JP3130368B2 - Liver function improving drugs - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an agent for improving liver function, and more particularly to a liver function containing lysospermic acid B and / or a pharmaceutically acceptable salt thereof as an active ingredient, which is isolated and purified from an extract of ginseng root. It provides an improving drug.

[0002]

2. Description of the Related Art Salvia m.
Studies on the root extract of iltiorrhiza Bunge have revealed the stereochemical structure of lysospermic acid B, which is the active ingredient, and it is known that lysospermic acid B and its salts have remarkable renal function improving effects. (JP-A-1-268682).

[0003] Also, it has been reported that lysospermic acid B and its salts increase the amount of prostaglandin in urine, suggesting that it affects the arachidonic acid cascade.

On the other hand, leukotriene (LT), which is a lipoxygenase metabolite of arachidonic acid, has an inflammatory reaction,
It is involved not only in allergic reactions but also in immune reactions and plays an important role in the pathogenesis of various liver diseases. When an LT synthesis inhibitor is administered to an experimentally induced hepatocellular injury model, hepatocellular injury occurs. Is reported to be reduced (Mizoguchi, Y. et al . Gastroenterologia Jpn. 23,
263-267, 1988).

[0005] However, there is no known effect of lysospermic acid B or a salt thereof on the liver function.

[0006]

There are no known conventional liver function improving drugs which are effective for both acute and chronic liver damage. In addition, it enhances the reaction of the cyclooxygenase system in the arachidonic acid cascade as a pharmacological action,
-No drug that suppresses the reaction of the lipoxygenase system is known. An object of the present invention is to provide a liver function improving drug effective for both acute and chronic liver injury having the above advantages.

[0007]

Means for Solving the Problems The present inventors have found that lysospermic acid B and salts thereof have an anti-inflammatory effect and an anti-fibrotic effect, and have reached the present invention.

[0008] That is, the present invention is a liver function improving drug containing lysospermic acid B represented by Chemical Formula 1 and / or a pharmacologically acceptable salt thereof as an active ingredient. Hereinafter, the present invention will be described in detail.

<1> Liver Function Improving Agent of the Present Invention The liver function improving agent of the present invention contains lysospermic acid B represented by Chemical Formula 1 or a pharmacologically acceptable salt thereof as an active ingredient. Examples of the salt include a magnesium salt, a potassium salt, an ammonium salt, and a calcium salt represented by Chemical Formula 2, and these can be used alone or as an arbitrary mixture.

[0010] These compounds are used as they are as drugs, and the administration method, dosage and dosage form in this case are as follows. As an administration method, either oral administration or injection can be selected. The dose by oral administration varies depending on the age and symptoms of the patient, but it is considered that a predetermined effect can be expected by using 50 to 100 mg per day for one adult patient except in severe cases.

[0011] The dosage forms include lactose, potato starch, sodium alginate, pharmaceutically acceptable as oral preparations,
By compounding excipients such as precipitated calcium carbonate, aminoacetic acid, synthetic aluminum silicate, and crystalline cellulose,
Powders, granules, tablets, dragees and capsules can be prepared.

The lysospermic acid B salt is easily soluble in water, and therefore, is usually used as an injection in an amount of 1 to 10 mg per day for one adult patient. It is thought that the effect of can be expected. In this case, an injection can be prepared by adding an isotonic agent such as physiological saline, which is pharmaceutically acceptable, and a pH adjusting agent such as sodium hydroxide solution, in addition to distilled water for injection.

<2> Method for producing lithospermic acid B and its salt Lisospermic acid B and its salt are described in, for example,
It is obtained by the production method disclosed in US Pat.

The extract is obtained by separating a predetermined fraction from a ginseng extract by column chromatography. That is, for example, after adjusting the extract of Dansin to pH 2 to 4, the extract is subjected to column chromatography and washed with water, and then washed with an aqueous solution such as magnesium acetate or calcium acetate. After washing the column again with water, the column is eluted with water-containing methanol, and the eluted fraction is collected and further purified to obtain lysospermic acid B salt.

Similarly, the extract of red ginseng is subjected to column chromatography and monitored by thin layer chromatography to collect a fraction containing the main spot, which is again monitored by thin layer chromatography to include the main spot. Lisospermic acid B purified by fractionating the fraction by column chromatography
A magnesium salt and ammonium lysospermic acid B salt are obtained.

<3> Examination of liver function-improving effect The liver function-improving effect of lysospermic acid B and its salts can be determined by measuring serum transaminase activity in the case of acute liver injury, Considered by electron microscopic observation. In the case of chronic liver injury, measurement of hepatic reserve (values of serum albumin, serum protein, cholinesterase activity, etc.), fibrosis of liver tissue (hydroxyproline content, and liver histological examination by light microscopic or electron microscopic observation, etc.) ) As an index.

[0017]

The lysospermic acid B and its salts, which are the active ingredients of the agent for improving liver function of the present invention, inhibit the synthesis of leukotriene in liver tissue experimentally induced, and increase the production of prostaglandins.

In general, in experimentally induced liver failure, leukotriene, a lipoxygenase metabolite of arachidonic acid, acts impaired and prostaglandin, a cyclooxygenase metabolite, is thought to act protectively. .

Therefore, lysospermic acid B and its salts are considered to reduce and improve liver damage. Furthermore, lysospermic acid B and its salts have the effect of reducing the amount of liver collagen and reducing liver fibrosis, and from this effect also have the effect of improving liver damage.

[0020]

EXAMPLES Examples of the present invention and experimental examples showing the effects of lysospermic acid B and its salts will be described below.

[0021]

[Production Example] Production of magnesium salt of lyspermic acid B. Ginseng is extracted with water, and the extract is adjusted to about pH 3 with hydrochloric acid, and then subjected to MCI-gel CHP-20P column chromatography to convert lysospermic acid B into a column. Adsorbed on carrier. After washing the column with water, a 0.1 M magnesium acetate solution was poured and washed again with water, followed by a gradient elution of water-methanol, which was positive for the ferric chloride reagent eluted with 20 to 40% methanol. Fractions were collected and concentrated. The obtained fraction was purified by gel filtration using Sephadex LH-20 to obtain lyspermic acid B magnesium salt. The yield was 2-3%.

[0022]

[Experimental Example 1] <1> Effect of lysospermic acid B on 5-lipoxygenase activity The effect of lysospermic acid B salt on 5-lipoxygenase activity, an enzyme that induces arachidonic acid metabolism, was examined.

The metabolites of radiolabeled arachidonic acid by polynuclear leukocytes were measured as follows.

(1) Method of Preparing Rat Peritoneal Polynuclear Leukocyte Suspension 10 ml of sterile 8% casein was injected intraperitoneally into male Wistar rats (220 to 250 g). 12 ~
Fourteen hours later, the peritoneal cavity was perfused with Hanks' solution (HBSS), and peritoneal polynuclear leukocytes were collected. The perfusate was centrifuged at low temperature and the cells were collected. These cells were treated with 10% fetal calf serum (FCS), streptomycin 100 μg / ml.
And RPMI 164 containing penicillin 100 units / ml
After washing with medium 0, the cells were diluted with the same medium to prepare a cell suspension of 5 × 10 ⁶ cells / ml. Over 90% of the cells thus obtained were polynuclear leukocytes.

(2) Influence of lysospermic acid B on arachidonic acid metabolism by rat peritoneal polynuclear leukocytes To the peritoneal polynuclear leukocytes (5 × 10 ⁶ / ml) obtained above, various concentrations of magnesium lithosperminate B were added. And incubated at 37 ° C for 2 hours. After the culture, the cells were washed with phosphate buffered saline (PBS), and the cell concentration was adjusted to 5 × 10 ⁶ / ml with PBS containing 2 mM calcium chloride. 25 μl / ml [1- ¹⁴ C] -arachidonic acid (10 ⁴ cpm / nmol / μl, methanol solution) and 5 μl / ml calcium ionophore A23187 were added to the cell suspension.
(1 mM ethanol solution: hereinafter referred to as CAI), and the cells were further cultured for 20 minutes.

After the culture, the reaction solution was adjusted to pH 3 by adding formic acid, and twice the amount of chloroform was added to mix well. After centrifugation, the chloroform phase was concentrated by evaporation. This was analyzed by reversed phase high performance liquid chromatography (HPLC). The mobile phase was methanol: water: acetic acid = 75: 25: 0.01,
The flow rate was 1 ml / min, and the analytical column was TSK-gel 12
Using 0T (Tosoh), the absorbance of the eluate was simultaneously monitored at 270 nm and 235 nm. The results are shown in FIG.

The eluate from the column was collected every 30 seconds, and the radioactivity contained in each fraction was measured with a liquid scintillation counter. Was calculated.

In the control group, the same procedure was carried out without adding magnesium lyspermate B. As a result, it was found that LTB ₄ and 5-HETE (hydroxyeicosatetraenoic acid) are mainly produced as metabolites of arachidonic acid, and these are produced in the presence of about 10 ⁻⁸ M magnesium lithospermate B. Was found to be suppressed.

Further, we conducted experiments in the same manner by changing the concentration of Risosuperumin acid B magnesium, and LTB ₄ 5-HET
The 5-lipoxygenase activity was measured from the amount of E produced, and as shown in FIG. 2, it was found that the activity was suppressed by magnesium lyspermate B.

[0030] (3) subjected to the same experiment shadow Risosuperumin acid B magnesium on peritoneal polymorphonuclear leukocytes 5-lipoxygenase activity <br/> Symphony Then, the centrifuged supernatant of homogenate of peritoneal polymorphonuclear cells as an enzyme solution Was. Peritoneal multinucleated leukocytes prepared in the same manner as above were treated with PBS containing 2 mM calcium chloride for 5 hours.
× 10 ⁷ / ml and sonicated (Branson sonicat
or model 350S), 10,000 xg,
The mixture was centrifuged at 4 ° C. for 10 minutes, and the obtained supernatant was used as a 5-lipoxygenase enzyme solution.

2 mM ATP was added to 100 μl of this enzyme solution.
0.05 M Tris-HCl buffer (pH 7.5) containing 2 mM calcium chloride and various concentrations of lysospermic acid B
After adding magnesium and culturing at 37 ° C for 5 minutes, [1
- ¹⁴ C] - arachidonic acid ^{5μl (10 5 cpm / nmol /} μl
(Ethanol solution) was added, and the cells were cultured at 30 ° C. for 3 minutes.
After the cultivation, the reaction stop solution (diethyl ether: methanol:
300 μl of 1M citric acid = 30: 4: 1) was added, mixed well, and deproteinized by centrifugation (3000 rpm, 4 ° C., 15 minutes) to obtain a supernatant. The supernatant was concentrated by evaporation, dissolved in 50 μl of methanol, and subjected to reverse phase HPLC to measure the 5-lipoxygenase product. In the control group, the measurement was performed in the same manner without adding magnesium lithospermate B.

As a result, the main product was 5-HETE without adding magnesium lyspermate B (FIG. 3). Furthermore, it was found that the production of 5-HETE was suppressed by magnesium magnesium lyspermate.

Therefore, various concentrations of magnesium B-lysospermate were added directly to the peritoneal polynuclear leukocyte suspension, and 5-HETE was quantified in the same manner as described above. Was found to be suppressed by magnesium lyspermate B.

[0034]

[Table 1]

From the above results, it was clarified that magnesium lyspermate B suppresses leukotriene production and 5-lipoxygenase activity of peritoneal polynuclear leukocytes.

[0036]

[Experimental Example 2] Next, the effects of magnesium lysospermate B on acute liver injury were examined using experimental animals having acute liver injury.

<1> Induction of D-galactosamine hepatocellular injury model D-galactosamine (purchased from Wako Pure Chemical) at 700 mg / kg was added to male Wistar rats (170 to 180 g).
And lipopolysaccharide (LSP, derived from Salmonella enteritidis ,
Difco) (1 μg / rat).

Four hours after the administration, the rats were laparotomized under ether anesthesia, and blood was collected from the inferior vena cava. Liver tissues were taken at the same time as measuring serum transaminase levels, and the liver tissues were examined under a light microscope.

Furthermore, liver infiltrating polynuclear leukocytes were separated. In addition, when examining the effect of magnesium lithospermate, 10 mg / mg magnesium lithospermate was used.
Oral administration was performed at a rate of kg body weight.

<2> Effect of magnesium lysospermate B on serum transaminase (hereinafter referred to as “LSA”)
Sometimes. Affect the 4 hours after administration of the), and abdominal rat under ether anesthesia,
Blood was collected from the inferior vena cava and serum transaminase levels were measured. Table 2 shows ALT (alanine transaminase) values and serum AST (aspartate transaminase) values.

[0041]

[Table 2]

From these results, it was found that magnesium lysospermate B suppresses an increase in serum transaminase level.

<3> Liver histological examination Liver tissues were fixed with formalin, stained with hematoxylin and eosin, and examined under a light microscope.

As a result, in the non-administered group of magnesium lysospermate B, a decrease in hepatocyte staining and degeneration of empty cells were observed. In addition, cell necrosis around the Gleason sheath and infiltration of mononuclear cells were observed.

On the other hand, infiltration of mononuclear cells was observed in the group administered with magnesium lysospermate B, but necrosis or degeneration of hepatocytes was hardly observed.

<4> Influence of hepatic infiltrated polynuclear leukocytes on prostaglandin (PG) produced (1) Isolation of rat hepatic infiltrated polynuclear leukocytes Rats were anesthetized by injecting pentobarbital intraperitoneally into the abdominal cavity. The pulse was exposed and an 18 gauge indwelling needle was inserted. Calcium-free N-2-hydroxyethylpiperazine-
Calcium N'-2-ethanesulfonate (HEPES,
Sigma) Hank's solution (HBSS) solution 100m
1 followed by 0.05% collagenase (type I
V, HBES100 containing HEPES containing Wako Pure Chemical Industries, Ltd.)
ml, HEP containing 0.05% pronase E
Flow 50 ml of the ES-added HBSS solution at a flow rate of 12 ml /
Shed in minutes.

After perfusion, the liver was removed, minced with ophthalmic scissors, and HEPES containing 0.05% pronase E.
Hepatocytes were digested by shaking for 20 minutes at 37 ° C. in 50 ml of the added HBSS solution.

After filtering the undigested substance with gauze, centrifugation (4
(00 × G, 3 minutes, 4 ° C.) to remove hepatocytes, and the mixed red blood cells were lysed by hypotonic treatment. The obtained cell pellet was further washed three times with HBSS, and then subjected to the following experiment.

(2) Measurement of prostaglandin (PG) The liver-infiltrated polynuclear leukocytes separated as described above were suspended in PBS containing 2 mM calcium chloride, and the cell concentration was adjusted to 1 × 1.
Adjusted to 0 ⁷ cells / ml.

The cell suspension was added with 25 μl / ml of [1-
¹⁴ C] -arachidonic acid (10 ⁴ cpm / nM / μl, ethanol solution) and 5 μl / ml CaΙ were added,
Incubated for 30 minutes.

After the completion of the reaction, formic acid is added to adjust the pH of the reaction solution to pH.
It was adjusted to 3.0, and twice the amount of chloroform was added and mixed well. This solution was centrifuged (1500 × G, 10 minutes,
4 ° C.), and the chloroform phase was separated, evaporated and concentrated. Then, the mobile phase (acetonitrile:
Water: phosphoric acid = 33: 67: 0.1) at a flow rate of 1.0 ml /
The eluate was monitored at 195 nm using an analytical column (TSK-Gel 120T, Tosoh) to analyze the generated cyclooxygenase product.

Simultaneously, the eluate of the column was collected every 30 seconds, and the radioactivity contained in each fraction was measured with a liquid scintillation counter to quantify the amount of prostaglandin.

When the effect of magnesium lyspermate B was examined, samples were collected in the same manner after culturing liver-infiltrated polynuclear leukocytes for 2 hours in the presence of various concentrations of magnesium lithospermate.

As a result, as shown in FIGS. 4 and 5, non-parenchymal liver cells of normal rats consisted of PGD ₂ , PGF ₂ α, 6-keto-
PGF ₁ α, it was confirmed that the production of PGE _2, TXB _2. Compared with normal rats, the amount of all prostaglandins and TXB ₂ was significantly reduced in the liver-impaired rats. On the other hand, in the liver injury rats administered Risosuperumin acid B magnesium, PGD ₂ and 6-keto-PGF ₁ alpha
The amount was significantly increased as compared with the non-administration group.

From the above results, magnesium lysospermate B suppresses the induction of acute liver injury by D-galactosamine, and further suppresses the decrease in prostaglandin production from hepatic infiltrating leukocytes, in other words, prostaglandin. Was found to enhance the production of.

[0056]

[Experimental Example 3] Next, using a chronic liver injury model, the effect of magnesium lithospermate B on liver injury was examined.

<1> Induction of Carbon Tetrachloride Hepatopathy Model Male Wistar rats (170-180 g) were initially injected subcutaneously with 0.5 ml / body weight of 100% carbon tetrachloride (purchased from Wako Pure Chemical Industries). From the first time, 0.3 ml / body weight of 40% carbon tetrachloride (dissolved in olive oil) was injected subcutaneously three times a week.

Seven weeks later, serum transaminase was measured, and at the same time, the amount of hydroxyproline in the liver tissue and the liver tissue were examined under a light microscope. In addition, liver infiltrating polynuclear leukocytes were isolated.

When examining the effect of magnesium lysospermate, magnesium lysospermate B should be used for 1
0 mg / kg body weight was orally administered once a day.

<2> Effect of magnesium lysospermate B on serum transaminase As in Experimental Example 1, serum transaminase levels were measured. Table 3 shows the serum ALT value and the serum AST value.

[0061]

[Table 3]

As is evident from the results, an increase in serum transaminase level, particularly serum AST level was suppressed.

<3> Histological Examination of Liver Liver tissue was fixed with formalin, stained with hematoxin-eosin and Azan-Mallory, and examined under a light microscope.

As a result, in the hematoxylin / eosin stained non-administered group of magnesium lyspermate B,
In the liver tissue, fat deposition and necrosis including bridging necrosis from the area around the Gleason sheath to the intralobular area were observed. On the other hand, fat deposition and focal necrosis in the lobules were observed in the magnesium lysospermate B administration group, but the necrosis was lighter than in the non-administration group.

When examined by Azan Mallory staining, high fibrosis such as bridging fibrosis and pericella fibrosis was observed in the group not administered with magnesium lyspermate B. On the other hand, in the group administered with magnesium lithospermate B, only slight fibrosis extending in a star cap shape from the Gleason sheath was observed.

<4> Effect of Liver-Infiltrated Polynuclear Leukocytes on Prostaglandins Produced In the same manner as in Experimental Example 2, rat liver-infiltrated polynuclear leukocytes were separated and analyzed for brostaglandin production.

As a result, as shown in FIGS. 6 and 7, non-hepatic parenchymal cells of normal rats consisted of PGD ₂ , PGF ₂ α, 6-keto-
PGF ₁ alpha, it was confirmed that produce PGE _2, TXB _2.

In particular, normal rat liver non-parenchymal cells are PGD
Mainly produce ₂ and TXB _2, further UV- peak absorbance peak with radioactivity match. On the other hand, the liver failure rats amount of all prostaglandins and TXB ₂ were significantly reduced. However, the liver injury rats administered Risosuperumin acid B magnesium, PGD ₂ and TXB ₂ content is increased significantly compared to the untreated group, as a result, decreased production of total prostaglandin amount suppressed markedly Sa
Was .

<5> Effect of magnesium lysospermate B on the amount of liver collagen in liver tissue In order to examine the effect of lysospermic acid B salt on hepatic fibrosis, collagen in liver tissue was examined according to the method of Jamal et al. The amount was measured as the content of hydroxyproline, the main constituent amino acid of collagen.

As a result, the hydroxyproline content was 0.913 in the group not administered with magnesium lyspermate B.
It increased to ± 0.143 mg / g. On the other hand, 0.673 ± 0.112 in the magnesium lysospermate B administration group.
mg / g. From this, it was found that lysospermic acid B salt has an effect of reducing liver fibrosis.

From the above results, magnesium lysospermate B suppresses the induction of chronic liver injury by carbon tetrachloride, reduces liver fibrosis, and further reduces the production of prostaglandin produced from hepatic infiltrating leukocytes. And it is considered to be effective not only for acute liver injury but also for chronic liver injury.

[0072]

Experiment 4] Risosuperumin acid B magnesium results of measurement of LD ₅₀ for are as follows. (1) Intraperitoneal administration (ddY male mouse, 6 weeks old, body weight 3
1～34G, measured by Up and Down _{method) LD 50> 2195mg / kg (} 2) Oral administration (ddY male mice, 6 weeks old, body weight 31
~35g, measured by the Up and Down method) LD _50> 3000mg / kg

[0073]

[Formulation Examples] The following are formulation examples.

[0074]

Formulation Example 1 (Production method) The composition shown in Table 4 is converted into a tablet weighing 240 mg per tablet according to the tablet production method of the Japanese Pharmacopoeia. (Dosage and administration) Usually, for adults, oral administration of 2 tablets at a time, 3 times a day (increase or decrease) can be expected.

[0075]

[Table 4]

[0076]

Formulation Example 2 (Preparation method) 2 g of magnesium lithospermate B is dissolved in physiological saline in Japan to make up to 1000 ml. (Usage and dosage) Usually, one tube (5 ml) once a day for adults
Is expected to be effective by subcutaneous or intravenous administration.

[0077]

EFFECT OF THE INVENTION Lysospermic acid B as an active ingredient and
/ Or liver function improving agent of the present invention having free a salt thereof, also has the effect of improving these in either acute liver disorders and chronic liver failure. It is considered that this effect is based on the action of inhibiting 5-lipoxygenase activity in the liver to inhibit the synthesis of leukotriene, and further suppressing the decrease in production of prostaglandin from hepatic infiltrating leukocytes.

[Brief description of the drawings]

FIG. 1 is a view showing an elution pattern of arachidonic acid metabolite by reverse phase HPLC. (B) untreated magnesium lithospermate B, (C) treated, (A) standard compound.

FIG. 2 is a graph showing suppression of lipoxygenase activity by magnesium lyspermate B.

FIG. 3 is a view showing an elution pattern of arachidonic acid metabolite by reverse phase HPLC. (B) untreated magnesium lithospermate B, (C) treated, (A) standard compound.

FIG. 4 is a view showing an elution pattern of arachidonic acid metabolite by reverse phase HPLC. (B) is a normal rat, (C) is a hepatopathy rat induced by galactosamine, (D) is a hepatopathy rat treated with magnesium lysospermate B,
(A) is a standard compound.

FIG. 5 is a graph showing the amount of arachidonic acid metabolites.

FIG. 6 is a view showing an elution pattern of arachidonic acid metabolite by reverse phase HPLC. (B) is a normal rat, (C) is a liver injury rat induced by carbon tetrachloride, (D) is a liver injury rat treated with magnesium magnesium lysospermate, and (A) is a standard compound.

FIG. 7 is a graph showing the amount of arachidonic acid metabolites.

[Explanation of symbols]

In Figure 1, 3, A is LTB _4, B represents a 5-HETE.
4 and 6, 1 is 6-keto-PGF ₁ α, 2 is TXB ₂ ,
3 indicates PGF ₂ α, 4 indicates PGE ₂ , and 5 indicates PGD ₂ . In FIG. 5, GAL represents D-galactosamine, and in FIGS.
SA stands for magnesium magnesium lithospermate. FIG.
PGs represents the total prostaglandin amount.

Continuation of the front page (56) References Biochemical Pharmology, 43 (2), (1992), pp. 147-152 (58) Fields investigated (Int. Cl. ⁷ , DB name) A61K 31/343 A61P 1/16 A61K 35/78 C07D 307/86 CA (STN) REGISTRY (STN)

Claims (2)

(57) [Claims] A hepatic function-improving agent comprising lysospermic acid B represented by Chemical Formula 1 and / or a pharmacologically acceptable salt thereof as an active ingredient. Embedded image 2. The hepatic function improving drug according to claim 1, wherein the pharmacologically acceptable salt is a salt represented by Chemical formula 2. Embedded image In the formula, M ²⁺ is Mg ²⁺ , 2K ⁺ , 2NH ₄ ⁺ or Ca ²⁺
Represents