KR100599249B1 - Nitric oxide production inhibiting compound purified from Forsythiae fructus - Google Patents

Abstract

The present invention relates to a compound having a nitric oxide production inhibitory activity isolated from duct bridge, and more particularly, the compound represented by the following formula (1) after separation from duct bridge ( Forsythiae fructus ) is nitric oxide by iNOS (inducible nitric oxide synthase) The present invention relates to an inflammatory disease, an immune disease therapeutic composition, and a health food caused by increased production of nitric oxide containing the compound as an active ingredient. The present invention also relates to a method for separating the compound of formula (1) from duct bridge.

 Forsythiae fructus, iNOS, nitric oxide

Description

Nitric oxide production inhibiting compound purified from Forsythiae fructus}             

1 is a result of measuring the effect of inhibiting the production of nitric oxide by the compound represented by the formula (1) of the present invention.

2 is a result of analysis of Western blot measuring the protein expression inhibitory effect of iNOS by the compound represented by Formula 1 of the present invention.

The present invention relates to a compound having a nitric oxide production inhibitory activity isolated from duct bridge, more specifically, the compound represented by the formula (1) isolated from Forsythiae fructus (nitrogen oxide) by iNOS (inducible nitric oxide synthase) The present invention relates to an inflammatory disease, an immune disease therapeutic composition, and a health food caused by an increase in the production of nitric oxide containing the compound as an active ingredient.

Nitric oxide (NO), one of the inflammatory mediators, is produced in endothelial cells or macrophages in a normal state and exhibits various physiological activities in addition to cell killing and bactericidal action. It is known to play a role in maintaining homeostasis. Stimulations such as LPS (lipopolysaccharide; `` LPS ''), inflammation-inducing factors and irradiation, in particular, induce the expression of iNOS, an inducible nitric oxide synthase, continuously producing large amounts of NO for 4-6 hours. The excessive amount of NO thus produced causes such diseases.

Therefore, the development of an iNOS activity inhibitor is highly likely to be developed as a therapeutic agent for the above-mentioned diseases, and in this respect, the compounds that inhibit NO production by iNOS are therapeutic agents for inflammatory diseases such as arthritis, sepsis and autoimmune diseases such as diabetes and diabetes. Can be used as [AS Baldwin Jr. J. Clin. Invest. , 2001, 107, 241-2460.

Research into substances that inhibit the production of NO has been mainly focused on the development of substances that specifically inhibit the enzymatic activity of iNOS, derivatives of L-arginin, precursors, derivatives of L-citrulline, Development studies of aminoguanidine derivatives, isothiourea derivatives, and the like are being conducted [Babu, BRB, OW, Current Opinion in Chemical Biology, 2: 491-500, 1998].

On the other hand, many compounds have been reported in ducts and natural plants, but there have been no reports that these compounds inhibit the production of NO in LPS-stimulated cell lines or the expression of iNOS.

 Therefore, the present inventors focused on the fact that inhibiting the activity of iNOS can be used for the treatment of inflammatory diseases and immune diseases, and as a result of the study, by separating the compound comprising the formula (1) of the iNOS which is an inflammation inducing enzyme It was confirmed that the inhibition of expression strongly inhibits the production of nitric oxide and completed the present invention by confirming that it is a safe substance through toxicological experiments.

Therefore, an object of the present invention is to provide a compound represented by the following formula (1).

[Formula 1]

Another object of the present invention is to provide an inflammatory disease or immune disease therapeutic composition and health food due to the increased production of nitric oxide containing the compound of Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

Another object of the present invention is to provide a method for separating a compound having a nitric oxide inhibitory activity from the duct bridge.

The present invention is characterized by an inflammatory disease or immune disease therapeutic composition and health foods due to the increased production of nitric oxide containing a compound of formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

[Formula 1]

In addition, the present invention

1) extracting Forsythiae fructus with methanol, concentrating under reduced pressure, and then extracting with ethyl acetate to obtain ethyl acetate extract;

2) concentrating the ethyl acetate extract, dissolving it in a small amount of mixed solvent, and performing silica gel column adsorption chromatography to concentrate the active fraction to obtain a concentrate;

 3) collecting the active fraction by dissolving the concentrate in methanol and then performing Sephadex LH-20 gel column chromatography; And

4) separating the active fraction by high pressure liquid chromatography, removing the solvent with a reduced pressure dryer, and freeze drying to obtain a compound represented by Formula 1 below.

Another aspect of the method for separating the nitric oxide production inhibitors from the bridge comprising a.

The present invention will be described in more detail as follows.

The present invention is to increase the production of nitric oxide containing the compound as an active ingredient by confirming that the compound represented by the formula (1) after separation from Forsythiae fructus has an inhibitory activity of nitric oxide production by inducible nitric oxide synthase (iNOS) It relates to inflammatory diseases, immunological diseases therapeutic composition and health food. The present invention also relates to a method for separating the compound of formula (1) from duct bridge.

First, a method of separating and extracting an apoptosis inhibitory active substance from ducts will be described in detail.

Take ducts, reflux for 48 hours with methanol, concentrate under reduced pressure, and extract with ethyl acetate.

The active fraction is concentrated by performing silica gel column adsorption chromatography using the concentrated ethyl acetate extract as a elution solvent using a mixed solvent of chloroform and methanol (volume ratio of 100 to 10: 1).

After dissolving the concentrated active fraction in methanol, the active fractions are collected by performing Sephadex LH-20 gel column chromatography.

Holding time by separating the active fraction by high pressure liquid chromatography using a C18 column and an elution solvent of water-acetonitrile (20% acetonitrile to 100% acetonitrile, 30 minutes) The compound of formula 1 is obtained from an active fraction of 12 components.

In addition, the compounds according to the present invention can be extracted and separated from duct bridge as described above, and can be prepared by organic synthesis.

The compound of the present invention represented by the formula (1) can be used in the form of a pharmaceutically acceptable salt, the salt is an acid addition salt formed by a pharmaceutically acceptable free acid is useful. The compound of Formula 1 may form an acid addition salt which is pharmaceutically acceptable according to a conventional method in the art. Free acid and inorganic acid may be used as the free acid, and hydrochloric acid, bromic acid, sulfuric acid, phosphoric acid, etc. may be used as the inorganic acid, and citric acid, acetic acid, lactic acid, tartaric acid, maleic acid, and fumaric acid may be used as the organic acid. (fumaric acid), formic acid, propionic acid, oxalic acid, trifluoroacetic acid, benzoic acid, gluconic acid, methanesulfonic acid, glycolic acid, succinic acid, 4-toluenesulfonic acid, galluxuronic acid, embonic acid, glutamic acid or aspartic acid Can be used.

As a result of measuring the nitric oxide inhibitory activity and the iNOS expression inhibitory effect of the compound of Formula 1 or a pharmaceutically acceptable salt thereof, it was confirmed that the compound having the excellent nitric oxide inhibitory activity.

Therefore, the present invention contains a compound represented by the formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient to inhibit the abnormally induced nitric oxide production mechanisms for the treatment of inflammatory diseases and immune diseases caused by increased nitric oxide production And health foods.

The compositions of the present invention may be administered orally or parenterally, for example, intravenously, subcutaneously, intraperitoneally or topically, during clinical administration and may be used in the form of general pharmaceuticals and health foods.

Compositions of the invention may be formulated for oral administration such as tablets, troches, lozenges, aqueous or oily suspensions, prepared powders or granules, emulsions, hard or soft capsules, syrups or elixirs Formulated). Binders such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose or gelatin for preparation in formulations such as tablets and capsules; Excipients such as dicalcium phosphate; Disintegrants such as corn starch or sweet potato starch; Lubricants such as magnesium stearate, calcium stearate, sodium stearyl fumarate or polyethylene glycol wax. Capsules contain liquid carriers, such as fatty oils, in addition to the substances mentioned above.

In addition, the composition of the present invention can be administered parenterally, parenteral administration is by subcutaneous injection, intravenous injection, intramuscular injection or intrathoracic injection injection method. To formulate into a non-oral dosage form, the compound of Formula 1 or a pharmaceutically acceptable salt thereof is mixed in water with a stabilizer or buffer to prepare a solution or suspension, which is formulated in unit dosage forms of ampoules or vials.

In addition, the active ingredient of the present invention is generally 1 to 50 mg / day per 1 kg of adult patient weight, preferably has an effective dose of 5 to 20 mg / day, at regular intervals according to the judgment of the doctor or pharmacist It may be administered several times a day, preferably two to three times a day.

In addition, the health food is a food prepared by adding the active ingredient to food materials such as beverages, teas, spices, gums, confectionery, or the like, encapsulated, powdered, or suspension, and when ingested, has a specific health effect. It means coming, but unlike the general medicine has the advantage that there is no side effect that can occur when using the drug as a raw material for long-term use.

Hereinafter, the present invention will be described in more detail based on the following examples, but the present invention is not limited thereto.

Example 1 Isolation of Nitric Oxide Production Inhibitor from Fed Bridge

1 kg of duct bridge purchased from Ilshin Chemical was extracted with 100% methanol for 48 hours, concentrated under reduced pressure and extracted with ethyl acetate. The ethyl acetate layer was concentrated and dissolved in a small amount of a mixed solvent of chloroform and methanol (100: 1), followed by silica gel column chromatography (20-230 mes, Merck) to concentrate the active fraction.

High-performance liquid chromatography (column: C18, flow rate: 1.5 ml / min, 220 nm detection) using the concentrated active fraction as an eluent for water-acetonitrile (20% acetonitrile to 100% acetonitrile, 30 minutes) The active fraction of 12 minutes of holding time was separated and the solvent was removed by a vacuum dryer, and the residue obtained was lyophilized to obtain 7.1 mg of nitric oxide inhibitor.

Example 2: Physicochemical Characterization of Separation Materials

In order to analyze the physicochemical properties of the separated compounds, the inventors used methods such as electrospray ionization mass spectrometry (ESI-MS), Fisons VG Quattro 400 mass spectrometer (USA), hydrogen and carbon nuclear magnetic resonance spectra. . In the NMR experiment, each sample was dissolved in methanol (CD ₃ OD) solvent and measured in a 5 mm NMR tube. The chemical shift was measured based on the peak of each solvent as the internal standard or the peak of TMS (tetramethylsilane). . As a result of analyzing the property, molecular weight, molecular formula and mass of the compound, it was confirmed that the compound of the present invention had the following physical and chemical properties.

As a result, the material property of the compound was powder, the molecular weight was 154, the molecular formula was confirmed as C ₈ H ₁₀ O ₃ , and the mass spectrometry (MH) ⁻ of the compound was 153 ( m / z ). Hydrogen nuclear magnetic resonance spectra and carbon nuclear magnetic resonance spectra of methanol (CD ₃ OD) as a solvent, together with the physicochemical properties, were analyzed. As a result, the compound of the present invention represented by the following Chemical Formula 1 has reported its function to date. It turned out to be a compound that exhibits an inhibitory effect on nitric oxide production.

<NMR data>

Compound of Formula 1

¹ H NMR (CD ₃ OD) δ 2.22, 2.58, 2.75, 3.6, 3.82, 3.95, 5.95, 6.78

¹³ C NMR (CD ₃ OD) δ 40.38, 40.54, 67.1, 75.55, 82.3, 128.8, 150.7, 199.1

[Formula 1]

Experimental Example 1: Determination of nitric oxide production inhibitory effect of the compound of formula 1

In order to measure the nitric oxide production inhibitory effect of the compound represented by the formula (1), the inventors carried out as follows to investigate the inhibition of NO production.

To measure the amount of NO produced by adding the compound of Formula 1 isolated from the duct of Example 1 to the murine macrophage stimulated with LPS, more specifically, Raw 264.7 murine macrophages of Dulbecco Suspended in Dulbecco's Modified Essential Medium (hereinafter referred to as 'DMEM') and dispensed in 96 well plates at a concentration of 1 × 10 ⁵ cells / ml and for 3 hours at 37 ° C, 5% CO ₂ . Cells were attached by incubation. In order to induce NO production in the macrophages, LPS was added at a final concentration of 1 μg / ml, and the compounds separated at the ducts of Example 1 were added at 25, 12.5, 6.25, 3, and 1.5 μg / ml, respectively. And incubated for 24 hours.

In order to measure the concentration of NO induced production by LPS in the culture, the culture was recovered and centrifuged, and 100 µl of the grease reagent was added to 100 µl of each culture, and left at room temperature for 10 minutes, and the absorbance was measured at 550 nm. The grease reagent was prepared by mixing 1: 1 solution of 1% sulfanilamide in 5% phosphoric acid and an aqueous solution of 0.1% naphthylethylenediamine-HCl (naphthyethldiamine-HCl).

In order to measure the standard NO production amount, NaNO ₂ was diluted by concentration, and then reacted with the grease reagent to measure absorbance to prepare a calibration curve. Therefore, FIG. 1 shows the results of measuring the amount of NO ₂ (nitrite) to obtain the amount of NO produced by the addition of the compound separated in the bridge of Example 1 by using the calibration curve. 1 expressing the results, it was observed that the compound of Formula 1 inhibits the production of NO in LPS-induced macrophages in a concentration-dependent manner.

Inhibition concentration of NO was observed as IC ₅₀ (inhibition concentration) was 5.45 ㎍ / ㎖ of the compound of formula 1, the addition of a small amount of the compound was excellent in inhibiting NO production.

Experimental Example 2: Confirmation of the iNOS expression inhibitory effect of the compound of Formula 1

In the present invention, the compound of Formula 1 was tested as follows to confirm the inhibitory effect of iNOS expression through Western blot analysis.

The raw 264.7 murine macrophages were treated with 1 μg / ml LPS and at the same time the compounds of Formula 1 extracted from the ducts of the present invention were treated with 25, 12.5, 6.25, 3, 1.5 μg / ml concentrations at 37 ° C., 5% CO ₂ Incubated for 18 hours at. Total cell lysates were prepared from the cultured cells using protein extraction buffer (N. Suh et al, Cancer Res., 1998, 58, 712-723) and analyzed by Western blot.

FIG. 2 is a result of Western blot analysis of the protein inhibition effect of iNOS by the compound of Formula 1, which is 130 kDa iNOS protein in LPS-induced macrophages in the control group not treated with Formula 1 Expression was inhibited. In particular, protein expression of iNOS was completely inhibited when the compound of Formula 1 was treated at a concentration of 25 μg / ml. Results of inhibiting protein expression of iNOS by the addition of the compound of Formula 1 are shown in FIG. 2.

Experimental Example 3: Acute Toxicity of Oral Administration in Rats

Acute toxicity test was performed using 6-week-old SPF SD rats. Two animals per group were suspended orally in a dose of 1 g / kg / 1 ml by suspending the compound of formula 1 in 0.5% methylcellulose solution, respectively. After administration of the test substance, mortality, clinical symptoms, and changes in body weight were observed. Hematological and hematological examinations were performed. Necropsy was performed to visually observe the abdominal and thoracic organ abnormalities. As a result, there were no clinical symptoms or deaths in all animals treated with the test substance, and no toxicity change was observed in weight change, blood test, blood biochemistry test, autopsy findings, etc. As a result, all of the tested compounds did not show toxic changes up to 500 mg / kg in rats, and the minimum lethal dose (LD ₅₀ ) was determined to be a safe substance of 500 mg / kg or more.

As confirmed above, the compound of the present invention represented by the formula (1) exhibits an excellent effect of inhibiting the production of nitric oxide, and may be prepared as a composition for treating inflammatory diseases or for treating immunological diseases.

Formulation Example 1 Preparation of Syrup

Syrup containing the compound of Formula 1 of the present invention or a pharmaceutically acceptable salt thereof as an active ingredient of 2% (weight / volume) is prepared by the following method.

Acid addition salts, saccharin and sugars of the compound of formula 1 were dissolved in 80 g of warm water. After the solution was cooled, a solution consisting of glycerin, saccharin, spices, ethanol, sorbic acid and distilled water was prepared and mixed. Water was added to this mixture to 100 ml. The addition salt can be replaced with other salts according to the examples.

The components of the syrup are as follows.

Hydrochloride of a compound of formula 1

Saccharin 0.8 g

25.4 g of sugar

Glycerin ... 8.0 g

Spices ··················· 0.04 g

Ethanol 4.0 g

Sorbic acid0.4 g

Distilled water ·····················

Formulation Example 2: Preparation of Tablet

A tablet containing 15 mg of active ingredient is prepared by the following method.

250 g of hydrochloride of the compound of formula 1 were mixed with 175.9 g of lactose, 180 g of potato starch and 32 g of colloidal silicic acid. 10% gelatin solution was added to the mixture, which was then ground and passed through a 14 mesh sieve. It was dried and the mixture obtained by adding 160 g of potato starch, 50 g of talc and 5 g of magnesium stearate was made into a tablet.

The components of the tablet are as follows.

Hydrochloride of the compound of formula 1 ... 250 g

Lactose ···················· 175.9 g

Potato starch ·············· 180 g

Colloidal silicic acid 32 g

10% gelatin solution

Potato starch · 160 g

Talc · 50 g

Magnesium stearate 5 g

Formulation Example 3: Preparation of Injection

Injection solution containing 10 mg of the active ingredient was prepared by the following method.

1 g of hydrochloride, 0.6 g of sodium chloride and 0.1 g of ascorbic acid of the compound of formula 1 were dissolved in distilled water to make 100 ml. The solution was bottled and sterilized by heating at 20 ° C. for 30 minutes.

The components of the injection solution are as follows.

Hydrochloride of the compound of formula 1 ... 1 g

Sodium Chloride ・ ・ ・ ・ 0.6 g

0.1 g of ascorbic acid

Distilled water ··················

As described above, the compound of Formula 1 obtained from the duct bridge according to the present invention is not toxic and exhibits an excellent inhibitory effect on nitric oxide production, and thus is useful as a medicine or health food for treating inflammatory diseases and immune diseases due to increased nitric oxide production. Can be used.

Claims (5)

A composition for treating inflammatory diseases due to an increase in nitric oxide production, which comprises a compound represented by the following Chemical Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient. [Formula 1]

The composition for treating immune diseases due to the increased production of nitric oxide, characterized in that it comprises a compound represented by the formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient. [Formula 1]

1) extracting Forsythiae fructus with methanol, concentrating under reduced pressure, and then extracting with ethyl acetate to obtain ethyl acetate extract; 2) concentrating the ethyl acetate extract, dissolving it in a small amount of mixed solvent, and performing silica gel column adsorption chromatography to concentrate the active fraction to obtain a concentrate;  3) collecting the active fraction by dissolving the concentrate in methanol and then performing Sephadex LH-20 gel column chromatography; And 4) separating the active fraction by high pressure liquid chromatography, removing the solvent with a reduced pressure dryer, and freeze drying to obtain a compound represented by Formula 1 below. Separation method of nitric oxide production inhibitors from duct bridge comprising a. The method of claim 3, wherein the nitric oxide production inhibitor is a compound represented by the following formula (1). [Formula 1]

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