Description

FOMITOPSIS PINICOLA EXTRACT HAVING SUPPRESSIVE ACTIVITY AGAINST PANCREATIC CELL DAMAGE 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 is effective in suppressing pancreatic injury. Background Art

[2] The pancreas, running horizontally between the duodenum and the spleen, is an organ located in the digestive and endocrine system that serves to secrete several important hormones including insulin and glucagon into the blood and also serves to secrete pancreatic juice containing digestive enzymes such as amylase, lipase, trypsin, etc., and alkaline juices into the duodenum. Hence, pancreatic injury results in the interruption of secretion of the digestive enzymes and the hormones responsible for the regulation of blood sugar level. Particularly, when beta cells of the pancreatic islets of Langerhans are injured, diabetes mellitus and complications are likely to occur.

[3] There are many factors that harm the pancreas, such as infection, autoimmune response, etc. Various efforts have been made to treat injured pancreas, including the differentiation of stem cells into beta cells and the transplantation of pancreatic islets, but these are difficult to apply in practice.

[4] Pancreatic islet transplantation encounters difficulties related to the undersupply of islets, side effects caused by the long-term administration of immunosuppressive agents, and destruction due to autoimmune response. In the case of differentiation and proliferation using embryonic stem cells, ethical problems arise, and there is the possibility of the transplant, even if successfully achieved, differentiating into undesired cells and further into cancerous cells in other tissues or organs.

[5] Therefore, there is a need for a method of suppressing pancreatic injury without side effects occurring as a result of the pancreatic islet transplantation and without the differentiation of embryonic cells. Disclosure of Invention Technical Problem

[6] Leading to the present invention, intensive and thorough research into a natural material capable of protecting pancreatic cells, conducted by the present inventors,

resulted in the finding that rats, in which diabetes mellitus was induced with STZ, underwent pancreatic injury to a lesser extent when they were administered with extracts from the fruit body and cultured mycelia of Fomitopsis pinicola than with other control materials. In addition, the principle which brought about these protective effects was determined to be 'β-l,3-glucano-β-l,6-heterogalactomannan-protein complex'.

[7] It is therefore an object of the present invention to provide a fruit body extract or a cultured mycelial extract of Fomitopsis pinicola which has protective activity against pancreatic injury.

[8] 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 effective in suppressing pancreatic injury.

Technical Solution

[9] In order to accomplish the above objects, there is provided a composition, effective in suppressing pancreatic injury induced by diabetes mellitus, comprising a fruit body extract or cultured mycelial extract of Fomitopsis pinicola.

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

[11] 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.

[12] 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.

[13] 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.

[14] 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.

[15] 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.

[16] 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. Advantageous Effects

[17] Fruit body extracts and cultured mycelial extracts of Fomitopsis pinicola according to the present invention effectively inhibit the pancreatic injury induced by diabetes mellitus, thereby finding various applications for the development of functional foods for preventing pancreatic injury resulting from infection or autoimmune response. Brief Description of the Drawings

[18] FIG. 1 shows optical microphotographs, magnified 200 times, of a normal control group (NC) of mice raised for 4 weeks on different diets, a pancreatic injury group (DM), a pancreatic injury-induced, hot-water extract-administered group (DM-WE) which was administered with 1% of the hot- water extract after treatment with STZ, a pancreatic injury-induced, alkali extract-administered group (DM-AE) which was administered with 1% of the alkali extract after treatment with STZ, and a pancreatic injury-induced, cultured mycelial extract-administered group (DM-CM) which was administered with 1 % of the cultured mycelial extract after treatment with STZ. Best Mode for Carrying Out the Invention

[19] 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 suppressing pancreatic injury.

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

[21] 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 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.

[22] 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.

[23] 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.

[24] The extract from Fomitopsis pinicola according to the present invention was assayed for suppressive activity against diabetes mellitus-caused pancreatic injury 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 pancreas and adjacent tissues, which were then fixed with a 10% formalin solution, dehydrated with alcohol, embedded in paraffin, and stained with HE. Under an optical microscope, the samples were observed to determine the suppressive effect of the extracts on pancreatic injury.

[25] Furthermore, in order to identify the principle 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.

[26] 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

[27] EXAMPLES

[28]

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

[30]

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

Water

[32]

[33] 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 yield 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.

[34]

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

Alkali

[36]

[37] 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.

[38]

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

[40]

[41] 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 (150rpm).

[42] 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 150rpm with sterile air (10cm /min) provided thereto. Then, neutralization with sodium hydrogen carbonate (NaHCO ) to a pH of 6.5 preceded precipitation with 10 volumes of ethanol.

[43] 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.

[44]

[45] EXAMPLE 2: Suppressive Effect of Fomitopsis pinicola Extract on Pancreatic

Injury

[46]

[47] EXAMPLE 2- 1 : Experiment Animal and Method

[48]

[49] SD rats, each having a 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 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 groups was reared for four weeks according to the dietary schedules given in 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 had pancreatic injury induced therein. Blood sugar levels were measured using a bio-sensor and a kit.

[50] [51] Table 1 Basic Dietary Composition for Animal Test (g/kg)

[52] 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

[53] 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.

[54] [55] 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 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. [56] 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

[57] ^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

[58] ²⁾ feed efficiency ratio: weight gain/dietary intake

[59]

[60] After being fed with the experimental diets for 4 weeks, the animals were starved for 16 hrs with only water given thereto. They were etherized and subjected to laparotomy along the ventral median line to excise the pancreas and adjacent tissues which were then fixed with a 10% formalin solution, dehydrated with alcohol, embedded in paraffin, stained with HE, and observed under an optical microscope.

[61]

[62] EXAMPLE 2-2: Suppressive Effect of Fomitopsis pinicola Extracts on Diabetes

Mellitus-Induced Pancreatic Injury

[63]

[64] Pancreatic tissues, excised from the experimental animals which had been fed different diets for 4 weeks, were observed under an optical microscope, and the results are shown in FIG. 1.

[65] The pancreas was observed to have typical morphology in the NC group which was fed the basic diet, as shown in FIG. 1. The functional arrangement of secretory cells around blood vessels was normal with no injury detected therein. The DM group, which was treated only with STZ, was observed to show a homogeneous nucleus density in marginal cells (red), but to have serious swelling in central cells (black). Further, some cells underwent necrosis (blue).

[66] In the DM-WE group, which was treated with STZ and then with the hot-water extract of Fomitopsis pinicola, type A cells were maintained in their normal state (red), but the central cells were observed to have swellings (black) and undergo necrosis (blue). The DM-AE group, which was treated with STZ and then with the alkali extract of Fomitopsis pinicola, was homogeneous in nucleus and cytoplasm size density with no morphological change. Cell swellings, along with an abnormal functional arrangement of cells, were found in the DM-CM group, which was treated with STZ and then with the cultured mycelial extract. However, all of the groups which were treated with the extracts from Fomitopsis pinicola were found to experience hepatic injury to a much lesser extent, compared to the DM group, which was not treated with the extract of the present invention.

[67]

[68] EXAMPLE 3: Qualitative Assay for Fomitopsis pinicola Extracts

[70] Fomitopsis pinicola extracts of the present invention were identified as suppressors of pancreatic injury in diabetes mellitus-induced rats, as evident in Example 2. In order to examine the principle of suppressing diabetes mellitus-induced pancreatic injury, the Fomitopsis pinicola extracts were qualitatively analyzed.

[71] The Fomitopsis pinicola fruit body 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)).

[72] 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 3, 2,3,4,6-tetramethyl glucose and 2,4,6-trimethyl glucose were detected, evincing the presence of β-l,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- 1,3-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.

[73] Table 3

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

[74]

Industrial Applicability [75] Fruit body extracts and cultured mycelial extracts of Fomitopsis pinicola according to the present invention effectively suppress the pancreatic injury induced by diabetes mellitus, thereby finding various applications for the development of functional foods for preventing the pancreatic injury resulting from infection or autoimmune response.

[76] [77] 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 other than as specifically described.

[78]