64] 5) DM-GT: diabetes mellitus-induced group administered with 2% of the concentrate(l/20) from a submerged culture of Fomitopsis pinicola in the green tea(GT) medium

[65] 6) DM-WE: diabetes mellitus-induced group administered with 2% of hot-water extract(WE) from fruit bodies of Fomitopsis pinicola

[66] 7) DM-WC: diabetes mellitus-induced group administered with 2% of a mixture of

1 : 1 the hot- water extract from fruit bodies : the concentrate from a submerged culture in the citrus peel medium

[67] Table 1

Basic Dietary Cure for Animal Test (g/kg)

[68] AIN-mineral mix(g/kg): Calcium lactate 620.0, sodium chloride 74.0, dibasic potassium phosphate 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.

[69] 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. [70] [71] EXAMPLE 3-2: Anti-Diabetic Activity

[72] [73] Weight Gain. Dietary Intake and Food Efficiency [74] [75] A solution of streptozotocin(STZ, Sigma Chem. Co. MO, U.S. A.) in 0.1 M citrate buffer(pH 4.3) was injected in a dose of 40mg/kg into the thigh. Animals which had a blood sugar level of 300 mg/dL, measured with a blood sample taken from the tail vein 48 hrs after injection with STZ, were regarded as having diabetes mellitus induced therein.

[76] Blood sugar levels were measured using Gluco-Tester(Life Scan Inc., U.S.A.) at 10-12 a.m. Blood sampling was performed with lancet from the tail vein of rats which had been starved until the sampling time.

[77] Over the experiment period, body weight, dietary intake, and water intake were measured at a predetermined time each day, and the food efficiency ratio(FER) was defined as the ratio of weight gain to dietary intake in the same time period. The weight gain, dietary intake, and FER are given in Table 2, below.

[78] The DM group remarkably lost weight(58% decreased) but increased 14% in dietary intake, 84% in water intake and 108% in urine output compared with the NC group. Likewise, the other groups showed an increase in the three aspects and a decrease in weight gain. However, these symptoms were largely alleviated, demonstrating the anti-diabetic activity of the extracts. High anti-diabetic effects, far superior to those obtained in the conventional potato medium, were observed particularly in the DM-WC group and the DM-GT group. In detail, weight loss was measured to be 17% for the DM-GT group, 16% for the DM-CP group, 32% for the DM-PD group, 23% for the DM-WE group and 15% for the DM-WC group, with about two-fold greater effect of the submerged cultures in the green tea medium, the citrus peel medium, or a mixture of fruit hot- water extract and citrus peel medium than those in the conventional potato medium. The strongest effects on dietary intake, FER, water intake and urine output were detected in the DM-CP group and the DM-WC group(see: Table 2).

[79] Table 2

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

[80] ^NC: normal control, DM: diabetes mellitus control, DM-PD: 2% of the concentrate(l/20) from the submerged culture in the potato(PD) medium was administered after the induction of diabetes mellitus, DM-CP: 2% of the concentrate(l/20) from the submerged culture in the citrus peel(CP) medium was administered after the induction of diabetes mellitus, DM-GT: 2% of the concentrate(l/20) from the submerged culture in the green tea(GT) medium was administered after the induction of diabetes mellitus, DM-WE: 2% of hot-water extract(WE) from fruit bodies was administered after the induction of diabetes mellitus, DM-WC: 2% of a mixture of 1 : 1 the hot-water extract from fruit bodies : the concentrate from the submerged culture in the citrus peel medium was administered after the induction of diabetes mellitus

[81] food efficiency ratio: weight gain/dietary intake [82]

[83] Organ Weight [84] [85] When afflicted with diabetes mellitus, cells fail to conduct normal glucose metabolism due to low insulin levels and high insulin resistance while lipogenesis occurs with the aid of acetyl-CoA, resulting in the accumulation of lipids in the liver. Particularly, rats in which diabetes mellitus was induced with STZ were found to increase the activity of hepatic glucose-6-phosphatase, involved in carbohydrate metabolism, resulting in liver enlargement. Kidney enlargement was also found because when increased in level due to diabetes mellitus, plasma glucose is metabolized into glycogen which is accumulated in mesangial cells of the aglomerulus.

[86] During rearing for 4 weeks after the induction of diabetes mellitus, the rats were monitored for weight of the liver and kidneys. The DM group was measured to increase 48% in liver weight compared to the NC group as expressed as a weight% on the basis of body weight. Thus, liver enlargement was caused by diabetes mellitus. The livers of the experimental groups fed with the extracts of the present invention were smaller than those of the DM group. The largest alleviation effects were measured in the DM-CP group and the DM-WC group. The same phenomena were found for the kidneys(see: Table 3).

[87] Table 3

Effect of 4- Week Administration with Fomitopsis pinicola Extracts on Liver and Kidney Weights (% of Body Weight)

[88] the same as in Table 2 [89] Avg.+SD obtained from 10 rats of each group, different letters indicating a significance within 5%.

[90] [91] Blood Glucose [92] [93] While being fed for 4 weeks with the experimental diets, the diabetes mellitus- induced rats were monitored for blood glucose level. The DM group maintained a blood glucose level from 523.5 to 546.5 mg/dL for the period. Lower blood glucose levels were measured in the groups fed with the extracts of the present invention. In Week 4, the blood glucose level was reduced by 69% in the DM-GT group, 74% in the DM-CP group, 53% in the DM-PD group, 58% in the DM-WE group, and 75% in the DM-WC group. Depending on the culture condition of the mycelia, the blood glucose reduction efficiency varied. Better effects were shown when using concentrates obtained from cultures in the citrus peel medium or the green tea medium or using mixtures of hot- water extract therewith than when using the concentrates obtained from cultures in the conventional potato medium(see: Table 4). After the extract from the submerged culture in the conventional potato medium was fed for 4 weeks, the blood glucose level was decreased by about 292 mg/dl, which corresponded to a rate of about 53%. On the other hand, the difference of blood glucose level between pre- and post-administration of the extract from the submerged culture in the citrus peel medium amounted to about 406 mg/dl, which corresponded to a decrease of about 74%.

[94] Table 4

Blood Glucose Levels of Diabetes Mellitus-Induced Rats During Administration with Fomitopsis pinicola Extract

[95] ''the same as in Table 2 [96] Avg.+SD obtained from 10 rats of each group, different letters showing a significance within 5%.

[97] [98] EXAMPLE 4: Suppressive Effect of Fomitopsis pinicola Extract on Diabetes Mellitus-Induced Hyperlipidemia and on Hepatic Xanthine Oxidase Activity

[99] [100] The same experimental groups and diets as in Example 3 were conducted. After being fed with the experimental diets for 4 weeks, the animals were starved for 24 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 perfused with iced physiological saline and excised. The sampled blood was left at a cold temperature for 7 hrs, followed by cen- trifugation at 3,000 rpm for 10 min to separate sera. The excised liver was immersed in 4 volumes of a 0.25M sucrose solution and homogenized on ice, followed by cen- trifugation at 10,000x g for 30 min. The cytosolic fraction obtained as the supernatant was stored, along with other samples, in a deep freezer maintained at -70°C.

[101]

[102] Analysis of serum lipid level

[103] 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 triglyceride level was determined by measuring absorbance at 550 nm after 0.02 ml of the serum was mixed and reacted at 37°C for 10 min with 3.0 ml of the enzyme solution. To determine the total cholesterol level according to the formula mg/dL = (Absorbance of standard solution/Absorbance of test sample) x 300, the serum was reacted with the enzyme solution, as in triglycerides at 37°C, but for 5 min, and absorbance at 500 nm was obtained. As for the HLD- cholesterol value, it was calculated according to the following formula mg/dL = (Absorbance of standard solution/Absorbance of test sample) x 100. In this regard, 0.1 ml of the serum was reacted with 3.0 ml of the enzyme solution at 37°C for 5 min and absorbance at 500nm was measured. Blood LDL-cholesterol level could be calculated according to Total Cholesterol level - (HDL-Cholesterol level) - (Triglyceride level/5).

[104] Compared to the NC group, the blood triglyceride level increased remarkably, by about 73%, in the DM group, but slightly, by 16% in the DM-GT group, by 8% in the DM-CP group, and by 7% in the DM-WC group, and moderately, by 47% in the DM- PD group and by 39% by the DM-WE group. As seen, the DM-WC group and the DM-CP group, both comparable, were lowest in level increase.

[105] The total cholesterol level showed the same tendency as in the triglyceride level, with no significance.

[106] The HDL-cholesterol level of the DM group was 19% lower than that of the NC group, but there was no significant difference in HDL-cholesterol level between the experimental groups fed with the extracts and the NC group.

[107] Compared to the NC group, the DM group was 42% higher in LDL-cholesterol level. In this level, however, the DM-WC group, the DM-CP group and the DM-GT group were comparable to or lower than the NC group. Thus, the extracts from the submerged cultures in the media according to the present invention were found to decrease blood lipid levels efficiently compared to the extract from the submerged culture in the conventional potato medium, (see Table 5).

[108] Table 5

Effect of 4 Week- Administration with Fomitopsis pinicola Extracts on Blood Lipid Level

[109] ''the same as in Table 2. [HO] ²⁾Total cholesterol-(HDL-Cholesterol+Triglyceride/5). [111] ³⁾Avg.+SD obtained from 10 rats of each group, different letters (a-c)showing a significance within 5%.

[112] [113] Hepatic xanthine oxidase activity [114] [115] Hepatic xanthine oxidase(XOD) activity was determined according to the method of Strirpe in which xanthine is used as a substrate for reaction at 30°C for 10 min to produce uric acid and absorbance at 292nm is measured. The activity of the enzyme was expressed as the concentration(nmol) of the uric acid produced as a result of the reaction of lmg of the liver tissue protein with the substrate for 1 min.

[116] Upon hepatic injury, XOD activity increases. The administration of toxic materials, such as ethanol, carbon tetrachloride, STZ, etc., causes XOD activity to increase to promote the oxidation of xanthine. Higher O type activity and O/T ratios(%) indicate the generation of more reactive oxygen species.

[117] After being fed for 4 weeks with the experimental feeds, the diabetes mellitus-induc ed rats were analyzed for XOD activity and the results are given in Table 6, below. As seen in Table 6, there is no significant difference in XOD activity among them, but the O type activity and O/T(%) of the DM-CP group, the DM-WC group and the DM-GT group were remarkably low compared to that of the DM-PD group, and comparable to that of the NC group, implying that the extract from the submerged cultures in the media according to the present invention has great inhibitory activity against the generation of ROS.

[118] Table 6 Hepatic XOD Activity of Diabetes Mellitus-Induced Rats After Administration with Experimental Diets for 4 Weeks

[119] ^1)rrhe same as in Table 2. [120] Avg.+SD obtained from 10 rats of each group, different letters (a-c)showing a significance within 5%.

[121]

Industrial Applicability [122] As described hitherto, the extracts from submerged cultures of Fomitopsis pinicola in media containing green tea or citrus peel extracts are superior in anti-diabetic activity. With great hypoglycemic activity, the extracts of the present invention find various applications in the functional food industry.

[123] [124] 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.

[125]

Claims

[1] An extract from a submerged culture of Fomitopsis pinicola cultured in a green tea medium or a citrus peel medium.

[2] The extract according to claim 1, wherein the green tea medium comprises

0.5%-3.0%(v/v) of a green tea extract, 3.0%-7.0%(v/v) of glucose, 0.5%-1.5% (w/v) of a yeast extract, 0.1%-1.5%(w/v) of peptone, 0.1%-0.7%(w/v) of a malt extract, and 86.3%-95.8%(v/v) of purified water, said green tea extract being obtained by infusing green tea powder in hot water and filtering the solution.

[3] The extract according to claim 1, wherein the citrus peel medium comprises

0.5%-3.0%(v/v) of a citrus peel extract, 3.0%-7.0%(v/v) of glucose, 0.5%-1.5%(w/v) of a yeast extract, 0.1%-1.5%(w/v) of peptone, 0.1%-0.7%(w/v) of a malt extract, and 86.3%-95.8%(v/v) of purified water, said citrus peel extract being obtained by infusing citrus peel powder in hot water and filtering the solution.

[4] A hypoglycemic composition, comprising the extract of claim 1.

[5] The hypoglycemic composition according to claim 4, further comprising a hot- water extract from fruit bodies of Fomitopsis pinicola.

[6] The hypoglycemic composition according to claim 5, wherein the hot- water extract is obtained by mincing and pulverizing the fruit bodies of Fomitopsis pinicola, immersing the pulverized fruit bodies in distilled water, heating the water for 24 hrs in an extraction bath equipped with a cooling tube, and concentrating the extract collected in the cooling tube.

[7] The hypoglycemic composition according to claim 4, further comprising a filtered, submerged culture of Fomitopsis pinicola in the green tea medium or the citrus peel medium.