KR100780664B1 - Biopolymer having hypolipidemic effect extracted from the fruiting bodies of auricularia auricula-judae and method for preparing the same - Google Patents

Abstract

A biopolymer derived from fruit bodies of Auricularia auricula-judae is provided to show excellent hyperlipidemia treating effect, decrease the total cholesterol, triglyceride, atherogenic index and low density lipoprotein(LDL) cholesterol in plasma and maintain the concentration of high density lipoprotein(HDL) cholesterol high, thereby excellently preventing atherosclerosis. A method for preparing a biopolymer derived from fruit bodies of Auricularia auricula-judae comprises the steps of: (a) after finely crushing the fruit bodies of Auricularia auricula-judae and homogenizing them, hot water-extracting them at a temperature of 121 deg.C for 2 hours under high temperature and high pressure sterile state; (b) after centrifuging the hot water extract obtained from the step(a) under 10,4477x g/20minutes and precipitating a supernatant obtained therefrom in ethanol, centrifuging the precipitated supernatant; and (c) dissolving the precipitated material obtained from the step(b) in water and dialyzing it for 3 days, freeze-drying it to obtain the biopolymer. A composition for treating hyperlipidemia or preventing atherosclerosis comprises the biopolymer prepared by the method as an effective ingredient.

Description

Biopolymer having hypolipidemic effect extracted from the fruiting bodies of Auricularia auricula--judae and method for preparing the same}

1 is a graph showing the components of the polysaccharide derived from the fruiting body of Thirsty mushroom using Separators CL-6B chromatography.

Figure 2 is a graph showing the molecular weight of the polysaccharide derived from the fruiting body of the neck mushroom using gel chromatography.

The present invention is a fungus ( Auricularia) Auricula - judae ) relates to a polysaccharide (bipolymer) derived from the fruiting body, and more particularly, to a polysaccharide-derived polysaccharide derived from the fruiting body having a hypolipemia effect and a method for producing the same.

Edible mushrooms contain high fiber, protein, trace elements, and low fat, making them an ideal ingredient for dietary prevention against atherosclerosis. There have already been studies of the effects of some mushrooms on hypercholesterolemia, and it has been reported that horticulture mushrooms and ganoderma lucidum have lower serum cholesterol levels in hyperlipidemic mice.

Thirsty mushrooms are also called trash, and they are 2-6 cm in size. They usually grow together and form irregular masses. When dried, it contracts and becomes hard cartilage. When water is eaten, it becomes round again. The whole body is gelatinous, translucent and has the shape of a crooked, wavy ear. The upper side is purple brown with dense microscopic hairs, and the bottom side is flat, glossy and covered with fruiting layers. Damsel disease (擔子 柄) is conical, divided into four chambers (가로) with a diaphragm, and projections emerge from each thread, with one spore attached to the end. Spores are colorless kidneys. When spores form, they appear to have been sprayed with white powder on their surface.

These sour mushrooms are well known for edible and medicinal purposes, and have been shown to contain many biologically active ingredients that exhibit interesting physiological activities such as anticancer activity, anti-inflammatory effect, hypoglycemic effect and anticoagulant activity.

Conventionally, as a method for using edible mushrooms for edible and medicinal purposes, the method for adding and using the edible mushroom extract to tea, beverages or foods was mostly used.

In addition, Korean Patent No. 10-0481880 discloses a method for extracting the adhesion inhibitory component of Helicobacter pylori from the woody mushroom and its Helicobacter pylori attachment inhibitory component in extracting the adhesion inhibitory component of Helicobacter pylori from the wood mushroom In addition, Korean Patent No. 10-0336848 relating to an antithrombotic component from a thirsty mushroom as a therapeutic agent for hypertension discloses a method for extracting an antithrombogenic component from a thirsty mushroom and its antithrombotic extract. However, no method has been attempted to use the polysaccharide isolated from the fruiting bodies of the wood mushrooms for food and medicinal purposes.

Accordingly, it is an object of the present invention to provide a polysaccharide having a hypolipidemic effect obtained from the fruiting body of the wood mushroom and a method for producing the same.

The object of the present invention is to obtain a polysaccharide from the fruiting body of the wood mushrooms, and to raise the blood from rats after oral administration of the polysaccharide to hyperlipidemic rats to obtain blood from the total cholesterol (total cholesterol), triglycerides (total cholesterol), Investigate the hypolipidemic effect of polysaccharide derived from the fruiting bodies of the woody mushroom by investigating the content of HDL (high concentration lipoprotein) cholesterol, LDL (low concentration lipoprotein) cholesterol and atherosclerosis index. Gel filtration was performed on a Ceuzas CL-6B column saturated with 0.2 M NaCl, followed by identification of the constituents and molecular weight of the polysaccharide derived from the fungus fruiting body and by analyzing the amino acid and sugar components of the polysaccharide.

The present invention is finely pulverized the fruiting body of the wood mushroom, hot water extraction (high temperature and high pressure sterilization) and centrifuged by centrifugation of the supernatant obtained by ethanol precipitation, the precipitate obtained by dilution and dialysis with water and then lyophilized to obtain a polysaccharide. The present invention provides a method for producing a polysaccharide derived from a fruiting body of a sour mushroom.

In addition, the present invention provides a polysaccharide derived from the fruit mushroom body of the bark mushroom prepared by the above method.

In another aspect, the present invention provides a composition for treating hyperlipidemia, characterized in that it comprises a polysaccharide derived from the fruiting body of the neck mushroom prepared by the above method as an active ingredient.

In another aspect, the present invention provides a composition for preventing atherosclerosis, characterized in that it comprises a polysaccharide derived from the fruiting body of the neck mushroom prepared by the above method as an active ingredient.

The present invention comprises a separation step of the polysaccharide derived from the fruiting body of the wood mushroom, the step of investigating the hypolipidemic effect of the polysaccharide in the hyperlipidemic mouse, and the component analysis step of the polysaccharide.

The polysaccharide derived from the fruiting body of the woody mushroom in the present invention is centrifuged by centrifugation of the supernatant obtained by centrifugation for 20 minutes at 10,447xg after hot water extraction (2 hours high temperature and high pressure sterilization at 121 ℃), the precipitate obtained above It can be obtained by diluting and dialysis with water followed by lyophilization.

In order to investigate the hypolipidemic effect of the polysaccharide obtained from the fruiting bodies of the soybeans obtained by centrifugation, the polysaccharides were raised by oral administration to hyperlipidemic rats derived from a high-fat diet, and then blood was obtained from the rats. Triglyceride, HDL (high concentration lipoprotein) cholesterol, LDL (low concentration lipoprotein) cholesterol content and arteriosclerosis index were investigated to investigate the hypolipidemic effect of polysaccharide derived from the fruiting body of the fungus.

Furthermore, in order to determine the hypolipidemic activity of the polysaccharide derived from the fruiting body of the present invention, the gel filtration was performed on a Sepahedz CL-6B column saturated with 0.2 M NaCl, and then the constituents and molecular weight of the polysaccharide were determined and the amino acid of the polysaccharide. Analyze fructose components.

The pharmaceutical composition may be prepared by mixing a composition for treating hyperlipidemia, or a composition for preventing atherosclerosis, comprising a polysaccharide having hypolipidemic effect derived from the fruiting body of the present invention, and a pharmaceutically acceptable carrier.

The pharmaceutical composition of the present invention may further include conventionally used excipients, disintegrants, sweeteners, glidants, anti-esthetics, etc., unit dosage forms such as oral or parenteral formulations by conventional methods Or in several dosage forms. The composition may be parenterally or orally administered as desired, and the exogenous polymer derived from the tree mushroom fruiting body per kg of body weight per day may be administered with 0.05g to 0.5g, preferably 0.15g 1 to several times. Can be administered in divided doses. Dosage levels for a particular patient may vary depending on the particular compound to be used, weight, the age, sex, health condition, diet, time of administration, method of administration, rate of excretion, severity of the disease, and the like.

The data values shown in each example are shown in the table expressed as mean ± standard deviation. The mean of each group was compared by univariate analysis and Duncan's multiple-range test. Statistical difference considered p <0.05 as a significant value.

Hereinafter, the specific method of the present invention will be described in detail with reference to Examples, but the scope of the present invention is not limited only to these Examples.

<Example>

Example  1: Separation of Polysaccharides from the Fruiting Body of the Wood

The fruiting body of the wood mushroom is purchased from the conventional market, finely crushed and homogenized, and then centrifuged (10,447x g / 20 minutes) of hot water extraction (high temperature and high pressure sterilization at 121 ° C for 2 hours). After centrifugation, the supernatant was collected, treated with ethanol (supernatant: ethanol = 1: 4, 24 hours), and centrifuged again. The ethanol precipitate was dissolved in water and dialyzed for 3 days, and then lyophilized to obtain a polysaccharide.

Example  2: of the polysaccharide derived from the fruit mushroom Hypolipemia  effect

The polysaccharide derived from the thirsty mushroom fruiting body obtained in Example 1 was orally administered to the diet-induced hyperlipidemic rats, and the growth response and hypolipidemic effects of the rats were examined.

First, Sprague-Dawley rats (4 weeks old, males) obtained from Daehan Biolink Co., Ltd. were placed in stainless steel cages, respectively, and maintained at a constant temperature (22 ± 2 ° C) and humidity (55 ± 5%) and contrasted every 12 hours. Breeding in this controlled breeding room. AIN-76 modified diets were fed to mice for 6 weeks.

The feed consists of 55.5% carbohydrates per weight, 14% fat (30% of total energy), 20% protein, 5% fiber and 0.5% cholesterol, increasing fat content to increase hyperlipidemia in mice Induced. Feed and water were provided to breeding animals sufficiently.

After breeding for 2 weeks in the nursery, each rat group was orally administered saline (control) and intracellular polysaccharides at a concentration of 50-150 mg / kg body weight daily for 8 weeks at 8 am to 9 am.

Dietary intake and body weight were recorded on different days each day. After the last oral administration, the rats were fasted for 14 hours and lightly anesthetized between 8 am and 12 am with anesthesia to incise the abdomen to obtain blood from the aorta, and the blood sample was placed in a heparinized tube and 1,100 xg, 20 Plasma was separated by centrifugation for minutes. Cold saline was sparged in the liver, dissected and frozen at -70 ° C. The total lipids of the liver were extracted by treating the chloroform / methanol (2: 1, v / v) mixture.

Plasma total cholesterol, triglycerides, and HDL (high concentration lipoprotein) cholesterol concentrations were evaluated by enzyme test kit (Asan Pharmaceutical Co., Ltd., Korea). LDL (low concentration lipoprotein) cholesterol and arteriosclerosis index were calculated according to the following equation:

LDL cholesterol = total cholesterol-HDL cholesterol-(triglycerides / 5)

Atherosclerosis index = (total cholesterol-HDL cholesterol) / HDL cholesterol

Liver total cholesterol and triglycerides were analyzed according to the same method as the method for measuring total cholesterol and triglycerides in plasma after treatment with Triton X-100.

Effects on Growth Response of Polysaccharides Derived from the Fruit Mushrooms in Hyperlipidemic Whites Group (polysaccharide, mg / kg / day) Weight gain (g / day) Dietary Intake (g / day) Control group ^{1 )} 5.52 ± 0.35 20.19 ± 0.52 50 5.31 ± 0.27 20.14 ± 0.68 100 4.97 ± 0.24 20.30 ± 0.80 150 5.14 ± 0.11 19.45 ± 0.44 ¹⁾ was administered as saline instead of the polysaccharide derived from the thirsty fruiting body of the present invention as a control. Each value is expressed as mean ± standard deviation (n = 9).

As shown in Table 1, the present invention was administered to the mice at different doses of the polysaccharide derived from the fruit mushroom body, all rats of the experimental group were healthy and showed normal weight gain for 4 weeks. In the three groups, weight gain and dietary intake were not significantly different compared to the control group. In addition, oral administration of polysaccharides did not affect the overall behavior of rats and no animals died. Therefore, oral administration of the polysaccharide derived from the thirsty fruiting body did not have a detrimental effect on hyperlipidemic rats.

Effects of Polysaccharide from Thirsty Fruit Fruit Body on Total Cholesterol and LDL Cholesterol in Hyperlipidemic Rats Group (polysaccharide, mg / kg / day) Total cholesterol (mg / dl) LDL cholesterol (mg / dl) ²⁾ Control group ^{1 )} 108.20 ± 8.14 80.44 ± 7.23 50 89.97 ± 5.29 61.14 ± 3.73 100 93.81 ± 3.16 65.94 ± 4.32 150 86.19 ± 8.43 55.01 ± 9.07 ¹⁾ Oral saline solution ²⁾ Total cholesterol-HDL cholesterol? (Triglycerides / 5)

As shown in Table 2, the effects of the polysaccharide derived from the thirsty mushroom fruiting body on the total cholesterol and LDL cholesterol of plasma were significantly decreased in all experimental groups. It was.

It is generally known that LDL cholesterol in plasma is more involved in atherosclerosis than total cholesterol. In this example, the concentration of 150 mg of polysaccharide per kg body weight was lowered to 20.34% and 31.61%, respectively, of plasma and LDL cholesterol concentrations compared to the control.

The hypolipidemic effect of the polysaccharide derived from the fruiting body of the fungus is thought to be due to the adhesiveness of the polysaccharide. The hypolipidemic effect continued to increase with increasing polysaccharide. This is associated with an increase in viscosity with increasing dose of polysaccharide. Because of these features, it is possible to reduce triglyceride and cholesterol absorption by inhibiting the formation of colloidal particles in the small intestine and altering the physical characteristics of the intestinal mucosa of mice.

However, the structural effect of the polysaccharide derived from the fruit mushroom body may not be excluded in showing the hypolipidemic effect. Furthermore, like total cholesterol, LDL cholesterol continued to decrease with increasing daily doses of polysaccharides. Therefore, it can be argued that the main reason for the decrease in the total cholesterol concentration in plasma is that the polysaccharide lowers the concentration of LDL cholesterol. In addition, the significant reduction of LDL and total cholesterol in plasma may be due to the inhibition of HMG-CoA reductase in liver tissues, resulting in reduced cholesterol production and sufficient removal of LDL cholesterol without subsequent regeneration in various tissues. LDL values in plasma modify VLDL metabolism, including the conversion of VLDL (ultra low concentration lipoprotein) to LDL (low concentration lipoprotein). Basically, the initial VLDL is converted to mature VLDL and to LDL as triglycerides are lost by the action of LPL.

Effect of Polysaccharide from Thirsty Fruit Fruit Body on Plasma HDL Cholesterol and Atherosclerosis in Hyperlipidemic Rats Group (polysaccharide, mg / kg / day) HDL cholesterol (mg / dl) Triglycerides (mg / dl) Arteriosclerosis Index ^{2 )} Control group ^{1 )} 19.45 ± 1.14 41.57 ± 5.26 4.56 ± 0.56 50 21.97 ± 0.86 34.32 ± 3.05 3.10 ± 0.18 100 22.28 ± 2.18 27.96 ± 2.44 3.21 ± 0.40 150 25.09 ± 1.60 30.46 ± 2.84 2.44 ± 0.48 ¹⁾ Oral saline solution ²⁾ (Total cholesterol-HDL cholesterol) / HDL cholesterol

Table 3 shows the effects on the HDL cholesterol and atherosclerosis index of the polysaccharide derived from the fruiting bodies of the fungus. Compared with the control group, the polysaccharide increased plasma HDL cholesterol concentration to 29.00% and reduced arteriosclerosis index to 46.50% at 150 mg / kg body weight.

As an indicator of anti-atherogenic atherosclerosis, HDL cholesterol in plasma is involved in excess cholesterol delivery for liver regeneration. Some types of dietary fiber have been reported to increase plasma HDL cholesterol levels. Kim et al. Reported that the fruiting bodies of Lentinus edodes increased plasma HDL cholesterol levels in hyperlipidemic rats.

Therefore, the increase in HDL in this embodiment is probably due to the slow conversion of HDL to LDL or some other method.

In addition, the present invention may be useful for preventing atherosclerosis because the polysaccharide derived from the fruiting body of the present invention increases the concentration of HDL cholesterol and significantly reduces the atherosclerosis index in a concentration-dependent manner.

As shown in Table 3, the polysaccharides of 100 and 150 mg per kg body weight decreased the plasma triglyceride concentrations to 32.74% and 26.77%, respectively, compared to the control group.

Effects of Polysaccharides from Thirsty Fruit Fruit Body on Liver Weight, Total Cholesterol, and Triglycerides in Hyperlipidemic Rats Group (polysaccharide, mg / kg / day) Weight (g / 100g body weight) Total cholesterol (mg / g) Triglycerides (mg / g) Control ^{1 )} 4.89 ± 0.22 5.36 ± 0.15 ^NS 7.06 ± 0.44 ^NS 50 4.38 ± 0.20 4.89 ± 0.06 7.84 ± 0.52 100 4.62 ± 0.44 5.06 ± 0.44 6.87 ± 0.24 150 4.38 ± 0.31 4.75 ± 0.26 6.97 ± 0.48 ¹⁾ Saline oral administration ^NS : No significance

As shown in Table 5, the triglyceride concentration of the liver was not significantly different from the control group in the polysaccharide-administered group, but the concentration tended to decrease in the 100 and 150 mg experimental groups. At the concentration of 150 mg / kg body weight, the polysaccharide reduced the total cholesterol concentration to 11.38% in the liver compared to the control.

Hepatic cholesterol levels are associated with the activity and esterification of hepatic cholesterol synthase and the expression of apo B / E receptors, a major determinant of plasma LDL levels. That is, the accumulation of cholesterol in the liver can reduce the increase and accumulation of esterification, increase the secretion of cholesterol in the liver lipoproteins and reduce the absorption of plasma cholesterol by the LDL receptor.

Example  3: Component Analysis of Polysaccharides Derived from the Fruiting Body of the Present Invention

As described in Example 2, the present invention has a hypoglycemic effect of the polysaccharide derived from the fruiting body of the neck mushroom in dietary hyperlipidemic mice. Therefore, in order to find out the active source of the polysaccharide, its components including molecular weight were analyzed.

First, in order to investigate the molecular weight of the polysaccharide, gel filtration was carried out at a flow rate of 30 drops per tube in a column of the Cefollows CL-6B diluted with 0.2 M NaCl and saturated with 0.2 M NaCl. The molecular weight of the polysaccharide was measured by gel chromatography.

As standard, standard dextran (T2000, 500, 70, 40, 10) and Glucose were used for molecular weight determination.

In addition, the sugar and amino acid components of the polysaccharide derived from the fruiting body of the present invention was analyzed. The total equivalent of polysaccharide was measured according to the phenol sulfonic acid method using mannose and glucose mixture (1: 1) as a standard. The sugar composition was analyzed by GS 3600 gas chromatography (Variang) according to the hydrolysis and acetylation methods of Jones and Albersheim.

The total protein content of the polysaccharide was measured according to Lowry's method using bovine serum albumin as a standard. The amino acid composition was analyzed by hydrochromic 20 amino acid automatic analyzer (manufactured by Pharmacia Biotech) equipped with Na-type column after hydrolysis of the protein.

As shown in Figs. 1 and 2, the polysaccharide derived from the thirsty mushroom fruiting body showed only one peak in the Ceuzers CL-6B column chromatography, and the molecular weight was determined to be about 3200 kDa in the gel chromatography.

In this case, the vertical axis Kav of FIG. 2 means (Ve-Vo) / (Vt-Vo), Vo is an empty volume, Vt is a total volume, and Ve is an elution volume.

The total sugar and protein content of the polysaccharide were 83.94% and 16.06%, respectively. Detailed chemical analysis of the polysaccharide derived from the fruit tree fruit body is shown in Table 5.

Composition of Sugars and Amino Acids of Polysaccharides Produced from Fruiting Body of the Wood Mushroom Party Composition (%) ^{1 )} amino acid Composition (%) ^{2 )} Fucose 0.8 Aspartic acid 5.0 Ribose 0.2 Threonine 5.2 Arabinose 0.8 Glutamic acid 4.7 Xylose 10.6 Glycine 12.5 Mannose 58.2 Alanine 9.6 Galactose 0.2 Cysteine 5.1 Glucose 29.2 Valine 10.1 Isoleucine 32.2 Leucine 8.3 Phenylalanine 7.2 Total sugar content 83.94% Total protein content 16.06% ¹⁾ : ratio to total sugar ²⁾ : ratio to total amino acids

As shown in Table 5, 10 kinds of amino acids constitute protein moiety, and major amino acids include glycine (12.5%), valine (10.1%), and isoleucine (32.2%). Xylose (10.6%), mannose (58.2%) and glucose (29.2%) are the major carbohydrates of sugar moieties.

From the above results, the mannose galactose content in the total sugars of the polysaccharide derived from the fruiting body of the woody mushroom occupies almost 90%. The polysaccharide derived from the fruiting body of the wood mushroom is close to glucomannan.

The difference in the molar ratio between glucose and mannose affects the hypocholesterolemic effect of glucomannan in rats. .

As described above, the present invention, as described through the examples, the fruiting body-derived polysaccharide of the present invention the neck mushroom has an excellent effect on the treatment of hyperlipidemia, reduces the concentration of plasma total cholesterol, triglycerides, arteriosclerosis index and LDL cholesterol and HDL Since cholesterol has an excellent effect of preventing the atherosclerosis by maintaining a high concentration, it is a very useful invention in the pharmaceutical industry because it can provide a composition for treating and preventing hyperlipidemia using the polysaccharide as an active ingredient.

Claims (4)

After pulverizing and homogenizing the fruiting body of the wood mushroom, centrifuged at 10,447xg / 20 minutes for hot water extraction under high temperature and high pressure sterilization at 121 ° C. for 2 hours, the supernatant obtained was ethanol precipitated and centrifuged. A method for producing a polysaccharide derived from the fruiting body of a fungi, characterized in that the precipitate is dissolved in water, dialyzed for 3 days, and then lyophilized to obtain a polysaccharide. It comprises 83.94% total sugar and 16.06% protein prepared by the method of claim 1, the molecular weight is 3200kDa, the content ratio of mannose and glucose of the major constituents is 58.2% and 29.2%, respectively Polysaccharide Derived from Thirsty Fruit Fruiting Body with Hypoglycemic Activity A composition for treating hyperlipidemia, comprising the polysaccharide derived from the fruiting body of the soybean mushroom prepared by the method of claim 1 as an active ingredient A composition for preventing atherosclerosis, comprising the polysaccharide derived from the fruiting body of the soybean mushroom prepared by the method of claim 1 as an active ingredient

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