WO2008105583A1

WO2008105583A1 - Crude exopolysaccharides produced from tremella fuciformis mycellium having hypoglycemic activity and preparation method thereof

Info

Publication number: WO2008105583A1
Application number: PCT/KR2007/005920
Authority: WO
Inventors: Jong Won Yun; Eun Jae Cho; Song Hwan Bae
Original assignee: SEROMBIO Co Ltd
Current assignee: SEROMBIO Co Ltd
Priority date: 2007-02-28
Filing date: 2007-11-22
Publication date: 2008-09-04
Anticipated expiration: 2009-08-28
Also published as: KR20080079962A

Abstract

This invention relates to the crude exopolysaccharides produced from Tremella fuciformis mycellium having hypoglycemic activity and preparation method thereof. In case type II diabetic model mice (ob/ob) are, treated with the exopolysaccharides produced from Tremella fuciformis mycellium, the plasma glucose levels in them are substantially reduced. In addition, the exopolysaccharides of this invention has excellent curative value on hyperlipidemia by reducing serum lipid levels. Thus, the exopolysaccharides of this invention are able to control the plasma glucose levels of the diabetic patients and improve hyperlipidemia.

Description

CRUDE EXOPOLYSACCHARIDES PRODUCED FROM TREMELLA FUCIFORMIS MYCELLIUM HAVING HYPOGLYCEMIC ACTIVITY AND PREPARATION METHOD

THEREOF Technical Field

[1] 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.

[2]

Background Art

[3] 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. Even though 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. With thousand years of medical practice, a great deal of valuable experiences have been accumulated in the traditional Chinese medical system for diabetes therapy. To date, there are about 33 traditional Chinese medicines that are most frequently used in traditional Chinese prescriptions for the clinical treatment of diabetes and its complications. The ailti-diabetebioactive compounds in Chinese medicinal plants include polysaccharides, terpenoids, flavonoids, sterols and alkanoids. In many countries, much attention has been paid to discover natural antidiabetic medicines from various medicinal plants.

[4] In addition, mushrooms and enthomopathogenic fungi are also exemplary sources of natural medicines having antidiabetic activity. With little scientific evidence, 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. investigated the hypoglycemic activity of a water-soluble polysaccharide from the fruiting bodies of Auricularia auricula-judae Que. Nevertheless, for this purpose, little effort has been made to use exopolysaccharides obtained by mycelial culture of medicinal mushrooms.

[5] On the other hand, 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.

[6]

Disclosure of Invention Technical Problem

[7] Therefore, 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.

[8] The present invention provide a pharmaceutical composition for preventing or treating diabete mellitus comprising the EPS as an effective ingredient.

[9] Further, the present invention provide a pharmaceutical composition for preventing or treating hyperlipidemia comprising the EPS as an effective ingredient.

[10] Further, the present invention provide a method for preparating EPS from submerged mycelial culture of Tremella fuciformis mycelium.

[11] 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-γ gene, a crucial factor of hypoglycemism. Hereinafter, the present invention is described in detail. [12]

Technical Solution

[13] 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).

[14] 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.

[15] The 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 4⁰C, 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).

[16] To investigate the hypoglycemic effect of EPS (exopoly saccharide) produced from myclial culture broth of Phellinus baumii, EPSs were orally administrated to diabetic mice at the level of 200mg/kg body weight per day. At the last day of experiment, adipose tissues were sampled from the mouse and the entire RNA of them were conducted with molecular biology experiment.

[17] The present invention will be described in detail by referring to the following examples. However, it will be appreciated that those skilled in the art, on consideration of this disclosure, may make modifications and improvements within the spirit and scope of the present invention.

[18]

Brief Description of the Drawings

[19] 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.).

[20] 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).

[21] Figure 3 is a graph showing glucose tolerance test of the exopolysaccharides produced from Tremella fuciformis against the plasma glucose in Type II diabetic πήce(ob/ob).

[22] Figure 4 is a graph that has proved the effect of reducing serum adiponectin levels with molecular biology by detecting Adipocyte PPAR-γ mRNA expressions separated from type II diabetic mice (ob/ob).

[23]

Mode for the Invention

[24] Example 1: Production of exopolysaccharides from my dial culture broth of

Tremella fuciformis

[25] The Tremella fuciformis materials to produce EPS with hypoglycemic effect from

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 25⁰C for seven (7) days, and used throughout the experiment. The stock culture was maintained by monthly subculture and the slants were stored at 4⁰C.

[26] To prepare inoculum, Tremella fuciformis was initially grown on a PDA medium

(2.4% potato dextrose broth and 2% agar) in a petri dish, and then transferred to the seed culture medium by punching out a portion with 5 mm of diameter of the agar plate with a sterilized house-developed cutter. 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 28⁰C with shaking at 150 rpm for 3 days.

[27] The submerged culture of Tremella fuciformis to produce exopolysaccharides, (EPS) was operated in a 5L stirred-type fermentation tank under the following culture conditions: 20 g/L of glucose, 20 g/L of tryptone, 0.5 g/1 of potassium sulfate, 1 g/1 of dibasic potassium phosphate, 0.46 g/1 of monobasic potassium phosphate; 3O⁰C of culture temperature, 2 vvm of aeration rate; 200 rpm of agitation speed; 5.0 of initial pH; 3L of working volume.

[28] To extract EPS, the culture broths were centrifuged at 10,000 x g for 20 minutes, and then the resulting supernatant was filtered through a Whatman filter paper No. 2 (Whatman International Ltd., Maids tone, England).

[29] The resulting culture filtrate was mixed with four (4) times of volumes for absolute ethanol, stirred vigorously and left overnight at 4⁰C. 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 9O⁰C overnight to a constant weight.

[30] The ethanol precipitates of the crude EPS components were dissolved in 0.2 M NaCl buffer to a concentration of 10 g/L, and loaded onto a Sepharose CL-column (2.4 cmxlOO cm, Sigma Chemical Co., Louis, MO, USA). The column was eluted with the same buffer as above at a flow rate of 0.6 mL/min. The total carbohydrate content of the EPS produced from Tremella fuciformis was determined by phenol sulfuric acid methodusing glucose as the standard. Total protein was determined by the Lowry method with bovine serum albumin as the standard. The protein moiety in the EPS was monitored by absorbance at 280 nm, whilstthe carbohydrate moiety was monitored at 480 nm.

[31] When mycelial growth and EPS production were achieved in a 5 L stirred- type fermentation tank, the maximum mycelial concentration reached about 8.8 g/L on 5th day, whereas the maximum EPS production was achieved about 5 g/L (data not shown on the Figure 1).

[32] 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).

[33]

[34] Example 2: Investigation on Hypoglycemic effect of the EPS produced from

Tremella fuciformis mycelium

[35] The present inventors used a Type II diabetic mouse (ob/ob) model to investigate the hypoglycemic effect of EPS Tremella fuciformis earned from Example 1 above, 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.

[36] The male C57BL/6 J Ham SIc-ob/ob mice with weight of 25 ~ 35g purchased from

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+2⁰C 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.

[37] Since ob/ob mouse is a genetically modified mouse, it shows obese phenomenon after 7 weeks from birth.

[38] After measuring plasma glucose levels of the fasting mice at 7th week age, the mice with glucose content more than 300 mg/dl were included into diabetic group.

[39] All the animals 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.

[40] Based on the other experimental design (normal administration concentration:

100-300 mg/kg) of some researchers using natural substances to treat diabetes, 200 mg/kg of administration concentration had been selected for this experiment appropriately.

[41] Next, the body weight gain and food intake were periodically measured. Blood samples of the experimental animals were collected in tubes treated with 0.1 M ethylene diaminetetra-acetic acid (EDTA) as anticoagulant, and plasma was separated by centrifugation at 3,000 x g for 10 min. The total plasma glucose, cholesterol and triglyceride levels were measured using an enzymatic colorimetric assay (YD Diagnostics, Seoul, Korea).

[42]

[43] Example 3: Determination of gene expression on adipose tissues of ob/ob mouse

[44] The adipose tissues of ob/ob mouse 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 PPARγ had been investigated using RT-PCR technique.

[45] The results were analyzed for statistical significance by one way analysis of variance

(ANOVA) test using the Statistical Package of the Social Science (SPSS) program. All data were expressed as mean ± SE (P<0.05). Group means were considered to be significantly different at P<0.05, as determined by the technique of protective least- significant difference (LSD).

[46] Effect of Tremella fuciformis EPS on body weight gain and food intake in the Type

II ob/ob mice after treatments is indicated on Table 1.

[47] Table 1 [Table 1]

[Table ]

Effect of Tremella fiiciformis EPS on body weight gain and food intake in the Type II ob/ob mice after treatments for 52 days

[48] [49] It was founded that the body weight gain had not a big difference between control group and treated group but the food intake had significant difference between the said groups as shown on Table 1. The food intake of the control group was increased by 37% comparing to the on of the treated group. Moreover, the control group showed significantly low food efficiency ration than the treated group did. This restrained intake would be considered due to some specific substance of EPS that has not been revealed.

[50] Figure 2 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. However, the plasma glucose levels in the control group were continuously increased to finally reach 442 mg/L during the experiment period. On the other hand, 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.

[51] Figure 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.

[52] [53] Table 2 [Table 2]

[Table ]

Effect of Tremella fiiciformis EPS on plasma total cholesterol and triglyceride in the ob/ob mice after treatments for 52 days

[54] [55] As shown in Table 3, the plasma total cholesterol levels in the treated groups were were within the normal range during the experiment period. Although the plasma total triglyceride levels were significantly decreased in the orally treated mice with Tremella fuciformis EPS within a normal range, the control group recored higher values than the normal ones.

[56] Figure 4 shows a graph investigating PPARγ gene expression in the adipose tissues after separating them from the mice treated with Tremella fuciformis EPS and those of the control group. In case of TZD-series medicines for Type II diabetes, they cause blood glucose decrease phenomenon by acting on PPARγ of the adipose tissue as a ligand to activate PPARγ, and increasing sensitivity of insulin. As shown on the Figure 4, the Tremella fuciformis EPS has also increased the expression of PPARγ 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.

[57] Based on the results above, 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.

[59]

Industrial Applicability [60] As mentioned on the examples above, the present invention relates to crude exopoly- saccharides produced from Tremella fuciformis mycelium having hypoglycemic activity and preparation method thereof. When 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. Also, 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. [61]

Claims

[1] A composition for preventing or treating diabete mellitus comprising expolysac- charides(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.

[2] A pharmaceutical composition for preventing or treating diabete mellitus comprising the EPS of claim 1 as an effective ingredient.

[3] A composition for preventing or treating hyperlipidemia comprising expolysac- charides(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.

[4] A pharmaceutical composition for preventing or treating hyperlipidemia comprising the EPS of Claim 3 as an effective ingredient .

[5] A method for preparating EPS comprising, a) adding ethanol to submerged culture of Tremella fuciformis and incubating to obtain mycelia pellet from the culture; b) dissolving the pellet into NaCl buffer; c) chromatographic separating the EPS by loading the dissolved solution onto Sepharose CL-column.