JP2001058969A - Nitrogen monoxide production inhibitor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a nitrogen monoxide production inhibitor that is useful for occlusive cerebrovascular disorder and the like with side-effect reduced by extraction-treating the plant that has been used as a legendary herb in Indonesia. SOLUTION: A compound represented by formula I(R1 and R2 are each H or a 1-6C alkyl) or formula II (R3 is H or a residue of monosaccharide) or their salts or their hydrates is used as an active ingredient. The compound represented by formula I bearing methyl groups as R1 and R2 is new and is isolated from the body of a plant called Kaya secang in Indonesia (Caesalpinia sappan) by solvent extraction and the like. The compound of formula II where the R3 is a glucose residue is also obtained from a plant called Sambi boto in Indonesia (Andrographis paniculaata) by the extraction form the plant body with a solvent or the like.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a nitric oxide production inhibitor. More specifically, nitric oxide synthase (Nitr
ic Oxide Synthase (hereinafter abbreviated as NOS) inhibitory action, and nitric oxide (Nitric Ox
ide) By suppressing the production, cerebrovascular disorder in which nitric oxide or a metabolite of nitric oxide is considered to be involved, particularly in the acute phase of obstructive cerebrovascular disorder and hypotension in sepsis The present invention relates to a nitric oxide production inhibitor containing a useful compound as an active ingredient.

[0002]

BACKGROUND OF THE INVENTION The discovery that macrophages, one of the immunocompetent cells, produce a large amount of nitrate, has revealed that nitric oxide is produced in vivo [Pr
oc. Natl. Acad. Sci. USA, 82, 7
738-7742 (1985); Immun
ol. , 138, 550-565 (1987)]. In the circulatory system field, a substance having a relaxing action released from vascular endothelial cells was discovered, and was named vascular endothelium-derived relaxing factor (EDRF). Furthermore, the body of this EDRF was found to be nitric oxide [Natur
e, 327, 524-526 (1987)].

[0003] Since then, the role of nitric oxide as a signal transmitting substance in many physiological functions in the living body, such as the nervous system and the immune system, other than the circulatory system, has been revealed one after another. That is, in the nervous system, it acts as a neurotransmitter and is involved in the establishment of synaptic plasticity (cerebellar long-term depression and hippocampal long-term potentiation) [Katsuei Shibuki: NO and nervous system plasticity-regulation of cerebellar long-term depression. Experimental medicine 11, 24
51-2456, 1993. In addition, in the immune system, the importance of nitric oxide produced from macrophages acting as effector cells in the body's defense mechanism against tumor cells and pathogens has been pointed out [Tatsuo Taki, Masayasu Nakano: Arginine and macrophages in macrophages Antibacterial and antitumor effects of its metabolites. History of Medicine 156
194-197, 1991. ]. Even if it is limited to the vasodilator action, it has been proved that it acts not only from the intima side as EDRF but also from the outer membrane side as a non-adrenergic / non-cholinergic vasodilator transmitter. It has been pointed out that nitric oxide regulates various physiological functions by a complicated mechanism.

[0004] At present, the relationship of nitric oxide to physiological functions and disease states has been clarified in a wide range of fields such as circulation, immunity, and nervous system. Among them, it has been elucidated, for example, that nitric oxide constantly produced in the body plays an important role in maintaining homeostasis of circulatory dynamics. On the other hand, in sepsis, a large amount of nitric oxide is produced by the action of cytokines activated by endotoxin, which is said to cause an endotoxin shock state such as endothelial cell damage and reduced myocardial contractility.

Nitric oxide, which has been found to be produced in vivo as described above, is produced by nitric oxide synthase (NOS) using L-arginine as a substrate. NO
S is at least non-inducible (vascular endothelial and neural)
And inducible isozymes. Vascular endothelium type N
OS is mainly present in vascular endothelial cells, and its activity is controlled by the intracellular calcium concentration. Neuronal NOS is present in central nerve cells, peripheral nerve cells, or pancreatic islet β cells, gastrointestinal nerves, adrenal medulla, renal compact plaques, etc.
As in OS, the activity is controlled by the intracellular calcium concentration.

The vascular endothelial NOS and the neural NOS (c
(NOS, abbreviated as cNOS) is constantly present in cells, and changes in the amount of enzymes due to physiological changes are hardly observed. Inductive NOS (ind
UNO (abbreviated as iNOS) is present in hepatic parenchymal cells, neutrophils, macrophages, smooth muscle, fibroblasts, renal mesangial cells, gastrointestinal epithelium, pancreatic islet β cells, vascular smooth muscle cells, glial cells, etc. I do. This is not usually observed in cells, but is induced by stimulation with endotoxin, various cytokines, and the like.

[0007] The action of nitric oxide produced by NOS is diverse and includes, for example, vasorelaxant action, platelet aggregation inhibitory action, adhesion inhibition, leukocyte adhesion / migration inhibition, sympathetic nerve activity inhibition, endotoxin shock, endotoxin or cytokine. Hypotension disorder, action as a signal transmitter between nerve cells, ischemic brain cell disorder, antitumor, bactericidal action, autoimmune disease, insulin-dependent diabetes, arthritis,
Tissue disorders after transplantation, rejection and the like.

[0008] In analyzing the biological activity of nitric oxide in vivo, NOS inhibitors are useful and may be used as therapeutic agents for shock and ischemic diseases. Development of NOS inhibitors is currently underway. For example, there is an arginine analog as a substrate competitor, and Nω-monomethyl-L-arginine (L-NMM
A), Nω-nitro-L-arginine (L-NNA),
Nω-amino-L-arginine (L-NAA), Nω-
Iminoethyl-ornithine (L-NIO) and the like correspond thereto. In addition, diphenylene iodonium (DPI), di-
2-thienyl iodonium (DTI), calcineurin and the like. In addition, those inhibiting gene transcription induction include corticosteroids, TGFβ, IL-4, IL-
10 and the like.

[0009]

However, most of the known NOS inhibitors, including the above representative examples, inhibit not only iNOS but also cNOS.
According to the use as these therapeutic agents, even the regulation of constant circulatory dynamics is suppressed, and it is impossible to avoid side effects such as an increase in blood pressure and a decrease in organ blood flow. Furthermore, when these are used, there are concerns about problems such as effects on the central nervous system and impotence. As described above, conventionally known NOS inhibitors cannot be evaluated as pharmaceuticals. Instead of these, there is a need for the development of a drug having a nitric oxide production inhibitory action capable of selectively inhibiting iNOS. Was sought. Adrenocortical hormones such as dexamethasone have been used as drugs that selectively inhibit iNOS, but serious side effects often afflicted patients and could not be a drug that should be used universally. There is a need for a drug that is not a corticosteroid but has a nitric oxide production inhibitory action with few side effects.

[0010]

Means for Solving the Problems The present inventors have developed iNOS.
As a result of diligent studies to find a drug that has a nitric oxide production inhibitory action that can selectively inhibit the production of nitric oxide, the plant extract that has been used as a herbal medicine in Indonesia is safer and more The present inventors have found a compound having a high nitrogen production suppressing effect and completed the present invention. Therefore, the present invention
The present invention relates to a novel nitric oxide inhibitor having high safety and few side effects.

That is, the present invention provides a compound represented by the following general formula I:
The present invention relates to a nitric oxide production inhibitor comprising a compound represented by I, a salt thereof or a hydrate thereof as an active ingredient.

Embedded image [In the formula, R ₁ and R ₂ represent hydrogen or linear or branched lower alkyl having 1 to 6 carbon atoms. ]

Embedded image [Wherein, R ₃ represents hydrogen or a residue of a monosaccharide. Furthermore, the present invention relates to a compound represented by the following general formula I ′ or II ′, a salt thereof or a hydrate thereof.

Embedded image [Wherein, R ′ ₁ and R ′ ₂ represent a linear or branched lower alkyl having 1 to 6 carbon atoms. ]

Embedded image [Wherein, R ′ ₃ represents hydrogen. ]

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION The nitric oxide production inhibitor of the present invention
In the compound of the general formula I used as an active ingredient, R₁
And R_TwoIs hydrogen or a linear or branched carbon
Represents lower alkyl having a prime number of 1 to 6. Where the linear
Or a branched lower alkyl having 1 to 6 carbon atoms
Is, for example, methyl, ethyl, propyl, isopropyl
Butyl, isobutyl, pentyl, hexyl, etc.
Can be In general formula I, R ₁And R_TwoWith
Is a known compound called Butein
is there. In general formula I, R₁And R_TwoAre both linear
Or a branched lower alkyl having 1 to 6 carbon atoms
The compound represented by the general formula I 'is a novel compound
It is. As an active ingredient of the nitric oxide production inhibitor of the present invention
In the compound of general formula II used for_ThreeIs hydrogen
Or the residue of a monosaccharide. Here, the residue of the monosaccharide is
For example, glucose residues, xylose residues,
And a fructose residue. In general formula II:
R_ThreeIs a glucose residue, neoandrographol
It is a known compound called ide. In general formula II
R_ThreeIs a compound represented by the general formula II '
The compound is a new compound. In the present invention, these
The compound may be a salt or a hydrate thereof. With salt
The sodium and potassium salts of the compound of general formula I
Examples of the hydrate include hydrates of these salts.
No.

As shown in the following reaction scheme, a compound represented by the general formula I is obtained by using an acetophenone derivative represented by the general formula III and a benzaldehyde derivative represented by the general formula IV as raw materials. It can be obtained by condensation with an alkali. This reaction is a known reaction, and detailed reaction conditions and the like can be easily determined by those skilled in the art. Reaction scheme

Embedded image A novel compound in which both R ₁ and R ₂ in Formula I are methyl groups (hereinafter sometimes referred to as Cs-1)
Can be isolated and extracted by a solvent extraction method from a plant having a scientific name of Caesalpinia sappan , Indonesian name (Kaya secang) or a supercritical extraction method using carbon dioxide gas. Similarly, the general formula II
Wherein the compound in which R ₃ is a glucose residue (neoandr
ographolide) is the scientific name: Andrographis
paniculata Indonesian name (Sambi
bot)) can be isolated and extracted from a plant by a solvent extraction method or a supercritical extraction method using carbon dioxide gas.

In the case of solvent extraction from a plant, the plant may be used without being cut as it is, but it is more preferable to use a plant cut before the extraction operation in that the extraction time can be reduced. As the extraction method, a known method can be adopted. Examples of the extraction method using a solvent include a method in which a solvent is added to a raw material and heat treatment is performed at a reflux temperature of the solvent. In general, this heat treatment is preferably carried out at a temperature of 80 ° C. or lower.
An extract can be obtained by performing heat treatment for up to 6 hours. Alternatively, an extract can be obtained by digesting the dry powder of the raw material in a solvent. The extraction operation using a solvent can be repeatedly performed on the raw material residue after the first extraction operation. The amount of the solvent used for the extraction is 100 to 10,000 per 100 parts by weight of the raw material.
Parts by weight are appropriate, and more preferably 300 to 5,0.
00 parts by weight.

As the above-mentioned extraction solvent, lower alcohols such as methanol, ethanol, propyl alcohol,
Isopropyl alcohol, butanol, isobutanol, etc., or polyhydric alcohols such as propylene glycol, 1,3 butylene glycol, acetone, dioxane,
Examples include methyl ethyl ketone, acetonitrile, ethyl acetate, butyl methyl ketone, diethyl ether, dichloromethane, xylene and toluene. Particularly, ethyl acetate is preferable. Even if the above solvents are used alone, 2
A mixture of two or more species may be used, or water and an organic solvent may be used in combination. The extract thus obtained is concentrated under reduced pressure, and then subjected to silica gel chromatography or the like, whereby the target compound can be isolated.

The supercritical extraction method is a method using a supercritical fluid, and since the dissolving power of the supercritical fluid can be easily and continuously controlled by pressure and temperature over a wide range, a supercritical fluid utilizing this property is used. The criticality extraction method is expected as a new separation method. A supercritical extraction method and an extraction method using the above-mentioned solvent may be used in combination. Supercritical extraction is an extraction technique that has attracted attention in a wide range of fields such as the food, chemical, pharmaceutical, and cosmetic industries. In particular, when carbon dioxide is used as an extraction solvent, its critical point (75 kg / cm ² , 31
C) is relatively low and the safety is high, so that components unstable to heat and components having high volatility can be efficiently extracted and separated. In order to extract the desired components from the raw materials,
This can be achieved by setting the pressure and temperature of carbon dioxide above the critical point. Generally, at the same temperature, the higher the pressure, the higher the dissolving power of supercritical carbon dioxide. For example, using a supercritical extraction device, the temperature of the high-pressure cell section 33 ~ 40 ℃,
Preferably, the temperature is 35 ° C., the pressure is 75 to 300 atm, preferably 150 atm, and the flow rate of carbon dioxide is 0.5 to 5.0.
dm ³ / min, preferably at a condition of 4.0dm ³ / min. Thereby, the extract containing the nitric oxide production inhibitor can be efficiently recovered. The desired compound can be isolated by subjecting the thus obtained extract to silica gel chromatography or the like, if necessary.

The compound in which R ₃ is hydrogen in the general formula II is a compound which is a glucose residue in the general formula II obtained by the above-mentioned extraction method (neoandrographolide)
Thus, it can be obtained by removing glucose residues by a known hydrolysis reaction or the like. Also,
A compound in which R ₃ is a residue of a monosaccharide other than a glucose residue in the general formula II can be obtained by reacting a compound in which R ₃ is hydrogen in the general formula II with a halide of a monosaccharide or the like. .

The plant used in the present invention has a long history as a constituent plant of a traditional herbal medicine, and its safety has been confirmed. Therefore, compounds obtained therefrom can be used with confidence. For example, the crude extract of the above plant was administered to mice and rats at a dosage limit of 15 g.
It is extremely safe, as is clear from the fact that no fatal cases or abnormal findings were observed after oral administration of / kg.

The nitric oxide production inhibitor of the present invention may be appropriately added to the above-mentioned compound as an active ingredient, unless the compound is not harmful to the compound and is not unsuitable for the product used as the nitric oxide production inhibitor. It is possible to mix the agent according to a conventional method, glycyrrhizic acid, dipotassium glycyrrhizinate, lysozyme chloride, lytic enzyme, mutanase,
Chlorhexidine, sorbic acid, alexidine, hinokitiol, cetylpyridinium chloride, alkyl glycine, alkyl diaminoethyl glycine salt, allantoin, ε-aminocaproic acid, azulene, vitamin E and its derivatives, sodium monofluorophosphate, sodium fluoride, fluoride Other active ingredients such as tin, water-soluble primary or secondary phosphates, quaternary ammonium compounds, and sodium chloride can also be added.

When the nitric oxide production inhibitor of the present invention is used as a pharmaceutical, it can be administered as an oral preparation or a parenteral preparation such as an injection or an infusion.

These preparations can be prepared by an ordinary method using a suitable pharmaceutical carrier. Pharmaceutical carriers can be selected according to the above-mentioned administration forms and dosage forms. In the case of oral preparations, for example, starch, lactose, sucrose, mannitol, carboxymethylcellulose, corn starch, inorganic salts and the like are used. In preparing the oral preparation, a binder, a disintegrant, a surfactant, a lubricant, a fluidity promoter, a flavoring agent, a coloring agent, a flavor, and the like can be further blended. Specific examples thereof include the following.

Examples of the binder include starch, dextrin, gum arabic, gelatin, hydroxypropyl starch, methylcellulose, sodium carboxymethylcellulose, hydroxypropylcellulose,
Examples include crystalline cellulose, ethyl cellulose, polyvinylpyrrolidone, macrogol and the like.

Examples of the disintegrant include starch, hydroxypropyl starch, sodium carboxymethylcellulose, calcium carboxymethylcellulose,
Examples include carboxymethyl cellulose and low-substituted hydroxypropyl cellulose.

Examples of the surfactant include sodium lauryl sulfate, soybean lecithin, sucrose fatty acid ester and the like.

Examples of the lubricant include talc, waxes, hydrogenated vegetable oil, sucrose fatty acid ester, magnesium stearate, calcium stearate, aluminum stearate, polyethylene glycol and the like.

Examples of the fluidity promoter include light anhydrous silicic acid, dried aluminum hydroxide gel, synthetic aluminum silicate, magnesium silicate and the like.

Further, the nitric oxide production inhibitor of the present invention comprises:
Oral liquids such as suspensions, emulsions, syrups and elixirs can also be administered, and flavoring agents and coloring agents can be added to these various dosage forms.

On the other hand, in the case of parenteral preparations, they are produced according to a conventional method, and are generally used as diluents such as distilled water for injection, physiological saline, aqueous glucose solution, vegetable oil for injection, sesame oil, peanut oil, soybean oil, corn oil, propylene oil. Glycol, polyethylene glycol and the like can be used.
Further, if necessary, a bactericide, a preservative, and a stabilizer may be added. In addition, from the viewpoint of stability, this parenteral preparation can be frozen after filling into a vial or the like, water can be removed by a usual freeze-drying technique, and a liquid preparation can be prepared from the freeze-dried product immediately before use. Further, if necessary, an isotonic agent, a stabilizer, a preservative, a soothing agent and the like can be appropriately added.

The dose of the nitric oxide production inhibitor of the present invention may vary depending on the subject to be administered, the administration route, and the like. Usually, when orally administered to an adult, the total amount of the compound of the present invention is 1%. 1 mg to 10 g per day, preferably 1 mg to 2 g, more preferably 1 mg to 200 mg
In the case of parenteral administration, the total amount of the compound of the present invention is 0.1 mg to 1 g per day, preferably 0.1 mg to 1 g.
mg to 200 mg, more preferably 0.1 mg to 10 mg
The dose can be adjusted appropriately within the range of 0 mg.

The nitric oxide production inhibitor of the present invention is
It can be added to a feed or the like according to a conventional method, and the amount of addition can be appropriately selected according to the type of product. Specific examples of products to which the nitric oxide production inhibitor of the present invention is applied include foods such as pasta, chewing gum, chewing jelly, coffee beverages, and feeds such as dog foods and cat foods.

[0031]

EXAMPLES The production of the compound of the present invention will be described in more detail with reference to the following Examples, but it should not be construed that the invention is limited thereto. Furthermore, the selective inhibitory effect on nitric oxide production of the compound of the present invention is shown by test examples.

Example 1 In the general formula I, both R ₁ and R ₂ are methyl groups.
Caesalpinia sappan of the compound of the present invention
The core (dry weight 300 g) extracted by the production Caesalpinia Sappan from extracted 3 times immersed in methanol (3 liters) at room temperature. The methanol was removed from the extract by concentration under reduced pressure to obtain 19 g of a methanol extract. Distilled water was added to the obtained methanol extract, and the mixture was extracted with ethyl acetate. The ethyl acetate-soluble fraction (9.3 g) and the water-soluble fraction, and the residue (1.3 g) not eluted in both, were obtained. Obtained. For 450 g of silica gel, 9.0 of ethyl acetate-soluble fractions
g and eluted in a mixed solvent of n-hexane and ethyl acetate in a successively varying ratio, followed by washing with methanol, and Fr1 (10% ethyl acetate, 45.7 m 2).
g), Fr2 (20% ethyl acetate, 2.15 g), Fr
3 (30% ethyl acetate, 1.17 g), Fr4 (50%
Ethyl acetate, 1.71 g), Fr5 (70% ethyl acetate, 1.71 g), Fr6 (90% ethyl acetate, 1.2%
0g) and Fr7 (methanol, 0.90 g). Furthermore, Fr2, which had the strongest effect of suppressing nitric oxide production,
(1.70 g) was applied to 85 g of silica gel to give n-he
The mixture was sequentially eluted with a mixed solvent of xane and ethyl acetate and methanol, and Fr2-1 (50% ethyl acetate, 339.7 m
g), Fr2-2 (70% ethyl acetate, 1.13 g),
Fr2-3 (ethyl acetate, 100.9 mg), Fr2-
4 (methanol, 86.5 mg) was obtained. Fr2-2 was applied to 50 g of silica gel and eluted with chloroform / methanol (95: 5) to obtain Cs-1. Cs-1 is a substance in which R ₁ and R ₂ in Formula I are both methyl groups, and is a novel compound that has not been reported before. In addition, various properties of Cs-1 are shown below. UV (methanol) λmax (ε): 230
(1600), 348 (7700) ¹ H-NMR (C ₅ D ₅ N) δ: 3.35 (3
H, s), 3.46 (3H, s), 6.71.
(1H, dd, J = 8.4, 2.1 Hz),
6.68 (1H.d, J = 2.1 Hz),
6.09 (1H, d, J = 8.4 Hz);
13 (1H, dd, J = 8.4, 2.2 H
z), 7.57 (1H, d, J = 2.2H
z), 7.70 (1H, d, J = 15.8H
z), 7.97 (1H, d, J = 8.4H
z), 7.98 (1H, d, J = 15.8 H)
z). FAB-MS m / z 93, 123, 135,
151, 286

Test Example 1 Suppression of nitric oxide produced by BCG-induced peritoneal macrophages
Braking test (1) BCG-induced peritoneal macrophages collected ICR mice (5 weeks old, male, Kumagai) using physiological saline for injection (the Otsuka) After acclimation 5-7 days 5 mg / mL
BCG (Japan BCG) solution suspended at a concentration of
0.2 mL was administered intraperitoneally per mouse. Four days later, the head was decapitated under ether anesthesia, and the blood was blocked.
The MI-1640 medium was injected and massaged 50 times, and the abdomen was opened to collect wet cells together with the medium. The collected cell suspension was centrifuged at 1200 g for 5 minutes. After removing the supernatant, the RPMI-1640 medium was added again to suspend, and 1
This was centrifuged at 200 g for 5 minutes, and this was repeated once again to wash the cells. Finally, RPMI-1640 containing 5% FCS (Bioserum) was added to adjust to an appropriate concentration, and the number of cells was counted using a hemocytometer.

(2) Monoacid in macrophage culture supernatant
The cell concentration for measuring the amount of nitrogen iodide was adjusted to 1 × 10 ⁶ ce11s / mL.
Adjusted using PMI-1640 (5% FCS), 96
140 mL of the mixture was spread on a flat-bottomed plate and cultured in an incubator (37 ° C., 5% CO ₂ ). After 2 hours, the specimen and LPS were added. The sample is 10 μg per 1000 μg.
The sample is dissolved in 0 μL of ethanol, and this solution is
Dilute 20-fold with MI-1640 (5% FCS)
μg / mL. This was used as a stock solution and further RPMI-1
Dilute to the required concentration with 640 (5% FCS) and add 20 μL
Each was added to a macrophage culture solution. Next, LPS (Lipopo) was added so that the final concentration was 10 μg / mL.
1ysaccaride, Escherishia c
oil 055: B5, Sigma) was added to make the total volume of the culture solution 200 μL. 24
After culturing for a period of time, the amount of nitric oxide in the supernatant of the macrophage culture solution was measured by the following procedure. The nitric oxide concentration was measured by a color reaction using a Griess reagent as the NO ₂ salt. That is, 100 μL of the culture supernatant was added to 96
Take a well plate and add an equal amount of Griess reagent (1
% Su1faniamide, 0.1% naphty
Laethylene diamine is dissolved in 97 mL of double distilled water, and 3 mL of phosphoric acid is added).
After leaving it at room temperature for 0 minutes to develop a red color, 510 mm
The absorbance was measured. At the same time, an aqueous solution of NaNO ₂ was colored as a standard solution, a calibration curve was prepared from the absorbance, and the amount of nitric oxide was calculated from the concentration of NO ₂ salt produced in the medium.

(3) Measurement of Cell Viability by MTT Method The cell viability of the plate from which the supernatant was removed for measuring nitric oxide was measured by the MTT method. First, the remaining 100μ
L medium was completely removed and RPMI-1640 (5
% FCS) in 100 μL and 0.5 w / v% MTT
(NAKARAITESQUE) 20 μL was added. Thereafter, it was handled under light shielding. Incubate for 5 hours (37 ° C, 5
% CO ₂ , then SDS (50% DMF solution) 100μ
L was added to dissolve the MTT formazan formed, and 590 m
The absorbance at m was measured, and the absorbance with respect to the control was determined as a percentage, which was defined as the cell viability.

(4) Results FIGS. 1 and 2 show the results of measuring the inhibitory effect of nitric oxide production and cell viability by using Cs-1 as a sample and changing the concentration in the range of 0-50 μg / mL. . 8 μg / mL as judged from the results shown in FIGS.
In the vicinity, a strong nitric oxide production inhibitory effect on macrophage cells was observed. However, higher concentrations reduced cell viability and developed toxicity. The effect of suppressing the production of nitric oxide, Cs-1, which is a novel compound used in this experiment, was comparable to that of known isoflavonoid Genistein, which was previously found to exhibit a high effect. The value of ₅₀ is 9.6 μM for Genistein,
Cs-1 is 16.2 μM, and Cs-1 has an IC ₅₀
Was large, and the cytotoxicity was judged to be less.

Example 2 In the general formula II, R ₃ is a glucose residue.
Andrographis panicul
Production by extraction from ata Compound of the present invention Andrographis panic
Extraction fractionation from urata was performed according to the procedure shown in FIG. That is, dried Andrographis pa
niculata whole plant (300 g) was immersed in 4 L of methanol at room temperature for 7 days. After the extract was collected by filtration, 4 L of methanol was newly added to the residue of the extraction, and the mixture was cold-immersed at room temperature for 1 day, followed by filtration to obtain an extract. The filtrates obtained by the two extractions were combined, concentrated under reduced pressure, and extracted with methanol (2).
0.5 g). This was then combined with ethyl acetate-water, n-
Partitioned sequentially with butanol-water. During the concentration of the ethyl acetate-soluble fraction and the n-butanol-soluble fraction under reduced pressure, crystals were precipitated. The crystals were collected by filtration to obtain crude crystals. By the above operations, the ethyl acetate-soluble fraction (7.789 g), the n-butanol-soluble fraction (2.675 g), and the water-soluble fraction (6.0)
10 g) and the crude crystal 1 (2
64 mg), n-butanol layer soluble fraction crude crystals 2 (1.
958 g) of five crude fractions. Then, the soluble fraction crude crystals 1 (264 mg) were applied to a silica gel column (25 g) and eluted with chloroform / methanol (20: 1) while confirming the eluate by TLC. A fraction containing a spot with a remarkably high content was concentrated under reduced pressure to obtain crystals (212 mg). This crystal was prepared using the known compound neoandrographide, TL
The Rf values in C are matched with various solvents, as well as ¹ and neoandrographo1ide preparation H-
By matching the NMR spectra, neoand
rographoide (R ₃ in general formula II is a glucose residue). Similarly, from the soluble fraction crude crystals 2, a known compound, andrographo
Iide was identified.

Test Example 2 BCG-induced peritoneal macrophages are produced by oral administration
That nitrogen monoxide inhibition test (1) in vitro against macrophages collected from the process Test Example 1, mice were examined nitric oxide production inhibitory effect of the compounds of the present invention, in this test example 2, to mouse After oral administration of the compound of the present invention in advance, the effect of suppressing the nitric oxide production of the collected macrophages was examined. Preparation of a specimen and administration to a mouse were performed by the following methods. That is, among the specimens to be administered to the mice, the non-water-soluble specimens were used by suspending them in a saline solution containing 0.5% carboxymethylcellulose and thoroughly polished with a mortar. The mice were orally administered once a day from three days before the intraperitoneal injection of BCG for a total of three days. The oral dose per mouse was 50 mg / kg / day for the methanol layer, ethyl acetate layer, butanol layer and aqueous layer obtained in Example 2, and the soluble fraction crude crystal 1
30 mg / kg / day, neoa for
5 mg / kg for ndrographoide
/ Day. The collection of BCG-induced peritoneal macrophages, the measurement of the amount of nitric oxide in the macrophage culture supernatant, and the measurement of cell viability by the MTT method were performed in the same manner as in Test Example 1, and the degree of suppression of nitric oxide produced by macrophages was measured. And cell viability were measured.

(2) Results FIG. 4 shows the results when the components transferred to the methanol layer, ethyl acetate layer, butanol layer and aqueous layer were administered at a dose of 50 mg / kg / day. Crude crystal of soluble fraction 1
The results when and 2 were administered at a dose of 30 mg / kg / day are shown in FIG. neoandrogra
FIG. 6 shows the results when phoide was administered at a dose of 5 mg / kg / day. As shown in FIG. 4, the components transferred to the methanol layer and the ethyl acetate layer effectively suppressed the production of nitric oxide by oral administration. As shown in FIG. 5, the soluble fraction crude crystals 1
Upon oral administration at the time of addition, production of nitric oxide was efficiently suppressed. As shown in FIG. 6, neoandrog
Rapho1ide, when administered orally at 5 mg / kg / day, efficiently suppressed the production of nitric oxide.

[0040]

Industrial Applicability According to the present invention, an extremely safe nitric oxide production inhibitor which does not show harmful effects on humans and animals is provided. When the nitric oxide inhibitor of the present invention is used as a pharmaceutical or when used in foods and feeds, etc., it effectively inhibits the production of macrophage nitric oxide, and therefore has shock, hypotension, chronic joint It is useful for treating and / or preventing rheumatism, ulcerative colitis, ischemic encephalopathy, tumor, insulin-dependent diabetes, and the like.

[Brief description of the drawings]

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows a compound of the formula (I) in which both R ₁ and R ₂ are methyl groups.
It is a graph which shows the result of having measured the nitric oxide production suppression effect and the cell viability by changing the density | concentration in the range of -50 microgram / mL. In FIG. 1, * represents p <0.0 in the significance test.
5, *** indicates p <0.005 in the significance test.

FIG. 2 shows that the compound (Cs-1) of the present invention
It is a graph which shows the result of having measured the nitric oxide production suppression effect and the cell viability by changing the density | concentration in the range of -50 microgram / mL.

FIG. 3 shows a compound of the present invention in which R ₃ is a glucose residue in general formula II (neoandrograph).
ho1ide) Andrographis pani
5 shows an extraction procedure from C. culata.

FIG. 4 is a graph showing the effect of inhibiting the production of nitric oxide when the components transferred to the methanol layer, ethyl acetate layer, butanol layer and aqueous layer obtained in Example 2 are administered at a dose of 50 mg / kg / day. 4 is a graph showing the results obtained by measuring cell viability. In FIG. 4, ** represents p in the significance test.
<0.01, *** indicates p <0.005 in the significance test.

FIG. 5 shows a soluble fraction crude crystal 1 obtained in Example 2.
6 is a graph showing the results of measuring the nitric oxide production inhibitory effect and the cell viability when administering No. 2 and No. 2 at a dose of 30 mg / kg / day. In FIG. 5, *** represents p <0.005 in the significance test.

FIG. 6 shows neoandro obtained in Example 2.
It is a graph which shows the result of having measured the nitric oxide production inhibitory effect and cell viability at the time of administration of 5 mg / kg / day of graphoide. In FIG. 6, *
* Indicates p <0.01 in significance test and *** indicates p <0.005 in significance test.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 3/10 A61P 3/10 9/10 9/10 29/00 101 29/00 101 35/00 35 / 00 43/00 111 43/00 111 C07D 307/58 C07D 307/58 // C07C 45/74 C07C 45/74 C07H 15/26 C07H 15/26 (72) Inventor Ryo Soda Kitahama 4, Chuo-ku, Osaka-shi, Osaka 7-28, Sumitomo Forestry Co., Ltd. (72) Inventor Fumihide Takano 8-2-231-104, Nagamachi, Taihaku-ku, Sendai-city, Miyagi Prefecture (72) Inventor Shinji Fushiya 2-8-, Gobashi, Aoba-ku, Sendai City, Miyagi Prefecture 7-601 F term (reference) 4C037 JA04 4C057 BB02 DD01 KK02 4C086 AA01 AA02 AA03 BA03 EA11 GA17 MA01 MA04 NA14 ZA36 ZA43 ZA66 ZB15 ZB26 ZB35 ZC20 ZC35 4C206 AA01 AA02 AA03 CB19 ZA36 ZA35 Z35B 4H006 AA01 AA03 AB23 BJ50 BN30 BP30 BR30 GP03

Claims (2)

[Claims] 1. A nitric oxide production inhibitor comprising a compound represented by the following general formula I or II, a salt thereof or a hydrate thereof as an active ingredient. Embedded image [In the formula, R ₁ and R ₂ represent hydrogen or linear or branched lower alkyl having 1 to 6 carbon atoms. ] [Wherein, R ₃ represents hydrogen or a residue of a monosaccharide. ] 2. A compound represented by the following general formula I ′ or II ′. Embedded image [Wherein, R ′ ₁ and R ′ ₂ represent a linear or branched lower alkyl having 1 to 6 carbon atoms. ] [Wherein, R ′ ₃ represents hydrogen. ]