KR100641867B1 - Compound showing apoptosis-inhibitive effect purified from Xanthium strumarium - Google Patents

Abstract

The present invention relates to a compound exhibiting an apoptosis inhibitory effect isolated from docomari , and more specifically, Xanthium strumarium extract, and isolated from it and then confirmed that the compound of formula 1 has apoptosis inhibitory activity It relates to a inflammatory disease, neurodegenerative disease or liver disease therapeutic composition and health foods due to an increase in apoptosis containing the compound as an active ingredient. The present invention also relates to a method for separating the compound of formula

Apoptosis, Xanthium strumarium

Description

Compound showing apoptosis-inhibitive effect purified from Xanthium strumarium}

The present invention relates to a compound exhibiting an apoptosis inhibitory effect isolated from docomari , and more specifically, Xanthium strumarium extract, and isolated from it and then confirmed that the compound of formula 1 has apoptosis inhibitory activity It relates to a inflammatory disease, neurodegenerative disease or liver disease therapeutic composition and health foods due to an increase in apoptosis containing the compound as an active ingredient.

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 released extracellularly and do not damage other cells.

As mentioned above, the apoptosis process, which plays an important role in living organisms, must be precisely regulated according to a predetermined program. If apoptosis is inhibited, diseases such as cancer, autoimmune disease and viral infectious diseases are induced. Kerr et al., Br. J. Cancer, 26, 239-257, 1972], Inappropriately increasing apoptosis leads to diseases such as acquired immunodeficiency, various neurodegenerative diseases, ischemic diseases (strokes) and liver diseases caused by toxic substances such as alcohol.

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 revealed 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 evolutionary tree, three types of interleukin 1 converting enzyme (ICE), CPP32 and ICH-1 can be classified.

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, such as poly (ADP-ribose) polymerase (RARP), lamine, Induces apoptosis formation by breaking down cellular proteins such as cytokeratins and caspase-activated DNase inhibitors (ICADs) [Cryns, V., and Yuan, J., Genes Dev., 12, 1551-1570, 1998].

As a result of active research on apoptosis, caspases belonging to at least 14 different classes so far have been identified in mammals, all of which are peptides after aspartic acid residues that play a pivotal role in the initiation and execution of apoptosis. Induction of apoptosis by cleaving substrates has been reported (Nunez et al., Oncogene, 17, 3237-3245, 1998).

There are many studies on natural substances that inhibit apoptosis for the above reasons, but the search for natural substances is insufficient.

Accordingly, the present inventors have tried to search for compounds capable of modulating apoptosis in natural substances, and have isolated compounds exhibiting apoptosis inhibitory effects from lizards to identify their physicochemical properties and said compounds have excellent apoptosis in apoptosis-induced cell lines. The present invention was completed by confirming the inhibitory effect.

Accordingly, the present invention is an apoptosis inhibitor composition containing Xanthium strumarium extract, and the compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient, that is, inflammatory diseases due to increased apoptosis, It is an object of the present invention to provide a composition for treating liver disease caused by neurodegenerative diseases and toxic substances.

[Formula 1]

It is another object of the present invention to provide a method for separating a compound having apoptosis inhibitory activity from a lizard.

The present invention is characterized by a composition for treating liver disease caused by inflammatory diseases, neurodegenerative diseases and toxic substances containing Xanthium strumarium extract as an active ingredient.

In addition, a compound represented by the following formula (1) having an excellent apoptosis inhibitory activity or a pharmaceutically acceptable salt thereof, and a therapeutic agent composition for treating liver disease by inflammatory diseases, neurodegenerative diseases and toxic substances containing the same as an active ingredient .

[Formula 1]

In addition, the method of extracting and separating apoptosis inhibitory activator from docomari includes the following steps:

 1) extracting the macaw horse with methanol, concentrated under reduced pressure and extracted with ethyl acetate to obtain an ethyl acetate extract;

 2) concentrating the ethyl acetate extract, dissolving it in a mixed solvent of chloroform and methanol, and performing chromatography to concentrate the active fraction to obtain a concentrate;

3) collecting the active fraction by dissolving the concentrated solution in methanol and performing chromatography; And

4) separating the active fraction by chromatography, removing the solvent with a reduced pressure dryer, and freeze drying to obtain an apoptosis inhibitory active material.

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

The present invention relates to an extract of Xanthium strumarium having apoptosis inhibitory activity, a compound isolated therefrom, a separation method thereof, and an apoptosis inhibitor composition containing the compound as an active ingredient.

As mentioned above, the compounds according to the present invention can be synthesized by organic synthesis or can be extracted and separated from the larvae according to the method of the present invention.

First, a method of separating and extracting an apoptosis inhibitory active substance from docomari is described in detail as follows.

Extract the coconut, extract it under reflux for 40-50 hours with 100% methanol, filter under reduced pressure, and extract with ethyl acetate.

The concentrated ethyl acetate extract was dissolved in a mixed solvent of chloroform and methanol (100: 1), followed by silica gel column adsorption using a mixed solvent of chloroform and methanol (100: 1, 50: 1 and 10: 1) as an elution solvent. The active fractions are concentrated by chromatography (silica gel column adsorption chromatography).

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

The active fraction is subjected to high pressure liquid chromatography using a C18 column and 20 to 100% acetonitrile aqueous solution to separate the active fraction to obtain a compound of Chemical Formula 1 from the active fraction having a holding time of 26 minutes.

As a result of analyzing the chemical properties of the compound of the present invention represented by Chemical Formula 1 by using a method such as electron spray ionization mass spectrometer (ESI-MS), nuclear magnetic resonance spectrum, etc., the compound of the present invention is as follows: It was confirmed to have characteristics.

<Compound of Formula 1>

i) Material Properties: Powder ii) Molecular Weight: 234

iii) molecular formula: C ₁₄ H ₁₈ O ₃ iv) mass spectrometry (MH)-: 233 (m / z)

In addition, hydrogen nuclear magnetic resonance spectra and carbon nuclear magnetic resonance spectra were analyzed using chloroform (CDCl ₃ ) as a solvent. As a result, it was found to be a compound represented by Chemical Formula 1.

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. 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, 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, 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 Etc. can be used.

Finally, the present invention provides an apoptosis inhibitor composition containing as an active ingredient extract of Docomari, a compound of Formula 1 or a pharmaceutically acceptable salt thereof.

Since the pharmaceutical composition contains an extract of Docomari, a compound represented by Formula 1 showing an apoptosis inhibitory effect, or a pharmaceutically acceptable salt thereof as an active ingredient, inflammatory diseases, cancers, and the like caused by an increase in apoptosis by inhibiting apoptotic mechanisms that are abnormally induced. It can be usefully used to treat or prevent AIDS, various neurodegenerative diseases, ischemic diseases (strokes) and liver diseases caused by toxic substances such as alcohol.

The pharmaceutical composition 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 medicines and health foods.

Pharmaceutical 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 into elixirs). 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 pharmaceutical composition of the present invention may be administered parenterally, and parenteral administration may be by subcutaneous injection, intravenous injection, intramuscular injection, or intrathoracic injection injection. To formulate into a parenteral formulation, the compound of Formula 1 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 dosage of the active ingredient is generally 1 to 50 mg / day per kg body weight of an adult patient, preferably 5 to 20 mg / day, 1 to several times at regular intervals according to the judgment of the doctor or pharmacist Preferably, it can be divided into 2-3 times a day.

In addition, the composition according to the present invention includes a health food containing the extract of Docomari, the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

The health food is a food prepared by adding a compound represented by the formula (1) or a pharmaceutically acceptable salt thereof to food materials such as beverages, teas, spices, gums, confectionery, or the like, encapsulated, powdered, suspensions, etc. When ingested, it means that it brings a specific effect on health, but unlike general medicine, it has the advantage that there is no side effect that can occur when the long-term use of the drug with food as a raw material.

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 and Purification of Apoptosis Inhibitors

In order to search for compounds capable of modulating apoptosis mechanisms, the inventors used extraction and chromatography to separate and purify the compounds from the larvae.

First, 2 kg of docomari 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. The concentrated material was dissolved in 2 ml of methanol, and then Sephadex LH gel column chromatography was performed to collect the active fractions. The active fractions were subjected to high-pressure liquid chromatography (column: C18, flow rate: 1.5 ml / min, 220 nm detection) for 30 minutes with 60% aqueous acetonitrile solution to separate the active fractions for 26 minutes of holding time, The residue obtained after removing the solvent was freeze-dried to obtain 3.0 mg of the compound represented by the formula (1).

Example 2: Physicochemical Characterization of Compounds

In order to analyze the physicochemical properties of the compound obtained in Example 1, the present inventors used a method such as electrospray ionization mass spectrometry, Fisons VG Quattro 400 mass spectrometer (USA), hydrogen and carbon nuclear magnetic resonance spectra Was used. In the NMR experiment, each sample was dissolved in a chloroform (CDCl ₃ ) solvent and measured in a 5 mm NMR tube, and 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 obtained in Example 1 was powder, the molecular weight was 234, the molecular formula was confirmed as C ₁₄ H ₁₈ O ₃ , and the mass spectrometry (MH) ⁻ of the compound was 233 (m / z). . As a result of analyzing hydrogen nuclear magnetic resonance spectra and carbon nuclear magnetic resonance spectra with chloroform (CDCl ₃ ) as a solvent with the physicochemical properties, compounds of the present invention represented by the following formula (1) have not been reported to inhibit apoptosis until now It turned out to be a compound which produces an effect.

<NMR data>

¹³ C NMR (CDCl ₃ ) δ 13.5, 17.0, 19.4, 27.6, 28.9, 35.2, 41.1, 85.4, 121.4, 136.4, 145.9, 146.0, 165.0, 197.6

[Formula 1]

Test Example 1 Determination of Apoptosis Inhibitory Effect of Compounds

In order to measure the apoptosis inhibitory effect of the compound represented by Formula 1, the present inventors performed the following in vitro experiments using human blood cancer cell line U937 cells.

U937 cells were cultured in 96-well microplate until 5 × 10 ⁶ cells / well, and then 10 μg / ml of etoposide, an apoptosis inducer, and 1-100 μg / 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, and the cells obtained by centrifugation were used for 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 (IC₅₀, Μg / ml) Compound of Formula 1 1.5 Pyrrolidine dithiocarbonate (PDTC) 15.0

As shown in Table 1, the inhibitory activity concentration of the compound represented by the formula (1) on the apoptosis of etoposide-induced U937 cells (IC ₅₀ , the concentration required to exhibit an inhibitory effect of 50%) was 1.5 µg / ml. In addition, the inhibitory concentration of 15.0 μg / ml of PDTC (pyrrolidine dithiocarbonate), which is a standard comparator, was found to be 15.0 μg / ml. From the above results, it was confirmed that the compound of formula 1 of the present invention is a compound derived from natural products that can inhibit apoptosis in vitro.

Test 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 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 examined. Hematological and hematological examinations were performed. Necropsy was performed to observe 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) shows an excellent apoptosis inhibitory effect to toxic substances such as inflammatory diseases, acquired immunodeficiency, various neurodegenerative diseases, ischemic diseases (strokes) and alcohols due to increased apoptosis It can be usefully used to treat or prevent liver disease.

Preparation Example 1 Preparation of Syrup

A syrup containing the compound of Formula 1 of the present invention and a pharmaceutically acceptable salt thereof as an active ingredient of 2% (weight / volume) was 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 thereto. 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.

Compound of Chemical Formula 1, hydrochloride 2 g

Saccharin 0.8 g

25.4 g of sugar

Glycerin ... 8.0 g

Spices ··················· 0.04 g

Ethanol 4.0 g

0.4 g of sorbic acid

Distilled water ·····················

Preparation Example 2 Preparation of Tablet

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

250 g of the compound of the formula (1) and hydrochloride 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.

Compound of formula (1), hydrochloride salt 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

Preparation Example 3 Preparation of Injection Solution

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

1 g of a compound of the formula (1), 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.

Compound of formula (1), 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 the novel compound according to the present invention in an appropriate amount of water, vitamin C as an auxiliary component, citric acid, sodium citrate, and oligosaccharide as an auxiliary component were added, and an appropriate amount of sodium benzoate was added as a preservative, followed by adding water to the whole amount. 100 ml was prepared 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 extract of Docomari and the compounds isolated therefrom exhibited an excellent apoptosis inhibitory effect, so that inflammatory diseases, acquired immunodeficiency diseases, various neurodegenerative diseases, ischemic diseases (strokes), alcohols, etc. due to increased apoptosis It can be usefully used to inhibit or prevent liver disease caused by toxic substances.

Claims (11)

delete delete delete A compound represented by the following formula (1) having apoptosis inhibitory activity. [Formula 1]

delete delete A therapeutic agent composition for treating liver disease due to toxic substances due to an increase in apoptosis, comprising a compound represented by the following Chemical Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient. [Formula 1]

1) extracting Xanthium strumarium with methanol, concentrated under reduced pressure, and extracted with ethyl acetate to obtain ethyl acetate extract; 2) concentrating the ethyl acetate extract, dissolving in a mixed solvent of chloroform and methanol, and performing silica gel column adsorption chromatography to concentrate the apoptosis inhibitory active fraction to obtain a concentrate; 3) dissolving the concentrate in methanol and then performing a Sephadex LH-20 gel column chromatography to collect an apoptosis inhibitory activity fraction; 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 the following Chemical Formula 1 from Chemical Formula 1; Method for separating the compound represented by. [Formula 1]

delete Health food for the prevention and improvement of liver disease caused by toxic substances due to the increase in apoptosis, characterized in that the main component of the extract of Docomari. The health food for the prevention and improvement of liver disease caused by toxic substances due to an increase in apoptosis, characterized in that it contains a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as a main component. [Formula 1]