JPH0477427A - Theradeutic agent for hepatic disease containing alisol a-relating compound as active component - Google Patents

Abstract

PURPOSE:To obtain a therapeutic agent for hepatic diseases containing 16- ketoalisol A or 13,17-epoxyalisol A as an active component. CONSTITUTION:The objective agent for hepatic diseases contains the compound of formula I or formula II as an active component. The compound of formula I or formula II can be produced by extracting TAKUSHA (a drug prepared from bulb of Alisma plantago-aquatica) with chloroform or 60% ethanol and purifying the extract by a stepwise elution with a proper mixed eluent using silica gel chromatography, ODS column chromatography, ODS preparative high-performance liquid chromatography, etc. The eluent is selected from a hydrophobic organic solvent (e.g. hexane, benzene, chloroform and ethyl acetate), a hydrophilic organic solvent (e.g. acetone, n-butanol and ethanol) and water, and mixing the components at specific ratios. The drug is administered by oral administration at a daily dose of 10-600mg in 1-3 divided doses for adult.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application 1] The present invention relates to a drug for treating liver diseases, which contains 16-ketoalisol A or 13.17-epoxyalisol A obtained from Sawabori as an active ingredient.

13.17-Epoxy Alisole A [Prior Art] Sawa-sara is a plantago that belongs to the family Al i smataceae.
-aquatica Linne varOrlenta
This herbal medicine is prepared from the rhizome of Le Samuel SS On) and is said to have diuretic and laxative effects in ancient Chinese medicine, and is an important herbal medicine that is included in some prescriptions. In addition to starch, organic acids, potassium salts, vitamins, and volatile oil, Sawasha contains alisol-related compounds, which are protostane-type triterpenes, and alismol, a sesquiterpene.The active ingredients include choline, alisol A, Alisol A24-acetate, Alisol 823
- Acetate, Alisol C23-acetate (anti-fatty liver effect), Alisol B (diuretic effect), 16-medoxyallysole B23-acetate, 16-hydroxyallysole 823-acetate (coronary artery dilator effect)
, Arithmol (hypertensive effect), etc. have been reported, but its therapeutic effect on liver diseases is still unknown.

[Problems to be Solved by the Invention] The present inventors have been conducting studies on the active ingredients contained in crude drugs for the purpose of searching for therapeutic drugs for liver diseases, and found that extracts from Sawabori or primary cultured rat hepatocytes. It was discovered that the active ingredient 16-tro was effective in both the D-galactosamine-induced hepatotoxicity-induced group v+tro system and the 10-vivo system using D-galactosamine liver-impaired rats.
It was determined that it was ketoalisol A or 13.17-epoxyalisol A, and the present invention was achieved.

[Means for Solving the Problems] According to the present invention, 16-ketoalisole A or 1311
- A liver disease therapeutic agent containing epoxy alysole A as an active ingredient is provided.

16-Ketoalisol A or 13.1 used in the present invention
7-Epoxy alysole A can be obtained using a stepwise elution method (silica gel column chromatography, [) DS column] in which sawawa is extracted with chloroform or 60% ethanol, and an appropriate mixture of these extracts is used as the eluent. Chromatography or 005 preparative high performance liquid chromatography (
It can be produced by subjecting it to HPLC (hereinafter referred to as HPLC) and purifying it.

The eluate is selected from hydrophobic organic solvents such as hexane, pentene, chloroform or ethyl acetate, hydrophilic organic solvents such as acetone, D-butanol, ethanol or methanol, or water, and mixed in a suitable combination. It goes without saying that in the present invention, 16-ketoalisol A or 13.17-epoxyalisol Δ extracted from other crude drugs or chemically synthesized can also be used.

[Actions and Effects of the Invention] The therapeutic action of 16-ketoalisol A or 13.17-epoxyalisol A on liver diseases will be described in detail below.

-1nVitrO- system D-galactosamine injury model of rat primary cultured hepatocytes Rat hepatocytes were isolated and incubated with the test compound in a 002 incubator in the presence of 1mM D-galactosamine.
The cells were cultured at ℃ for 42 hours. After the culture was completed, the GOT activity released in the culture solution was measured using a Centrificem 660 automatic analyzer (manufactured by Baker).

The hepatocyte damage inhibition rate was determined using the following formula, and the results are shown in Table 1.

[+C0nt]GOT-[S]GOT Suppression rate (%)・X 100[+
contlGOT-[-contlGOT[+cont
lGOT: GOT with O-galactosamine lnM addition control system [-contlGOT: D-galactosamine 1mM
Additive-free control system GOT [5lGOT: D-galactosamine 1mM + sample addition system GOT (blank below) -in vivo system D-galactosamine liver-damaged rats 1 star male rats fasted for 24 hours (7 years old,
D-galactosamine (pH 7) dissolved in physiological saline (manufactured by Nippon Talea Co., Ltd.) was intraperitoneally administered at a ratio of 400 #Irj/Ng. After a subsequent 24-hour fast, cardiac blood was collected under ether anesthesia, and the GOT and GPT activities of the obtained serum (centrifuged at 3000 rpm for 20 minutes) were measured using a Centrifuichem 600 automatic analyzer (manufactured by Baker).
It was measured with The test compound was intraperitoneally administered at a rate of 301 rlfl/Kl 2 hours before the administration of 0-force lactosamine. The results are shown in Table 2.

Table 2 Acute toxicity test animals used were ICR female mice (5-6 weeks old, manufactured by Charles River Japan), and 16-ketoalisole A 2
00.300.400.4BOmy/Kg, 13.1
7-Epoxy Alisole A 300.450I11g/
Kl was administered intraperitoneally, respectively. The test sample was a 1% Vienna 80/0.5% CHC mixture with a dose of 2.
Ornll/ was adjusted to 9. After administration, general symptoms were observed and body weight changes were measured for 3 days.

The lowest lethal dose (HL[)) value is 16-ketoalisole A.
400#l! iF/N# or above, 13.17-Epoxy Alisole A4501rl'J/'j or above'il).

Considering the above test results, it can be said that drugs containing 16-ketoalisol A or 13.17-epoxyalisol A as an active ingredient are excellent therapeutic agents for liver diseases. Dosage for patients varies depending on age, symptoms, etc.
Generally for adults, 1 to 1000 mg orally per day
, preferably from 10 to 600 cm, and is preferably divided into 1 to 6 times, preferably 1 to 3 times.

In the present invention, 16-ketoalisole A or 131
By blending 7-epoxy alysole A with a usual pharmaceutical carrier, tablets, hearts or soft capsules,
It can be prepared into solid preparations such as granules, powders, fine granules, powders, etc., or liquid preparations such as injections, syrups, solutions, suspensions, and emulsions. In the case of solid preparations, enteric-coated preparations or sustained-release preparations may be prepared. Depending on the desired dosage form, carriers for the formulation include, for example, excipients, binders, disintegrants, lubricants, coating agents, solubilizing agents, emulsifiers, suspending agents, surfactants, absorption aids, What is necessary is just to select and use a stabilizer, a solvent, etc. suitably. Specific pharmaceutical carriers include starches, dextrins, α, β or γ-cyclodextrin, glucose, lactose, sucrose, mannitol,
Sorbitol, partially pregelatinized starch, methylcellulose, ethylcellulose, hydroxyethylcellulose, carboxydimethylcellulose, carboxydimethylcellulose calcium thorium, crystalline cellulose, low-substituted hydroxypropylcellulose, calcium stearate, magnesium stearate, sodium alginate, magnesium silicate, Calcium hydrogen phosphate, calcium carbonate, magnesium carbonate, magnesium aluminate metasilicate, Witepsol W35, Witepsol E85, Witepsol H15, polyvinyl alcohol, silicic anhydride, synthetic aluminum silicate, titanium oxide, talc, waxes, hydroxypropyl cellulose, Hydroxypropyl methylcellulose, hydroxyethyl methylcellulose, carboxydimethylethylcellulose, cellulose acetate phthalate, cellulose acetate maleate,
Hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate succinate, polyvinyl alcohol phthalate, styrene maleic anhydride copolymer, polyvinyl acetal diethylamine acetate, methacrylic acid/methyl methacrylate copolymer, methacrylic acid/ac I ethyl norate copolymer , butyl methacrylate and dimethylamine ethyl methacrylate copolymer, ethyl acrylate/methyl methacrylate and trimethylammonium ethyl methacrylate copolymer, gelatin,
Glycerin, propylene glycol, sodium lauryl FjM acid, medium chain fatty acid triglyceride, lecithin,
Sorbitan monooleate, sorbitan sesquioleate, polyoxyethylene sorbitan monooleate, glyceryl monostearate, glyceryl monooleate, polyoxyethylene cetyl ether, polyoxyethylene hydrogenated castor oil, sucrose fatty acid ester, polyglycerin fatty acid ester , polyoxyethylene/polyoxypropylene block polymer, polyethylene glycol,
Examples include polygonylpyrrolidone, crosslinked polyvinylpyrrolidone, and fats and oils such as cacao butter, lauric fat, and glycerogelatin.

The present invention will be further explained using production examples and specific formulation examples of 16-ketoalisol A and 13.17-epoxyalisol A.

[Production example] Pouring water (Kinokuniya Chinese Pharmacy Lot IH940905Y and IH282826Y > 1.5 Hg was extracted twice with 7 Jl of chloroform each time at 60°C for 60 minutes to prepare a chloroform extract. Then, concentrated to dryness. A chloroform extracted fraction of 54!iF was obtained. This was further subjected to primary silica gel column chromatography, and benzene (2) and benzene 7/acetone (volume ratio 10.
Sequentially eluted from the mixture of 1 to 5.1), 7.9 g of crude material
I got it. This was subjected to secondary silica gel column chromatography and sequentially eluted from a mixture of ethyl acetate and ethanol (volume ratio 3:2) to ethyl acetate to obtain 2.79 of a crude substance eluted with ethyl acetate. This was subjected to ODS column chromatography and sequentially eluted with 60 to 100% methanol to obtain a fraction 68 containing 16 to 1-ketoalinosole A.
A fraction 440η containing 0Irtg and 13.17-■ poxyalisol A was obtained. Add these to ODS preparative HPLC and develop them! 11; 70% Meta/
), 16-ketoalisole A 340m5 and 1
3.17-Epoxy Alisole A230! I got q.

Physical properties of 16-ketoalisol A: Colorless fine cubic crystals Melting point: 101.3-102.9°C Optical rotation [(X]'t0: +72.5° (c: 0
．． 51% in MeOH).

Mass spectrometry spectrum (m/z): 504 (M"), 486 (1), 468 (5L450
(45), 409 (36) 396 (4B), 367 (6
2).

Infrared absorption spectrum (KBr, C771): 343
62961.292216901642,1463,1
380°1169, 1045.1008.679°1H
-Nuclear magnetic resonance spectrum (CDCl3.ppm):
0.90(3H,S), 1.08~1.34(3
Hx7).

2.72 (IH, d, J=9.281-12), 2
．． 88 (IH, brs).

3.02 (d, J=8.3Hz), 3.21 (1N
, q, J=14.16°5.86H2), 3.52
(IH, d, J=2.93H2), 3.62 (1N
.

q, J=10.25, 2.93Hz), 4.06(
IH, m).

13C-Nuclear Magnetic Resonance Spectrum (CDCl3.pl)m
:19.34 q, 19.88 t), 1
9.98 q), 23.09 (Q.

23.41 a, 25.30 q), 25.83
d), 26.22 (q.

26.97 Q, 29.47 q), 30.7
5t), 33.53ft.

34.86 t, 35.97 t), 36.86
5), 39.56 (t.

39.90 s, 45.31 t), 46.91
s), 48.18 (d.

4B, 38 d, 49.97 S), 69.25
6), 69.43 (d.

73.92(S), 77.23(d), 139.
95(S).

177.86 (s), 209.45 (s), 2
20.06(s).

13. Physical properties of 17-Epoxy Alisole A: Colorless fine crystals Melting point = 111-112.5°C Optical rotation [a] io: +100.4° (C; 0.4
4. in CDCl3).

Mass spectrometry spectrum (n+/z): 506 (M), 488 (1), 47 ([1), 45
2(3), 450(6).

434(10), 380(23), 338(19).

Infrared absorption spectrum (KBr, (,7): 3450
298017001460.13751240.116
0°1100, 1000,950.910.800°1
H-nuclear magnetic resonance spectrum (CDCl3.ppm)
:1.05~1.12(3Hx6), 1.26(3
H,s).

1.30 (3H, s), 2.70 (IH, m),
3.09 (2H, brs) 3.50 (IH, brs)
, 3.65 (1H, brs) 4.04 (IH, br
Q), 4.16 (IN,brd)13C-nuclear magnetic resonance spectrum (CDCl3.Dpm): 17.48
q, 19.25 G, 19.93 a
, 20.06 t).

24.62 Q, 25.67 Q, 26.4
6 t, 26.59 Q.

26.72 Q, 29.57 Q, 30.5
7ft, 30.78d.

30.85 t, 33.53 t, 34.
73 t, 35.53 t.

36.89 s, 3B, 69 t, 40
．． 34 S, 46.89 S.

4B, 71 d, 48.88 d, 50.
02s, 6B, 39d.

68.53 d), 73.31 S), 73.
54 [S, 77.00 d.

78.455), 220.09 (S).

[Formulation Example 1] (Tablet) Weight (%) (1) 16-ketoalisole A 40.0
(2) Lactose 25.0 (3) Corn starch 15.0 (4) Crystalline cellulose 15.0 (5) Hydroxypropyl cellulose 3.0 (6) Magnesium stearate 1.0 (7) Polyoxyethylene (40) Monostearate 1.0 ioo.

The above (1) to (5) were mixed, water in which (7) was dissolved was added to form granules, and then dried. After sizing the obtained granules, (6) was added and mixed, and these were compression molded to prepare tablets each weighing 250 IIPJ.

[Formulation Example 2 (Hard Capsule) 16-Ketoalisole A Lactose Corn Starch! Powdered hydroxypropyl cellulose weight (%) 33.0 41.5 20,0 3.0 (5) Synthetic aluminum silicate 1.0 (6) Magnesium stearate 1.0 (7) Polyoxyethylene (20) Sorbitan mono Oleate
0.5100.0 According to a conventional method, the above (1) to (4) were mixed, ethanol in which (7) was dissolved was added and mixed, and then dried to form granules. (5) and (6) were added to and mixed with the granules, and then filled into hard capsules.
Hard capsules of (2) were prepared.

[Formulation Example 3] (Granule) Weight (%) (1) 13.17-Nipoxyalisol A 10
．． 0(2) Lactose 73.0(
3) Low substituted hydroxypropyl cellulose io,.

(4) Polyvinylpyrrolidone 5.0 (5)
Sodium lauryl t1 acid 2.0100,0 The above (1) to (5) were mixed, water was added and kneaded, and then cylindrical granules were prepared using an extrusion granulator.

“Formulation Example 4” (Soft capsule) Heavy M (%) (1) 13.17-Nipoxyalisol A 25
．． 0(2) Polyethylene glycol 400 67.0
(3) Polyoxyethylene (20) Sorbitan monooleate 5.0 (4) Purified water
3.0100.0 The above-mentioned (1) to (4) are thoroughly mixed under heating, and the resulting dispersion is filled into soft capsules using a conventional method.
Soft capsules of 0.0 mm were prepared.

(4) Lecithin 10100.
0 The above (1) to (4) were thoroughly mixed under heating, and then cooled and solidified. Melt this at 50-60'C, and
After cooling to 35° C., the mixture was poured into a powder mold, left to cool, and solidified to prepare a powder weighing 2 g each.

Patent applicant: Tokyo Tanabe Seiso Co., Ltd.

Claims (2)

[Claims] (1) 16-ketoalisole A (2) A method for producing 16-ketoalisol A, characterized by obtaining 16-ketoalisol A from sawara (3) 16
- Liver disease treatment containing ketoalisol A or 13,17-epoxyalisol A as an active ingredient