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WO2007075069A1 - Extrait de fomitopsis pinicola et son utilisation pour traiter les diabetes - Google Patents

Extrait de fomitopsis pinicola et son utilisation pour traiter les diabetes Download PDF

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Publication number
WO2007075069A1
WO2007075069A1 PCT/KR2006/005877 KR2006005877W WO2007075069A1 WO 2007075069 A1 WO2007075069 A1 WO 2007075069A1 KR 2006005877 W KR2006005877 W KR 2006005877W WO 2007075069 A1 WO2007075069 A1 WO 2007075069A1
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WIPO (PCT)
Prior art keywords
extract
fomitopsis pinicola
pieces
composition according
fomitopsis
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Ceased
Application number
PCT/KR2006/005877
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English (en)
Inventor
Seung-Hee Oh
Soon-Dong Kim
Sang-Il Lee
Hyun-Goo Lee
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Eugene Biofarm Coltd
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Eugene Biofarm Coltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Application filed by Eugene Biofarm Coltd filed Critical Eugene Biofarm Coltd
Publication of WO2007075069A1 publication Critical patent/WO2007075069A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/06—Fungi, e.g. yeasts
    • A61K36/07—Basidiomycota, e.g. Cryptococcus

Definitions

  • the present invention relates to a Fomitopsis pinicola extract and the use thereof.
  • the present invention relates to the fruit body extracts and cultured mycelial extract of Fomitopsis pinicola, which effectively inhibit the reactive oxygen species production induced by diabetes mellitus.
  • AGEs advanced glycation end products
  • ROSs including superoxide radical (O " ), hydroxyl radical (HO ), hydrogen peroxide (H O ) and singlet oxygen ( O ), form as a natural byproduct of the normal metabolism of oxygen.
  • O superoxide radical
  • HO hydroxyl radical
  • H O hydrogen peroxide
  • O singlet oxygen
  • ROS levels can increase dramatically, which can result in significant damage to cell structures. That is, ROSs cause non-selective irreversible destructive damage to cell constituents, such as lipids, proteins, sugar, and DNA, and have been found to be involved in the occurrence of various diseases, such as cancer, apoplexy, Parkinson's disease, Alzheimer's disease, aging, heart failure, ischemia, arteriosclerosis, skin disease, inflammation, rheumatoid arthritis, autoimmune diseases, etc.
  • lipid peroxides which may be formed through the reaction of ROSs, such as free radicals, with phospholipids of cell membranes, have adverse effects on the fluidity and functionality of cell membranes, and thus on cellular functions, resulting in local defects of tissues, such as deformed cell structure. Therefore, the accumulation of lipid peroxides is now known to be a factor causative of various diseases, including cerebral apoplexy, myocardial infarction, hyperlipidemia, acute inflammation, rheumatoid arthritis, alcoholic hepatitis, etc.
  • compositions effective for the inhibition of diabetes mellitus-induced reactive oxygen species production, comprising a fruit body extract or cultured mycelial extract of Fomitopsis pinicola.
  • the fruit body extract is a hot- water extract or an alkali extract.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the present invention is directed to an extract from the fruit body or cultured mycelia of Fomitopsis pinicola, and a composition comprising the extract as an active ingredient for inhibiting the ROS production caused by diabetes mellitus.
  • the extract from the fruit body of Fomitopsis pinicola may be prepared with hot water or alkali.
  • a hot- water extract from the fruit body of Fomitopsis pinicola can be obtained as follows.
  • the fruit body is finely cut, pulverized, immersed in water, and heated, followed by concentration and precipitation with ethanol.
  • the precipitate is then freeze-dried.
  • Water is preferably used in an amount of about 30 times the weight of the pulverized fruit body, but is not limited thereto.
  • Heating is preferably conducted at 100°C for about 24 hrs, but is not limited thereto.
  • 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.
  • 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.
  • IN KOH it is preferable that IN KOH be used in a ratio of approximately 1:1 (w/v).
  • HAS homoogenization after alkali swelling
  • the extract from Fomitopsis pinicola according to the present invention is assayed for inhibitory activity against diabetes mellitus-caused ROS production as follows. First, the extract is orally administered to rats in which diabetes mellitus has been induced with STZ (streptozotocin). After being raised, each rat is sacrificed to excise the liver, which is then homogenized in ice. The homogenate is analyzed for the activity of enzymes having influence on ROS production and toxification. In addition, the levels of lipid peroxides, glutathione and ALT in the blood are measured to determine whether the extract of the present invention can inhibit the ROS production caused by diabetes mellitus.
  • STZ streptozotocin
  • the Fomitopsis pinicola extract is analyzed for ingredients and the molecular weights thereof.
  • 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.
  • the mycelia were inoculated in a sterilized (120°C, 30 min), cold potato medium (water: 16L, potato powder: 300g, glucose: 150g, peptone: O.lg) to an amount of 2%(v/v), and cultured at 30°C for 10 days in a rotary agitator operating at 150 rpm with sterile air (10cm 3 /min) provided thereto. Then, neutralization with sodium hydrogen carbonate (NaHCO ) to a pH of 6.5 preceded precipitation with 10 volumes of ethanol.
  • NaHCO sodium hydrogen carbonate
  • 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.
  • EXAMPLE 2 Inhibition Effect of Fomitopsis pinicola Extract on ROS Production
  • EXAMPLE 2-1 Experimental Animal and Method [45]
  • SD rats each having body weight of 200+5g, were divided into 5 groups, which were respectively set as a normal control group (NC), a diabetes mellitus control group (DM), in which diabetes mellitus was induced by STZ, a diabetes mellitus-induced, hot- water extract-administered group (DM-WE) which was administered with 1% of the hot-water extract after treatment with STZ, a diabetes mellitus-induced, alkali extract- administered group (DM-AE) which was administered with 1% of the alkali extract after treatment with STZ, and a diabetes mellitus-induced, cultured mycelial extract- administered group (DM-CM) which was administered with 1% of the cultured mycelial extract after treatment with STZ.
  • NC normal control group
  • DM-WE diabetes mellitus-induced, hot- water extract-ad
  • Each of the group has 7 rats and they were reared for four weeks according to the dietary schedules of Table 1, below.
  • STZ 55mg/kg was intramuscularly injected. Animals which had a blood sugar level of 300 mg/dL, measured 48 hrs after injection with STZ, were regarded as having ROSs induced in the hepatic tissues thereof. Blood sugar levels were measured using a bio-sensor and a kit. [47] Table 1
  • 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.
  • 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
  • feed efficiency ratio weight gain/dietary intake
  • the liver was also excised and homogenized in a homogenizer to obtain a postmitochondrial fraction, which was analyzed for the activity and level of xanthine oxidase (XOD), glutathione peroxidase (GPX), superoxide dismutase (SOD), glutathione, lipid peroxide, and blood alanine aminotransferase (ALT).
  • XOD xanthine oxidase
  • GPX glutathione peroxidase
  • SOD superoxide dismutase
  • glutathione glutathione
  • lipid peroxide lipid peroxide
  • ALT blood alanine aminotransferase
  • EXAMPLE 2-2 Inhibitory Effect of Fomitopsis pinicola Extracts on Diabetes Mellitus-Induced ROS Production
  • EXAMPLE 2-2-1 Activity change of xanthine oxidase (XOD) in hepatic tissue
  • XOD xanthine oxidase
  • Total XOD activity was measured according to the Stripe method, in which the uric acid, oxidized from xanthine in the presence of the enzyme and NAD+ at 30°C for 10 min in 0.1 M PBS (pH 7.4) is measured at 292 nm.
  • Type 0 activity of XOD obtained when NAD+ was absent, was measured.
  • XOD activity was expressed as nmoles of the uric acid produced from the substrate for 1 min by 1 mg of the hepatic protein.
  • Type 0 activity of XOD was observed to increase by approximately 68% for the DM group, compared to the NC group, and to decrease by approximately 32% for the DM-AE group, compared to the DM group. It was also observed that the DM-WE group and the DM-CM group both tended to decrease in type 0 activity (see Table 3).
  • EXAMPLE 2-2-2 Activity change of superoxide dismutase (SOD) and glutathione peroxidase (GPX) in hepatic tissue
  • SOD activity was analyzed according to the Martin method by which the extent to which the oxidation of hematoxylin in 5OmM PBS (pH 7.5) in the presence of 0.1 mM EDTA was inhibited was measured at 560nm.
  • GPX activity was determined by the method of Paglia, using NADPH-coupled reduction of GSSG, catalysed by glutathione reductase. H2O2 was used as a substrate. Oxidation of NADPH was conducted at 340 nm. One unit of enzyme activity is defined as the oxidation of 1 nmole NADPH/min/mg protein in a solution of 5 mM EDTA in 50 mM PBS (pH 7.0) at 25°C. The DM group was remarkably decreased in SOD and GPX activity compared to the NC group. The Fomitopsis pinicola extract- administered groups were increased in SOD and GPX activity compared to the DM group, but this difference was not significant (see Table 4).
  • EXAMPLE 2-2-3 Change in hepatic glutathione and lipid peroxide level and blood ALT level
  • the hepatic glutathione level was measured to be ap- proximately 21% and 13% larger in the DM-WE and DM-AE groups respectively, than in the DM group.
  • DM-CM group had little difference from the DM group.
  • the hepatic lipid peroxide level of the DM group increased by approximately 146% compared to that of the NC group, the hepatic lipid peroxide level of the DM-AE group was reduced by approximately 36% compared to that of the DM group.
  • the hot-water extract and the cultured mycelial extract did not significantly reduce the lipid peroxide level which was increased by STZ administration.
  • Blood ALT activity was measured to increase 2.97 times for the DM group compared to the NC group. A significant decrease in blood ALT activity was found in the DM- AE group and the DM-CM group. Also, the DM-WE group tended to decrease in blood ALT activity (see Table 5).
  • Fomitopsis pinicola extracts of the present invention were identified as inhibitors of ROS production in diabetes mellitus-induced rats, as evident in Example 2. In order to examine the principle of inhibiting diabetes mellitus-induced ROS production, the Fomitopsis pinicola extracts were qualitatively analyzed.
  • 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)).
  • Fomitopsis pinicola extract one main component of the Fomitopsis pinicola extract was identified as ⁇ -l,3-glucano- ⁇ -l,6-heterogalactomannan, in which ⁇ -l,3-glucan is linked to ⁇ -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.

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  • Health & Medical Sciences (AREA)
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Abstract

L'invention concerne des extraits de Fomitopsis pinicola et leur utilisation. Des extraits de pulpe de Fomitopsis pinicola et des extraits mycéliens de culture de ce dernier inhibent efficacement la production de ROS induite par le diabète sucré, trouvant ainsi diverses applications pour le dévelopmement d'aliments fonctionnels destinés à la prévention des lésions tissulaires dues au ROS.
PCT/KR2006/005877 2005-12-29 2006-12-29 Extrait de fomitopsis pinicola et son utilisation pour traiter les diabetes Ceased WO2007075069A1 (fr)

Applications Claiming Priority (2)

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KR1020050133990 2005-12-29
KR10-2005-0133990 2005-12-29

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS4830371B1 (fr) * 1968-09-07 1973-09-19
KR20050060726A (ko) * 2003-12-17 2005-06-22 김천환 소나무잔나비버섯 추출물이 포함된 혈당강하제 및 그제조방법
WO2005067955A1 (fr) * 2004-01-06 2005-07-28 Paul Stamets Activite antimicrobienne de champignons medicinaux

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS4830371B1 (fr) * 1968-09-07 1973-09-19
KR20050060726A (ko) * 2003-12-17 2005-06-22 김천환 소나무잔나비버섯 추출물이 포함된 혈당강하제 및 그제조방법
WO2005067955A1 (fr) * 2004-01-06 2005-07-28 Paul Stamets Activite antimicrobienne de champignons medicinaux

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE MEDLINE [online] USUI T. ET AL.: "Investigation of the heterogeneity of heterogalactan from the fruit bodies of Fomitopsis pinicola, by employing cancanavalin A-Sepharose affinity chromatography", XP003014873, Database accession no. (NLM6894749) *
J. OF BIOCHEMISTRY, JAPAN, vol. 89, no. 4, April 1981 (1981-04-01), pages 1029 - 1037 *

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