KR0178097B1 - Composition for the treatment of liver diseases containing proteoglycan goo9 - Google Patents

Abstract

The present invention relates to a liver disease therapeutic composition. More specifically, the present invention relates to a liver disease therapeutic composition containing protein polysaccharide G009 extracted from Ganoderma lucidum strain. Protein polysaccharide G009, an active ingredient of the composition according to the present invention, has excellent hepatoprotective effect, lipid peroxidation inhibitory effect, hepatic fibrosis inhibitory effect, and DNA synthesis promoting effect, which can be usefully used for the treatment of liver disease, and has little toxicity and can be safely used. It can be a drug.

Description

Liver disease therapeutic composition containing protein polysaccharide G009

Figure 1 shows the I.R. Analysis diagram.

2 is a graph showing the effect of G009 on the blood concentration of ethanol administered orally to rats [-○-control,-●-experimental group].

3 is a graph showing the effect of G009 on ethanol-induced hepatic triglyceride accumulation [N: normal, C: control, T: experimental group].

4 is a graph showing the effect of G009 on serum ALT in rats induced by acute hepatitis by CCl ₄ [N: normal group, C: control group, G2.5: G009: 2.5 mg / kg administration test Group 1, G10: Test administered with G009 10 mg / kg Group 2, G25: Test administered with G009 25 mg / kg, Group 3].

FIG. 5 is a graph showing the effect of G009 on serum AST in rats induced by acute hepatitis by CCl ₄ [N: normal, C: control, G2.5: G009: 2.5 mg / kg Group 1, G10: Test administered with G009 10 mg / kg Group 2, G25: Test administered with G009 25 mg / kg, Group 3].

FIG. 6 is a photograph showing the effect of G009 on liver tissue damage induced by CCl ₄ [200 magnification, FIG. 6 (a): normal group, FIG. 6 (b): control group, FIG. 6 (c) : G009 25 mg / kg test group 3].

FIG. 7 is a graph showing the effect of G009 on serum ALT in rats induced by acute hepatitis by thioacetamide [N: Normal, C: Control, G10: G009: 10 mg / kg Group 1, G25: test group 2 administered with G009 25 mg / kg, G100: test group 3 administered with G009 100 mg / kg].

FIG. 8 is a graph showing the effect of G009 on serum AST in rats induced with acute hepatitis by thioacetamide [N: normal, C: control, G10: G009: 10 mg / kg Group 1, G25: test group 2 administered with G009 25 mg / kg, G100: test group 3 administered with G009 100 mg / kg].

FIG. 9 is a photograph showing the effect of G009 on hepatic damage of acute hepatitis caused by thioacetamide [200 magnification, FIG. 9 (a): normal group, FIG. 9 (b): control group, 9 (c ): Test group 3 administered G009 100 mg / kg].

10 is a graph showing the concentration of serum PNIIIP in the simulated surgery control group and the experimental group, the BDL control group and the experimental group [■: control group, ▨: experimental group].

FIG. 11 is an optical micrograph of liver tissues of BDL control group and experimental group 4 weeks after BDL surgery (FIG. 11 (a): control group, FIG. 11 (b): experimental group).

The present invention relates to a liver disease therapeutic composition. More specifically, the present invention relates to a liver disease therapeutic composition containing protein polysaccharide G009 extracted from Ganoderma lucidum strain.

Protein polysaccharide G009 used as an active ingredient in the present invention is a substance isolated from Ganoderma lucidom, Ganoderma lucidom, Ganoderma lucidum strain, in particular, Ganoderma lucidum IY009 (KFCC-10709), and has an anticancer effect by an immune enhancing effect. It is known to indicate (Ref. Korean Patent Publication No. 92-10673 (1992.12.17)]. In addition to this, protein polysaccharide G009 has been reported to have various effects such as anti-inflammatory and anti-viral.

As a result of the intensive study on the protein polysaccharide G009 of Ganoderma lucidum, the present inventors have surprisingly shown that the protein polysaccharide G009 shows hepatoprotective action, lipid peroxidation inhibition, hepatic fibrosis inhibitory activity, and DNA synthesis promoting action in the treatment of liver disease. It was confirmed to exhibit a very excellent effect.

Accordingly, the present invention relates to a liver disease therapeutic composition containing protein polysaccharide G009 isolated from Ganoderma lucidum strain.

The protein polysaccharide G009 of Ganoderma lucidum used as an active ingredient in the composition of the present invention can be obtained from Ganoderma lucidum IY009 (KFCC-10709), which is a type of Ganoderma lucidum strain according to the method described in Korean Patent Publication No. 92-10763. Can be. In other words, the protein polysaccharide G009 was incubated with Ganoderma lucidum IY009 in Ganoderma strain, the mycelium was separated and infiltrated with sodium hydroxide solution, the supernatant was separated, neutralized with glacial acetic acid, and centrifuged. After running, the precipitate is obtained by dissolving in deionized water and lyophilizing.

Thus isolated protein polysaccharide G009 exhibits an excellent hepatoprotective effect, lipid peroxide inhibitory effect, hepatic fibrosis inhibitory effect and DNA synthesis promoting effect as demonstrated by the following experimental examples can be useful in the treatment of liver disease.

On the other hand, the protein polysaccharide G009 used as an active ingredient in the composition of the present invention, as already disclosed in Korean Patent Publication No. 92-10763, can be administered very safely regardless of the route of administration in acute toxicity experiments in mice, rats and rabbits. It has been proven to be an ingredient.

When clinically administered for the treatment of liver disease, the compositions of the present invention can be formulated into unit dosage forms suitable for oral or parenteral administration using conventional pharmaceutically acceptable carriers. . Suitable pharmaceutical formulations include tablets for oral administration, hard or soft capsules, granules, solutions or suspensions, and solutions for injection, suspensions for parenteral administration. Pharmaceutically acceptable carriers that can be used for the preparation of such formulations include, but are not limited to, excipients, binders, disintegrants, lubricants, solubilizers, suspending agents, preservatives, weighting agents, and the like in the case of oral preparations. Occasions include stabilizers, preservatives, dissolution aids, buffers, isotonic agents and the like.

The effective dose of the protein polysaccharide G009 for the treatment of liver disease may vary depending on the age, sex and health of the patient being treated and the severity of liver disease, but in general, 2 to 30 mg per kilogram of adult weight. Preferably, 5-10 mg is administered. Therefore, when preparing the composition of the present invention in unit dosage form, each unit dosage form may be formulated to contain 30 to 300 mg of G009, preferably 50 to 150 mg in consideration of the effective dose range of G009. . The unit dosage form so formulated may be administered to an adult once to several times a day, preferably one to six times.

The present invention is explained in more detail by the following examples and experimental examples, but the present invention is not limited in any way by these.

Example 1

[Isolation of Protein Polysaccharide G009]

1) strain

Ganoderma lucidum for the isolation of protein polysaccharide G009 was collected and identified in the area of Duryun-gun, Haenam-gun, Jeollanam-do, and identified.The strain was deposited with KFCC-10709 to the Korean spawn association on October 5, 1990. An undergrowth (Ganoderma lucidom) IY009 strain was used.

2) badge

Storage medium: Potato dextrose agar (Potato dextrose agar: PDA) Slope medium-Potato dextrose (Difco, USA) is dissolved in distilled water to make 1 liter and sterilized at 121 ℃ for 20 minutes under high pressure steam. Made and used.

Incubation medium: glucose 50g, peptone 20g, KH ₂ PO ₄ 0.87g, MgSO ₄ · 7H ₂ O 0.5g, CaCl ₂ 0.3g, FeSO ₄ · 7H ₂ O 10mg, MnCl ₂ · 4H ₂ O 7mg, Distilled water was added to 1 mg of ZnSO ₄ · 7H ₂ O and 1 mg of CuSO ₄ · 5H ₂ O to adjust the pH to 4.5, and was used by autoclaving at 121 ° C. for 20 minutes.

3) Culture

After culturing Ganoderma lucidum IY009 (KFCC-10709) strain on PDA slope medium and incubating for 7 days at 28 ± 1 ° C, the grown mycelia were aseptically separated for 15 seconds with 100 ml of incubation medium. While grinding with a microblender. This was transferred to a 500 ml Erlenmeyer flask and shaken at 28 ± 1 ° C. for 180 rpm for 6 days. The cultured mycelium was pulverized with a microblender for 10 seconds, and then inoculated by 500% containing 100 ml of culture medium with 10% (v / v) of an Erlenmeyer flask and shaken for 7 days under the same conditions. Incubated. The culture medium was used as a seed and inoculated at a concentration of 10% (v / v) in a 5 liter jar fermemter containing 3 liters of incubation medium and incubated at 28 ± 1 ° C. for one week.

4) Extraction and Separation of Protein Polysaccharides

After adding 2 times 2.0N NaOH solution to the incubated medium, it was left at room temperature for 24 hours, neutralized with pH 7.0 with glacial acetic acid, and then centrifuged to obtain a supernatant, and the supernatant was filtered through ultrafiltration system (Satorious). Co. molecular size 10,000) was used to concentrate up to 1/10 of the volume, followed by re-concentration with twice the addition of water. The concentrate was dried in a lyophilizer to give the desired protein polysaccharide G009. The obtained protein polysaccharide G009 shows an IR spectrum as shown in FIG. Using the thus obtained protein polysaccharide G009, the experiment of the following experimental example was carried out.

Experimental Example 1

[Inhibition of Lipid Peroxidation of Protein Polysaccharide G009]

In the following experiment, the lipid peroxidation inhibitory activity of the protein polysaccharide G009 which is the active ingredient of the composition according to the present invention was measured.

Superoxide radical present in the living body (O ₂ ^-), hydrogen peroxide (H ₂ O _2), hydroxyl radical (HO ^-) partial reduction active oxygen and singlet oxygen, such as ⁽¹ O _2), lipid peroxide radical (LOO ^-, LO ^- active oxygen, etc.) are known to represent a toxic cause lipid peroxidation in the case acts as the main concessions such as cancer, tissue damage, aging in vivo, they are many. In other words, these free radicals are believed to peroxidate the liver cell membrane, a detoxifying organ in vivo, and eventually cause liver disease. Therefore, in the following experiment, to determine the extent to which the protein polysaccharide G009 of the present invention can inhibit enzymatic or non-enzymatic lipid peroxidation induced by using microsomal membrane, to prove the usefulness of the protein polysaccharide G009 as a therapeutic agent for liver disease do.

1. Preparation of Microsomes

To separate microsomes, rats fasted for 24 hours (250 g body weight) were anesthetized using pentobarbital (40 mg / kg), and then opened, and 50 ml of Tris-Cl buffer (pH 7.4) was added with a 50 ml syringe. Perfusion was performed by injection from the portal vein to the inferior vena cava. The livers were then extracted, washed with 50 nM Tris-Cl / 150 nM KCI buffer, chopped and homogenized under ice cooling using polytrone. The homogenate thus obtained was centrifuged at 8000xg for 20 minutes to obtain a supernatant, and after ultracentrifugation at 105,000xg for 60 minutes while maintaining 4 ° C, the precipitated microsomes were subjected to 74.5mM Tris-Cl / 154mM KCI. Suspended in buffer (pH 8.0), the protein concentration was adjusted to 20 mg / ㎖ and used at the following experiments while stored at -20 ℃.

2. Induction of Lipid Peroxidation:

Induction of lipid peroxidation using microsomes was performed using two different systems.

1) Induction of Lipid Peroxidation by Ascorbate / Fe ²⁺

83.5 mM KCI, 37.2 mM Tris-Cl buffer (pH 8.0), 0.2 mM ascorbate, 10 μM FeSO ₄ .6H ₂ O), microsomal suspension (2 mg as protein) and a sample volume were added to the final 1 mL mixture. It was added to the test tube to react for 20 minutes at 37 ℃. Here, the test group 1 was added to the protein polysaccharide G009 of the present invention at a concentration of 0.5 and 1.0 mg / ㎖, and the test 2 group was a known antioxidant BHT (butylated hydoxytoluene) at a concentration of 0.5 and 1.0 mg / ㎖ In the test 3 group, vitamin E was added at a concentration of 0.5 and 10 mg / ml, and in the test 4 group, glycyrrhizin was added at a concentration of 1.0 mg / ml. Ascorbate, FeSO ₄ .6H ₂ O and a sample were not added to the normal group, and the reaction was performed without adding only the sample to the control group.

2) Induction of Lipid Peroxidation by CCl ₄ / NADPH

Add 83.5 mM KCI, 37.2 mM Tris-Cl buffer (pH8.0), 10 mM CCl ₄ , 2 nM NADPH, microsome suspension (2 mg as protein) and a sample volume to add 1 ml of the mixture to the test tube at 37 ° C. The reaction was carried out for 20 minutes. Here, test group 1 was added the protein polysaccharide G009 of the present invention at a concentration of 0.4 and 1.0 mg / ml, and test group 2 was added a known antioxidant BHT (butylated hydoxytoluene) at a concentration of 1.0 mg / ml. In the test 3 group, vitamin E was added at a concentration of 1.0 mg / ml, and in the test 4 group, glycyrrhizin was added at a concentration of 1.0 mg / ml. CCl ₄ / NADPH and the sample was not added to the normal group, the reaction was performed without adding only the sample to the control group.

3. Determination of lipid peroxide

After the reaction was completed, the degree of lipid peroxidation was measured by Okawa et al. [Hiroshi Ohkawa, Nobuko Ohishi and Kunio Yagi: Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction, Analytical Biochemistry 95, 351-358 (1979). ] And the specific method is as follows: 0.2 ml of 8.1% sodium dodecyl sulfate (SDS) in each test tube. 1.5 ml of 20% Acetgan solution containing 0.27M HCl and 1.5 ml of 0.8% TBA (thiobarbituric acid) were added and NaOH was added to adjust the pH to 3.5. The total reaction volume was adjusted to 5 ml. Thereafter, the mixture was developed at 95 DEG C for 60 minutes and cooled using running water. Then, 5 ml of a mixture of n-BuON-pyridine (15: 1, v / v) was added thereto, followed by vigorous shaking and centrifugation at 3800 rpm for 10 minutes. The absorbing stone was measured at OD 532 nm. A calibration curve was prepared using 1,1,3,3-tetramethoxypropane as a standard to measure the amount of malondialdehyde (MDA) in the sample. Inhibition rate of lipid peroxidation was calculated from the amount of MDA measured according to the following equation.

1) Effect of G009 on Lipid Peroxidation Induced by Ascorbate / Fe ²⁺

The effects of G009 and various samples on the production of lipid peroxide (MDA) induced by ascorbate / Fe ²⁺ from rat microsomes were investigated and the results are shown in Table 1 below.

From the results shown in Table 1, it can be seen that the protein polysaccharide G009 of the present invention exhibits a very high lipid peroxidation inhibitory effect. That is, the control group showed an MDA value of about 34.4 nM / ml, and when comparing the inhibition rate of lipid peroxidation, G009, BHT and vitamin E were 91.6% and 99.7%, respectively, when 0.5 mg / ml of the sample was added. And a high inhibition rate of 90.9%. From these results, it can be seen that the protein polysaccharide G009 of the present invention exhibits a lipid peroxidation inhibitory effect similar to that of BHT or vitamin E, which is known as a powerful antioxidant. In addition, when the sample was added at a high dose of 1.0 mg / ml, most of the samples showed over 97% inhibition of peroxide formation, and G009 exhibited 97.7% peroxide inhibitory effect, similar to vitamin E, a powerful antioxidant. Indicated.

2) Effect of G009 on CCl / NADPH-induced Lipid Peroxidation

The inhibitory effect of G009 on lipid peroxidation induced by CCl / NADPH is shown in Table 2 below.

As can be seen from the results described in Table 2, G009 showed lipid peroxide formation inhibition rates of 11.2% and 40.0%, respectively, at concentrations of 0.4 mg / ml and 1.0 mg / ml for lipid peroxidation induced by CCl / NADPH. BHT, vitamin E and glycyrrhizine showed inhibition rates of lipid peroxide formation of 54.1%, 35.1% and 12.2%, respectively, at concentrations of 1.0 mg / ml. Therefore, G009, the protein polysaccharide according to the present invention, shows a nearly equivalent lipid peroxide formation inhibitory effect when compared to BHT, vitamin E, and glycyrrazine, which are known to have strong antioxidant properties, and thus are clearly useful as a therapeutic agent for liver disease. It can be seen.

Experimental Example 2

[Liver protection action of G009]

In the experiments below, alcoholic fatty liver induced by ethanol administration and two acute hepatitis models derived from CCl and thioacetamide were used to confirm the hepatoprotective effect of G009, a protein polysaccharide according to the present invention.

A. Effect of G009 on Acute Fatty Liver by Ethanol

Thirteen Sprague-Dawley rats with a body weight of about 200 g were divided into three groups: five normal groups without any treatment, four control groups administered only with ethanol, and four test groups administered with G009 with ethanol. . In the test group, G009 was dissolved in deionized water and orally administered once daily at an amount of 25 mg / kg body weight for 4 days, and then fasted the day before the start of the experiment, and on the fifth day, G009 was orally administered at an amount of 25 mg / kg body weight. Two hours after administration, ethanol was orally administered at a 3 g / kg dose. In the control group, only ethanol was orally administered in an amount of 3 g / kg. Blood was taken from the tail vein at 2, 4, 6, 8 and 10 hours after administration of ethanol and the concentration of ethanol was measured according to the method as specifically mentioned below. In addition, 24 hours after the administration of ethanol, each group of animals was weighed, anesthetized with ether, collected by heart punture, left at room temperature for at least 1 hour, and centrifuged at 12,000xg for 2 minutes. And refrigerated and used within three days to measure the activity of serum asparatate transaminase (AST) and serum alanine transaminase (ALT) according to the method as detailed below. In addition, the liver was extracted, washed with 67 nM phosphate buffer (pH 7.0), weighed, and stored at -20 ° C. and then used for the determination of the content of liver triglycerides. Each measuring method described above is described in detail below.

1) Determination of Ethanol Concentration

Ethanol concentration was measured using the method of Bonichnsen and Theorell. Specifically, 800 μl of 0.3 N perchloric acid was added to blood μl taken from the tail vein of the rat, followed by centrifugation at 12,000 × g for 45 seconds. 50 µl of the centrifuged supernatant was taken and added to a test tube containing 2.4 ml of pH 8.7 buffer (75 mM NaPO-lOHO, 75 mM semicarbazide-HCl, 21 mM glycine). 50 µl of 0.33 N perchloric acid was added instead of the supernatant solution for the blank test. 50 μl of 25 mM NAD + and 10 μl of 15 kU / μl ADH were added thereto, followed by incubation at 25 ° C. for 70 minutes, followed by a test tube for blank test using a UV-VIS spectrophorometer (Schimadzu, Japan). The ethanol concentration was measured by measuring the absorbance at 340 nm.

2) AST and ALT activity measurement

AST and ALT activity were measured using a kit of Youngdong Pharmaceutical. 0.5 ml of AST and ALT substrate solution were added to the test tube and warmed at 37 ° C. for 2 to 3 minutes, and then 100 μl of the test serum was added. The reaction was performed at 37 ° C. for 60 minutes and ALT for 30 minutes. 0.5 ml of a color developing solution (2,3-ditrophenylhydrazine) was added to terminate the reaction, and the mixture was left at room temperature for 20 minutes, and then 5 ml of 0.4 N NaOH was added thereto, and the absorbance was measured at 505 nm as a control.

3) Determination of liver triglyceride content

The content of hepatic triglycerides is determined by Van Handel and Zilversmit (Van Handel, E. and Zilversimt, D.B. (1957) Micromethod for the direct determination of serum triglycerides, J. Lab. Clin. Med. 50, 152-157] by Butler et al. [Butler, W.M., Mairing, H.M., Horning, M.G. and Bordie, B.B. (1961), The direct determination of liver triglycerides, J. Lipid. Res. 2, 95-96]. To 1 g of liver, 9 ml of 67 mM phosphate buffer (pH 7.0) was added and ground. 1 ml of this milled product was added to a 50 ml test tube with a glass stopper with 4 g of activator olite containing 2 ml of chloroform. 18 ml of chloroform was added to the test tube, and the mixture was stirred for 10 minutes, followed by filtration with a No. 2 filter paper. 0.5 ml each of the filtrate was added to three test tubes, and 0.5 ml of chloroform was added to one test tube, which was then used for a blank test, and then heated at 80 ° C. for 30 minutes to remove chloroform. 0.5 ml of alcoholic KOH (0.4%) was added to one test tube (soap), 0.5 ml of 95% alcohol was added to another tube (soap), and warmed at 65 DEG C for 20 minutes, followed by 0.5 ml of 0.2 N H2SO4. Was added to warm for 15 minutes at 95 ℃ to remove the remaining alcohol. After cooling each test tube, 0.1 ml of 0.05 N sodium metaperiodate was added and left for 10 minutes, and then 0.1 ml of 2M sodium arsenite was added and left for 10 minutes. 5 ml of 0.2% chromotropic acid was added to each test tube and reacted in the dark for 30 minutes at 95 ° C., and then the absorbance was measured at 570 nm with the test tube for the blank test as a control.

Triglyceride content (mg per g tissue) was calculated by the following formula.

(Asu-Ansu) / (Ass-Anss) X 0.05 X (200 / F)

Asu: absorbance of saponified samples

Ansu: Absorbance of Unsaturated Specimens

Ass: absorbance of saponified standard

Anss: Absorbance of Unsaturated Standards

F: amount of chloroform extract (ml)

4) Ethanol Kinetic Parameter Calculation

The ethanol in blood of each animal to which ethanol was administered was calculated from the Weidmark's equation from the kinetic curve [Widmark, E.M.P (1993) Verteilung and Unwandltung des etyl alcohols in organismus des hundes, Biochem. Z. 267 (128-134)]. The slope (β) of the elimination phase was obtained by straightening the reduced portion of the ethanol curve. Initial concentration (Co) of ethanol was obtained from the linearized y-contact point. The distribution volume (Vd) was calculated by dividing the dose by the initial concentration, and the ethanol disappearance rate (βxVd) was obtained by multiplying the slope (β) by the distribution volume (Vd). Some of the areas were calculated using the trapezodial method.

All of the above test results are one way-ANOVA test [SAS Duncan's multiple range test [SAS] Statistical package]. Among the obtained results, it was judged that the p value was less than 0.05 in the statistical significance.

The results measured by the experiment as described above are as follows.

Changes in body weight and liver weight of experimental animals following G009 and ethanol administration are shown in Table 3 below.

As can be seen from the results in Table 3, no significant change in body weight or liver weight was observed by administration of ethanol and protein multi-group G009. On the other hand, Figure 2 shows the change in blood ethanol concentration according to G009 administration, and no significant difference was observed in the ethanol absorption rate and metabolic rate in the test group.

The results of calculating the kinetic constants of ethanol from FIG. 2 are shown in Table 4 below. In addition, the results of measuring the amount of triglycerides in order to determine how much G009 inhibits fatty liver production by ethanol administration are shown in Figure 3 and Table 4.

From the results in Table 4, it was found that there was no significant difference between the test group and the control group in all constants including several (bioavailability) in the hemodynamic parameters of ethanol. On the other hand, according to the amount of triglyceride measured in order to observe the inhibition of fatty liver production 24 hours after ethanol administration, in the ethanol-treated control group, the fatty liver increased by 2 times compared to the normal group, and G009 25 mg / kg administration test In the group, ethanol-induced fatty liver was suppressed to a level similar to that of the normal group.

Taken together, the results of Table 4 and FIG. 3 show that the protein polysaccharide G009 inhibits fatty liver without affecting the kinetic parameters of blood ethanol, which is directly responsible for the mobilization of fat from peripheral tissues by ethanol. Presumably due to inhibition of triglycerides to normal group levels.

Therefore, it was determined that G009 has an inhibitory effect at the dose of 25 mg / kg for ethanol-induced acute fatty liver formation.

B. Inhibitory effect of G009 on CCl-induced acute hepatitis

46 Sprague-Dawley rats weighing about 200 grams were fed 9 corns with corn oil only, 15 controls with CCl only, 5 tests with CCl and G009 2.5mg / kg, CCl and G009 The experiment was used by dividing into 5 groups of 5 trials 2 groups administered 10 mg / kg and 12 trials 3 groups administered CCl and G009 25 mg / kg. G009 is administered orally once a day or 4 days to the respective doses (2.5, 10, 25 mg / kg body weight) of each rat of the test group 1, 2 and 3, and fasted the day before the test. Two hours after oral administration of the corresponding dose of G009, CCl (0.1 or 0.15 ml / kg body weight, corn oil, intraperitoneal injection) was administered again. The control group was intraperitoneally injected at the same dose as the test group without administration of G009. 24 hours after CCl administration, animals in each group were weighed, anesthetized with ether, and drawn from the abdominal aorta. After leaving the collected blood at room temperature for 1 hour or more, the serum was obtained by centrifugation at 12,000xg for 2 minutes, and stored in refrigeration and used to measure ALT and AST in the same manner as in the experiment of A above within 3 days. . After 24 hours, the liver was extracted and weighed, and a portion of the liver was fixed in 10% formalin solution for histological examination, and then paraffin-treated after dehydration to make 4-6 μm thick tissue sections. Spectroscopy was performed after matocillin and eosin staining. The results of each experiment measured are as follows.

24 hours after intraperitoneal injection of CCl, body weight and liver weight did not change at all, as can be seen from the results shown in Table 5 below, and there was no significant change by G009 administration.

Changes in blood ALT, acute hepatitis toxicity indicators, are shown in FIG. 4. As can be seen from the results shown in FIG. 4, the ALT increased more than 10-fold in the control group administered with CCl only compared to the normal group. In addition, as shown in FIG. 4, the G009 was not different from the control group at the dose of 2.5 mg / kg, but in the test 2 group using G009 at a dose of 10 mg / kg, ALT was significantly reduced compared to the control group. It showed a sensitive dose-response line, and test 3 group administered G009 25 mg / kg had almost the same effect as test 2 group.

The results of measuring AST change in blood, another indicator of hepatotoxicity, are shown in FIG. 5. According to the results, AST increased 6-fold in the control group treated with CCl only compared to the normal group, and G009 was especially 10 mg / kg. It can be seen that the increase in AST significantly decreased in the test group administered at the dose of.

Taken together, the results of measuring the changes in ALT and AST shown in FIGS. 4 and 5 show that the effect of G009 shows a steep dose-response curve and has a significant effect from the G009 10 mg / kg dose. Therefore, it was confirmed that G009 has an inhibitory effect on hepatitis in CCl-induced acute hepatitis model. This effect of G009 was examined by liver biopsy (Figure 6). From the results shown in FIG. 6, CC1 treatment caused extensive necrosis, especially in the area around the portal vein, in the control group (FIG. 6 (b)). In Figure 6 (c)), hepatocyte necrosis is markedly reduced, indicating a similar histology to the normal group (Figure 6 (a)). These results were consistent with the blood ALT and AST results.

C. Inhibitory Effect of G009 on Acute Hepatitis Induced by Thioacetamide

25 Sprague-Dawley rats weighing about 200 g body weight, normal group administered with saline only, control treated with thioacetamide only, test group administered with thioacetamide and G009 10 mg / kg, thioacetamide and G009 25 Five groups of Test 2 group administered mg / kg and Test 3 group administered thioacetamide and G009 100 mg / kg were divided to include 5 rats in each group. Test animals in groups 1, 2, and 3 of the test were dosed orally with G009 at a corresponding dose (10, 25, 100 mg / kg body weight) once a day for 4 days, and on day 5, thioacetamide (kg body weight). 150 mg, saline, oral) was administered orally again at the dose of G009 2 hours prior to administration. The control group was orally administered with the same dose of thioacetamide as the test group without administration of G009. 48 hours after the administration of thioacetamide, the animals of each group were weighed, anesthetized with ether, and collected from the abdominal aorta. After leaving the collected blood at room temperature for 1 hour or more, the serum was obtained by centrifugation at 12,000xg for 2 minutes, and stored in refrigeration and used to measure ALT and AST in the same manner as in the experiment of A above within 3 days. . In addition, the liver was extracted and weighed, and the liver tissue was fixed in 10% formalin solution at a predetermined site, and liver biopsy was performed in the same manner as in B. The results obtained by the above experiments are as follows.

After 48 hours of oral administration of thioacetamide 150 mg / kg, the weight and the weight ratio of liver to liver were measured.

As can be seen from the results of Table 6, the three indicators, namely the weight, liver weight and liver weight / weight ratio, by administration of the protein polysaccharide G009 of the present invention were normal and control at all three doses. There was little change compared to.

The results of measuring ALT and AST levels in blood, which are indicative of acute hepatitis, after administration of diaacetamide are shown in FIGS. 7 and 8, respectively. As can be seen from the results shown in FIG. 7, the administration of thioacetamide resulted in an approximately 5-fold increase in the amount of ALT in the control group compared to the normal group. In the test group 1 administered G009 at a dose of 10 mg / kg, there was no effect of inhibiting the increase of ALT. However, the test group 2 and 3 administered G009 at doses of 25 and 100 mg / kg showed a significant increase in ALT. It was suppressed and showed little difference from the normal group, especially in the test group 3. On the other hand, as can be seen from the results of FIG. 8 showing the concentration of AST in the blood, the concentration of AST was increased by about five times in the control group treated with thioacetamide only. Indicated.

The effect of G009 on liver tissue damage caused by thioacetamide treatment is shown in FIG. As can be seen from the results shown in FIG. 9, in the control group treated only with thioacetamide (FIG. 9 (b)), a marked increase in necrosis and inflammatory cells was observed in the portal vein. In the third test group treated with G009 at a dose of 100 mg / kg, hepatocyte necrosis was not observed as shown in FIG. 9 (c), and the appearance was also reduced compared to the control group. It can be seen that the a) similar to the diagram). These results are similar to the changes in blood ALT and AST activity.

Therefore, the results of the three tests that tested the effects of G009 in acute hepatitis and fatty liver models with different mechanisms showed that G009 had a significant inhibitory effect on liver damage in ethanol, CCl and thioacetamide induction models. Able to know. The effect of G009 was slightly different according to the model used in the experiment, but it was judged that it showed the most effective effect at the dose of G009 10mg / kg sowl 100mg / kg.

Experimental Example 3

[Inhibitory Effect of G009 on Hepatic Fibrosis Induced by Bile Ligation]

Liver cirrhosis is associated with chronic damage, such as hepatitis virus, alcohol, drugs, metabolism, and biliary obstruction, resulting in repeated inflammation or other forms of regenerative nodule. Refers to the symptoms of hardening of the liver. Such cirrhosis eventually leads to functional changes in liver cells and structural changes in blood vessel tissues in the liver, resulting in deterioration of overall liver function, lack of production of various important substances such as albumin, and hypertension of hepatic pulse pressure. Causes worst case liver cancer. Therefore, by examining the inhibition of the protein polysaccharide G009 against hepatic fibrosis induced by biliary ligation, it was confirmed whether G009 could be usefully used for the treatment of liver disease.

In this experiment, 27 female Wistar rats (180-260 g in weight) were used as experimental animals. The experimental animals were divided into four groups, and the first group was a sham operation control group that performed sham operation, and the second group was a BDL control group which performed bile duct ligation / scission (hereinafter referred to as BDL) and a third group. The mock surgery group and the fourth group which performed mock surgery and administered G009 were divided into four groups of the BDL experimental group which performed BDL and administered G009. In the BDL control group and the experimental group, rats were anesthetized and opened, tied the distal and proximal degrees of laxity, cut between them, and then injected 2 ml of saline solution and sutured again. In the simulated control group and the experimental group, the experimental animals were opened as in the BDL group, and then 2 ml of saline was injected and resealed. The experimental animals of each group that performed mock surgery and BDL were closely observed for 4 weeks. At this time, the protein polysaccharide G009 of the present invention was dissolved in physiological saline from the day of mock surgery and BDL. Sonde was used orally at a dose of 5 mg per rat, and the same amount of saline was orally administered to the mock surgery and the BDL control group. During this period, the weights of liver animals were measured at weekly intervals. Four weeks later, the experimental animals of each group were anesthetized, opened, and cardiac punctured, blood was collected, and the time was extracted to measure the wet weight of the liver. Serum and liver tissue for hydroxyproline determination were stored at -20 ° C. Each observation and measurement result is as follows.

1. Observation of general condition of experimental animals

In the simulated control group and the experimental group, the animals were in good health during the observation period, and no abnormalities were observed in all organs at the time of autopsy.

Animals in the BDL control group developed strong jaundice three days after surgery, while bilirubinemia continued during the four week observation period. At necropsy, the liver was very hardened, granulated tissues formed, and the adhesions to the surrounding regularities were severe. The kidneys were darker green than those of the simulated surgical control. Severe and no ascites were observed.

Jaundice in animals in the BDL group was induced at the same time as the BDL control group, and the pigment of bilirubinuria was attenuated from 2 weeks after surgery, and adhesion with other organs was hardly observed.

2. Changes in weight and liver weight

Changes in body weight and liver weight measured in each group of experimental animals are shown in Table 7 below.

Note) Figures in the above table are mean ± standard error.

2. n = number of experimental animals

As can be seen from the results described in Table 7, the mock surgery control group and the animals in the experimental group showed normal weight gain without weight loss during the observation period. Animals in the BDL experimental group that received G009 showed slightly higher weight loss (control: 3%, experimental group: 5%) than those in the BDL control group after one week of surgery, but weight gain over time was 6.6%, respectively. The figure was as low as 11% and 9.1%.

The weight ratio of liver and body weight (final weight X 100 at 4 weeks post-surgery) was almost equal to 3.5% and 3.9% in the mock and control animals, but 7.5% in the BDL experimental animals. Slightly lower than 8.3% of the BDL control.

5. Procollagen Type III Fab Tide (PNIIIP) in Serum and Hydroxyproline in Liver Tissue

PNIIIP concentration measurement in serum Schuppan et al. Using I-labeled antigen (PNIIIP). Schuppan, D., Dumont, JM, Kim, KY, Hennings, G. and Hahn, EG: Serum concentration of the aminoterminal procollagen III Peptides in the rat reflects early formation of connective tisssue in expermental liver cirrhosis, J. Hepatol., 3, 27 (1986)] were measured by radioimmunossay. In other words, anti-serum (Musha-PNIIIP serum) diluted to 50% concentration of the antigen-binding peak was mixed with standard antigen solution (PNIIIP) or serum obtained above and incubated at 4 ° C. for 12 hours. Here I-labeled antigen was added and left. Bound and free forms were determined by precipitation with goat anti-rabbit-IgG antiserum and measuring the radioactivity of the precipitate.

On the other hand, the amount of hydroxyproline in liver tissue was measured by Jamall et al. [Jamall, IS, Finelli, V. N and Que Hee, SS: A simple method to determine nanogram levels of 4-hydroxyproline in biological tissuem Anal. Biochem. 112, 70 (1981). The amount of hydroxyproline was determined by hydrolyzing 5% liver homogenate in 6N HCl at 110 ° C, oxidizing it with chloramine-T, developing it with Ehrlich's reagent soln, and measuring the absorbance at 558 nm. It was.

The results measured by the above method are shown in the following Table 8 and FIG.

As can be seen from the results shown in Table 8 and the results shown in FIG. 10, in the simulated animals, low PNIIIP (control: 1.9 ng / ml ± 0.18, experimental: 2.0 ng / ml ± 0.18) in both control and experimental groups. ) And Hardidoxyproline (control: 239 ng / ml ± 7.5, experimental: 242 ng / ml ± 14.1). In BDL animals, PNIIIP was significantly lower in the experimental group by 50% than in the control group. In addition, hydroxyproline, which represents the total amount of collagen in liver tissue, was 17% lower in the BDL experimental group than in the BDL control group. From these results, G009 seems to have an effect of inhibiting liver fibrosis by inhibiting or inhibiting the synthesis of connective tissue, that is, fibrogenesis, and inhibiting the accumulation of collagen (connected tissue) in liver tissue.

4. Clinical and Chemical Findings of Serum

Using the stored serum, clinical kit (Ciba-Corning) was used to analyze GOT (glutamate-oxaloacetate transaminase), GPT (glutamate-pyruvate transaminase), ALP (alkaline phosphatase), total bilirubin, cholesterol, and creatinine in serum. It was measured by a chemical chemistry analyzer (Gliford 400E). The measured results are shown in Table 9 below.

As can be seen from the results shown in Table 9, the cholesterol level of the mock surgery group was significantly reduced by 31% compared to the mock surgery control group, and the GOT, GPT and total bilirubin levels were about 14%, 35 in the mock surgery group. % And 33% were increased, but there was no significant increase, and ALP level increased by about 140%. In the BDL group, the GOT and cholesterol levels decreased by about 10% and 16%, but there was no significant increase, and the ALP level increased by 71%. Other parameters were similar in the control and experimental groups or slightly higher in the experimental group.

5. Liver biopsy

Liver biopsy was performed as follows. Part of the liver tissues of rats of the BDL control group and the experimental group were collected and placed in a 10% neutral formalin solution, stored at 4 ° C, and stained with hematoxylin and eosin to observe histological changes under an optical microscope (80 magnification). It is shown in Figure 11.

As can be seen from the photograph of FIG. 11, fibrous tissue proliferation involving moderate hepatocellular necrosis, inflammation and severe bile duct proliferation was observed in the animals of the BDL control group (FIG. 11 (a)). Hepatocellular necrosis and inflammation were observed in the BDL group compared to the BDL control group, and bile duct proliferation was also weak compared to the control group, and only a slight degree of fibrous tissue growth was observed (Fig. 11 (b)).

From all the above experimental results, it was confirmed that the protein polysaccharide G009 of the present invention exhibits a clear fibrosis inhibitory effect upon oral administration in BDL experimental animals induced by hepatic fibrosis by cholestasis.

Experimental Example 4

[DNA Synthesis Effect of Protein Polysaccharide G009]

In order to expect a therapeutic effect on livers damaged by various causes such as alcohol or drugs, drugs that can promote the recovery or regeneration of liver cells are needed. Therefore, in this experiment, in general, CCl, which destroys the structure of unsaturated fatty acids and proteins present in the hepatocellular membrane and eventually leads to necrosis, damages the hepatocytes and is treated with protein polysaccharide G009, followed by BrDU (5-bromo-2). 'Deoxyuridine) reagent was added and incubated for 2 hours to introduce BrDU into the DNA, and the effect of G009 on DNA synthesis was examined. The specific method is as described below.

1. Isolation and Culture of Hepatocytes

Hepatocytes were isolated by modifying the method of Seglen. A pentobarbital (40 mg / kg) was intraperitoneally injected into Wistar rats (250 g), anesthetized with 70% ethanol, and the catheter (18Gx2 '') was attached to a pre-tethered portal vein. . At this time, check the blood coming out and supply the Ca2 +, Mg2 + -non-milking HBSS (Hank's ballanced salt solution, pH7.2: Sigma) preheated to 37 ℃ while starting pumping (30ml / min) and at the same time The cut was made to allow blood to flow out. In the meantime, relative vein was perfused with HBSS containing collagenase. Circulation for about 10 minutes breaks the Glisson's capsule, causing the fluid to flow out. After the end of the perfusion, the liver tissue was removed, washed with HBSS, added with an appropriate amount of HBSS, cut with scissors, and filtered through a nylon net (250㎛) to separate the liver cells.

Isolated hepatocytes were added to WEM (William's E Medium: Gibco) medium and washed by centrifugation at 50 × g for 3 min at 4 ° C. for 3 min. Cell viability was measured with 0.4% trypan blue solution and used as initial cultured hepatocytes when the survival rate was 85% or more.

10% fetal calf serum, penicillin (100 IU / ml), streptomycin (100 μg / ml), 10 in hepatocyte suspension M dexamethasone and 10 4x10 by adding WEM containing M insulin After adjusting to cells / ml, 1 ml was placed in a 24-well tissue culture plate (Corning Co.) and pre-incubated for 3 hours in a CO incubator (37 ° C., 5% CO). The medium was changed after the previous embryo and 18 hours later, it was used as an experiment with hepatocytes.

2. DNA Synthesis Effect of Protein Polysaccharide G009 on CCl-induced Hepatotoxicity

A 24-well culture plate (4 × 10 5 cells / ml) in which hepatocytes were cultured was divided into three sections, which were divided into three groups: the normal group, the second section, and the CCl treatment group, and the third section, the CCl4 + G009 treatment group. 10 µm of protein polysaccharide G009 (final concentration: 1 mg / ml) dissolved in saline was added to 3 sections of the plate, and equal amounts of physiological saline were added to 1 and 2 sections. Thereafter, CCl dissolved in ethanol in the 2nd and 3rd zones was adjusted to a final concentration of 10 mM. The treated plates were incubated at 5% CO, 37 ° C. for 24 hours, and then BrDU reagent was added to each well, followed by incubation for 2 hours to introduce BrDU into the DNA, and the DNA synthesis ability was measured (see Gratzer, HG, 218, 474-475 (1982)] The measured results are shown in Table 10 below.

As can be seen from the results shown in Table 10 above, the CCI alone-induced stimulation index for hepatocytes is 0.57 of the normal group, resulting in about 60% hepatocellular necrosis. On the other hand, when CCl and G009 were added simultaneously, the stimulation index was 1.03 times higher than that of the normal group, indicating that hepatocyte proliferation was increased.

From these results, the present invention, the protein polysaccharide G009 is a component that plays an important role in the proliferation and protection of hepatocytes, and thus not only inhibits hepatocellular membrane damage due to lipid peroxidation, which is a series of reactions occurring in hepatocytes by CCl, It also seems to have a protective and proliferative effect.

Judging from the experimental results as described above, the protein polysaccharide G009 of the present invention effectively inhibits acute hepatitis, fatty liver formation, liver fibrosis, etc. and protects the liver and stimulates liver tissue by stimulating DNA synthesis in the damaged liver site. It has been proven to be useful as a therapeutic agent for liver disease by promoting regeneration and recovery.

Composition Example

[Composition 1 (tablet)]

G009 100 mg

D-mannitol 50mg

Crospovidone 10mg

Calcium Dihydrogen Phosphate 35mg

Magnesium Tearate 5mg

G009, D-mannitol and crospovidone were mixed to prepare granules, and the remaining ingredients were mixed to tablet tablets by the tablet manufacturing method.

[Composition 2 (made of hard calcel)]

G009 100 mg

Crospovidone 5.5mg

Light anhydrous silicic acid 0.5mg

Calcium Dihydrogen Phosphate 9.0mg

Granules were prepared by mixing the above components, such as G009 and crospovidone, and the capsules were tableted by a conventional capsule preparation method.

Composition 3 (soft capsule)

G009 100 mg

Soybean oil 400 mg

Lecithin 50mg

White lead 50mg

Concentrated glycerin 100 mg

The above-mentioned ingredients, such as G009 and soybean oil, were mixed, and the soft capsule agent was manufactured by the conventional soft capsule agent manufacturing method.

Composition 4 (Liquid)

G009 100 mg

Polysorbate 80 10mg

Mannitol 1mg

Stevioside 10mg

Polyvinylprolidone 500mg

Citric acid 50mg

Sodium benzoate 30mg

Purified water

A 50 ml capacity liquid solution was prepared by the usual liquid production method using the above components as the solution phase.

[Composition 5 (syringe)]

G009 10mg

Polysorbate 80 5mg

Polyoxyethylene Glycol 6000 15mg

Injection water volume

Injecting the above-mentioned components into a solution phase and filling into 5 ml ampoules by the usual injection preparation method.

Claims (6)

A liver disease therapeutic composition containing protein polysaccharide G009. The composition of claim 1, wherein the protein polysaccharide G009 is an ingredient extracted from a mycelium of Ganoderma lucidom IY-009 (KFCC-10709). The therapeutic agent composition for treating liver disease according to claim 1, which is formulated in a unit dosage form with a pharmaceutically acceptable carrier. The therapeutic agent composition for treating liver disease according to claim 3, wherein the unit dosage form is a tablet, hard or soft capsule, granule, liquid, suspension, injectable solution or suspension. The therapeutic agent composition for liver disease according to claim 4, wherein the unit dosage form is formulated to contain 30 to 300 mg of G009. 6. The liver disease therapeutic composition according to claim 5, wherein the unit dosage form is formulated to contain 50 to 150 mg of G009.