KR100553329B1 - Flavipucine showing apoptosis-inhibitive effect - Google Patents

Abstract

The present invention relates to flavifucin having apoptosis inhibitory activity, and more particularly, to determine the flavifusin by confirming that the flavifusin of formula (1) or a pharmaceutically acceptable salt thereof has apoptosis inhibitory activity The present invention relates to a composition for treating liver disease caused by an inflammatory disease, a neurodegenerative disease or a toxic substance due to an increase in apoptosis, which is contained as an active ingredient, and a health food.

Apoptosis, Flavifusin

Description

Flavipucine showing apoptosis-inhibitive effect

The present invention relates to flavifucin having apoptosis inhibitory activity, and more particularly, it is effective to confirm that flavifusin (flavipucin) of formula 1 or a pharmaceutically acceptable salt thereof has apoptosis inhibitory activity. The present invention relates to a composition for treating liver disease caused by an inflammatory disease, a neurodegenerative disease or a toxic substance due to increased apoptosis, and a health food.

Apoptosis or programmed cell death (PCD) is the basic intracellular process for spontaneous self-elimination of damaged or unwanted cells in a wide range of biological systems. Apoptosis is an essential mechanism for organ development, tissue remodeling, maintenance of intracellular homeostasis and removal of abnormal and damaged cells because the contents of cells that have died through the process of apoptosis are not extracellularly released and do not damage other cells.

As mentioned above, the apoptosis process, which plays an important role in the organism, must be precisely regulated according to a predetermined program. If apoptosis is inhibited, diseases such as cancer, autoimmune disease and viral infections are induced. Kerr et al ., Br. J. Cancer , 26, 239-257, 1972], when apoptosis is inappropriately increased, diseases such as acquired immunodeficiency syndrome, inflammatory diseases, various neurodegenerative diseases, ischemic diseases (strokes), and liver diseases caused by toxic substances such as alcohol Induced (Thompson et al., Science, 267, 1456-1462, 1995).

Early cells undergoing apoptosis develop chromatin aggregation, cytoplasm, and the like, which are induced during late stages of damage such as cell junctions, blebbing of cell membranes, and cell shrinkage. Nuclear enrichment (cytoplasm and nuclei condensation), loss of mitochondrial membrane potential, changes in plasma membrane composition, and apoptotic body formation are induced to undergo morphological and biochemical changes throughout. DNA fragmentation in the form of oligonucleosomes, the final result of apoptosis, is a typical biochemical feature of cells undergoing apoptosis [Green DR, and Reed, JC, Science , 281, 1309-1312, 1998].

Many apoptosis-related studies have now identified key players and mechanisms involved in apoptosis in a variety of organisms. Representatively, in mammalian cells, caspase, a type of cellular cysteine protease, is known to play an important role in regulating apoptosis mechanism, which is estimated by homology of amino acid sequence. By the number of evolutionary strains can be divided into three types centered on ICE (interlukin 1 converting enzyme), CPP32, ICH-1.

Caspases may be receptor-mediated signal transduction, depletion of growth factors, oxidative stress, DNA damage and cell-cells. ) Or activated by a variety of stimuli, including disruption of cell-matrix interactions, poly (ADP-ribose) polymerase (RARP), lamine, Induces apoptosis formation by degrading cellular proteins such as cytokeratins and caspase-activated DNase inhibitors (ICAD) [Cryns, V., and Yuan, J., Genes Dev ., 12, 1551-1570, 1998].

Active studies of apoptosis have revealed that in mammals caspases belonging to at least 14 different classes so far, all of which are peptide substrates after aspartic acid residues, which play a pivotal role in the initiation and execution of apoptosis. Has been reported to induce apoptosis by cutting (Nunez et al ., Oncogene, 17, 3237-3245, 1998).

Although flavifucin is already known as a secondary metabolite produced by microorganisms, it was first confirmed by the inventors that it has apoptosis inhibitory activity.

Accordingly, the present inventors have tried to search for apoptosis inhibitors in order to effectively treat diseases caused by excessive apoptosis. As a result, the present inventors confirmed that flavifucin represented by the following formula (1) exhibited an excellent apoptosis inhibitory effect in apoptosis-induced cell lines. The invention was completed.

[Formula 1]

Therefore, the present invention is a liver disease caused by inflammatory diseases, neurodegenerative diseases or toxic substances due to increased apoptosis containing flavifusin or a pharmaceutically acceptable salt thereof as an active ingredient, and a health food The purpose is to provide.

The present invention provides a therapeutic composition and a health food for liver disease caused by inflammatory diseases, neurodegenerative diseases or toxic substances due to increased apoptosis containing flavifucin represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient It features.

[Formula 1]

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

The present invention is to determine whether the flavifusin (flavipucin) of the formula (1) or a pharmaceutically acceptable salt thereof has apoptosis inhibitory activity, inflammatory diseases, neurodegenerative diseases caused by increased apoptosis containing flavifucin as an active ingredient or It relates to a composition for treating liver disease caused by a toxic substance and a health food.

In the present invention, the apoptosis inhibitory effect of the blood cancer cell line of flavifusin having the following physicochemical properties was tested, it was confirmed that the apoptosis inhibitory activity than the reference material PDTC (pyrrolidine dithiocarbonate).

1) Compound of Formula 1

i) Material Properties: Powder ii) Molecular Weight: 237

iii) Molecular formula: C ₁₂ H ₁₅ O ₄ iv) Mass spectrometry (MH) ^- : 236 ( m / z )

On the other hand, flavifusin having excellent apoptosis inhibitory activity of the present invention can be used in the form of a pharmaceutically acceptable salt, and acid addition salts formed by pharmaceutically acceptable free acid are useful as salts. . Compounds of formula (1) can form pharmaceutically acceptable acid addition salts according to methods conventional 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.

Therefore, the active ingredient of the present invention flavifusin or a pharmaceutically acceptable salt thereof is liver disease caused by toxic substances such as inflammatory diseases, acquired immunodeficiency, various neurodegenerative diseases, ischemic diseases (stroke) and alcohols due to increased apoptosis Can be treated or prevented.

That is, a composition for treating inflammatory diseases, neurodegenerative diseases and liver diseases containing flavifucin or a pharmaceutically acceptable salt thereof as an active ingredient.

The composition according to the invention can be administered orally or parenterally, for example, intravenously, subcutaneously, intraperitoneally or topically, during clinical administration and can be used in the form of general medicines and health foods.

In addition, the compositions 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 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, flavifucin 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 flavifucin or a pharmaceutically acceptable salt thereof is generally 1 to 50 mg / day per kg body weight of an adult patient, preferably has an effective dose of 5 to 20 mg / day In accordance with the judgment of the doctor or pharmacist, it may be administered several times a day, preferably divided into two to three times a day at regular intervals.

On the other hand, the above-mentioned health food is a food prepared by adding the active ingredient to food materials such as beverages, teas, spices, gums, confections, or the like, encapsulated, powdered, suspensions, etc. Means to bring a certain effect, 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.

Reference Example 1: Physicochemical Characterization of Flavifucin

In order to analyze the physicochemical properties of the flavifusin obtained from the fungus, we used methods such as electrospray ionization mass spectrometry (ESI-MS), Fisons VG Quattro 400 mass spectrometer (USA), hydrogen and carbon nuclear magnetic resonance spectra. It was. In the NMR experiment, each sample was dissolved in methanol (CD ₃ CD) solvent and measured in a 5 mm NMR tube, and the chemical shift was measured based on the peak of each solvent or the peak of TMS (tetramethylsilane). . As a result of analyzing the property, molecular weight, molecular formula and mass of the flavifucin, the compound of the present invention was found to have the following physical and chemical properties.

As a result, the material property of flavifucin was powder, the molecular weight was 237, the molecular formula was confirmed as C ₁₂ H ₁₅ O ₄ , and the mass spectrometry (MH) ⁻ of this compound was 236 ( m / z ). Hydrogen nuclear magnetic resonance spectra and carbon nuclear magnetic resonance spectra of methanol (CD ₃ CD) as a solvent were measured together with the physicochemical properties, and are represented by the following Chemical Formula 1.

<NMR data>

Compound of Formula 1

¹³ C NMR (CD ₃ CD) δ 21.8, 21.8, 22.4, 24.0, 43.3, 63.3, 80.9, 104.4, 145.4, 171.8, 197.6, 208.2,

[Formula 1]

Example 1: Determination of apoptosis inhibitory effect of flavifucin

In order to measure the apoptosis inhibitory effect of flavifucin, the present inventors performed the following in vitro experiment using U937 cells, a human blood cancer cell line.

U937 cells were cultured in 96-well microplates until they were 5 × 10 ⁶ cells / well, and then 10 μg / ml of apoptotic inducer etoposide and 1 to 100 μg / of the compound of the present invention. Treatment with the U937 cells at a concentration of ㎖ and incubated for 5 hours at 37 ℃ under 5% CO ₂ -95% air conditions to compare the apoptosis inhibitory effect. At this time, cells treated with only etoposide were used as the positive control group, and cells not treated with both etoposide and the compound of the present invention were used as the negative control group. After 5 hours, the shape of U937 cells was observed under a microscope to determine apoptosis. The cells obtained by centrifugation were treated with DEVD-AFC (Z-Asp-Glu-Val-Asp-7-amino-4-trifluoromethyl). coumarin) was used to measure caspase-3 activity. Determine the apoptosis inhibitory effect exhibited by the compounds of the present invention based on the caspase-3 activity of the positive control (only treated with etoposide) and the caspase-3 activity of the negative control (untreated with etoposide and compounds) It is shown in Table 1 below.

Comparison of Apoptosis Inhibitory Effects in U937 Cell Lines compound Inhibition rate (IC ₅₀ , μg / ml) Flavifucin 0.35 Pyrrolidine dithiocarbonate (PDTC) 15.0

As shown in Table 1 above, the inhibitory activity concentration of flavifucin (a concentration required to exhibit an inhibitory effect of IC ₅₀ , 50%) on the apoptosis of etoposide-induced U937 cells is 0.35 μg / ml and is a standard comparative material. The inhibitory activity concentration of phosphorus PDTC (pyrrolidine dithiocarbonate) was shown to be 15.0 μg / ml, indicating that the flavifucin of the present invention had a superior apoptosis inhibitory effect than that of the PDTC standard. From the above results, it was confirmed that the flavifucin of the present invention is a compound capable of inhibiting apoptosis in vitro.

Example 2: Oral Administration of Rats Acute Toxicity Test

Acute toxicity test was performed using 6-week-old SPF SD rats. Two animals per group were individually administered orally at a dose of 1 g / kg / 1 ml suspended in 0.5% methylcellulose solution in flavifucin. After administration of the test substance, mortality, clinical symptoms, and changes in body weight were observed. Hematological and hematological examinations were performed. The 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, the tested compound 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, flavifucin exhibits an excellent apoptosis inhibitory effect to treat inflammatory diseases, acquired immunodeficiency, various neurodegenerative diseases, ischemic diseases (strokes), and liver diseases caused by toxic substances such as alcohol. It can be formulated into a pharmaceutical composition.

Formulation Example 1 Preparation of Syrup

A syrup containing flavifucin of the present invention or a pharmaceutically acceptable salt thereof as an active ingredient of 2% (weight / volume) was prepared by the following method.

Acid addition salts of flavifucin, saccharin, and sugar 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.

Flavifucin hydrochloride 2 g

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 was prepared by the following method.

250 g of flavifucin hydrochloride were mixed with 175.9 g lactose, 180 g potato starch and 32 g 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.

Flavifucin hydrochloride 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 flavifucin hydrochloride, 0.6 g of sodium chloride and 0.1 g of ascorbic acid 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.

Flavifucin hydrochloride 1 g

Sodium Chloride ・ ・ ・ ・ 0.6 g

0.1 g of ascorbic acid

Distilled water ··················

Formulation Example 4: Beverage Preparation

After dissolving 500 mg of flavifucin in an appropriate amount of water, vitamin C as an auxiliary component, citric acid, sodium citrate, oligosaccharides as an auxiliary component, and an appropriate amount of sodium benzoate as a preservative are added, followed by water, and the total amount is 100 ml. To prepare a beverage composition. At this time, taurine, myo-inositol, folic acid, pantothenic acid, etc. may be added alone or together.

As described above, the flavifucin according to the present invention exhibits an excellent apoptosis inhibitory effect, and thus is caused by toxic substances such as inflammatory diseases, acquired immunodeficiency diseases, various neurodegenerative diseases, ischemic diseases (strokes) and alcohols due to increased apoptosis. It can be usefully used to treat or prevent liver disease.

Claims (4)

The composition for treating inflammatory diseases due to an increase in apoptosis containing a compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient. [Formula 1]

A neurodegenerative disease therapeutic composition due to an increase in apoptosis containing a compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient. [Formula 1]

Next, a composition for treating liver disease due to an increase in apoptosis containing a compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient. [Formula 1]

A health food comprising the compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as a main component. [Formula 1]