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WO2008105583A1 - Exopolysaccharides bruts produits à partir de tremella fuciformis mycellium ayant une activité hypoglycémique et procédé de préparation correspondant - Google Patents

Exopolysaccharides bruts produits à partir de tremella fuciformis mycellium ayant une activité hypoglycémique et procédé de préparation correspondant Download PDF

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Publication number
WO2008105583A1
WO2008105583A1 PCT/KR2007/005920 KR2007005920W WO2008105583A1 WO 2008105583 A1 WO2008105583 A1 WO 2008105583A1 KR 2007005920 W KR2007005920 W KR 2007005920W WO 2008105583 A1 WO2008105583 A1 WO 2008105583A1
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Prior art keywords
eps
tremella fuciformis
tremella
produced
exopolysaccharides
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Ceased
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PCT/KR2007/005920
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English (en)
Inventor
Jong Won Yun
Eun Jae Cho
Song Hwan Bae
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SEROMBIO Co Ltd
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SEROMBIO Co Ltd
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Classifications

    • 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
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L31/00—Edible extracts or preparations of fungi; Preparation or treatment thereof
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00—Function of food ingredients
    • A23V2200/30—Foods, ingredients or supplements having a functional effect on health
    • A23V2200/3262—Foods, ingredients or supplements having a functional effect on health having an effect on blood cholesterol
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00—Function of food ingredients
    • A23V2200/30—Foods, ingredients or supplements having a functional effect on health
    • A23V2200/328—Foods, ingredients or supplements having a functional effect on health having effect on glycaemic control and diabetes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2250/00—Food ingredients
    • A23V2250/20—Natural extracts
    • A23V2250/208—Fungi extracts

Definitions

  • the present invention relates to the crude exopolysaccharides produced from
  • Tremella fuciformis mycellium having hypoglycemic activity and preparation method thereof. More particularly, the present invention relates to the exopolysaccharides from submerged culture of Tremella fuciformis mycellium, having an activity of reducing the level of glucose and cholesterol in blood, and the use thereof as medicament or health food.
  • the Type II diabetes has been contracted on 135 million peoples, i.e. 5 ⁇ 7% of the world population today, and it is expected that the number will be explosively increased up to 300 million peoples within 20 years in future.
  • various kinds of oral antidiabetic drugs have been used to treat the Type II diabetes, there is increasing demand by patients who want to use those from natural substances without any side effect. Because continuous use of the synthetic antidiabetic medicine may cause side effects and toxicity. Therefore, various kinds of natural substances such as herbal drugs are widely prescribed even though their biological activation compounds are unknown, because of their effectiveness, limited side effect, and relatively lower cost comparing to synthetic antidiatic medicine.
  • mushrooms and enthomopathogenic fungi are also exemplary sources of natural medicines having antidiabetic activity.
  • many researchers have endeavored to study the hypoglycemic effect from ether the fruiting body or mycelia of various edible/medicinal fungi including Tremella aurantia, Cordyceps sinensis, and Lentinus edodes.
  • Lo et al. reported that the fruiting body of Cordyceps has a potential to be a functional food for diabetes.
  • Kiho et al. stated that the hot water extract of the fruiting body of Agrocybe cylindracea has antidiabetic effect. Yuan et al.
  • Tremella fuciformis is a mushroom that has been used as a folk medicine for a variety of human diseases in several Asian countries. Guo et al. reported that extract of Tremella fuciformis might be useful for strengthening immune reaction of human body. Moreover, Ukai et al. reported that extract of Tremella fuciformis indicated 37 - 64 % of activity after testing its antioncogenic activity. To investigate the biological activities, most researchers have used the fruiting body of Tremella fuciformis rather than exopolysaccharides obtained from submerged mycelial culture broth, but there is no report on the hypoglycemic effect of exopolysaccharides obtained from mycelial cultures.
  • the present invention provide a composition for preventing or treating diabete mellitus or hyperlipidemia comprising expolysaccharides(EPS) as an effective ingredient wherein the EPS is produced from mycelial culture broth of Tremella fuciformis mycelium, consisting of protein moiety containing eleven (11) amino acids including serine, and carbohydrate moiety containing mainly mannose (56.10%), xylose (21.67%) and fucose (19.60%) and arabinose.
  • EPS expolysaccharides
  • the present invention provide a pharmaceutical composition for preventing or treating diabete mellitus comprising the EPS as an effective ingredient.
  • the present invention provide a pharmaceutical composition for preventing or treating hyperlipidemia comprising the EPS as an effective ingredient.
  • the present invention provide a method for preparating EPS from submerged mycelial culture of Tremella fuciformis mycelium.
  • the objects of the present invention have been achieved by cultivating mycelia with submerged culture of Tremella fuciformis, obtaining demarcations of exopolysaccharides through conducting sephalose column chromatography of ethanol extract from mycelial culture filtrate, and measuring concentrations of plasma glucose, cholesterol and triglyceride levels through oral intake to the Type II diabetic mice (ob/ob). Furthermore, the present invention has examined the molecular mechanism of hy- poglycemism by separating fat cell of the mouse and analyzing PPAR- ⁇ gene, a crucial factor of hypoglycemism. Hereinafter, the present invention is described in detail. [12]
  • the present invention consists of several steps to obtain exopoly saccharides from submerged mycelial culture broth of Tremella fuciformis mycelium and to measure the concentrations of plasma glucose, cholesterol and triglyceride levels through oral intake of the said exopoly saccharides to the Type II diabetic mice (ob/ob).
  • One aspect of the present invention is to provide exopoly saccharides which is produced from submerged mycelial culture broth of Tremella fuciformis, having hypoglycemic activity, composed of carbohydrate (89.12%) consisting of mannose, xylose and fucose, and protein (10.88%) consisting of eleven (11) amino acids including serine.
  • exopolysaccharides of the present invention extracted from myclial culture broth of Tremella fuciformis, can be obtained through several steps to get mycelial pellet from the submerged culture filtrated mixed with four (4) times of volumes of absolute ethanol, stirred vigorously and left overnight at 4 0 C, to dissolve the pellet into 0.2M NaCl buffer to a concentration of 10g/L, and to elute EPS loaded onto Separose C1-4B column (2.4cm x 100cm).
  • EPS exopoly saccharide
  • Figure 1 illustrates eruption pattern of exopolysaccharides produced from Tremella fuciformis mycellium on the cephalose CL-4B chromatography (Individual demarcations are indicated in Fr 1, 2, 3 and so on.).
  • Figure 2 is a graph showing effect of blood glucose levels for the exopolysaccharides produced from Tremella fuciformis versus plasma glucose levels in type II diabetic mice (ob/ob).
  • Figure 3 is a graph showing glucose tolerance test of the exopolysaccharides produced from Tremella fuciformis against the plasma glucose in Type II diabetic ⁇ ce(ob/ob).
  • Figure 4 is a graph that has proved the effect of reducing serum adiponectin levels with molecular biology by detecting Adipocyte PPAR- ⁇ mRNA expressions separated from type II diabetic mice (ob/ob).
  • Tremella fuciformis had been extracted from the fresh mycelia by the inventors (Laboratory of Functional Materials, Department of Biotechnology, Daegu University, Kyungbuk, Korea).
  • the stock culture was inoculated on a potato dextrose agar (potato dextrose agar, PDA) slant, incubated at 25 0 C for seven (7) days, and used throughout the experiment.
  • the stock culture was maintained by monthly subculture and the slants were stored at 4 0 C.
  • Tremella fuciformis was initially grown on a PDA medium
  • the seed culture was grown in a 250 ml flask containing 50 ml of MCM medium (20 g/1 of glucose, 2 g/1 of malt extract, 2 g/1 of peptone, 0.5 g/1 of potassium sulfate, 1 g/1 of dibasic potassium phosphate, 0.46 g/1 of monobasic potassium phosphate) at 28 0 C with shaking at 150 rpm for 3 days.
  • MCM medium 20 g/1 of glucose, 2 g/1 of malt extract, 2 g/1 of peptone, 0.5 g/1 of potassium sulfate, 1 g/1 of dibasic potassium phosphate, 0.46 g/1 of monobasic potassium phosphate
  • the resulting culture filtrate was mixed with four (4) times of volumes for absolute ethanol, stirred vigorously and left overnight at 4 0 C.
  • the precipitated EPS was centrifuged at 10,000 x g for 20 min and the supernatant was discarded.
  • the precipitates of crude EPS were lyophilized and its weight was estimated. Dry weight of the mycelium was measured after repeated washing of the mycelial pellet with distilled water and drying at 9O 0 C overnight to a constant weight.
  • the crude EPS was monitored by a gel filtration in a Sepharose CL4B column, by which two different heteropoly saccharides and two proteoglycans were eluted as shown on Figure 1.
  • the carbohydrate and protein contents in the crude EPS were 89.12 % and 10.88 %, respectively.
  • the crude EPS consisted of mainly 11 amino acids including serine in protein moiety and mainly mannose (56.10%), xylose (21.67%) and fucose (19.60%) in carbohydrate moiety (data not shown on Figure 1).
  • ob/ob mouse has similat metabolic disorders such as obesity and high plasma glucose due to genetic deficiency of leptin, which are indicated on the patients with noninsulin dependent diabetes (Type II diabetes). Hence, it is widely used for many researchers to verify the effect on Type II diabetes in experimental animal models.
  • Japan SLC (Hamamatsu, Japan) were bred within the dedicated case and left to acclimatize for 2 weeks under a constant 12-h light: 12-h dark cycle with a temperature of 23+2 0 C and relative humidity of 55+5%, given free access to feed stuff and water. They were given free access to standard pellets (Sam Yang, Seoul, Korea) and water throughout the experimental period.
  • ob/ob mouse is a genetically modified mouse, it shows obese phenomenon after 7 weeks from birth.
  • mice with glucose content more than 300 mg/dl were included into diabetic group.
  • mice were randomly divided into three groups with seven animals in each group: The control group, a Type II diabetic group received 0.9% NaCl solution; the treated group, a Type II diabetic groups treated with EPS from Tremella fuciformis at the level of 200 mg/kg body weight using an oral zoned daily for 54 days. Oral glucose tolerance test was performed on 5th day by fasting the mice for 15 h, followed by an oral administration of glucose (2 g glucose/kg body weight), and plasma glucose levels were determined at 30, 0, 30, 60, and 120 min after glucose administration.
  • Example 3 Determination of gene expression on adipose tissues of ob/ob mouse
  • RNA of adipose tissues were separated on the last day of animal experiment. After separating RNA of adipose tissues using reagent to separate the whole RNA, the expression of PPAR ⁇ had been investigated using RT-PCR technique.
  • Tremella fiiciformis EPS Effect of Tremella fiiciformis EPS on body weight gain and food intake in the Type II ob/ob mice after treatments for 52 days
  • FIG. 1 shows the effect of Tremella fiiciformis EPS on the plasma glucose levels in Type II mice (ob/ob) for 54 days.
  • the initial plasma glucose levels in two diabetic groups were almost same within an error range.
  • the plasma glucose levels in the control group were continuously increased to finally reach 442 mg/L during the experiment period.
  • the mice treated with Tremella fiiciformis EPS significantly reduced the plasma glucose levels to 52% on the 52nd day comparing to those of the control group.
  • FIG. 3 shows the glucose tolerance test executed to retest the plasma reducing phenomenon of the Tremella fiiciformis EPS. From the results, it was noted that both control and treated groups showed high blood glucose levels after 30 min, however the glucose levels of the treated group were significantly decreased from 60 min and returned approximately to baseline after 120 min. But it was confirmed that in case of the control group maintained a blood glucose levels higher than 500 mg/dl. Based on the results the blood glucose decrease phenomenon of Tremella fiiciformis EPS had been reconfirmed.
  • Tremella fiiciformis EPS Effect of Tremella fiiciformis EPS on plasma total cholesterol and triglyceride in the ob/ob mice after treatments for 52 days
  • Figure 4 shows a graph investigating PPAR ⁇ gene expression in the adipose tissues after separating them from the mice treated with Tremella fuciformis EPS and those of the control group.
  • Tremella fuciformis EPS In case of TZD-series medicines for Type II diabetes, they cause blood glucose decrease phenomenon by acting on PPAR ⁇ of the adipose tissue as a ligand to activate PPAR ⁇ , and increasing sensitivity of insulin.
  • the Tremella fuciformis EPS has also increased the expression of PPAR ⁇ gene comparing to the control group. Accordingly, we can see that the said effect of reducing blood glucose levels had been caused form this molecular mechanism.
  • the present invention found that Tremella fuciformis EPS administration led to the significantly reduced degree of diabetes of Type II diabetic mice (ob/ob). [58] Therefore, the EPS from Tremella fuciformis of the present invention can be effectively used for the production of health food or a medicine for preventing or treating diabete mellitus.
  • the present invention relates to crude exopoly- saccharides produced from Tremella fuciformis mycelium having hypoglycemic activity and preparation method thereof.
  • the crude exopolysaccharide(EPS) produced from Tremella fuciformis mycelium medicates for the Type II diabetic mice ( ob/ob)
  • it has excellent effect on reducing the plasma glucose level in blood sub- stantially.
  • the EPS of the present invention has a therapeutic effect on hyper- lipidemia by reducing serum lipids. Consequently the EPS of the present invention would be very useful invention in the medicinal industry for controlling blood glucose of diabetic patients or improving hyperlipidemia due to obesity and so on.

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  • Health & Medical Sciences (AREA)
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Abstract

L'invention concerne des exopolysaccharides bruts produits à partir de Tremella fuciformis mycellium à activité hypoglycémique et un procédé de préparation correspondant. Lorsque des souris modèles atteintes du diabète de type II (ob/ob) sont traitées avec les exopolysaccharides produits à partir de Tremella fuciformis mycellium, leurs niveaux de glycémie sont sensiblement réduits. En outre, les exopolysaccharides de l'invention possèdent d'excellentes propriétés thérapeutiques sur l'hyperlipidémie par réduction des taux de lipides sériques. Ils permettent ainsi de réguler le niveau de glycémie des patients diabétiques et de stabiliser l'hyperlipidémie.
PCT/KR2007/005920 2007-02-28 2007-11-22 Exopolysaccharides bruts produits à partir de tremella fuciformis mycellium ayant une activité hypoglycémique et procédé de préparation correspondant Ceased WO2008105583A1 (fr)

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KR1020070020650A KR20080079962A (ko) 2007-02-28 2007-02-28 흰목이 버섯 균사체 유래의 항당뇨 활성 세포외다당체 및그 제조방법
KR10-2007-0020650 2007-02-28

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CN110964761B (zh) * 2019-12-13 2021-07-30 江苏远大仙乐药业有限公司 一株银耳菌及其应用
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Citations (4)

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JPS6420070A (en) * 1987-07-14 1989-01-24 Mitsui Sugar Co Food and beverage
JPH11193244A (ja) * 1997-12-26 1999-07-21 New Food Creation Gijutsu Kenkyu Kumiai コレステロール上昇抑制剤、コレステロール低下剤および動脈硬化予防治療剤
WO2001032830A2 (fr) * 1999-10-15 2001-05-10 Medmyco, Inc. Compositions de glucuronoxylomannane comme agent nutritif obtenu a partir de champignons basidiomycetes superieurs, techniques et procede de production correspondants
KR20060020548A (ko) * 2004-08-31 2006-03-06 주식회사풀무원 혈당강하 효과를 나타내는 흰목이 버섯(Tremellafuciformis) 조다당체 추출물과 이를 포함하는 당뇨병예방 및 치료를 위한 조성물

Patent Citations (4)

* Cited by examiner, † Cited by third party
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JPS6420070A (en) * 1987-07-14 1989-01-24 Mitsui Sugar Co Food and beverage
JPH11193244A (ja) * 1997-12-26 1999-07-21 New Food Creation Gijutsu Kenkyu Kumiai コレステロール上昇抑制剤、コレステロール低下剤および動脈硬化予防治療剤
WO2001032830A2 (fr) * 1999-10-15 2001-05-10 Medmyco, Inc. Compositions de glucuronoxylomannane comme agent nutritif obtenu a partir de champignons basidiomycetes superieurs, techniques et procede de production correspondants
KR20060020548A (ko) * 2004-08-31 2006-03-06 주식회사풀무원 혈당강하 효과를 나타내는 흰목이 버섯(Tremellafuciformis) 조다당체 추출물과 이를 포함하는 당뇨병예방 및 치료를 위한 조성물

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Title
CHENG H.H. ET AL.: "Interactions of lipid metabolism and intestinal physiology with Tremella fuciformis Berk edible mushroom in rats fed a high-cholesterol diet with or without Nebacitin", J. ANGRIC. FOOD CHEM., vol. 50, no. 25, 2002, pages 7438 - 7443 *
CHO E.J. ET AL.: "Production of exopolysaccharides by submerged mycelial culture of a mushroom Tremella fuciformis", J. BIOTECHNOL., vol. 127, no. 1, 2006, pages 129 - 140, XP005754685 *
KIHO T. ET AL.: "Polysaccharides in fungi. XXXIII. Hypoglycemic activity of anacidic polysaccharide (AC) from Tremella fuciformis", YAKUGAKU ZASSHI, vol. 114, no. 5, 1994, pages 308 - 315 *
PETER C.K. ET AL.: "The hypocholesterolemic effect of two edible mushrooms: Auricularia auricula (Tre-ear) and Tremella fuciformis (White jelly-leaf) in hypercholesterolemic rats", NUTR. RES., vol. 16, no. 10, 1996, pages 1721 - 1725 *

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