KR19990066786A - Liver function improver using Bergenin and its derivatives as active ingredients - Google Patents

Abstract

The present invention relates to a liver function improving agent comprising an active ingredient of Bergenin (bergenin) and its derivatives, specifically, as a liver function improving agent of Bergenin and its derivatives and pharmaceutically acceptable salts thereof represented by the following general formula (1) It is about a use.

(Wherein R _1, R _2, R ₃ represent hydrogen or acetyl group; R ₄ and R ₆ represent hydrogen, methyl or acetyl group; R ₅ represents hydrogen or methyl.)

Description

Liver function improver using Bergenin and its derivatives as active ingredients

The present invention relates to a hepatic function improving agent comprising a bergenin and a derivative thereof or a pharmaceutically acceptable salt thereof represented by the following formula (1).

(Wherein R _1, R _2, R ₃ represent hydrogen or acetyl group; R ₄ and R ₆ represent hydrogen, methyl or acetyl group; R ₅ represents hydrogen or methyl.)

In modern times, liver function is impaired due to irregular eating, stress, excessive drinking and smoking, or worse, liver disease develops frequently. Such damage of liver function and liver disease has a fatal effect on the human body and is difficult to recover. There are many kinds of such liver diseases, for example, acute hepatitis and chronic hepatitis, liver cirrhosis, alcoholic fatty liver, hepatitis B virus and the like. In particular, hepatitis B virus is contagious, requiring isolation from society and making it difficult for group life. In addition, chronic hepatitis, cirrhosis, and liver cancer often develop, and these liver diseases are almost incurable and are a real threat to modern people. Therefore, there is a further demand for the development of a medicament capable of effectively treating the liver function impairment and liver disease without side effects or recurrence.

Conventional therapeutic agents for liver disease currently include liver function aids, antiviral agents such as acyclovir, immunosuppressive agents such as corticosteroids, 6-mercaptopurine, azathioprine and urosodeoxycholine acid, silymarin, glutathione, glycyrrhizin , Hepatohydrates, multivitamins, hepatocyte regeneration accelerators, fibrosis inhibitors such as D-penicillamine, biphenyldimethyldicarboxylate, interferon, etc. are used, but they do not show sufficient effects in the treatment of liver disease and have side effects and recurrence. There is a high risk that new drugs for liver disease are strongly required.

Bergennin of the present invention has anti-ulcer activity (Kang et al., Japanese pharmacopeia 69 (2), 369-78, 1973), anti-inflammatory action (Swarnalakshmi et al., Curr Sci., 53 (17), 917, 1984), anti Hyperlipidemic activity (Jahromi et al. Phytother. Res 6, 180 ~ 183, 1992), etc., and its derivatives are coronary vasodilation and anti-ulcer activity [Japanese Patent Publication No. 59-186988, Suntory LTD 771-780 (1984). )] Is known.

The bergennin can be obtained by extracting the bark of ordinary wood bark with an organic solvent such as methanol or water, and even in clownfish and cornflower by using a method such as Ramaiah (JCS, 2313-2316, 1979). Can be separated.

Bergenin is known to be not toxic when LD ₅₀ is more than 13000 mg / kg when administered orally to mice in acute toxicity test, and also little to no toxicity in subacute and chronic toxicity tests (Kang et al., Basic and Clinical). (Japan) 9 (5), 96-106, 1975).

In order to solve the above problems, the present inventors have completed the present invention by finding that bergennin and its derivatives, which are conventionally used as therapeutic agents for gastric ulcers, are effective in preventing or treating liver disease while searching for herbal substances for improving liver function. It was.

SUMMARY OF THE INVENTION An object of the present invention is to provide an agent for improving liver function, wherein the agent contains Bergenin and its derivatives as an active ingredient.

The present invention relates to a hepatic function-improving agent comprising Bergenin and its derivatives represented by the following general formula (1) or a pharmaceutically acceptable salt thereof.

(Wherein R _1, R _2, R ₃ represent hydrogen or acetyl group; R ₄ and R ₆ represent hydrogen, methyl or acetyl group; R ₅ represents hydrogen or methyl.)

Bergenin derivatives in the present invention may be dimethylbergenin (dimethylbergenin), norbergenin (norbergenin), Bergenin pentaacetate (bergenin pentaacetate) and the like, the structure is shown in Table 1 below .

division R ₁ R ₂ R ₃ R ₄ R ₅ R ₆ Bergennin Hydrogen Hydrogen Hydrogen Hydrogen methyl Hydrogen Dimethylbergennin Hydrogen Hydrogen Hydrogen methyl methyl methyl Norbergennin Hydrogen Hydrogen Hydrogen Hydrogen Hydrogen Hydrogen Bergennin pentaacetate Acetyl Acetyl Acetyl Acetyl methyl Acetyl

The liver function improving agent of the present invention may include conventionally used excipients and preservatives such as methyl paraoxybenzoate, propyl paraoxybenzoate, sodium benzoate and the like.

Bergenin of the present invention can be obtained through the following conventional extraction method (Japanese Pharmacy Out-of-Pharmaceutical Standard, P 151-152, Pharmacopoeia, 1993).

10 parts by weight of distilled water is added to 1 part by weight of dried yeokpi dried poultry, and 2 times each for 2 hours, and 5 parts by weight for 3 times. After drying to obtain Xerox bark, it was dissolved in methanol, the insoluble portion was filtered off, and the filtrate was concentrated under reduced pressure, and then hung on a silica gel column to develop with a chloroform: methanol (4: 1) solvent, which was then recrystallized and purified. Bergenin (C ₁₄ H ₁₆ O ₉ · H ₂ O, mp 140 ° C., drying, C ₁₄ H ₁₆ O ₉ , mp 238 ° C.) can be obtained.

Bergennin derivatives of the present invention can be synthesized or isolated using the conventional method for the above-mentioned Bergennin.

Bergenin and its derivatives of the present invention has a prophylactic and therapeutic action (Tables 2-9, 10-16) of liver disease, and its mechanism of action is glutathione sulfur transferase and a biological body involved in the detoxification process through glutathione. It is believed that this is due to the activity induction of glutathione reductase (Table 4, 6, 8, 10, 12) involved in maintaining the content of glutathione within.

The dose of the liver function improving agent of the present invention is Bergenin, which is administered 20 to 200 mg three times a day for adults, and the dosage may be changed depending on the degree of symptoms or the age and weight of the patient. .

The liver function improving agent of the present invention may be prepared in a form suitable for oral administration or parenteral administration according to a conventional preparation method. That is, in the case of oral administration, it may be prepared in the form of tablets, capsules, powders, granules, syrups, liquids, elecils, etc., and in the case of parenteral administration, in the form of injections for abdominal cavity, subcutaneous, muscle, and transdermal. Can be prepared.

Reference Examples and Experimental Examples below describe the contents of the present invention in more detail, but the present invention is not limited thereto.

Reference Manufacturing Example 1

10 kg of distilled water was added to 1 kg of dried Yeokpi dried poultry, and 2 times each for 2 hours, and 5 liters for the third time. The mixture was extracted by heating in a water bath at 90 ~ 100 ℃ for 2 hours, filtered, and the filtrate was concentrated under reduced pressure in a water bath and dried. After obtaining 130 g (13%) of Xerox bark, it was dissolved in methanol, the insoluble portion was filtered off, the filtrate was concentrated under reduced pressure, and then, the filtrate was placed on a silica gel column and developed with a chloroform: methanol (4: 1) solvent. Repeated recrystallization gave 19.5 g of pure bergennin.

Reference Production Example 2

2 g of bergennin extracted in Reference Example 1 was dissolved in 1 L of methyl alcohol, and 500 ml of ether solution of diazomethane was added, followed by Raimaiah et al. (JCS 2313-2316) at 0 ° C. , 1979 and JE Hay et al. J. Chem. Soc., 2231, 1958) were synthesized 1.1 g of dimethylbergennin.

Reference Production Example 3

Column chromatography on acetone extraction concentrates of leaves of Securinega suffruticosa according to Lee et al., Kor. J. Pharmacogn., 25 (2), 105-112, 1994 Norbergennin was isolated.

Reference Production Example 4

2 g of bergennin extracted in Reference Example 1 was dissolved in 100 ml of acetic anhydride (Ac ₂ O) and 2 ml of anhydrous pyridine, and heated for 6 hours in a water bath to prepare a method such as Ramiaiah (Raimaiah et al., JCS 2313). -2316, 1979) and then recrystallized in benzene to give 2.4 g of bergennin pentaacetate.

Experimental Example 1. Hepatoprotective effect of bergennin on carbon tetrachloride-induced hepatotoxicity

SD rats of 150 ± 20g purchased from the Korea Experimental Animal Center were adapted for a week or more under a certain condition (temperature: 20 ± 2 ℃, humidity: 60%, contrast: 12 hours Light / dark cycle). As a result, 50, 100 and 200 mg / kg of Bergennin prepared in Reference Production Example 1 was orally challenged daily for 7 days, and 0.5 ml / kg of carbon tetrachloride 12 hours and 36 hours after the 7th day of administration (the same amount of olive oil Dissolved in the abdominal cavity, respectively, and 12 hours after the last administration of carbon tetrachloride, anesthetized with carbonic acid gas (CO ₂ gas) and opened to collect blood and liver tissue. At this time, the experiment was also performed on the group not administered with the Bergenin and carbon tetrachloride (normal group), the group administered with only carbon tetrachloride without the administration of Bergenin (control). The battle of the samples was performed in the context of protection against hepatotoxicity.

For the blood and liver tissues taken above, s-AST (serum aspartic acid aminotransferase), s-ALT (serum alanine aminotransferase), SDH (sorbitol dehydrogenase), γ-GT ( The activity of γ-glutamic acid transferase and the content of LPO (lipid peroxide) and GSH (glutathione) and the activities of GST (glutathione sulfur transferase) and GR (glutathione reductase) in liver tissue were measured (see Table 2). At this time, the enzyme source was prepared by preparing a cytosol fraction by a general method, and protein quantification was carried out using Lowry method (Lowry, OH, Rosebrough, Bovine serum albumin (Sigma Fr. IV) as a standard). NJ and Randall, RJ (1951) J. Biol. Chem., 193, 265). When liver failure is induced, enzymes such as AST, ALT, γ-GT, and SDH, which are selectively present in hepatocytes, are leaked into the blood to increase activity, and the content of lipid peroxides due to toxic substances increases in the liver. In addition, in the liver, the activities of GR and GST, which are involved in the detoxification of toxic substances, and the content of GSH, which is detoxified by being combined with toxic substances, are reduced. Thus, drugs that reduce or restore the activity and content of all or a portion of the blood and liver indices increased or decreased due to liver dysfunction have a liver improving effect.

How to measure liver function index division How to measure unit activity of s-AST Reitman, S. and Frankel, S. (1957) Am. J. Clin. Pathol. 28, 56-63 Karmen unit / ml activity of s-ALT Reitman, S. and Frankel, S. (1957) Am. J. Clin. Pathol. 28, 56-63 Karmen unit / ml γ-GT activity Szasa, G. (1969) Clin. Chem. 15, 124-136 mU / ml Activity of SDH Gerlach, U. (1965) In Methods of Enzymatic Anlaysis (Bergmyer, H. U., Ed), 112-117, Verlag Chemic Press. Weitheim. Germany Sigma unit / ml LPO content Okawa et al. (Ohkawa, H., Ohishi, N and Yaki, K. (1979) Anal. Biochem., 95, 351) Nmol of malondialdehyde per gram of tissue GSH content Elman et al. (Ellman, G. L. (1959) Arch. Biophys., 30, 2409) Μmol per gram of tissue Activity of GST Habig et al. (Habig, W. H., Pabist, M. J. and Jakoby, W. B. (1974) J. Biol. Chem., 249, 7130) Nmole of 1-chloro-2,4-dinitrobenzene per mg of protein for 1 minute Activity of GR Mize, C. E. and Langdon, R. G. (1962) J. Biol. Chem., 237, 1589 Nmole of GSH produced per mg of protein for 1 min

The results of the experiments are shown in Table 3 and Table 4 as mean ± standard error, and the statistical significance test was performed using T-test.

Hepatoprotective effect of bergennin on carbon tetrachloride-induced hepatotoxicity (in serum) division Bergennin (mg / kg) Carbon tetrachloride (ml / kg) Indicators in serum activity of s-AST activity of s-ALT γ-GT activity Activity of SDH Normal 0 0 58.9 ± 1.66 32.6 ± 1.99 25.3 ± 1.97 18.7 ± 1.23 Control 0 0.5 192.3 ± 4.81 ^@ 88.6 ± 1.87 ^@ 194.6 ± 6.08 ^@ 67.4 ± 1.63 ^@ Sample Group 1 50 0.5 132.3 ± 2.82 ^# 70.4 ± 2.80 ^# 152.3 ± 2.95 ^# 37.4 ± 1.11 ^# Sample Group 2 100 0.5 92.8 ± 2.90 ^# 58.4 ± 1.94 ^# 99.0 ± 2.32 ^# 29.6 ± 1.19 ^# Sample Group 3 200 0.5 96.9 ± 2.40 ^# 55.5 ± 1.44 ^# 93.8 ± 1.67 ^# 27.8 ± 0.69 ^#

Hepatoprotective effect of bergennin on carbon tetrachloride-induced hepatotoxicity (indicator in liver) division Bergennin (mg / kg) Carbon tetrachloride (ml / kg) Indicators in the liver LPO content GSH content Activity of GST Activity of GR Normal 0 0 22.6 ± 1.97 5.39 ± 0.17 249.6 ± 7.46 25.8 ± 0.80 Control 0 0.5 66.8 ± 1.25 ^@ 2.87 ± 0.15 ^@ 130.9 ± 6.12 ^@ 11.8 ± 0.50 ^@ Sample Group 1 50 0.5 45.8 ± 1.26 ^# 3.87 ± 0.13 ^# 204.7 ± 6.33 ^# 19.9 ± 0.56 ^# Sample Group 2 100 0.5 38.3 ± 1.41 ^# 4.29 ± 0.19 ^# 217.2 ± 5.73 ^# 21.3 ± 1.16 ^# Sample Group 3 200 0.5 35.7 ± 1.87 ^# 4.52 ± 0.26 ^# 221.2 ± 5.80 ^# 22.4 ± 0.67 ^#

@: P <0.01 was significant when compared with normal group.

#: P <0.01, which is significant when compared with control group.

The administration of carbon tetrachloride increased s-AST, s-ALT, γ-GT, SDH activity and LPO content in the liver, and markedly decreased GSH content, GST and GR activity in the liver. Dose-induced dose-dependent decreases in serum s-AST, s-ALT, γ-GT, and SDH activity and hepatic LPO content in serum, and dose-dependently increased GSH content, GST and GR activity in liver .

Experimental Example 2 Hepatoprotective Effect of Bergenin on Galactosamine-Induced Hepatotoxicity

50, 100 and 200 mg / kg of Bergenin prepared in Reference Example 1 was orally battled daily in rats for 7 days and then administered on day 7 and then 400 mg / kg of galactosamine 400 mg / kg after 7 days. Dissolved in the abdominal cavity, respectively, and after the last administration of galactosamine, anesthetized and reopened with carbon dioxide gas 72 hours later, and blood samples and liver tissues were extracted and used for the experiment. The results are shown in Tables 5 and 6 below.

Hepatoprotective effect of bergennin on galactosamine-induced hepatotoxicity (in serum) division Bergennin (mg / kg) Galactosamine (mg / kg) Indicators in serum activity of s-AST activity of s-ALT γ-GT activity Activity of SDH Normal 0 0 57.9 ± 1.63 34.6 ± 1.24 25.8 ± 1.53 19.5 ± 1.97 Control 0 400 107.2 ± 4.45 ^@ 60.1 ± 2.07 ^@ 85.8 ± 3.39 ^@ 53.9 ± 2.37 ^@ Sample Group 1 50 400 80.3 ± 1.59 ^# 43.5 ± 1.34 ^# 48.4 ± 1.59 ^# 38.6 ± 1.00 ^# Sample Group 2 100 400 70.2 ± 1.19 ^# 35.6 ± 1.34 ^# 37.0 ± 1.93 ^# 30.4 ± 1.62 ^# Sample Group 3 200 400 68.2 ± 1.52 ^# 37.0 ± 0.99 ^# 39.9 ± 0.92 ^# 31.9 ± 1.30 ^#

Hepatoprotective effect of bergennin on galactosamine-induced hepatotoxicity (indicator in liver) division Bergennin (mg / kg) Galactosamine (mg / kg) Indicators in the liver LPO content GSH content GST content Content of GR Normal 0 0 21.6 ± 1.63 5.33 ± 0.11 251.2 ± 8.38 22.9 ± 1.20 Control 0 400 57.5 ± 2.37 ^@ 3.40 ± 0.20 ^@ 166.8 ± 7.39 ^@ 16.5 ± 0.75 ^@ Sample Group 1 50 400 40.5 ± 0.94 ^# 4.27 ± 0.11 ^# 212.9 ± 5.20 ^# 20.3 ± 0.56 ^# Sample Group 2 100 400 30.6 ± 1.03 ^# 4.53 ± 0.14 ^# 217.9 ± 6.93 ^# 21.5 ± 1.01 ^# Sample Group 3 200 400 28.7 ± 1.36 ^# 4.61 ± 0.17 ^# 220.3 ± 5.98 ^# 22.0 ± 0.91 ^#

@: P <0.01, which is significant when compared with normal group.

#: P <0.01, which is significant when compared with control group.

The administration of galactosamine increased the s-AST, s-ALT, γ-GT, SDH activity in the serum, and the LPO content in the liver, and significantly decreased the GSH content, GST and GR activity in the liver. In the case of pre-administration of bergennin, the serum-sponsored doses of s-AST, s-ALT, γ-GT, SDH and LPO in liver were dose-dependently reduced. Increased.

Experimental Example 3 Effect of Bergennin on the Hepatotoxicity of Carbon Tetrachloride-Induced Hepatotoxicity

0.5 ml / kg of carbon tetrachloride (dissolved in the same amount of olive oil) was intraperitoneally administered to rats, respectively, at the beginning of the experiment (0 hours) and 12 hours, and the Bergennin 50, 100 and 100 prepared in Reference Preparation Example 1 from 24 hours after the initial administration. 200 mg / kg was orally administered daily for 7 days, followed by anesthesia for 24 hours, and then open blood and liver tissue were extracted and used for the experiment. The rest was the same as in Experiment 1, and the results are shown in Tables 7 and 8. Post-administration of the samples was carried out for the purpose of treating hepatotoxicity.

Hepatic Effects of Bergenin on Carbon Tetrachloride-Induced Hepatotoxicity (indices in Serum) division Carbon tetrachloride (ml / kg) Bergennin (mg / kg) Indicators in serum activity of s-AST activity of s-ALT γ-GT activity Activity of SDH Normal 0 0 53.9 ± 2.37 39.6 ± 2.01 26.9 ± 1.73 16.9 ± 1.58 Control 0.5 0 183.6 ± 6.19 ^@ 90.3 ± 3.45 ^@ 211.4 ± 6.89 ^@ 61.3 ± 2.75 ^@ Sample Group 1 0.5 50 94.7 ± 3.02 ^# 63.5 ± 1.83 ^# 87.2 ± 3.05 ^# 25.7 ± 1.17 ^# Sample Group 2 0.5 100 89.3 ± 3.02 ^# 61.9 ± 2.35 ^# 85.3 ± 2.92 ^# 27.8 ± 1.36 ^# Sample Group 3 0.5 200 86.1 ± 4.38 ^# 58.2 ± 3.67 ^# 81.9 ± 1.56 ^# 24.6 ± 0.82 ^#

Hepatic Effects of Bergenin on Carbon Tetrachloride-Induced Hepatotoxicity (indicator in liver) division Carbon tetrachloride (ml / kg) Bergennin (mg / kg) Indicators in the liver LPO content GSH content Activity of GST Activity of GR Normal 0 0 18.7 ± 1.63 5.48 ± 0.14 240.6 ± 7.67 25.6 ± 0.80 Control 0.5 0 61.2 ± 3.24 ^@ 2.75 ± 0.15 ^@ 119.8 ± 5.73 ^@ 14.2 ± 0.48 ^@ Sample Group 1 0.5 50 25.7 ± 0.83 ^# 4.47 ± 0.13 ^# 210.4 ± 4.63 ^# 22.3 ± 0.60 ^# Sample Group 2 0.5 100 23.5 ± 1.24 ^# 4.67 ± 0.13 ^# 223.2 ± 7.11 ^# 22.8 ± 0.80 ^# Sample Group 3 0.5 200 28.6 ± 1.63 ^# 4.74 ± 0.24 ^# 204.6 ± 5.20 ^# 21.9 ± 0.56 ^#

@: P <0.01, which is significant when compared with normal group.

#: P <0.01, which is significant when compared with control group.

The administration of carbon tetrachloride increased s-AST, s-ALT, γ-GT, SDH activity and LPO content in the liver, and markedly decreased GSH content, GST and GR activity in the liver. Dose-dependent administration of s-AST, s-ALT, γ-GT, and SDH in the serum and LPO content in the liver decreased dose-dependently, and GSH content, GST and GR activity in the liver increased dose-dependently. .

Experimental Example 4 Effect of Bergennin on the Hepatotoxicity of Galactosamine-Induced Hepatotoxicity

Galactosamine 400 mg / kg was intraperitoneally administered to rats after starting experiment (0 hours) and 72 hours, respectively. After oral dosing every day for 24 hours, after 24 hours of anesthesia, open blood and liver tissue were extracted and used for the experiment. The rest is the same as Experimental Example 1, the results are shown in Table 9, Table 10 below.

Hepatic Effects of Bergenin on Galactosamine-induced Hepatotoxicity (in Serum Markers) division Galactosamine (mg / kg) Bergennin (mg / kg) Indicators in serum activity of s-AST activity of s-ALT γ-GT activity Activity of SDH Normal 0 0 59.3 ± 3.09 40.4 ± 2.00 29.7 ± 2.05 16.9 ± 1.09 Control 400 0 117.4 ± 4.63 ^@ 76.9 ± 3.75 ^@ 87.2 ± 2.38 ^@ 49.8 ± 2.05 ^@ Sample Group 1 400 50 75.9 ± 1.24 ^# 46.0 ± 1.14 ^# 42.4 ± 1.61 ^# 29.6 ± 0.94 ^# Sample Group 2 400 100 74.2 ± 1.46 ^# 44.1 ± 1.17 ^# 40.6 ± 1.17 ^# 31.6 ± 0.90 ^# Sample Group 3 400 200 65.4 ± 0.85 ^# 48.0 ± 1.77 ^# 39.1 ± 1.05 ^# 27.7 ± 1.22 ^#

Hepatic Effects of Bergenin on Galactosamine-induced Hepatotoxicity (indicator in liver) division Galactosamine (mg / kg) Bergennin (mg / kg) Indicators in the liver LPO content GSH content Activity of GST Activity of GR Normal 0 0 21.3 ± 1.59 5.45 ± 0.15 240.6 ± 7.14 26.9 ± 0.83 Control 400 0 50.0 ± 2.40 ^@ 3.16 ± 0.08 ^@ 167.2 ± 3.26 ^@ 18.0 ± 0.58 ^@ Sample Group 1 400 50 29.6 ± 1.50 ^# 4.48 ± 0.20 ^# 221.1 ± 7.21 ^# 21.9 ± 0.98 ^# Sample Group 2 400 100 25.4 ± 1.11 ^# 4.52 ± 0.18 ^# 219.6 ± 5.80 ^# 22.8 ± 1.26 ^# Sample Group 3 400 200 26.7 ± 1.55 ^# 4.72 ± 0.11 ^# 210.7 ± 5.87 ^# 22.1 ± 0.69 ^#

@: P <0.01, which is significant when compared with normal group.

#: P <0.01, which is significant when compared with control group.

The administration of galactosamine increased the s-AST, s-ALT, γ-GT, SDH activity in the serum, and the LPO content in the liver, and significantly decreased the GSH content, GST and GR activity in the liver. Post-administration of bergennin decreased dose-dependently the serum s-AST, s-ALT, γ-GT, SDH activity and LPO content in the liver, and the dose of GSH and GST and GR in the liver Increased.

Experimental Example 5. Blocking Effect of Bergenin on Carbon Tetrachloride-Induced Hepatotoxicity

Berry and Friend's method (Berry & Friend, J. Cell Biol., 43, 5006, 1969) was modified to incubate liver cells isolated from rats for 24 hours, remove the culture medium, and then cultured without carbon tetrachloride and bergenin ( Normal), 10 mM carbon tetrachloride, culture medium containing no Bergenin (control), and culture medium containing 10 mM carbon tetrachloride and bergenin at each concentration (sample group) were further incubated for 1.5 hours. Each culture was taken to determine the activity of free alanine aminotransferase (ALT), sorbitol dehydrogenase (SDH) and glutathione sulfur transferase (GST), glutathione (GSH) content and GSSG reductase activity in hepatocytes. Was measured twice (see Table 11), and converted into% protection (Coi et al., J. Appli. Pharmacol., Korea, 4, 291-294, 1996) in Table 1 Shown in At this time, the experimental results were expressed as mean ± standard error, and statistical significance test was done by T-test.

How to measure division How to measure Activity of ALT Reitman & Frankel, (1957) Am. J. Cli. Pathol., 28, 56 Activity of SDH Gerlach, (1965) Sorbitol dehydrogenase In: Bergmeyer HU, eds.Method in Enzymology NY .: Elsevier pp. 761-767) Activity of GST Habig et al. (Habig et al., (1974) J. Biol. Chem., 249, 7130) Total glutathione (GSH and GSSG) content Prasada & Harihara, (1991) Biochemical methods of studying hepatoxicity.In: Robert GM, Steadman DH and Richard JB, eds. Hepatoxicology.Florida: CRC Press. Pp 241-325 Content of Glutathione (oxidized, GSSG) Prasada & Harihara, (1991) Biochemical methods of studying hepatoxicity. In: Robert GM, Steadman DH and Richard JB, eds. Hepatoxicology.Florida: CRC Press. Pp 241-325) Activity of GSSG Reductase Carlberg & Mannervik, (1975) Purification and characterization of the flavoenzyme glutathione reductase frome rat liver. J. Biol. Chem., 250 (14), 5475-80)

* Preparation of post mitochondrial supernatants is described by Gibson & Skett, (1986) Technique and experiments illustrating drug metabolism.In: Chapman and Hall, eds.Introduction to drug metabolism.Cambridge: University Press.pp 239-281).

The protein content of the cultured cells was measured by Lowry et al., (1951) J. Biol. Chem., 193, 265.

* Reduced glutathione content (GSH) = total glutathione content (GSH + GSSG)-oxidized glutathione content (GSSG)

Blocking Effect of Bergenin on Carbon Tetrachloride-induced Hepatotoxicity division Carbon tetrachloride (mM) Bergennin (μM) % Protection Activity of ALT Activity of SDH Activity of GST GSH content GSSG Reductase Activity Normal 0 0 100.0 100.0 100.0 100.0 100.0 Control 10 0 0.0 0.0 0.0 0.0 0.0 Sample Group 1 10 One 6.7 ± 2.1 ^# 8.2 ± 2.4 ^@ 11.6 ± 3.5 ^# 1.0 ± 3.0 2.5 ± 2.7 Sample Group 2 10 10 30.7 ± 4.0 ^@ 20.5 ± 4.4 ^@ 30.0 ± 4.6 ^@ 10.4 ± 2.8 ^@ 10.2 ± 3.5 Sample Group 3 10 100 62.1 ± 5.9 ^@ 49.9 ± 5.2 ^@ 49.1 ± 2.5 ^@ 24.2 ± 3.6 ^@ 28.1 ± 2.3 ^@ Sample Group 4 10 300 57.8 ± 5.9 ^@ 40.9 ± 5.5 ^@ 51.5 ± 3.2 ^@ 25.3 ± 2.1 ^@ 29.1 ± 3.3 ^@

#: P <0.05, which is significant when compared with control group.

@: P <0.01, significant compared to control.

As the concentration of bergennin was increased to 100 μM, the activity of ALT and SDH liberated into the culture decreased.

* As the concentration of bergennin was increased to 300μM, the activities of GST and GSSG reductase and GSH contents in hepatocytes increased.

Experimental Example 6. Blocking Effect of Bergenin on Galactosamine-induced Hepatotoxicity

After culturing the isolated hepatocytes in the same manner as in Experimental Example 5, after 1.5 hours, the culture medium was changed to the culture medium containing 1.5 mM galactosamine, and the cells were further cultured for 14 hours to induce cytotoxicity (Kiso et al. , (1983) Planta Med., 49, 222), the group was added to the new culture medium again, the concentration of bergennin prepared in Reference Preparation Example 1 (sample group) and the group without addition of bergennin (control) Incubated for 24 hours. At this time, each culture medium was also taken for the hepatocyte group (normal group) which did not cause toxicity with galactosamine, and the activity of ALT and SDH liberated as the culture medium was measured and converted into the protection rate of Equation 1, and RNA biosynthesis was measured. The measurement is shown in Table 13. At this time, the activities of ALT and SDH were measured by the same method as Experimental Example 5, and RNA biosynthesis was measured by Karner's method (Karner, (1964) Biochem. J., 92, 449).

Blocking Effect of Bergenin on Galactosamine-induced Hepatotoxicity division Galactosamine (mM) Bergennin (μM) % Protection RNA synthesis (cpm) Activity of ALT Activity of SDH Normal 0 0 100.0 100.0 615 ± 48 Control 1.5 0 0.0 0.0 125 ± 11 Sample Group 1 1.5 One 15.1 ± 4.4 ^@ 12.2 ± 3.2 ^@ 243 ± 28 ^@ Sample Group 2 1.5 10 50.9 ± 5.8 ^@ 45.1 ± 3.5 ^@ 281 ± 32 ^$ Sample Group 3 1.5 100 49.1 ± 5.0 ^@ 44.9 ± 3.4 ^@ 309 ± 35 ^$ Sample Group 4 1.5 300 22.6 ± 4.0 ^@ 20.9 ± 7.1 ^@ 231 ± 15 ^@

@: P <0.01, significant compared to control.

$: P <0.001 compared with control group, significant.

Increasing the concentration of bergenin up to 100μM showed hepatoprotective activity against ALT and SDH liberated in the culture, and increased the biosynthesis of RNA in hepatocytes in a concentration-dependent manner.

Experimental Example 7. Blocking Effect of Bergennin, Dimethylbergennin, Norbergennin, and Bergennin Pentaacetate on Carbon Tetrachloride-induced Hepatotoxicity

For Bergennin and dimethyl Bergennin, Norbergennin, Bergennin pentaacetate, ALT and SDH activity were measured in the same manner as in Experimental Example 5, and the protection rate of Equation 1 (Choi et al., J. Appli.Pharmacol , Korea, 4, 291-294, 1996), the results are shown in Table 14, Table 15. At this time, the measurement was carried out three times and the mean value was expressed as a standard error. The statistical significance was verified by T-test.

Blocking Effect of ALT Activity on Carbon Tetrachloride-Induced Hepatotoxicity of Bergennin, Dimethylbergennin, Norbergennin, and Bergenin Pentaacetate Concentration (μM) Protection rate (%) Bergennin Dimethylbergennin Norbergennin Bergennin pentaacetate Normal 100 100 100 100 Control 0 0 0 0 One 6.7 ± 2.1 ^# 8.7 ± 2.4 ^@ 11.3 ± 1.8 ^@ 23.8 ± 5.0 ^@ 10 30.7 ± 4.0 ^@ 15.0 ± 3.5 ^@ 57.5 ± 5.8 ^@ 27.6 ± 4.3 ^@ 100 62.1 ± 5.9 ^@ 34.3 ± 4.5 ^@ 96.5 ± 6.1 ^@ 39.0 ± 5.1 ^@ 300 57.8 ± 5.8 ^@ 39.9 ± 3.2 ^@ 80.0 ± 5.0 ^@ 49.2 ± 5.3 ^@

#: P <0.05, which is significant when compared with control group.

@: P <0.01, significant compared to control.

When the concentration of the sample was increased from 1 μM to 300 μM, bergennin and norbergennin at 100 μM and dimethylbergennin and bergennin pentaacetate at 300 μM, respectively, showed ALT activity blocking effects on maximal carbon tetrachloride-induced hepatotoxicity.

Blocking Effect of SDH Activity on Carbon Tetrachloride-induced Hepatotoxicity of Bergennin, Dimethylbergennin, Norbergennin, and Bergenin Pentaacetate Concentration (μM) Protection rate (%) Bergennin Dimethylbergennin Norbergennin Bergennin pentaacetate Normal 100 100 100 100 Control 0 0 0 0 One 8.2 ± 2.4 ^@ 9.7 ± 2.1 ^@ 7.4 ± 3.9 ^@ 21.8 ± 4.2 ^@ 10 20.5 ± 4.4 ^@ 20.2 ± 3.5 ^@ 21.1 ± 3.3 ^@ 24.2 ± 4.8 ^@ 100 49.9 ± 5.2 ^@ 35.2 ± 4.6 ^@ 49.6 ± 4.4 ^@ 35.1 ± 4.1 ^@ 300 40.9 ± 5.5 ^@ 40.1 ± 5.5 ^@ 55.3 ± 4.7 ^@ 44.8 ± 4.0 ^@

@: P <0.01, significant compared to control.

When increasing the concentration of the sample from 1 μM to 300 μM, bergennin at 100 μM concentration, dimethylbergennin, norbergennin, and bergennin pentaacetate exhibited the maximum blocking effect on SDH activity against carbon tetrachloride-induced hepatotoxicity at 300μM concentration, respectively. It was.

Experimental Example 8. Blocking Effect of Bergenin, Dimethylbergennin, Norbergennin, and Bergenin Pentaacetate on Galactosamine-induced Hepatotoxicity

Bergennin and dimethylbergennin, norbergennin, and Bergenin pentaacetate were tested in the same manner as in Experimental Example 6 ALT and SDH activity in terms of the protection rate of Equation 1, the results are shown in Table 16 ~ It is shown in 17. At this time, the measurement was carried out three times and the mean value was expressed as a standard error. The statistical significance was verified by T-test.

Blocking Effect of ALT Activity on Galactosamine-induced Hepatotoxicity of Bergennin, Dimethylbergennin, Norbergennin, and Bergenin Pentaacetate Concentration (μM) Protection rate (%) Bergennin Dimethylbergennin Norbergennin Bergennin pentaacetate Normal 100 100 100 100 Control 0 0 0 0 One 15.1 ± 4.4 ^@ 42.0 ± 4.1 ^@ 60.9 ± 6.4 ^@ 23.3 ± 3.5 ^@ 10 50.9 ± 5.8 ^@ 64.7 ± 3.1 ^@ 68.8 ± 5.7 ^@ 19.7 ± 3.6 ^@ 100 49.1 ± 5.0 ^@ 19.1 ± 3.7 ^@ 60.5 ± 3.4 ^@ 14.4 ± 2.7 ^@ 300 22.6 ± 4.0 ^@ 15.6 ± 4.6 ^@ 51.4 ± 4.9 ^@ 8.2 ± 3.2

#: P <0.05, which is significant when compared with control group.

@: P <0.01, significant compared to control.

Increasing the concentration of the sample from 1 μM to 300 μM, maximal blocking effect on ALT activity against galactosamine-induced hepatotoxicity at the concentration of Bergenin, Dimethylbergenin and Norbergenin at 10μM and Bergenin Pentaacetate at 1μM Indicated.

Blocking Effect of SDH Activity on Galactosamine-induced Hepatotoxicity of Bergennin, Dimethylbergennin, Norbergennin, and Bergenin Pentaacetate Concentration (μM) Protection rate (%) Bergennin Dimethylbergennin Norbergennin Bergennin pentaacetate Normal 100 100 100 100 Control 0 0 0 0 One 12.2 ± 3.2 ^@ 40.0 ± 3.1 ^@ 50.2 ± 5.3 ^@ 12.2 ± 3.1 ^@ 10 45.1 ± 3.5 ^@ 60.2 ± 5.9 ^@ 62.8 ± 4.8 ^@ 17.5 ± 2.7 ^@ 100 44.9 ± 3.4 ^@ 20.1 ± 5.3 ^@ 58.5 ± 5.9 ^@ 15.4 ± 5.0 ^@ 300 20.9 ± 4.1 ^@ 15.3 ± 4.0 ^@ 48.5 ± 3.8 ^@ 6.6 ± 2.6 ^@

@: P <0.01, significant compared to control.

When increasing the concentration of the sample from 1 μM to 300 μM, bergennin, dimethylbergennin and norbergennin, and bergennin pentaacetate showed the maximum blocking effect on SDH activity against galactosamine induced hepatotoxicity at 10μM concentration, respectively.

Experimental Example 9. Hepatic cirrhosis (fibrosis) inhibitory effect of the bergenin derivative

Biliary ligation model according to the method of Kountouras, Biling, BH and Scheuer, PJ (1984) Br. J. Exp. Pathol. 65, 305-311) can be used to determine the causes, mechanisms, and changes in cirrhosis. It was widely used as an experimental animal model of cirrhosis (fibrosis) for the study.

150 ± 20g SD rats purchased from the Korea Experimental Animal Center were acclimated for a week or more under the same conditions (temperature: 20 ± 2 ℃, humidity: 60%, contrast: 12 hours light / dark cycle). The group was opened under anesthesia, ligation of the biliary tract, the muscles and skin were sutured, and then stabilized for 3 days. Bergennin and acetylbergennin 50, 100 and 200 mg / kg, and silymarin 40 mg / kg as a positive control for 4 weeks, respectively. In this case, the group was also opened and closed (do not biliary ligation) but did not receive either bergennin or acetylbergennin (normal group), but also the group that did biliary ligation but not bergennin and acetylbergennin (control). It was. Fasting for 16 hours after the last drug administration, blood was collected from the abdominal aorta under ether anesthesia, and liver tissue was extracted and used for the experiment.

The activity of serum s-AST (aspartic acid aminotransferase) and s-ALT (alanine aminotransferase) in the blood as indicators of hepatic function in the blood and liver tissues taken above was measured by the lateman and Frankel method (Reitman, S. and Frankel, S. (1957) Am. J. Clin. Pathol. 28, 56-63), and the degree of cirrhosis (fibrosis) is an indicator of hydroxyproline content as Jamal et al. , IS, Finelli, VN and Que Hee, SS (1981) A simple method to determine nanogram levels of 4-hydroxyproline in biological tissue.Anal. Biochem. 117, 70). When the biliary tract causes liver failure, enzymes such as AST and ALT, which are selectively present in hepatocytes, are leaked into the blood to increase activity, and liver tissue is hardened (fibrosis) to increase the hydroxyproline content. Therefore, drugs that reduce the activity of AST and ALT increased due to hepatic injuries due to biliary tract ligation and decrease the hydroxyproline content in liver tissues have the effect of preventing and preventing cirrhosis (fibrosis).

The results of the experiments were expressed in the mean value ± standard error, and are shown in Tables 18 and 19, and the statistical significance test was performed using t-test.

division Bergennin (mg / kg) Liver cirrhosis (fibrosis) indicator Hydroxyproline (μg / 0.2g) AST ALT Normal 0 71.1 ± 2.7 72.8 ± 6.3 38.8 ± 1.8 Control 0 272.2 ± 13.9 ^# 1228.3 ± 30.1 ^# 109.6 ± 2.0 ^# Sample Group 1 50 218.8 ± 8.3 ^*** 1138.9 ± 56.9 97.5 ± 5.1 Sample Group 2 100 202.1 ± 11.5 ^*** 969.7 ± 42.5 ^*** 77.3 ± 5.5 ^*** Sample Group 3 200 198.2 ± 6.1 ^*** 938.4 ± 34.7 ^*** 74.8 ± 5.1 ^*** Silymarin 40 234.1 ± 13.0 ^** 1071.3 ± 40.2 ^*** 92.8 ± 4.1 ^*

#: P <0.01, which is significant when compared with normal group.

*: P <0.05, **: P <0.02, ***: P <0.01, which is significant when compared with the control group.

division Acetyl Bergenin (mg / kg) Liver cirrhosis (fibrosis) indicator Hydroxyproline (μg / 0.2g) AST ALT Normal 0 71.1 ± 2.7 72.8 ± 6.3 38.8 ± 1.8 Control 0 272.2 ± 13.9 ^# 1228.3 ± 30.1 ^# 109.6 ± 2.0 ^# Sample Group 1 50 212.3 ± 9.4 ^*** 1183.2 ± 34.7 93.5 ± 2.7 Sample Group 2 100 205.5 ± 7.0 ^*** 1033.8 ± 78.1 ^*** 79.2 ± 2.7 ^*** Sample Group 3 200 190.6 ± 9.9 ^*** 842.6 ± 62.5 ^*** 75.6 ± 2.2 ^*** Silymarin 40 234.1 ± 13.0 ^** 1071.3 ± 40.2 ^*** 92.8 ± 4.1 ^*

#: P <0.01, which is significant when compared with normal group.

*: P <0.05, **: P <0.02, ***: P <0.01, which is significant when compared with the control group.

In the control group, the content of hydroxyproline, an indicator of liver cirrhosis (fibrosis), was 272.2 ㎍ / 0.2 g, which was about 3.8 times higher than that of the normal group of 71.1 ㎍ / 0.2 g, and the AST was 16.9 times higher and the ALT 2.9 times higher. However, the Bergenin and acetylbergennin-administered groups decreased hydroxyproline content and AST and ALT activity in a dose-dependent manner. Therefore, it can be seen that bergennin and acetylbergennin effectively inhibit cirrhosis (fibrosis).

Formulation Preparation Example 1 : Tablet

Bergenin 100.0mg

Lactose BP 150.0mg

Starch BP 30.0mg

Pregelatinized Corn Starch BP 15.0mg

Magnesium Stearate 1.0mg

Bergennin was mixed with lactose, starch and pregelatinized corn starch, and then a suitable volume of purified water was added and granulated into a powder. The granules were dried and then mixed with magnesium stearate and compressed to obtain tablets.

Formulation Preparation Example 2 : Coated Tablet

Bergenin 100.0 mg

Lactose 60.0 mg

Corn starch 300.0 mg

Hydroxypropyl cellulose 3.0 mg

Magnesium stearate 3.0 mg

The mixture of bergennin, lactose and corn starch was granulated through a 1 mm mesh sieve using a hydroxypropyl cellulose solution, dried at 40 ^° C. and sieved again. The granules thus obtained were mixed and compressed with magnesium stearate. The resulting central tablet was coated with sugar by an aqueous suspension of sucrose, titanium oxide, talc and gum arabic in a conventional manner. The coated tablets were bred with beeswax.

Formulation Preparation Example 3 : Capsule

Bergenin 100.0mg

Starch 1500 100.0mg

Magnesium Stearate BP 2.0mg

Bergennin was mixed with excipients and then filled into gelatin capsules to obtain capsules.

Formulation Preparation Example 4 Injection

Bergenin 70.0mg

Dilute sodium hydroxide pH 7.5-8.5

Injectable sodium chloride BP up to 2 ml

Bergenin was dissolved in a suitable volume of dilute sodium hydroxide solution to adjust to pH 7.5-8.5, followed by volume control and thoroughly mixing with sodium chloride BP for injection. The solution was filled into a 2 ml type ampoule of clear glass, dissolved under glass and enclosed under an upper grid of air and then sterilized in an autoclave at 120 ° C. for at least 15 minutes to obtain an injection.

As can be seen in the above experimental examples, the Bergenin and its derivatives have a prophylactic and therapeutic effect of liver disease.

Claims (2)

Liver function improving agent using as an active ingredient bergenin and its derivatives or pharmaceutically acceptable salts thereof represented by the following general formula (1). (Wherein R _1, R _2, R ₃ represent hydrogen or acetyl group; R ₄ and R ₆ represent hydrogen, methyl or acetyl group; R ₅ represents hydrogen or methyl.) The liver function improving agent according to claim 1, wherein the bergennin derivative is selected from the group consisting of norbergenin, dimethyl bergennin and bergennin pentaacetate.