JPH045235A - Preventive/therapeutic agent for hepatitis - Google Patents

Abstract

PURPOSE:To provide the title nontoxic agent having anti-hepatitis activity at least equivalent to those of conventional antihepatitis agents such as glycyrrhetinic acid, glycyrrhizic acid oleanolic acid, containing, as active ingredient, ginsenoside R0. CONSTITUTION:The objective agent having the above-mentioned effects, containing, as active ingredient, ginsenoside R0 of the formula which is a saponin component found in medicinal carrot, and has been known to have anti- inflammatory and anti-thrombogenic activities etc., but has not been known to have anti-hepatitis activity. The dosage of the above-mentioned compound, depending on the ages and symptoms of patients, is pref. 10 - 100 mg/day for adult (totaling 60 - 500 mg) in the case of a medicine for peroral use. The above-mentioned compound can be obtained by either extraction from the whole of Panax ginseng or its root or synthesis.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a prophylactic/therapeutic agent for hepatitis containing ginsenoside R6, which is a saponin component present in medicinal ginseng, as an active ingredient.

Among medicinal ginseng, especially Araliaceae
Panax ginseng C
, A. Meyer) is known as Panax ginseng, and it is widely known that it has been used as a tonic, tonic, anti-inflammatory, diuretic, blood pressure lowering, and antidiabetic drug since ancient times. Recently, research has progressed on the saponins contained in ginseng, including Rb+, Rbt, and ginseng containing ginseng.
Rbs, Re, Rd, Re, Rf.

Rg, , Rgt, Rh, Ro, etc. are known.

It is represented by the formula: It has a structure of a pentavalent triterpene derivative, and its structure is completely different from other ginsenosides which have a structure of a tetracyclic triterpene derivative. It has been reported that ginzenoside R0 has anti-inflammatory and antithrombotic effects (for example, Japanese Patent Application Laid-Open No. 163315/1982).
It is not known to have anti-hepatitis activity.

The inventors of the present application measured the anti-hepatitis activity of ginsenoside R using various experimental models, and found that glycylresin acid, its aglycone, and ginsenoside R8, which are conventionally used as anti-hepatitis agents. It was discovered that it has an antihepatitis activity equal to or higher than that of oleanolic acid, which is an aglycone, and is substantially free of toxicity, leading to the development of this invention.

Ginsenoside R6 used in the present invention is produced by extraction from the whole plant of Panax ginseng or its roots (tree buds, white ginseng, red ginseng, and tail ginseng), or by synthesis.

Extraction methods include, for example, the following methods. That is, the whole body of Panax ginseng or its roots is extracted with a water-containing lower alcohol (for example, 70% methanol), and the extract is concentrated to dryness to obtain an extract. This was extracted with a mixture of ethyl acetate and water, the aqueous layer was extracted with water-saturated n-butyl alcohol, the organic layer was subjected to column chromatography using silica gel, and the fraction containing ginsenoside R8 was concentrated to dryness. R0
is obtained.

As a synthetic method, it is produced, for example, by bonding a sugar component to oleanolic acid (formula below) contained in sugar beet, olive leaves, apple peel, etc. by a known method. At this time, if a derivative of glucose or glucuronic acid (for example, one in which the hydroxy group is protected with a protective group) is used as the sugar component, a derivative of ginsenoside R0 can be obtained, which is also the prophylactic/therapeutic agent for hepatitis of the present invention. It can be used as an active ingredient. As the above protecting group,
It is desirable that the compound be easily desorbed by chemical treatment or in vivo. Furthermore, when other sugars or sugar acids are used in place of glucose or glucuronic acid, derivatives of ginsenoside R0 are obtained, which can also be used as active ingredients in the prophylactic and therapeutic agent for hepatitis of the present invention. Furthermore, the active ingredients include other hepatitis treatment drugs, such as glycyrrhetinic acid, glycyresinic acid, steroids, etc.
It can be used in combination with Shiko, licorice, and Chinese herbal preparations containing these (for example, Shoshikoto).

The anti-inflammatory prophylactic/therapeutic agent of the present invention contains ginsenoside R6 as a main component, to which is added a pharmaceutically acceptable carrier, such as an organic or inorganic, solid or liquid excipient for oral or parenteral use. A formulation is used. Specifically, oral preparations include powders, tablets, granules, capsules, solutions, tinctures, liquid extracts, spirits, suspensions,
Includes limonade, syrup, etc. In addition, parenteral preparations include injections, drips, suppositories, and the like.

Excipients used here include water, lactose, starch,
Dextrin, calcium phosphate, calcium carbonate, aluminum silicate, magnesium oxide, magnesium stearate, dry aluminum hydroxide, etc. are used. Further, the above-mentioned formulation may contain stabilizers, emulsifiers, buffers, etc.

Ginzenoside R used in the hepatitis preventive/therapeutic agent of this invention
The dosage of o varies depending on the patient's age, medical condition, etc., but for example, in the case of an oral drug, 0 to 1000 mg of ginzenoside R0 is used per day for an adult, preferably about 60 to 500 u.

Next, Examples of this invention will be shown, and the effects of this invention will be clarified through reagent examples and comparative examples.

Formulation Example 1 Ginsenoside Ro5011g was aseptically placed in a sterilized vial, and water was removed to obtain a vial of a drug for injection.

The basic rule is to add distilled water for injection at the time of use to make an injection solution.

Formulation Example 2 Lactose is added to Ginzenoside R0, mixed uniformly, and tableted using a tablet machine, resulting in 980 mg of Ginzenoside R in one tablet.
A tablet containing the following was obtained.

Next, the antihepatitis activity of ginsenoside R8 used in this invention was investigated using various experimental models, and compared with known antihepatitis agents glycyresinic acid, glycyrrhetinic acid, and oleanolic acid.

1. The test drug and reagent ginsenoside R6 are Korean red ginseng (Panaxgins).
(Eng C, A, Meyer) and submitted to the test subjects.

Oleanolic acid was obtained by hydrolyzing ginsenoside R0 with hydrochloric acid according to the method of Shoji et al. Glycyrrhetinic acid and glycyrrhesin acid were commercially available products (Tokyo Kasei Kogyo) that were recrystallized and then provided to the test subjects. In addition, D-galactosamine, carbon tetrachloride (CC1,), and α-naphthylisothiocyanate (ANIT, Nacalai Tesque) were used as hepatitis-inducing agents.

2. Experimental animal Slc; 11 star male rat (body weight 180-2
009) was used. The breeding environment was a breeding room with a temperature of 23±2°C, humidity of 50-60%, and a cycle of 12 hours of light and 712 hours of darkness.The animals were fed commercially available solid feed (Labo MR Stock, Nippon Nosan), and were given free access to water. After purchase, the animals were preliminarily reared for one week before being used for experiments.

! D-galactosamine-induced acute hepatitis test D-galactosamine 400m9/9 was intraperitoneally administered to rats, and 24 hours later, cardiac blood was collected under bendoparbital anesthesia, and 5-G
OT and 5-GPT values were measured. The test substances (ginsenoside R0 and oleanolic acid) were suspended in 0.2% CMC-Na solution or in physiological saline and administered orally or intraperitoneally 1 hour before administration of D-galactosamine.

The results are as follows.

1) Effects of oral administration: As shown in Table 1, when D-galactosamine 400xg/9 was administered intraperitoneally to rats, 5-GOT and 5-GPT values increased. An effect of significantly suppressing the increase in 5-GOT and 5-GPT values was observed in the ginzenoside Ro20019/9 administration group, and a tendency to suppress was observed in the 50119/kg administration group. Although this tendency was observed in the oleanolic acid 200H/9 administration group, the activity was not significant.

(Left below) Table 1 Effect of ginzenoside R0 and oleanolic acid on D-galactosamine-induced acute hepatitis in rats (oral administration) Dose Number of rats 5-GOT 5-G
PT” intact control 6 Kinzenocide R8 intact control al Oleanolic acid 7127 ± 10 10 1084 ± 88 10 938 soil 140 10 747 + 82” 7116 ± 3 8755 ± 111 7 742 ± 149 7 668 ± 89 70 ± 6 1054 soil 151 730 ± 122 678 Sat96C1 40±2 451±73 475±93 410±25 Subjects were orally administered D-galactosamine 1 hour before the injection, and then blood was collected 24 hours after the injection.

One control received 0.2% CMCNa solution. 1 Each measurement value is the average value ± S, E.

Indicated by CゝThere is a significant difference compared to the control (p<0.05
).

2) Effects of intraperitoneal administration: As shown in Table 1, when ginsenoside R6, oleanolic acid, glycyrrhesin acid, and glycyrrhetinic acid were intraperitoneally administered at a dose of 30 x g/kg, all subjects had 5- Although the effect of significantly suppressing the increase in GOTS 5-GPT value was observed, the activity of ginsenoside R6 was the strongest.

Table ■ Effects of ginsenoside R0, oleanolic acid, glinorrenonic acid, and glycyrrhetinic acid on D-galactosamine-induced acute hepatitis in rats (intraperitoneal administration) Dose Number of rafts 5-GOT
5-GOT” treatment (island/to 9) (U/L
) (U/L) Unharmed 7 139:14 57
24 contrasts” 9
758±123 458:92 Kinzeno Dimension ID R0106372:56C' 221±40Cゝ30 6 243±43"
126 soil 27dl oleanolic acid
10 7 526±6
2 340:6630 7
412±56C' 217 soil 36C'gui'y
hW acid 10 7
414t39°' 216+3QC+30
7 357±167°'
198±60°'glinorretinoic acid to
7 511±68
244±2930 7
373±33” 17!J soil 22C1 subject is D-
Galactosamine was administered intraperitoneally 1 hour before injection, and blood was then collected 24 hours after D-galactosamine injection.

1′ control received saline intraperitoneally. ') Each measured value is expressed as mean value ± SE.

1. There is a significant difference from the control group (p<0.05).

There is a significant difference from the dl control group (p<0.01).

4. α-Naphthylisothiocyanate (ANIT)-induced chromatic hepatitis test ANIT (dissolved in olive oil) 75 was orally administered to rats in the shoulder 9/24 hours later, cardiac blood was collected under bendoparbital anesthesia, and 5-GOT and 5-GPT.

Direct and indirect bilirubin levels were measured.

The specimen (in 0.2% CMC-Nλ solution @#J) was ANI
It was orally administered 1 hour before T administration.

Oral administration of AN[T75 my/kg to rats increased 5-GOT and 5-GPT, as well as direct and indirect bilirubin levels.

No effect of suppressing the increase in these biochemical parameters was observed in the Ginsenoside Ro50.200x9/ni9 administration group. (Results omitted) 5.0C1, induced acute hepatitis test in rats CC1, (dissolved in olive oil) 1-u/9
was administered subcutaneously, and 24 hours later, cardiac blood was collected under bendoparbital anesthesia and 5-GOT and 5-GPT were measured.

The specimen (suspended in 0.2% CMC-Na solution) was CC1,
The drug was orally administered 1 hour after administration.

The results are shown in Table 1. CCI was administered to rats.

When administered subcutaneously, 5-GOT and 5-GOT
-GOT value increased. Ginzenocide R, 200m9
An effect of significantly suppressing the increase in 5-GOT and 5-GPT values was observed in the 9 administration group.

Table 1 Rat CC1, Ginzeno intact against induced acute hepatitis 7117±12 75=10 controls”
10 874±54 783±5
2X Zenocide R, 501075B±37 5
80 Saturday 45 subjects were orally administered CCl4 1 hour after injection, and blood was collected 24 hours after the injection.

As a control, 0.2% CMC-Na solution was orally administered.

1 Each measured value is shown as the average value ±S, E.

e′ There is a significant difference from the control group (p<0.01).

6. CC1, Induced Chronic Hepatitis Test This injection was induced in rats by inhaling CC14 gas once a day for 15 minutes, three times a week for 8 weeks. Test drug (0.2%
CMC-Na (suspended in CC1) was orally administered daily from the day of gas inhalation, and 8 weeks later, cardiac blood was collected under bendoparbital anesthesia, and 5-CIOT, S-GPT, and ALPSLD were administered.
H, triglycerides, total cholesterol content, and niglobulin lysis time (ELT) were measured. Immediately after blood collection, the liver and thoracic aorta were removed to measure the amount of tissue hydroxyproline.

■) Measurement results of blood biochemical parameters
When C1 is inhaled for 8 weeks, as shown in Table ■,
5-GOT, sGPT, ALP, LDH, and total cholesterol levels increased, but triglyceride levels decreased. In the Ginsenoside R8200x9/Kg administration group, an effect of significantly suppressing the increase in total cholesterol level was observed, but in s-G.

No effect on suppressing the increase in T,5-GPT values was observed.

(The following is a blank space) 2) Measurement of the amount of hydroxyproline The amount of hydroxyproline in the excised liver and blood vessels was measured by Woessner's method.

When examining the effect of hydroxyproline levels on connective tissue proliferation in the liver and blood vessel walls, as shown in Table V, the levels of hydroxyproline in the liver and blood vessels of CC1-induced hepatitis rats were similar to those of the intact group. It increased compared to the previous year. In the Ginsenoside Ro200ff9/Kg administration group, an effect of significantly suppressing the increase in the amount of hydroxyproline in blood vessels was observed, and a tendency to suppress the increase in hydroxyproline in the liver was also observed.

Table V Tissues of CCl4-induced chronic hepatitis in rats Hydroxyproline subjects were orally administered daily for 8 weeks, and tissue hydroxyproline content was measured.

For al control, 0.2% CMC-Na solution was orally administered.

b) Each measured value is shown as an average value.

There is a significant difference compared to the el control group (p<0.OL
).

3) Measurement of the dissolution time (ELT) of niglobulin Blood collected under Bendoparbital anesthesia was cooled with water at 3.8 mL.
Add 1/10% sodium citrate solution and incubate at 4°C.
Using plasma obtained by centrifugation at 000Xg for 10 minutes,
ELT was measured by the method of Kaul et al.

As shown in Table 3, the ELT of CC1 treated rats was longer than that of uninjured rats. In the Ginzenoside R6200y9/Kg administration group, an effect of significantly suppressing the prolongation was observed.

(Left below) 200 10 0.86 soil 0
．． 10824±1.09" Table ■ Dissolution time of Neeglobulin in CC1,i all chronic hepatitis in rats Dose Dissolution time of Neeglobulin 1 Injured 7143±19 Control" 10
347 Sat 35 Kinzenocide R6501027g±27200
to 248±30C1 subject to 8
Niglobulin was orally administered every day for a week, and the dissolution time of Niglobulin was measured.

1' control was orally administered with 0.2% CMC-Na solution.

bl Each measured value shows the average value = S, E.

C' There is a significant difference from the control group (p<0.00°7,
A method for measuring biochemical parameters in the blood.

5-GOT, 5-GPT, ALP, L in the above experiment
DH,) liglyceride, total cholesterol level, direct bilirubin level, and indirect bilirubin level were measured using a multi-item automatic analyzer (SMAC, Toshiba 60S autoanalyzer).

8. Statistical processing of experimental results The above experimental results were calculated using mean standard difference (S, E,), and significance was determined using Student's test method.

The anti-hepatitis activity of ginsenoside R0 was investigated using various experimental models, and the strength of its action was compared with that of oleanolic acid, glinnuresic acid, and glinnuresic acid.The results are summarized as follows.

First, when the effects of oral administration of ginsenoside R6 and oleanolic acid on D-galactosamine-induced acute hepatitis were investigated, ginsenoside Ro showed an effect of suppressing D-galactosamine-induced acute hepatitis. However, oleanolic acid, which is an aglycone of ginsenoside R6, showed a tendency to suppress the effect, but the activity was not significant. Furthermore, when we examined the effects of intraperitoneal administration of ginsenoside R0 and oleanolic acid on D-galactosamine-induced acute hepatitis, both ginsenoside R8 and oleanolic acid showed significant inhibitory activity, but the strength of the action was greater than that of ginsenoside R8. This was followed by leanolic acid.

In addition, when we examined the effects of glycylresin acid, which is currently in clinical use, and glycylresin acid, which is an aglycone, on D-galactosamine-induced acute hepatitis, we found that these also had the effect of significantly suppressing 5GOT and 5-GPT activities. When compared on a molar basis, ginsenoside R0 showed the strongest activity, and its potency was about three times that of glycylresin acid.

The effect of ginsenoside Re on hepatitis caused by bile duct damage was investigated in an ANIT-induced acute hepatitis model, but no activity was found in ginsenoside R0 to suppress AN[T-induced acute hepatitis.

Next, the effect of ginsenoside R0 on CCl4-induced hepatitis was investigated. Ginsenoside R0 was found to have the effect of significantly suppressing the increase in 5-GOT and 5-GPT values in CC1-induced hepatitis, but it was not effective in suppressing the increase in 5-GOT and 5-GPT values in CCt-induced chronic hepatitis. No inhibitory effect was found. However, when we examined the effect on connective tissue proliferation induced during chronic hepatitis using histology and hydroquineproline levels as indicators, we found that ginzenoside R6 suppresses the proliferation of connective tissue in the liver and blood vessels. r2.

Furthermore, it is said that the blood coagulation-fibrinolysis system becomes abnormal during chronic hepatitis. Therefore, when the fibrinolytic ability of rats with chronic hepatitis was examined using ELT, the ELT of rats with chronic hepatitis was longer than that of intact rats. In the Ginsenoside R8 administration group, an effect of suppressing the prolongation was observed.

Based on the above, ginzenoside R0 was found to be effective against D~galactosamine and CC1-induced hepatic model, and its mechanism of action seems to be a membrane stabilizing effect on hepatocytes, but the detailed mechanism of action is unclear. The order is currently unknown. In addition, CC1, 5-GOT and 5- during induced chronic hepatitis
Although the GPT level did not increase, the effect of suppressing the growth of connective tissue and the effect of suppressing the prolongation of ELT1 were observed.
This is thought to be due to the action mediated by the fibrinolytic activity found in

In any case, it is clear that ginsenoside R0 has an antihepatitis activity that is equivalent to or greater than the three compounds compared.

Next, the results of an acute toxicity test of Ginsenoside R6 will be shown.

Claims (1)

[Claims] 1. A prophylactic/therapeutic agent for hepatitis containing ginsenoside R_0 as an active ingredient.