KR100190756B1 - Novel strain of ganodrema lucidum, polysaccharides from it, and a liver protecting composition containing the polysaccharides - Google Patents

Abstract

The present invention is Ganoderma lucidum WK-003 (KCTC 0179BP), secreted therefrom, provides a polysaccharide, a production method thereof, and a composition for inhibiting hepatic fibrosis, which is excellent in protecting liver function and does not cause hepatotoxicity. do.

The polysaccharide according to the present invention can be used safely because it exhibits excellent hepatoprotective action and does not cause liver toxicity.

Description

Novel ganoderma lucidum mushroom, polysaccharide secreted therefrom, and liver fibrosis inhibiting composition containing same

The present invention relates to a novel ganoderma lucidum mushroom, polysaccharide secreted therefrom, and a liver hardening inhibiting composition containing the same as an active ingredient.

Liver fibrosis or cirrhosis is caused by excessive intake of alcohol, abuse of chemicals, viral hepatitis, cholestasis due to biliary abnormalities, etc. The treatment is not easy, and the death rate is high, causing social problems.

Liver cirrhosis is a histologically unbalanced metabolic process that produces and breaks down collagen, resulting in liver fibrosis resulting in excessive accumulation of connective tissue in liver tissue, accompanied by necrosis or inflammation (Popper, Leber Magen). Darm, 8, 65, 1978; Schuppan et al., Z. Gastro., 26, Suppl. 3, 28, 1988).

Drugs that inhibit hepatic fibrosis include penicillamine, 16, 16-dimethyl prostaglandin E2, biphenyl dimethyl dicarboxylic acid, colchicine, glucocorticoids, malotilate, gamma interferon, pentoxifylline, pyridine- 2,4-dicarboxylic-diethylamide, pyridine-2, 4-dicarboxylic-di (2-methoxyethyl) amide, etc. have been reported. It can be said that there is no treatment for liver cirrhosis (Boker et al., J. Hepato 1., 13, s35, 1991; Tamayo et al., Ibid , 3, 112, 1983; Jenkins et al., Ibid , 1, 489, 1985). Kershenobich et al., Ibid , 7, 1104, 1987; Svegliati et al., 18, 209A, 1993; Guzelian et a1., Gastro., 86, 897, 1984; Ala-Kokko et a1., J. Lab. Clin. Med., 113, 177, 1989).

On the other hand, Ganoderma lucidum , which has been known to have blood pressure control, antitussive expectoration, analgesic calm, hypoglycemic effect and anticancer activity, has been reported to show hepatoprotective action. Pathological damage and liver detoxification when acute toxicity is induced by carbon tetrachloride It has been reported to alleviate impairment of function and promote the regeneration of hepatocytes (Shakhakkan, Chinese Medicine Dictionary, Shanghai Science and Technology Press, Tokyo, 1985).

Korean Patent Publication No. 92-10763 discloses that the polysaccharide of Ganoderma lucidum mushroom (Garoderma lucidum IY009) KFCC 10709 has a function of inhibiting liver fibrosis.

The polysaccharide derived from Ganoderma lucidum IY009 (KFCC 10709) described in this document is a kind of protein polysaccharide whose composition contains beta-glucose, alpha-glucose, galactose, alpha-mannose, and fructose as sugar components. As a protein component, glycine, lysine, alanine, hastidine, arginine, varine, aspartic acid, threonine, isoleucine, serine, leucine, glutamic acid, tyrosine, proline, phenylalanine and methionine are contained. However, the polysaccharide derived from the Ganoderma lucidum mushroom KFCC 10709 has a problem of causing hepatotoxicity due to an increase in ALT (Alanine transaminase) levels in the normal rat group to which the polysaccharide was administered (Pak Eun-Jeon et al., 38, 338, 1994).

Accordingly, the present inventors who screened ganoderma lucidum producing polysaccharides to protect the liver and studied its components in depth, unlike the above Ganoderma lucidum mushrooms, produced a new Ganoderma lucidum strain that secretes polysaccharides that do not cause liver toxicity to the medium. It was isolated, confirmed the hepatoprotective action of the polysaccharide and did not cause liver toxicity by animal experiments and came to complete the present invention.

1A-1D to 3A-3D are morphological observations of Ganoderma lucidum promotion No. 1 (A), promotion No. 2 (B), IY-009 (C) and strain WK-003 (D) of the present invention, respectively. As a photograph, FIG. 1 shows the result observed from the side, FIG. 2 shows the result observed from the bottom, and FIG. 3 shows the result observed from the top.

4 is a gene homology test of Ganoderma lucidum promotion No. 1 (A), promotion No. 2 (B), IY-009 (C) and strain WK-003 (D) of the present invention using a RADP experiment through PCR. An electrophoretic gel picture showing one result.

That is, a first object of the present invention is to provide a new Ganoderma ludicum WK-003 (KCTC 0179 BP).

Another object of the present invention is to provide a new polysaccharide derived from Ganoderma lucidum that has excellent liver protection and does not exhibit liver toxicity.

Another object of the present invention is also secreted from Ganoderma ludicum WK-003 (KCTC 0179 BP), 0-2.5% fucose, 0-5% fructose, 18-80.2% glucose, mannose To provide a water-soluble polysaccharide consisting of 5-60% by weight, galactose 0-10.5% by weight, arabinose 0-15% by weight, rhamnose 0-20% by weight and xylose 0-10% by weight.

Another object of the present invention is characterized by culturing the Ganoderma lucidum WK-003 (KCTC 0179 BP) in a culture medium, and recovering the water-soluble polysaccharide from the medium, the water-soluble polysaccharide does not cause liver toxicity It is to provide a production method of.

It is still another object of the present invention to provide a liver fibrosis inhibiting composition comprising the water-soluble polysaccharide as an active ingredient in an amount effective to inhibit liver fibrosis.

Other objects, features, and applications of the present invention will become apparent to those skilled in the art by the following detailed description.

Hereinafter, the present invention will be described in more detail.

(1) Selection of Ganoderma lucidum

Ganoderma lucidum strain WK-003 (KCTC 0179 BP) according to the present invention is isolated from wild acids of Hoengseong-gun, Gangwon-do, and the present inventors extract polysaccharides from them and induce their hepatoprotective action and hepatotoxicity in several ganoderma lucidum mushrooms. Check for presence. As a result, the polysaccharides isolated from the mycelium of the protein polysaccharide and Ganoderma lucidum WK-003 (KCTC 0179 BP) isolated according to the prior art show hepatotoxicity, whereas the polysaccharides secreted by Ganoderma lucidum WK-003 (KCTC 0179 BP) are liver. The present inventors have found that the protective action is remarkably excellent but does not cause hepatotoxicity.

(2) production of secreted polysaccharides

In the present invention, ordinary polysaccharides containing mycelium of Ganoderma lucidum mushrooms are called mycelium polysaccharides, and polysaccharides secreted into the medium at the time of culture are called secretory polysaccharides.

The secreted polysaccharide according to the present invention can be produced as follows. The Ganoderma lucidum WK-003 (KCTC 0179 BP) was incubated in a medium for producing polysaccharides (Example 3) at pH 2-5, temperature 25-35 ° C. for 15-20 days, and the culture was centrifuged to remove the mycelium. Only the supernatant is recovered. Four times of ethanol was added thereto, and the precipitate obtained by precipitating at 5 ° C. for 24 hours was dialyzed for 48 hours. 40 mg of the precipitate was dissolved in 50 ml of Tris buffer solution (pH 7.9) added with 10 mM calcium chloride, pronase was added to react for 48 hours at 37 ° C, and dialyzed at 4 ° C for 3 days to remove protein. The polysaccharide thus separated was hydrolyzed at 121 ° C. for 1.5 hours in 2M trifluoroacetic acid and analyzed by gas chromatography. The composition of the monosaccharides is shown in Table 3 below.

(3) liver protection test

Exposure to experimental animals to carbon tetrachloride, ethanol, methotrexate, dimethylnitrosamine or deficiency of vitamin B ₁₂ results in experimental animals (called liver cirrhosis), mainly treated with carbon tetrachloride. In the present invention, rats treated with carbon tetrachloride were used to examine liver toxicity. Polysaccharides were administered to rats treated with carbon tetrachloride, and serum ALT (Alanine transaminase) and AST (Aspartate transaminase) were measured. The hepatoprotective effect of the polysaccharides according to the invention in carbon tetrachloride-derived cirrhosis animals was excellent.

However, when exposed to carbon tetrachloride, the mortality rate of experimental animals is high (30-60%), liver cirrhosis occurs 8-12 weeks later, and liver histological changes are observed in the clinical stages such as atrophy of liver and little increase in total collagen level. There are many differences from disease findings (Tamayo et al., J. Hepatol., 3, 112, 1983).

On the other hand, according to the method of ligation of the biliary tract to induce hepatic fibrosis, more than 90% of experimental animals induce cirrhosis in 4 weeks, and the amount of collagen is relatively increased, so that metabolic-regulation of connective tissue can be studied. At the same time, histological findings of the liver are reported to be similar to those of liver disease (Kountouras et al., Br. J. Exp. Path., 65, 305, 1984).

In the present invention, both the carbon tetrachloride induction method (Example 5) and the bile duct ligation method (Example 6) were adopted to measure the hepatoprotective effect of the polysaccharide of the present invention.

(4) animal experiment

In liver cirrhosis animals obtained by biliary ligating surgery, the drug group was orally administered 10-50 mg / kg / day in sonde by dissolving polysaccharides in saline for 4 weeks from the day of surgery. An equal amount of saline is administered orally. Four weeks later, rats are anesthetized, blood is drawn from the heart, and the liver is removed immediately to determine wet weight. Serum and liver tissues were stored at minus 20 ℃.

(5) liver fibrosis test

Determination of hydroxyproline, which makes up 10% of the collagen constituting the connective tissue of the liver, can predict the amount of collagen in the liver tissue (Schuppan, J. Hepatol., 13, s17, 1991), Jamal et al. hydroxyproline was quantified according to (al., Anal. Biochem., 112, 70, 1981). That is, the 5% liver homogenate homogenized with 6N hydrochloric acid was hydrolyzed at 110 ° C., and oxidized with chloramine-T. Color development was carried out with the Erlich reaction solution, and the absorbance at 558 nm was measured.

(6) Serum test

Serum levels of ALT, AST, ALP (alkaline phosphatase), and total bilirubin were measured.

As a result of these experiments, water-soluble polysaccharides derived from Ganoderma lucidum KCTC 0179BP significantly reduced hepatotoxicity in carbon tetrachloride-induced hepatotoxicity experiments, did not induce hepatotoxicity in normal rats, and was induced by biliary ligation Animals also had the effect of protecting the liver and simultaneously inhibiting fibrosis.

The present invention also provides a composition for the treatment and prevention of liver cirrhosis by liver fibrosis containing the water-soluble polysaccharide as an active ingredient, the effective amount of the water-soluble polysaccharide contained in the composition is the route of administration, formulation, purpose of use, patient It may be determined within a wide range depending on the extent of the disease and the like. In general, however, the water-soluble polysaccharide may be formulated to be administered at a concentration of 1-1000 mg / kg / day, preferably 10-100 mg / kg / day. Formulations for administering water-soluble polysaccharides within the above ranges may have a conventional form, for example, can be used in the form of pills, drinks, medicines or dietary supplements. They can be administered for the prevention and treatment of liver cirrhosis by liver fibrosis via oral or various parenteral routes of administration, and are suitable for the dosage form and are well known to those skilled in the art and can be easily selected by those skilled in the art. Excipients, carriers, diluents and the like.

The present invention will be described in detail through examples. However, the present invention is not limited to the embodiment.

(Example 1)

Several species of ganoderma lucidum were isolated from Yasan, Anheung-myeon, Hoengseong-gun, Gangwon-do, and secreted polysaccharides were separated from them according to the methods of Examples 3 and 4 below.

In order to measure the hepatoprotective effect on the polysaccharides isolated from various Ganoderma lucidum mushrooms, carbon tetrachloride was administered to experimental rats according to the method of Example 5 below to cause liver damage, and then these polysaccharides were added in amounts of 20 mg / kg / day, respectively. Administered. And hulls were collected from each experimental animal receiving polysaccharide, and the amount of AST and ALT was calculated by measuring the absorbance at 505 nm.

Table 1 shows the measurement results for nine kinds of Ganoderma lucidum mushrooms, which show significant AST and ALT reduction effects among several Ganoderma lucidum mushrooms.

Table 1

Note: Reliable in the interval a = p0.01, b = p0.05

From the above results, it was confirmed that Ganoderma lucidum exhibited a different hepatic therapeutic effect from strain to strain, which was due to the difference in the sugar composition of the secreted polysaccharides.

Ganoderma lucidum WK-003, the eighth most effective liver healing treatment among Ganoderma lucidum , was named WK-003 and deposited on July 2, 1995 at the Gene Bank in the Biotechnology Research Institute, Daedeok. I received the accession number of KCTC 0179BP.

(Example 2-1)

Morphological and genetic characteristics of Ganoderma lucidum WK-003 according to the present invention were compared with the conventional Ganoderma lucidum mushroom. That is, as a conventional mushroom strain Ganoderma lucidum Promotion No. 1 (presale from Rural Development Administration), Promotion No. 2 (presale from Rural Development Administration), and IY-009 (KFCC-10709), and according to the present invention Strain WK-003 (KCTC 0179BP) was incubated for 7 days at 30 ° C. in a potato-dextrose liquid medium, respectively, and 500 ml were transferred to a seed culture vessel for 10 days. Subsequently, photographs were taken after 60 days of incubation in a chamber at a temperature of 24-26 ° C. where 80-40% humidity was maintained. The results are shown in Figures 1-3.

1 to 3 it was confirmed that the strain according to the present invention is morphologically different from the conventional strain. On the other hand, the morphological differences of the fruiting bodies of these estates are summarized in Table 2.

[Table 2] Morphological comparison of the fruiting body of the strain according to the invention and the conventional strain

From the results of Table 2 above, it is determined that the strain of the present invention is a strain that is morphologically different from the conventional strain.

(Example 2-2)

To compare the gene homology of each strain of Example 2-1, RAPD (Random Amplified Polymorphic DNA marker) using three synthetic oligonucleotide primers (AP 8, AP 11, AP12) having a random sequence ) Experiments were carried out via polymerase chain reaction (PCR).

The sequence of the synthetic oligonucleotide primer is as follows:

AP 8: 5'-GGATTGTGCG-3 '

AP 11: 5'-CAGACCGTTC-3 '

AP 12: 5'-TGCTGACCTG-3 '

92 ng of genomic DNA extracted from the fruiting body was used as a template for PCR reaction, 0.4 micromoles of each of the three primers in 10 μl of the total reaction solution, and 4 mM of a mixture of dATP, dCTP, dGTP and dTTP were added, followed by Taq polymerase 2 Units were added. PCR reaction conditions were 1 cycle of denaturation at 94 ° C. for 1 minute, annealing at 37 ° C. for 1 minute, and polymerization reaction at 72 ° C. for 1.5 minutes to perform a total of 30 cycles, and 2% agarose gel electrophoresis was performed. .

The results are shown in FIG. In FIG. 4, AP 8, AP11, and AP12 represent types of primers, and lanes A, B, C, and D each represent promotion 1, promotion 2, IY-009, and WK-003 strain of the present invention. .

From the results of FIG. 4, all four strains show different sizes and numbers of amplified gene fragments, which have different gene sequences, and thus, may be distinct strains.

(Example 3)

Medium for polysaccharide production (glucose 0.9%, sugar 0.9%, galactose 0.1%, gyros 0.1%, yeast extract 0.05%, peptone 0.2%, potato glucose 0.2%, ammonium dihydrogen phosphate 0.05%, DL-serine 0.05%, phosphoric acid Kalihydrogen 0.1%, calcium chloride 0.06%, magnesium sulfate 0.2%, iron sulfate 0.002%, manganese sulfate 0.002%, thiamine 1 x 10 ^-4 g / l, biotin 1 x 10 ^-3 g / 1, pH 3.0) 30 The mycelium was removed by centrifugation of the Ganoderma lucidum strain WK-003 (KCTC 0179 BP) culture incubated for 18 days. The supernatant was collected, 4 times of ethanol was added, and the precipitate obtained by precipitating at 5 ° C. for 24 hours was dialyzed for 48 hours to obtain a secreted polysaccharide of 3.16 g / 1.

On the other hand, the mycelium was alkali treated to remove the residue, neutralized, and then precipitated with ethanol to obtain a mycelium polysaccharide.

(Example 4)

Sugars were quantified according to the method of Hyun-Kyung et al. (Korean Society for Industrial Microbiology, 22, p.360 (1994)). Total sugar was measured by the phenol-sulfuric acid method using glucose as a standard, chloroglucinol acetic acid method using arabinos as a standard, and protein by the Lowry method.

Twelve fractions were taken from the secreted polysaccharides of Example 3 and the sugars were analyzed. 5 mg of the polysaccharide precipitate obtained from each fraction was dissolved in 50 mM of 50 mM Tris buffer (pH 7.9) to which 10 mM calcium chloride was added, and 10 mg of pronase (Sigma) was added thereto, reacted at 37 ° C. for 48 hours, and heated for 5 minutes. Dialysis at 4 ° C. for 3 days, hydrolysis of the recovered polysaccharides at 121 ° C. for 1.5 hours in 2M trifluoroacetic acid, and treatment of neutral and acidic sugars with NaBH ₄ in the hydrolyzate were carried out by alditol and aldonic acid. After the reduction, the mixture was separated by passing through a Dowex-1 ^TM (anion exchange resin, acetic acid type) column, and reacted with acetic acid and sodium borate (Na ₂ B ₄ O ₇ ) to convert to an alditol acetate derivative.

The constituent sugars were identified using gas chromatography (GC 14A manufactured by Shimadzu Corporation), and the content of the constituent sugars was expressed in weight%. Gas chromatography conditions are as follows:

Dexter: FID

Column: Stainless Steel 0.2 × 200cm

Column material: 3% OV-255 uniport HP 100/200

Column temperature: 225 ° C, injector temperature: 250 ° C, decanter temperature: 250 ° C

Carrier gas; N ₂ (2kg / ㎠) + H ₂ (0.6kg / ㎠)

Integrator: Shimadzu C R6A Chromatography Pack

The analysis results are shown in Table 3.

Table 3 Sugar composition of the polysaccharide according to the present invention

From the above results, the water-soluble polysaccharide according to the present invention is 0-2.5% by weight of fucose, 0-5% by weight of fructose, 18-80.2% by weight of glucose, 5-60% by weight of mannose, 0-10.5% by weight of galactose, arabinose It was found to have a sugar composition of 0-15% by weight, 0-20% by weight of rhamnose and 0-10% by weight of xylose.

Comparative Example 1

Park, Eun-Jun et al. (38, 338, 1994) cultured ganoderma lucidum KFCC 10709 in the medium of Example 3, the solid obtained by centrifugation to remove the residue, neutralized and ethanol Precipitate was formed to obtain beta-1,3-linked glucan polysaccharide.

(Example 5)

Male rats (200-250 g body weight) (supplied from Osan three-breed experimental animals) were acclimated to the laboratory environment for one week, and were fed with sufficient feed and water.

Introduce the number of groups and experimental animals.

Group 1 (n = 6)

Group 2 (n = 4) The secreted polysaccharide (fraction 1) dose group of the present invention (10 mg / kg / day, p.o.)

Group 3 (n = 4) The secreted polysaccharide (fraction 1) dose group of the present invention (20 mg / kg / day, p.o.)

Group 4 (n = 4) The secreted polysaccharide (fraction 1) dose group of the present invention (50 mg / kg / day, p.o.)

Group 5 (n = 4) polysaccharide dosage group of Comparative Example (50 mg / kg / day, p.o.)

Group 6 (n = 12) Group causing liver damage by carbon tetrachloride

Group 7 (n = 11) group of the secreted polysaccharide (fraction 1) of the present invention after inducing liver disorder

(10 mg / kg / day, p.o.)

Group 8 (n = 11) group of the secreted polysaccharide (fraction 1) of the present invention after causing liver failure

(20 mg / kg / day, p.o.)

Group 9 (n = 12) secretion polysaccharide (fraction 1) administration group of the present invention after causing liver failure

(50 mg / kg / day, p.o.)

Group 10 (n = 10) polysaccharide dosage group of Comparative Example 1 after induction of liver failure (50 mg / kg / day, p.o.)

Group 11 (n = 10) mycelia polysaccharide administration group of Example 2 after the liver failure

(50 mg / kg / day, p.o.)

Groups 1 and 6 were orally administered the corresponding amount of distilled water, and the other group each sonde. On day 4, each drug was administered, and after 2 hours, 1-5 groups were mixed with corn oil (made by Dongyang Flow Co., Ltd.) and carbon tetrachloride was mixed in the same amount of sorghum oil to 0.35m1 / kg. Intraperitoneal injection. After 24 hours, rats were anesthetized, cardiac punctured, blood was collected, left at room temperature for at least 1 hour, centrifuged to obtain serum, and stored at minus 20 ° C.

0.5m1 of the substrate solution for measuring transaminase of Asan Pharmaceuticals was left to stand at 37 ° C. for 5 minutes to activate, mixed with 0.lml of serum, and maintained for AST for 60 minutes, ALT for 30 minutes, and 0.5 ml of color reagent. It was added and left at room temperature for 20 minutes. 5 m1 of 0.4 N caustic soda solution was added, and the mixture was left at room temperature for 10 minutes, and then absorbance was measured at 505 nm within 60 minutes. ALP (Tables 4 and 5) were measured for absorbance using an automatic analyzer (Gilford, Gilford Clinical Chemical Analyzer 400E) using a Shiva-Corning Git.

The results are shown in Table 4.

Table 4 Results of AST and ALT Analysis in Rats Treated with Carbon Tetrachloride

Reliable in the interval a = p0.01, b = p0.05

From the above results, it is confirmed that the secreted polysaccharides according to the present invention exhibit an excellent hepatoprotective effect against hepatic impairment caused by carbon tetrachloride, whereas the polysaccharides derived from the polysaccharides and mycelium of the comparative example induce hepatotoxicity.

(Example 6)

Male rats (200-250 g body weight) (supplied from Osan three-breed experimental animals) were acclimated to the laboratory environment for one week, and were fed with sufficient feed and water. The secreted polysaccharides were dissolved in physiological saline for 4 weeks from the day of surgery on liver cirrhosis animals who underwent biliary ligated surgery using Park Eun-jeon's method (Park Eun-jeon et al., 38, 338, 1994). 50mg / kg / day oral administration, anesthesia and laparotomy after the injection of 2ml physiological saline and the control group obtained by suture the same amount of physiological saline was orally administered. The number of experimental animals in each group was five.

Four weeks later, rats were anesthetized, blood was collected from the heart, and livers were taken immediately to determine wet weight. Serum and liver tissues were stored at minus 20 ℃.

Control in Group 12 Liver Cirrhosis Animals

20 mg / kg / day oral administration of secreted polysaccharides in group 13 liver cirrhosis animals

Control in Group 14 Control

20 mg / kg / day oral administration of secreted polysaccharide in group 15 control group

The control group showed weight loss for 1 week after surgery, but was in good health.

Groups 12 and 13 secreted dark yellow urine from 2 days after surgery, and jaundice was observed in ears and soles after 4 days. Body weight decreased for the first week. The initial weight loss observed in both the control and test groups is thought to be due to surgery.

Four weeks later, after measuring the weight, the liver was taken to measure the wet weight, and the liver wet weight value per 100 g of body weight was compared. The results are shown in Table 5.

Table 5

significance of a = p0.01, b = p0.05

In the results of Table 5, the weight of the liver was increased in the experimental animal group that induced cirrhosis, and when the secreted polysaccharide of the present invention was treated to the cirrhosis animal, the wet weight of the liver was slightly decreased, but there was little significance.

(Comparative Example 2)

In the same manner as in Example 6, the polysaccharide of Ganoderma lucidum KFCC 10709 obtained in Comparative Example 1 was orally administered. Prepared as in the experimental group set in Example 6, named as the ratio 12-15 group, the dose was 5mg / rat. This corresponds to about 20 mg / kg. After 4 weeks, liver wet weight per 100 g of body weight was compared.

The results are shown in Table 6, and the trend was consistent with Example 6.

TABLE 6

(Example 7)

0.3 g of liver tissue collected in Example 6 and Comparative Example 2 was homogenized with 6 ml of 6N hydrochloric acid, hydrolyzed at l10 ° C and filtered. 50 u1 of the filtrate was taken, evaporated to dryness at 80 ° C. under reduced pressure, dissolved in 1.2 m1 of a 50% isopropanol solution, and oxidized to 200 ul of chloramine-T (manufactured by Sigma). Hydroxyproline was quantified by color development with 0.1 ml of Erlich reaction solution and absorbance at 558 nm. The results are listed in Table 5 (secretory polysaccharide of Example 6) and Table 6 (polysaccharide of Comparative Example 2).

From these results, the amount of hydroxyproline in the experimental animal that caused cirrhosis was reduced by 28% by the secreted polysaccharide of the present invention and by 17% in the comparative example when each polysaccharide was administered in an amount of 20 mg to inhibit liver fibrosis. It was confirmed to let. On the other hand, in the control experimental animal group, the secreted polysaccharide of the present invention was observed to have a slight increase or decrease in the amount of hydroxyproline before or after the administration and was found to have little effect.

(Example 8)

Serum ALT, AST, ALP, and total bilirubin amounts measured in Example 6 and Comparative Example 2 were measured. ALT and AST were measured using a measuring kit of Asan Pharmaceutical as in Example 3, and ALP and total bilirubin were measured using a measuring kit of Ciba-Corning Corporation. The results are listed in Tables 5 and 6.

From the results of Table 5 and Table 6, it can be seen that the polysaccharide of the present invention does not exhibit hepatotoxicity in the control group, and decreases ALT, ALP, and AST for cirrhosis animals.

In the case of administration of the polysaccharide of Comparative Example 1, ALT and ALP were significantly increased compared to the non-dosed group, so that the mycelium extract polysaccharide was reconfirmed with the results of Table 4 of Example 5 showing that hepatotoxicity was observed in the experimental group. there was.

As described above, the secreted polysaccharide derived from Ganoderma lucidum KCTC 0179BP can be used as an agent for treating and preventing liver cirrhosis by protecting the liver and inhibiting liver fibrosis.

Example 9 Acute Toxicity Test

In the experimental group 4 group consisting of 5 mice per group for the mice were administered the polysaccharide of Example 3 of various doses through the oral route and the maximum allowable dose was measured, the results are shown in Table 7 below from the results of Table 7, It can be seen that the maximum tolerated dose at the time of oral administration is 5000 mg / kg or more, so that the biosafety composition of the present invention can be safely administered to the living body.

TABLE 7

According to the present invention, unlike the conventional ganoderma lucidum mushroom, a new ganoderma lucidum strain that secretes polysaccharides that do not cause liver toxicity into the medium, a water-soluble polysaccharide secreted therefrom, and a pharmaceutical agent having a hepatoprotective effect containing the polysaccharide as an active ingredient A composition is provided. The polysaccharides according to the invention can be used advantageously since they do not cause liver toxicity.

Claims (7)

Ganoderma lucidum (Knodec lucidum ) having polysaccharide production ability with liver protection WK-003 (KCTC 0179BP) According to claim 1, wherein the polysaccharide is 0-2.5% by weight of fucose, 0-5% by weight fructose, 18-80.2% by weight glucose, 5-60% by weight mannose, 0-10.5% by weight galactose, arabinose 0- Ganoderma lucidum WK-003 (KCTC 0179BP), characterized in that it has a sugar composition of 15% by weight, 0-20% by weight of rhamnose and 0-10% by weight of xylose. Ganoderma lucidum secreted from Ganoderma lucidum WK-003 (KCTC 0179BP), 0-2.5% by weight of fucose, 0-5% by weight of fructose, 18-80.2% by weight of glucose, mannose 5-60% by weight, galactose 0- A polysaccharide having a sugar composition of 10.5% by weight, 0-15% by weight of arabinose, 0-20% by weight of rhamnose and 0-10% by weight of xylose, and exhibits hepatoprotective action. According to claim 3, wherein 0-2.5% by weight of fucose, 0-4.5% by weight of fructose, 35-70% by weight of glucose, 0-20% by weight of mannose, 3-50% by weight of galactose, 0-3% by weight of arabinose. , A polysaccharide having a sugar composition of 0-5% by weight of rhamnose and 3-10% by weight of xylose. Culturing Ganoderma lucidum WK-003 (KCTC 0179BP) in a culture medium to accumulate polysaccharides in the medium; Removing the Ganoderma lucidum mycelium from the culture and recovering the supernatant: Separating polysaccharide from the culture supernatant Method for producing a polysaccharide having a liver fibrosis inhibiting ability, characterized in that it comprises a. The method of claim 5, wherein the culturing is conducted at a pH of about 2-5. A composition for inhibiting liver fibrosis, comprising the polysaccharide of claim 3 in an amount effective to inhibit liver fibrosis.