KR100571582B1 - Compound having apoptosis inhibitory activity isolated from Seogirincho and its isolation method - Google Patents

Abstract

The present invention relates to a compound having an apoptosis inhibitory activity isolated from Seogirincho, and to a method for separating the same, and more specifically, through the screening process of various natural products, it is revealed that Sedum takesimense extract has apoptosis inhibitory activity. By identifying and characterizing the apoptosis inhibitory properties of a known compound represented by formula (1) after being isolated and purified from an extract, the compound or a pharmaceutically acceptable salt thereof can result from an improperly increased apoptosis, acquired Compounds having apoptosis inhibitory activity isolated from island giraffe, which proved to be used as a medicament for treating liver diseases caused by toxic substances such as immunodeficiency, various neurodegenerative diseases, ischemic diseases (strokes) and alcohols It is about a method.

[Formula 1]

Island fish, inflammatory disease, AIDS, neurodegenerative disease, ischemic disease, liver disease

Description

Compound showing apoptosis-inhibitive effect purified from Sedum takesimense and a method for preparing the said compound}

The present invention relates to a compound having an apoptosis inhibitory activity isolated from island gilin herb and a method for separating the same, and more particularly, through the screening process of various natural products, it has been found that Sedum takesimense extract has apoptosis inhibitory activity. By characterizing the apoptosis inhibitory properties of a known compound represented by Formula 1 isolated and purified from an extract, the compound, or a pharmaceutically acceptable salt thereof, may result from an improperly increased apoptosis, acquired immunity. Compounds having apoptosis inhibitory activity isolated from island giraffe, which proved to be used as a medicament for treating liver diseases caused by deficiency, various neurodegenerative diseases, ischemic diseases (strokes) and alcohols It is about.

Seogirincho is a perennial plant of the dicotyledon rose rosaceae, which is native to Korea and grows on Ulleungdo and Seoraksan. In oriental medicine, it is effective to take advantage of dried blood on the ground because it is good for bruises when taking decoction of Seogirincho decoction.

Apoptosis or programmed cell death refers to intracellular processes for spontaneous self-elimination of damaged or unnecessary cells. Apoptosis is essential 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 outside the cell and do not damage other cells. It is mechanism.

This apoptosis process, which plays an important role in living organisms, must be precisely controlled according to a defined program. Normal cells produce a functional p53 protein that, if damaged in DNA, is inhibited from progressing to the S phase in the cell cycle G1. In the case of large DNA damage, the cell takes an apoptosis process that stops cell activity and kills itself in response to internal or external signals. If apoptosis fails to function properly, tumors, autoimmune disease, and Diseases such as viral infectious diseases are known to be induced [Kerr et al., Br. J. Cancer , 26, 239-257, 1972. Various attempts have been made to treat these diseases by isolating compounds that induce apoptosis from natural substances such as spp.

In contrast, improperly increased apoptosis can lead to diseases such as acquired immunodeficiency, inflammatory diseases, various neurodegenerative diseases, ischemic diseases (strokes), and liver diseases caused by toxic substances such as alcohols [Thompson et al. , Science, 267, 1456-1462, 1995].

Early cells, in which apoptosis progresses, induce phenomena such as damage to cell junctions, blistering of cell membranes, cell shrinkage, etc., and later, chromatin aggregation, cytoplasm and Nuclear enrichment (cytoplasm and nuclei condensation), loss of mitochondrial membrane potential, changes in plasma membrane composition, and apoptotic body formation are induced and undergo overall morphological and biochemical changes. 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].

As a result of constant research on apoptosis, key players and their mechanisms involved in apoptosis in various organisms have been identified. In mammalian cells, caspase, a type of cellular cysteine protease, has been found to be a major regulator of apoptosis.

Caspases can be classified into three types, centered on Interlukin 1 Converting Enzyme (ICE), CPP32, and ICH-1, based on the evolutionary phylogenetic tree estimated according to the homology of amino acid sequence. The mechanism by which caspase regulates apoptosis is as follows. Receptor-mediated signal transduction, depletion of growth factors, oxidative stress, DNA damage and cell-cell or cell-substrate Activated by a variety of stimuli, including disruption of cell-matrix interaction, poly (ADP-ribose) polymerase, lamin, cytokeratin and caspase -Induces apoptosis formation by breaking down cellular proteins such as activated diease inhibitors (ICAD) [Cryns, V., and Yuan, J., Genes Dev. , 12, 1551-1570, 1998].

There are at least 14 caspases belonging to different classes identified in mammals, all of which can attenuate apoptosis by cleaving peptide substrates after aspartic acid residues, which play a pivotal role in the initiation and execution of apoptosis. Induction has been reported [Nunez et al., Oncogene , 17, 3237-3245, 1998].

Therefore, researches on apoptosis inhibitors related to caspases have been actively conducted in the medicine to which the present invention belongs and its related fields. The inventors have isolated a novel compound exhibiting an inhibitory effect of apoptosis in the pool [Korean Patent No. 10-367115], or a novel compound exhibiting an inhibitory effect of apoptosis in the herbaceous plant [Korea Patent No. 10-420664]. We wanted to prevent and treat various diseases that could be caused by an inappropriate increase in apoptosis. However, the search for a natural substance having an apoptosis inhibitory effect is still insufficient, and therefore, it is necessary to study a variety of natural substances.

Therefore, the present inventors have tried to search for a compound capable of regulating apoptosis in various natural substances, and as a result, the physicochemical properties of the compound have been identified by separating a compound that exhibits apoptosis inhibitory effect from Sedum takesimense and the apoptosis The present invention was completed by confirming that the induced cell lines exhibited excellent apoptosis inhibitory activity.

Accordingly, an object of the present invention is to provide a pharmaceutical composition comprising a compound represented by the formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

In addition, the present invention is another object to provide a pharmaceutical composition characterized in that it contains an extract of island girincho containing the compound represented by the formula (1) as an active ingredient.

The present invention also provides oral or non-oral pharmaceutical formulations, food additives and health foods prepared from pharmaceutical compositions containing the compound represented by the formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient. There is another purpose.

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

The present invention is characterized by a pharmaceutical composition for treating diseases containing the compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

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

The present invention reveals that Sedum takesimense extract has apoptosis inhibitory activity through the screening process and is isolated and purified from the extract, and then characterized by apoptosis inhibitory properties of the known compound represented by Formula 1, To treat liver disease caused by toxic substances, such as inflammatory diseases, acquired immunodeficiency, various neurodegenerative diseases, ischemic diseases (strokes), and alcohols, in which pharmaceutically acceptable salts thereof can be caused by inappropriately increasing apoptosis The present invention relates to a compound having an apoptosis inhibitory activity isolated from Seogirincho and proved to be able to be used as a medicament.

The compound represented by the general formula (1) is a known compound, and can be synthesized by a conventional organic synthesis method, but can also be extracted and separated from a natural island giraffe, as found in the present invention.

The method for extracting and separating the apoptosis inhibitory active substance from sumirincho comprises the following steps i) to i). Iii) extracting syl girincho with methanol, concentrating under reduced pressure and then extracting with ethyl acetate to obtain ethyl acetate extract, ii) concentrating the ethyl acetate extract, dissolving in a mixed solvent of chloroform and methanol, and then silica gel column adsorption chromatography Performing active chromatography to collect the active fractions, and iii) separating the active fractions by high pressure liquid chromatography, removing the solvent with a reduced pressure drier, and freeze drying to obtain an apoptosis inhibitor.

When the separation method is described in more detail, it is preferable to perform reflux extraction for about 48 hours using 100% of methanol in step iii). In addition, as the mixed solvent of step ii), it is preferable to use a mixed solvent of chloroform and methanol (volume ratio of chloroform and methanol is 100: 1), and mix of chloroform and methanol (100: 1, 50: 1 and 10: 1). The active fraction is concentrated by silica gel column adsorption chromatography using a solvent as the eluent. Iii) step is performed by separating the active fraction by high pressure liquid chromatography using a C18 column and a water-acetonitrile (20-100% acetonitrile, 30 minutes) elution solvent. The desired compound is obtained from the active fraction of 26 components.

By using methods such as electron spray ionization mass spectrometer (ESI-MS) and nuclear magnetic resonance spectra, the physicochemical properties of the target compound, ie, the properties, molecular weight, molecular formula, and mass spectrometry of the target compound, can be identified, Hydrogen nuclear magnetic resonance spectra and carbon nuclear magnetic resonance spectra of ₃ OD) as solvents reveal the structural formulas of the target compounds.

The compound represented by Formula 1 of the present invention may be used in the form of a pharmaceutically acceptable salt, and may be usefully used as an acid addition salt formed by a pharmaceutically acceptable free acid. Organic acids and inorganic acids can be used as free acids, citric acid, acetic acid, lactic acid, tartaric acid, maleic acid, fumaric acid, formic acid, propionic acid, oxalic acid, and tree. Fluoroacetic acid, benzoic acid, gluconic acid, methanesulfonic acid, glycolic acid, succinic acid, 4-toluenesulfonic acid, galluxuronic acid, embonic acid, glutamic acid and aspartic acid, and the like. Representative.

Pharmaceutical compositions containing a compound represented by the formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient inhibit inflammatory diseases caused by abnormal apoptosis by inhibiting abnormally induced apoptosis mechanisms, acquired immunodeficiency diseases, various neurodegenerative diseases, ischemic diseases It can be usefully used to treat or prevent liver disease caused by toxic substances such as (stroke) and 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 present invention may be formulated for oral administration, eg, as tablets, troches, lozenges, aqueous or oily suspensions, prepared powders or granules, emulsions, hard or soft capsules, syrups or elixirs. Can be formulated. Lactose, saccharose, sorbitol, mannitol, starch, amylopectin, binders such as cellulose or gelatin, excipients such as dicalcium phosphate, disintegrants such as corn starch or sweet potato starch, magnesium stearate, for preparation in formulations such as tablets and capsules Lubricants such as calcium stearate, sodium stearyl fumarate or polyethylene glycol wax may be contained. Capsule formulations contain, in addition to the substances mentioned above, liquid carriers such as fatty oils.

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

The effective amount of the compound represented by the formula (1) or the pharmaceutically acceptable salt thereof according to the present invention is 1 to 50 mg / day per kg of body weight, preferably 5 to 20 mg / day for a general adult patient, preferably a doctor or Depending on the judgment of the pharmacist, it may be administered several times a day, preferably two to three times a day at regular intervals.

In addition, the compound represented by the formula (1) of the present invention or a pharmaceutically acceptable salt thereof may be included in the pharmaceutical composition as described above as well as used in the treatment of diseases as well as a food additive or a raw material of health food. That is, the compound represented by Chemical Formula 1 or a pharmaceutically acceptable salt thereof is added to food additives such as beverages, teas, spices, gums, confections, etc. Ingestion of the food containing the compound may bring a specific health effect. Unlike general medicines, these health foods have the advantage of minimizing the side effects that can occur due to long-term use of the drug as a raw material.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to Examples.

Example 1 Isolation and Purification of Apoptosis Inhibiting Substances

In order to search for compounds that can modulate the apoptosis mechanism, the present inventors extracted, separated and purified from sagilincho through the following procedure.

First, 3 kg of sagirincho was cut to a size of 5 cm, extracted with 100% methanol for 48 hours, concentrated under reduced pressure, and extracted with ethyl acetate. The ethyl acetate layer was concentrated, and the active fraction was concentrated by silica gel column adsorption chromatography dissolved in a mixed solvent of chloroform and methanol (100: 1). The active fraction was maintained by performing high-pressure liquid chromatography (column: C18, flow rate: 1.5 ml / min, 220 nm detection) using water-acetonitrile (20-100% acetonitrile, 30 minutes) as an elution solvent. An active fraction of 26 minutes was separated and the solvent was removed with a reduced pressure drier, and the resulting residue was freeze-dried to obtain 2.5 mg of the target compound.

Example 2 Physicochemical Characterization of Example 1 Compound

In order to analyze the physicochemical properties of the compound represented by the formula (1) obtained in Example 1, the inventors of the present invention, electrospray ionization mass spectrometry, Fisons VG Quattro 400 mass spectrometer (USA), hydrogen and carbon nuclear magnetic resonance spectrum Was used. In the NMR experiment, each sample was dissolved in methanol (CD ₃ OD) solvent and measured in a 5 mm NMR tube, and the chemical shift was determined based on the peak of each solvent or the peak of TMS (tetramethylsilane). Measured. As a result of analyzing the property, molecular weight, molecular formula and mass of the compound, the target compound was found to have physicochemical properties as shown in Table 1 below.

division Matter Molecular Weight Molecular formula Mass Spec. (MH) ^- Target compound powder 273 C ₁₇ H ₂₃ NO ₂ 272 ( m / z)

In addition, hydrogen and carbon nuclear magnetic resonance spectrometry of methanol (CD ₃ OD) as a solvent in combination with the above physicochemical properties revealed that the compound of Example 1 is a compound exhibiting apoptosis inhibitory activity not reported to date. It became.

NMR data of Example 1 compound were ¹³ C NMR (CD ₃ OD) δ 24.9, 33.9, 34.4, 44.2, 47.4, 49.3, 52.1, 70.7, 125.4, 125.7, 128.3, 132.2, 207.1.

Experimental Example 1 Measurement of Apoptosis Inhibitory Activity of the Compound of Example 1

In order to measure the apoptosis inhibitory activity of the compound represented by Formula 1 obtained in Example 1, 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, followed by 10 ug / ml of etoposide, an apoptosis-inducing substance, and 1-100 ug / ml of the compound of the present invention. Were treated in U937 cells and incubated at 37 ° C. for 5 hours at 5% CO ₂ -95% air to compare apoptosis inhibitory activity. At this time, cells treated with only etoposide were used as a positive control, and cells not treated with both etoposide and the compound of the present invention were used as a negative control. After 5 hours, the shape of each U937 cell was observed under a microscope to determine whether apoptosis occurred. In addition, the cells obtained by centrifugation were used as substrates of DEVD-AFC (Z-Asp-Glu-Val-). Aqueous-3 activity against Asp-7-amino-4-trifluoromethyl coumarin) was measured. The apoptosis inhibitory activity exhibited by the compounds of the present invention was measured based on the kephas-3 activity of the positive control treated with etoposide only and the kephas-3 activity of the negative control not treated with both etoposide and the compound of the present invention. It is shown in Table 2.

division Apoptosis Inhibition Rate (IC₅₀, Μg / ml)   Compound of the Invention 12.0   Pyrrolidine dithiocarbonate (PDTC) 15.0

As shown in Table 2, the inhibitory activity concentration (IC ₅₀ , concentration required to exhibit 50% inhibitory effect) of the compound represented by Formula 1 on apoptosis of etoposide-induced U937 cells was 12.0 μg / ml. The inhibitory activity concentration of PDTC, a standard comparator, was found to be 15.0 μg / ml, indicating that the compound of Formula 1 had better apoptosis inhibitory activity than PDTC, a standard comparator. From these results, it was confirmed that the compound of Formula 1 of the present invention is a compound derived from natural products that can effectively inhibit apoptosis.

Experimental Example 2 Oral Acute Toxicity in Rats

Acute toxicity experiments were performed using 6 week-old SPF rats. Two animals per group were suspended orally in a dose of 1 g / kg / 1 ml, each of the compounds represented by Formula 1, suspended in 0.5% methylcellulose solution. After administration of the test substance, mortality, clinical symptoms, and changes in body weight were observed. Hematological and blood biochemical tests were performed. Necropsy was performed to observe abdominal and thoracic organ abnormalities. As a result, no significant clinical symptoms or deaths occurred in all animals treated with the test substance, and no toxic changes were observed in weight changes, blood tests, blood biochemical tests, and autopsy findings. All of the above tested compounds showed no change in toxicity up to 500 mg / kg in rats and proved to be safe substances with a minimum oral dose (LD ₅₀ ) of 500 mg / kg or more.

Formulation Example 1 Preparation of Syrup

Syrup containing the compound of Formula 1 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 and then cooled. Here, 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 composition of this syrup is shown in Table 3 below.

Component Content (g) Compound of Chemical Formula 1-Hydrochloride 2.0 saccharin 0.8 Party 25.4 glycerin 8.0 Spice 0.04 ethanol 4.0 Sorbic acid 0.4 Distilled water dose

Formulation Example 2 Preparation of Tablet

A tablet containing 15 mg of the compound of Formula 1 and a pharmaceutically acceptable salt thereof as an 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 refined. The composition of the tablet is shown in Table 4 below.

Component Content (g) Compound of Chemical Formula 1-Hydrochloride 250 Lactose 175.9 Potato starch 340 Colloidal silicic acid 32 10% gelatin solution dose talc 50 Magnesium Stearate 5

Formulation Example 3 Preparation of Injection Solution

Injectable solution containing 10 mg of the compound of Formula 1 and a pharmaceutically acceptable salt thereof as an 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 composition of the injection solution is shown in Table 5 below.

Component Content (g) Compound of Chemical Formula 1-Hydrochloride One Sodium chloride 0.6 Ascorbic acid 0.1 Distilled water dose

Formulation Example 4 Preparation of Healthy Drinks

After dissolving 500 mg of the compound of 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 water. The composition for health drinks was prepared in ml. At this time, taurine, myo-inositol, folic acid, pantothenic acid, etc. may be added one kind or two or more kinds.

The present invention provides a pharmaceutical composition containing a compound having an apoptosis inhibitory activity as an active ingredient, by identifying non-toxic apoptosis inhibitory properties of a compound isolated and purified from Seogirincho extract, using the compound or a pharmaceutically acceptable salt thereof. , Health additives or health foods prevent liver disease caused by toxic substances such as inflammatory diseases, acquired immunodeficiency, various neurodegenerative diseases, ischemic diseases (strokes), and alcohol that can be caused by inappropriately increasing apoptosis Or it can be cured.

Claims (19)

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

delete delete delete delete delete A pharmaceutical formulation for oral administration, which is prepared from the pharmaceutical composition of claim 5. A pharmaceutical formulation for non-oral administration, which is prepared from the pharmaceutical composition of claim 5. delete delete A food additive comprising the compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as a main component. [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]

delete delete According to claim 5, wherein the compound of formula 1 is characterized in that the composition is separated from the island of giraffe.