JPH1135478A - Antihepatitis c viral agent and specific inhibitor of protease ns3 containing organic extract from plant of family compuestas - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an antihepatitis C viral agent and a specific inhibitor of an antihepatitis C viral protease NS3 by formulating an organic solvent extract from a plant belonging to a specific family as an active ingredient therein. SOLUTION: This agent is obtained by formulating an organic solvent extract from a plant belonging to the family Compuestas therein. For example, Achyroline alata DC. and Achyroline satureoides DC. can be cited as the plant belonging to the family Compuestas. Dichloromethane, methyl alcohol, etc., are preferred as the organic solvent. The amount of the formulated organic solvent extract (a concentrated dried substance) which is the active ingredient of the agent is 5-100 wt.% and the daily dose thereof is 0.1-200 mg/kg.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention
The present invention relates to an anti-hepatitis C virus agent and a specific inhibitor of hepatitis C virus protease NS3, which contain, as an active ingredient, an organic solvent extract of a plant belonging to stas).

[0002]

2. Description of the Related Art Hepatitis C virus (Hepatitis)
Research on substances having an effect on C Virus (hereinafter also referred to as HCV) has been actively conducted in recent years, and up to now, 2 ′, 3′-dideoxy-5-trifluoromethyluridines (Japanese Unexamined Patent Application Publication No. 148261),
Organic substances such as indole derivatives (JP-A-7-504673 and JP-A-7-504675) and thiadiazole derivatives (JP-A-7-188017);
Alternatively, antisense nucleotides (Japanese Unexamined Patent Publication No.
8026, JP-A-6-296492, JP-A-8-5
06479), IRES (Internal
Ribosome Entry Site) binding substance (JP-A-7-69899), thymosin (JP-A-6-69899)
No. 510998), and a mixed crude drug or an extract thereof (JP-A-7-206694).

[0003]

However, many of the above substances have unclear mechanisms of action.
In addition, antisense nucleotides have a problem that although their mechanism of action is clear, their efficacy is unstable. That is, an effective anti-HCV agent has not yet been developed. Therefore, an anti-HCV agent having a clear mechanism of action and exhibiting a stable drug effect has been desired.

[0004] On the other hand, a protease called NS3 exists as a protein constituting HCV [Vir.
logic, Vol. 171, p. 637-639,1
989; and Proc. Natl. Acad. Sci.
USA Vol. 87, p. 2057-2061,19
90]. It has recently become clear that HCV activity can be suppressed by inhibiting the protease NS3.
Use as a CV agent has been studied [Virus Vol. 45, no. 2, p. 105-115, 19
95; and Bio. Clin. Vol. 11, No.
9, p. 688-690, 1996]. However, since the above-mentioned protease NS3 is a kind of serine protease, even if it shows an inhibitory activity for the protease NS3, it also becomes severe if it simultaneously inhibits other serine proteases such as trypsin and elastase. May cause side effects. Therefore, when a protease NS3 inhibitor is used as an anti-HCV agent, it is required to specifically inhibit the protease NS3. However, no substance that specifically inhibits the protease NS3 has been known.

[0005] Incidentally, Yatei Ka, a medicinal herb from South America,

[Outside 1] (Generic name) is a plant belonging to the family Asteraceae (Compuestas), Acylorin alata DC (Ac)
hyroline alata DC. ) Or Acyrolin Satureoides DC
ine saureoides DC. ), Whose stems are on the South American market. Yatei Ka's hot or cold water decoction may cause indigestion, lower stomach discomfort,
It is said to be effective for chronic gastrointestinal disorders, constipation, appendicitis, colic, asthma or diabetes. However, almost no report has been made on the extracted components, and no particular information is known about its antiviral activity.

[0006] The present inventors have intensively searched for a specific inhibitor of protease NS3, and found that a plant belonging to the Asteraceae family, in particular, the organic solvent extract of Yatei mosquito, contained protease N
It has been found that there is an effect of specifically inhibiting S3. The present invention is based on these findings.

[0007]

Accordingly, the present invention relates to an anti-hepatitis C virus agent comprising an organic solvent extract of a plant belonging to the family Asteraceae (Compuestas). The present invention also relates to a specific inhibitor of hepatitis C virus protease NS3, which comprises an organic solvent extract of a plant belonging to the family Asteraceae (Compuestas).

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION The plant belonging to the family Asteraceae (Compuestas) from which the active ingredient of the anti-HCV agent of the present invention is extracted is not particularly limited.
plants belonging to the genus hyroline, for example, Acyroline a
lata DC. ) Or Acyroline Satureoides
Dee Cee (Acyroline satureo)
ids DC. ), That is, Yatei Ka

[Outside 2] It is preferable to use One or more of any part of the plant can be used in the extraction step in combination, but it is preferable to use leaves, stems or roots, and to dry those parts before use in the extraction step. Is particularly preferred.

As the organic solvent, alkyl halides or alcohols (particularly aqueous alcohols), or mixtures thereof can be used. The alkyl halide preferably has 1 to 4 carbon atoms and is substituted by one or more chlorine atoms (particularly, 1 or 2 chlorine atoms).
Lower alkyl can be used, specifically, chloromethane, dichloromethane, trichloromethane (chloroform), carbon tetrachloride, chloroethane, dichloroethane, trichloroethane, or a mixture thereof, particularly, dichloromethane or trichloromethane Can be. The alcohol is preferably a lower alcohol having 1 to 4 carbon atoms, for example, methyl alcohol, ethyl alcohol, n-propyl alcohol, i
-Propyl alcohol, n-butyl alcohol, i-butyl alcohol, s-butyl alcohol, t-butyl alcohol or mixtures thereof, in particular methyl alcohol, can be used.

In the extraction step, an appropriate part of the plant belonging to the above Asteraceae family (particularly, Yatei mosquito) is preferably ground after drying, preferably at room temperature, preferably at room temperature. Extraction is performed as described above, and the organic solvent extract is concentrated, preferably under reduced pressure. The concentrated solution obtained, or a crude extract obtained by further concentrating the concentrated solution to dryness, or a purified extract obtained by purifying the organic solvent extract or the crude extract, is treated with the anti-C form of the present invention. It can be used as an active ingredient of a hepatitis virus agent. As the above-mentioned purification treatment method, an ordinary method, for example, various column chromatography can be used, and preferably, silica gel column chromatography is used.

The anti-hepatitis C virus agent according to the present invention comprises a non-toxic and effective amount of the organic solvent extract described above and optionally a pharmaceutically or veterinarily acceptable carrier. Animal orally or parenterally,
Preferably, it can be administered to mammals (particularly humans). Oral administration includes sublingual administration, and parenteral administration includes topical (eg, subcutaneous or intramuscular administration), intravenous, rectal, or pulmonary administration.

The anti-hepatitis C virus agent of the present invention comprises a tablet,
It can be used in various dosage forms such as powders, granules, capsules, syrups, dispersants, injections, sublinguals, fine granules, external preparations, ointments, suppositories or tapes. Each of these preparations may contain a known carrier (eg, binder, excipient, lubricant, disintegrant, wetting agent, coloring agent, diluent, emulsifier, suspending agent, or filler) as necessary. And can be prepared by a conventional method. As a carrier, for example,
Corn starch for granules, crystalline cellulose for tablets, silicic anhydride for capsules, or glucose solution for injections can be used. In the anti-hepatitis C virus agent according to the present invention, the organic solvent extract (concentrated to dryness) as an active ingredient is 5 to 100% by weight, preferably 25 to 100% by weight.
It is contained in an amount of weight%. The dose varies depending on the administration subject, administration route and / or symptoms, but is usually 0.1
２００200 mg / kg / day, which can be administered once to five times a day.

[0013]

EXAMPLES The present invention will be described below in more detail with reference to examples, but these examples do not limit the scope of the present invention.

Production Example In this example, a process of obtaining an organic extract from Yatei mosquito and purifying it will be described. (1) Preparation of dichloromethane extract To about 1 kg of dried leaves and bark of yatai moss, a herb of Asteraceae from South America, about 1 liter of dichloromethane was added, and after immersion for about 1 month at room temperature, Filtration on filter paper gave a filtrate and a residue. The obtained filtrate was evaporated to dryness by a rotary evaporator to obtain a dichloromethane extract.

[0014]

[Example of activity evaluation test] In this example, the anti-HCV protease NS3 activity was evaluated. Specifically, the method of Saito et al. [Antiviral Res. Vol. 32, no.
1, p. 9-18, 1996].
That is, HC is used by E. coli by gene recombination technology.
Mass production of V protease NS3. On the other hand, a substrate containing a junction (junction) [NS5A-5B bond] known as a site where the protease NS3 is cleaved is artificially synthesized. A method for examining whether or not cleavage of the substrate by the protease NS3 is inhibited when a test sample is added to a reaction system containing the protease NS3 and the substrate includes:
S3 activity was evaluated.

(1) Mass Production of HCV Protease NS3
Amplified by CR method, plasmid pMAL-c2
(New England Biolabs In
c. Berly, Mass., USA). The plasmid was used for E. coli JM1.
09 and grown after isopropyl-β-D
-(-)-Thiogalacto-pyranoside (IPTG)
By adding 5 mM, the HCV protease NS
3 was produced. The cells were disrupted by ultrasonic waves and separated into solid and liquid by a centrifuge. The liquid phase was recovered and salted out with ammonium sulfate (70%). This precipitate was dissolved in a buffer (10 mM sodium phosphate buffer containing 30 mM NaCl, 2 mM-β-mercaptoethanol and 0.25% Tween 20; pH 7.2), passed through an amylose resin column, and washed. The bound substance was eluted (0.5
Eluted with 10 mM sodium phosphate buffer containing M-NaCl and 2 mM-β-mercaptoethanol; pH 7.8) to give HCV protease NS for activity measurement
3 was obtained.

(2) Synthesis of NS5A-5B bond Automatic peptide synthesizer (Model PSSM-8 Sh)
imazu, Kyoto, Japan) according to the formula: Dansyl-Gly-Glu-Ala-Gly-Asp-
Asp-Ile-Val-Pro-Cys-Ser-M
et-Ser-Tyr-Thr-Trp-Thr-Gl
2 consisting of the amino acid sequence of y-Ala-Leu-OH and containing NS5A-5B bond
A 0-mer artificial peptide was synthesized and used as an artificial substrate for activity measurement.

(3) Evaluation of anti-HCV protease NS3 activity HCV protease NS3 (final concentration = 0.0603 μm)
M) 1.4 μg of the extract from Yatei mosquito (final concentration = 1-100 μg / ml) was added to a buffer [30 mM-
50 mM Tris HCl containing NaCl, 1 mM CaCl ₂ and 2 mM (±) -dithiothreitol (DTT)
(PH 7.6) buffer], and the mixture was sufficiently stirred and kept at 37 ° C for 15 minutes. Then, 12 µg of an artificial substrate (final concentration 26 µM) was added, and the mixture was sufficiently stirred and kept at 37 ° C for 1 hour. Subsequently, the reaction was stopped by adding 4 μl of 2.5 M acetic acid, and the reaction solution was analyzed using an analytical HPLC apparatus (Type
eSC-8020, Tosoh, Tokyo, Japan). As a column in the analytical HPLC apparatus, an ODS-80Ts column (0.46 × 15 c
m) (Tosoh, Tokyo, Japan) using 0.1% trifluoroacetic acid (TFA) aqueous solution and 0.1% aqueous solution as eluents.
A mixture of 90% acetonitrile aqueous solution containing 09% TFA (gradient = 0: 1 to 1: 1) was used. 1.0ml /
The eluate was eluted at a flow rate of 20 minutes for 20 minutes and the absorbance of the eluate at 214 nm was continuously monitored.

The degradation product, ie, the peptide dansyl-Gly-Glu-Ala-Gly-Asp-
Asp-Ile-Val-Pro-Cys-OH) and the peptide H-Ser-Met-Ser-Tyr-Thr-Trp
-Thr-Gly-Ala-Leu-OH was detected from the difference in elution time, and the degree of degradation of the artificial substrate, that is, the degree of HCV protease NS3 activity was determined by examining the height of each peak.
For the extract, the concentration showing 50% inhibition was measured, and the results are shown in Table 1.

[0019]

Table 1 Test sample HCV protease NS3 inhibitory activity Yatei mosquito-derived extract 9.8 (μg / ml)

[0020]

Reference Example In this reference example, the inhibitory activity against other serine proteases was evaluated. Using the Yatei mosquito-derived extract prepared in the above production example, other serine proteases,
That is, the inhibitory activity on trypsin and elastase was evaluated by the following method using a fluorescent substrate specific to each of those proteases. The enzyme activity was measured (excitation 380 nm, fluorescence 460 n) using a multiplate fluorescence reader (Fluostar, Tecan Japan).
m).

(1) Evaluation of Inhibitory Activity against Trypsin Extracts of Yatei mosquito at various concentrations were placed in a 96-well microplate (MSD-8596, Sumitomo Bakelite), and Bz-Arg-MCA (formula) was used as a fluorescent substrate. Wherein Bz is a benzoyl group, and MCA is 4-methylcoumaryl-7-amide) [3092-v, Peptide Research Institute Co., Ltd.] to a final concentration of 0.1 mM. Was. 4 μl of an aqueous solution of trypsin (Funakoshi) (0.1 mg / ml) was added, and the final volume was adjusted to 200 μl with a buffer (10 mM sodium phosphate buffer containing 0.15 M-NaCl; pH 7.8).
After incubation at 37 ° C. for 1 hour, the enzyme activity was measured. The concentrations that showed 50% inhibition are shown in Table 2.

(2) Evaluation of inhibitory activity on elastase Suc-Ala-Pro-Ala-MCA as a fluorescent substrate (where Suc is a succinyl group and MCA is
Methylcumaryl-7-amide) [3100-v, Peptide Research Institute Co., Ltd.] was added to a final concentration of 0.1 mM, and an elastase (Sigma) aqueous solution (1.0 mg) was added. The procedure described in (1) above was repeated, except that 4 μl / ml) was added to measure the enzyme activity. The concentrations showing 50% inhibition are shown in Table 2.

[0023]

Table 2 Types of serine proteases Inhibitory activity (μg / ml) Trypsin> 50 Elastase> 50

[0024]

[Preparation example] 10 parts by weight of the dichloromethane extract of Yatei mosquito obtained in the above preparation example, 421.0 parts by weight of lactose and 50 parts by weight of potato starch are mixed well, and the mixture is placed in a fluidized bed granulator. Then, 18.75 parts by weight of hydroxypropylcellulose (binder) was made into a 5% aqueous solution and sprayed to obtain granules. Next, 20 parts by weight of carboxymethylcellulose calcium (disintegrant) and 15 parts by weight of magnesium stearate (lubricant) were added and mixed. The resulting mixture was press-molded so that the weight of one tablet became 125 mg, to obtain tablets. The tablet was sprayed with a coating solution having the following formulation to give an enteric tablet. Cellulose acetate phthalate 5.0 parts by weight 95% ethanol 47.5 parts by weight Ethyl acetate 47.5 parts by weight

[0025]

The anti-hepatitis C virus agent of the present invention has a specific inhibitory effect on the hepatitis C virus protease NS3, and thus can specifically inhibit the activity of hepatitis C virus. It is useful for the purpose of preventing or treating hepatitis C.

 ────────────────────────────────────────────────── ─── Continuing from the front page (72) Inventor Shoji Yamada 8-2-3 Mizukino, Moriya-cho, Kitasoma-gun, Ibaraki Prefecture (72) Inventor Hiroshi Nozaki 1-1, Oita-machi, Okayama-shi, Okayama Prefecture Okayama University of Science (72) Inventor Kenji Sudo 1-40 Kiyoake-cho, Fukushima City, Fukushima Prefecture Neo Heights Arakawa Park No. 302 (72) Inventor Tomoyuki Yokota 20-1 Kita-Satori, Hirano, Iizaka-cho, Fukushima City, Fukushima Prefecture (72) Inventor Kunada Shimo-Tono Kyoto Fushimi Joint Dormitory 245, Nishibongyo-cho Public Land, Fushimi-ku, Kyoto

Claims (2)

[Claims] 1. An organic solvent extract of a plant belonging to the family Asteraceae (Compuestas),
Anti-hepatitis C virus agent. 2. An organic solvent extract of a plant belonging to the family Asteraceae (Compuestas).
A specific inhibitor of the hepatitis C virus protease NS3.