JP2000191544A - Hepatic fibrogenetic inhibitor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject inhibitor having significant proliferation inhibitory effect against hepatic Ito cells and protein formation inhibitory effect by including crude-drug Scutellarial Radix-derived flavonoid compound(s). SOLUTION: This inhibitor contains at least one kind of crude-drug Scutellarial Radix-derived flavonoid compound (e.g. Baicalein of formula I, Baicalin of formula II, Wogonin of formula III, Wogonoside of formula IV). The inhibitor is pref. if the form of a crude drug, inhibiting hepatic fibrogenesis in chronic hepatopathy involving fibrosis such as chronic hepatitis, its daily dose being 1-2,000 mg/kg, pref. 1-1,000 mg/kg or so.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to an agent for suppressing hepatic fibrosis associated with fibrosis such as chronic hepatitis, which comprises one or more flaponoid compounds derived from the herbal medicine Ogon.

[0002]

BACKGROUND ART Chronic hepatitis (viral, alcoholic)
In many of the various liver diseases such as, Ito cells in the liver are activated into myofibroblast-like cells,
It is considered to be the main mechanism of liver fibrosis by actively growing and producing extracellular matrix such as collagen. Therefore, the activation of Ito cells into myofibroblast-like cells, that is, liver fibrosis in the liver, has been desired for the development of a drug capable of effectively suppressing this.

[0003] Therapeutic agents for viral chronic hepatitis include:
Conventionally, "interferon" has been used. The feature of this "interferon" is that it suppresses the viral load and activity against chronic viral hepatitis, and as a result, it has been confirmed that the inflammation is alleviated and the liver fibrosis is suppressed. However, the effect of this “interferon” varies greatly depending on the causative virus, and even in the hepatitis C virus, which is expected to be most effective, the effect on the virus varies greatly depending on the virus subtype.
At present, more than half of the patients can expect only ineffective or transient effects, and there is a problem that drug prices are relatively high.

As a Chinese herbal prescription for chronic hepatitis, Sho-saiko-to has been known for a long time. This "Sho-saiko-to" has been shown as a Chinese herbal prescription to suppress inflammation and suppress liver fibrosis in chronic hepatitis. However, the effect of Sho-saiko-to on liver fibrosis is clearly inadequate, and because Sho-saiko-to is a processed product of a complex crude drug, it is not only difficult to quantify the drug effect, but also It is difficult to change to various dosage forms such as changing the dosage route, adjusting the absorption rate, etc., and for this reason, there is no selectivity of the dosage and administration route according to each disease state. . Another major problem is that the drug price is high because it is a crude drug product.

On the other hand, Scutellaria (Labiatae Scutellari)
a baicalensis Georgi)
utellariae Radix) has been used in various Chinese herbal prescriptions for anti-inflammatory effects since ancient times. Above all, “Sho-saiko-to” has been widely used in Japan as a drug with a listed price, mainly for the purpose of suppressing inflammation of chronic hepatitis. In animal experiments in hepatitis models, it is known that Sho-saiko-to suppresses the expression and proliferation of myofibroblast-like cells derived from the activation of Ito cells in the liver. Since it is a processed product of a crude drug, it is not known which crude drug causes these effects.

[0006] Ougon is baicalein having a similar chemical structure, and its glycoside form baicalin (B
aicalin), wagonin, its glycoside form, wogonoside, and flavonoid compounds such as oroxylin-A and Skullcaflavone, which have slightly different side chains. ing. The chemical structure of baicalein is

[0007]

Embedded image

The chemical structural formula of baicalin is represented by the following formula:

[0009]

Embedded image

The chemical structural formula of Wogonin is represented by the following formula:

[0011]

Embedded image

The chemical structure of Wogonoside is represented by the following formula:

[0013]

Embedded image

## EQU1 ## These flavonoid compounds are known to suppress the growth of vascular smooth muscle cells and the like in cell culture experiments. Among these flavonoid compounds, baicalein is reported to have the strongest growth inhibitory effect on vascular smooth muscle cells. However, no report has been reported on its effect on Ito cells in the liver. In the present invention, for example, baicalein was used to clarify, for the first time, a remarkable growth-suppressing effect and a collagen-forming suppressing effect of a crude drug, a gogon-derived flaponoid compound, in cultured Ito cells.

[0015]

DISCLOSURE OF THE INVENTION The present inventors have found that flavonoid compounds derived from the herbal medicine Ogon, such as baicalein, have a remarkable growth inhibitory effect on liver hepatic Ito cells and a protein formation inhibitory effect. I found that. In chronic liver diseases such as chronic hepatitis (viral or alcoholic), Ito cells are activated into myofibroblast-like cells and actively proliferate to produce extracellular matrix such as collagen. And is known to be the main player in the mechanism of liver fibrosis, and therefore, the present invention relates to baicalein (Baicalei).
n), etc., to demonstrate that flavonoid compounds derived from the herbal medicine, Ogon, have a hepatic fibrosis inhibitory effect by directly acting on Ito cells. Is what you do.

[0016]

DISCLOSURE OF THE INVENTION The present invention relates to baicalein, wogonin, baicalin (Ba).
The present invention relates to a hepatic fibrosis inhibitor comprising one or two or more flaponoid compounds derived from the herbal medicine Ogon, such as icalin) or wogonoside.

The flaponoid compound derived from the herbal medicine Ogon such as baicalein, wogonin or baicalin of the present invention is a known compound and can be obtained by a known method. In addition, the flaponoid compound derived from the herb of the present invention may be used alone, or two or more of these may be used in an appropriate combination. Furthermore, crude drugs containing one or more of these can also be used as the active ingredient of the present invention. Further, the liver fibrosis inhibitor of the present invention is particularly preferably used as a liver fibrosis inhibitor for chronic liver diseases accompanied by fibrosis such as chronic hepatitis.

Next, the action of the liver fibrosis inhibitor of the present invention will be described more specifically. The inhibitory effect of baicalein on the proliferation of first-generation and second-generation subcultured cells and production of general proteins and collagen of Ito cells cultured from normal rat liver was examined. The inhibitory effect of baicalein on the proliferation was determined by conducting a DNA precursor ([ ³ H] thymidine) uptake experiment and a proliferating cell number measurement experiment.

In a DNA precursor uptake experiment, 10% fetal calf serum and platelet-derived growth factor (PDGF-BB), which activates Ito cells in hepatitis and changes in myofibroblasts and is involved in proliferation, are recognized. (25 ng / ml)
The effect of the concentration-dependent inhibition of baicalein on the uptake of DNA precursors of Ito cells stimulated by each of them was verified. The results are shown in Table 1 and FIGS. In this experiment, 10% fetal calf serum and 25 ng / ml PDGF
In both experiments using BB, a remarkable concentration-dependent effect of baicalein- [ ³ H] thymidine uptake inhibitory effect was observed. This inhibitory effect was also significantly significant at the lowest dose (3 μM).

In the experiment for measuring the number of proliferating cells, the effect of the concentration-dependent inhibition of baicalein on the increase in the number of cells in Ito cells over time by stimulation with 20% fetal calf serum was examined. In the results of the proliferating cell number measurement experiment shown in Table 2 and FIG.
A remarkable concentration-dependent cell growth inhibitory effect of baicalein was observed. The white circle in FIG. 3 indicates that the concentration of baicalein is 0 μm.
M, black circles indicate 3 μM, black triangles indicate 10 μM, and black squares indicate 30 μM.

The inhibitory effect of baicalein on the production of general proteins and collagen was tested by taking up amino acids ([ ³ H] leucine and [ ³ H] proline). In this experiment, the concentration-dependent inhibitory effect of baicalein on amino acid uptake by Ito cells, stimulated by 10% fetal calf serum, was examined. The results are shown in Table 3 and FIGS. From this experimental result, it can be seen that both [ ³ H] leucine and [ ³ H] proline have a small inhibitory amount, but a baicalein concentration-
The uptake inhibitory effect was significantly recognized. [ ³ H]
Baicalein showed a significant inhibitory effect at a concentration of 10 μM for leucine and ³ μM for [ ³ H] proline.

From the above experimental results of baicalein, it was proved that the flaponoid compound derived from the herbal medicine Hagone had a remarkable inhibitory effect on the growth of cultured Ito cells activated as a single substance and an inhibitory effect on the production of protein containing collagen. . This is because the flavonoid compound derived from the herbal medicine
In a chronic liver disease accompanied by fibrosis, by directly acting on the activated Ito cells, it is a result showing that the proliferation and the production of extracellular matrix such as collagen are strongly suppressed. The present invention strongly shows the usefulness as a liver fibrosis inhibitor.

The active ingredient of the present invention, the flaponoid compound derived from the herbal medicine Ougon, is a crude drug component and has been used as a crude drug since ancient times, and therefore has low toxicity.
It is an extremely safe medicinal ingredient. The hepatic fibrosis inhibitor of the present invention can be formulated into tablets, granules, injections and the like by a usual method, and can be orally or parenterally administered. The dosage of the active ingredient of the hepatic fibrosis inhibitor of the present invention varies depending on patients and symptoms, but is 1 m / day.
g / kg to 2000 mg / kg, preferably 1 mg / k
The dose is about g to 1000 mg / kg, but can be appropriately increased or decreased depending on the condition of the patient.

[0024]

The present invention will be described more specifically with reference to the following examples, but the present invention is not limited to these examples. In addition, Ito cells used in the following examples,
Cultured from normal rat liver.

Example 1 (Experiment of Uptake of DNA Precursor) Ito cells primarily cultured from rat (Sprague-Dawley) liver were implanted at 1 × 10 ⁴ cells / well in a 24-well culture plate, and a 20% embryo DME with bovine serum
The cells were cultured in M / F12 medium under standard cell culture conditions until confluence. The first-generation subcultured Ito cells thus monocultured were cultured in a thymidine-free culture solution (0.
Growth was stopped by culturing for 48 hours in a DMEM culture solution containing 5% albumin). Thus obtained,
The growth-arrested Ito cells were baicalein (0, 3, 10, 3)
0% or 100 μM), 10% fetal calf serum or 25 ng / ml platelet-derived growth factor (PDGF-B
B) Proliferation was stimulated for 24 hours in supplemented thymidine-free medium. These Ito cells were stimulated with 10% fetal calf serum in the latter 4 hours (from 20 to 24 hours), 25 ng.
/ Ml 12 hours late in PDGF-BB stimulation (up to 12 hours to 24 hours), ^- were added cultured [methyl 3 H] thymidine (1 [mu] Ci / ml). Some Ito cells were treated similarly except for fetal calf serum or PDGF-BB to confirm growth stimulation. Thereafter, these Ito cells were treated with a 20% trichloroacetic acid solution at 4 ° C. for 1 hour after washing with ice-cold phosphate buffer. After washing again with ice-cold phosphate buffer, these Ito cells were treated with 500 μl of 0.3 N NaOH / 0.1% sodium dodecyl sulfate.
Sulfate solution was collected, and the radioactivity was measured with a liquid scintillation counter. The experiment was repeated eight times each. The results are shown in Table 1 and FIGS. Significant concentration dependence of baicalein in both experiments
[ ³ H] thymidine uptake inhibitory effect was observed (On
e-factor ANOVA statistical processing; P <0.00
01). This inhibitory effect was significantly significant even at the lowest dose (3 μM) (Fisher PLSD te).
St statistical processing; P <0.0001).

[0026]

[Table 1]

Example 2 (Experiment for measuring the number of proliferating cells) Primary cultured rat liver Ito cells were seeded at 5 × 10 ³ cells / well in a 12-well culture plate, and were cultured in a DMEM / F12 culture medium supplemented with 20% fetal bovine serum. For 24 hours under standard cell culture conditions. Twenty-four hours later (day 0), these first generation subcultured Ito cells were cultured in 1 ml of DMEM / F12 medium supplemented with 1 ml of 20% fetal calf serum containing baicalein (0, 3, 10 or 30 μM). Was exchanged and cultured. The same procedure is performed every other day (2
Day 4 and Day 4) repeated. Over time (Day 0, 2
On days 4, 4 and 6), these Ito cells were treated with a 0.25% trypsin-EDTA solution and C
The number of cells was counted with an oulter counter. The experiment was repeated three times each. The results are shown in Table 2 and FIG.
In the proliferating cell number measurement experiment, a remarkable concentration-dependent cell growth inhibitory effect of baicalein was observed (Two-f).
actor ANOVA statistical processing; P <0.000
1).

[0028]

[Table 2]

Example 3 (Experiment of Uptake of Amino Acids ([ ³ H] Leucine and [ ³ H] Proline)) First-generation subcultured rat liver Ito cells were 24-well
1 × 10 ⁴ cells / well were implanted in a culture plate,
The cells were cultured in DMEM / F12 medium supplemented with 0% fetal calf serum under standard cell culture conditions until confluence. In the [ ³ H] leucine uptake experiment, the second generation subcultured Ito cells monolayer-cultured were treated with [ ³ H] proline uptake in a leucine-free culture solution (leucine-free DMEM culture solution supplemented with 0.5% albumin). In the experiment, leucine or proline was starved by culturing for 48 hours in a proline-free culture solution (DMEM culture solution containing 0.5% albumin). These Ito cells are baicalein (0,
[3H] in the presence of ( ³ , 10, 30 or 100 μM).
In the leucine uptake experiment, L- [4,5- ³ H] leucine (1
(Ci / ml) for 24 hours to stimulate protein biosynthesis. ^{Also, [3} H] with 10% fetal bovine serum added free proline culture in proline incorporation experiments, L- [2,3,
4,5- ³ H] proline (1 μCi / ml)
Protein biosynthesis was stimulated for 4 hours. Unstimulated Ito cell [ ³
Some Ito cells were similarly treated except fetal calf serum to observe [H] leucine or [ ³ H] proline incorporation. After this, these Ito cells were converted to DN
20% at 4 ° C., similar to treatment in A precursor uptake experiment
After treatment with a trichloroacetic acid solution for 1 hour, the cells were dissolved in 500 μl of 0.3N NaOH / 0.1% sodium dodecyl sulfate solution and collected, and the radioactivity was measured by a liquid scintillation counter. The experiment was repeated eight times each. The results are shown in Table 3 and FIGS. In the amino acid incorporation experiment, both [ ³ H] leucine and [ ³ H] proline exhibited a small amount of inhibition, but a significant effect of concentration-dependent uptake of baicalein was observed (One-way).
ANOVA statistical processing; P <0.0001). ^[3 H]
Baicalein showed a significant inhibitory effect at a concentration of 10 μM in leucine and ³ μM in [ ³ H] proline (Fisher PLSD test statistical processing; P <0.001 and P <0.0001).

[0030]

[Table 3]

[0031]

From the above experimental results of baicalein, it has been proved that the flavonoid compound derived from the crude drug, Ogon, has a remarkable growth inhibitory effect and an inhibitory effect on the production of collagen-containing protein on cultured Ito cells activated as a single substance. Was done. This is because the flavonoid compound derived from the herbal medicine
In a chronic liver disease accompanied by fibrosis, by directly acting on the activated Ito cells, it is a result showing that the proliferation and the production of extracellular matrix such as collagen are strongly suppressed. The present invention strongly shows the usefulness as a liver fibrosis inhibitor.

[Brief description of the drawings]

FIG. 1 shows the concentration-dependent effect of baicalein on rat liver Ito cells when stimulated with 25 ng / ml platelet-derived growth factor (PDGF-BB) —the results of the effect of inhibiting [ ³ H] thymidine incorporation.

FIG. 2 shows the concentration dependence of baicalein on rat liver Ito cells stimulated with 10% fetal calf serum.
The result of the [ ³ H] thymidine incorporation inhibitory effect is shown.

FIG. 3 shows the results of the concentration-dependent-inhibitory effect of baicalein on the proliferation of rat liver Ito cells stimulated with 20% fetal calf serum.

FIG. 4 shows the concentration dependence of baicalein on rat liver Ito cells when stimulated with 10% fetal calf serum.
The result of the [ ³ H] leucine uptake inhibitory effect is shown.

FIG. 5 shows concentration dependence of baicalein on rat liver Ito cells when stimulated with 10% fetal calf serum.
The results of the [ ³ H] proline uptake inhibitory effect are shown.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) // C07D 311/30 C07D 311/30 C07H 17/07 C07H 17/07

Claims (4)

[Claims] 1. A liver fibrosis-suppressing agent comprising one or more flaponoid compounds derived from the crude drug Ougon. 2. A flaponoid compound derived from a crude drug, Ogon, comprising baicalein and wogoni.
n), baicalin or wogonos
The hepatic fibrosis inhibitor according to claim 1, which is ide). 3. The hepatic fibrosis inhibitor according to claim 1 or 2, wherein the composition containing one or more flaponoid compounds derived from a crude drug of the pentagon is in the form of a crude drug. 4. The hepatic fibrosis inhibitor according to any one of claims 1 to 3, wherein the hepatic fibrosis inhibitor suppresses hepatic fibrosis for chronic liver disease accompanied by fibrosis such as chronic hepatitis.