KR100424392B1 - Antioxidative and anticancer compositions containing myxochelin a - Google Patents

Abstract

The invention mikso chelrin-A (myxochelin A) compound to that of the antioxidant and anti-cancer pharmaceutical composition for the active ingredient, specifically, the antioxidant activity is excellent and to excellent growth inhibitory activity against various cancer cell lines of human origin formula The present invention relates to a pharmaceutical composition for anti-oxidation and anti-cancer containing myxocellin-A compound as an active ingredient. The myxoclin-A of the present invention can be produced by a general method by culturing microorganisms, and can be industrially mass-produced by being easy to extract and purify. It can be usefully used as an antioxidant and an anticancer agent to regulate.

Description

Antioxidant and anticancer pharmaceutical composition comprising myxochelin-A as an active ingredient {ANTIOXIDATIVE AND ANTICANCER COMPOSITIONS CONTAINING MYXOCHELIN A}

The invention mikso chelrin-A (myxochelin A) compound to that of the antioxidant and anti-cancer pharmaceutical composition for the active ingredient, specifically, the antioxidant activity is excellent and to excellent growth inhibitory activity against various cancer cell lines of human origin formula The present invention relates to a pharmaceutical composition for anti-oxidation and anti-cancer containing the myxoclin-A compound as an active ingredient.

<Formula 1>

Oxygen is the most abundant element on earth, accounting for 21% of the dry atmosphere, and aerobic organisms all acquire energy through respiration using oxygen as an electron acceptor. Oxygen is absolutely necessary for life support, but the stable molecular state of the ground state triplet oxygen (ground state triplet oxygen) is due to various physical and chemical factors such as enzymes, reduction metabolism, chemicals, pollutants, photochemical reactions, etc. superoxide radicals ^{_{(superoxide radical, O 2 -)}} , hydroxy radicals (hydroxyl radical, HO ·), hydrogen peroxide (H ₂ O _2), the highly reactive free radicals, such as singlet oxygen (singlet oxygen, ¹ O ₂₎ When converted to a species, it has a double side effect that causes fatal toxicity to the living body. That is, these reactive oxygen species have non-selective and irreversible destruction effects on cell components such as lipids, proteins, sugars, deoxyribonucleic acid (DNA), and brain diseases such as cancer, stroke, Parkinson's disease, heart disease, ischemia, It is known to cause various diseases and aging, such as atherosclerosis, skin diseases, digestive diseases, inflammation, rheumatism and autoimmune diseases. In addition, lipid peroxides produced by the peroxidation of lipid components in the body by these free radicals, along with other peroxides in the body, destroy normal cells, causing various functional disorders (B. Halliwell, Drugs 42). : 569, 1991; K. Fukuzawa and Y. Takaishi, J. Act. Oxyg.Free Rad . 1:55, 1990; J. Neuzil, and JM Gebicki, J. Med . 320: 915, 1989).

Recently, studies on the development of antioxidants that can regulate such reactive oxygen species are actively conducted, including superoxide dismutase, peroxidase, catalase, glutathione peroxidase, and glutathione peroxidase. Studies on the Antioxidant Effects of Enzymes and Small Molecules Derived from Natural Products, such as Tocopherol, Ascorbate, Carotenoid, Glutathione, etc. (SS Chang et al., J. Food Sci 42: 1102, 1977; PA Hammerschmidt and DE Pratt, J. Food Sci . 43: 556, 1977; DE Pratt and BW Watts, J. Food Sci. 29:17, 1964). The development of effective antioxidants is important because it can lead to the development of new medicines as well as supplements for food and cosmetics.

In order to develop a novel antioxidant with high activity, the present inventors have developed Myxobacteria (G. Hofle and H. Reichenbach, GBF, Scient. Ann. Report 5-22, 1990; CJ Cowden and I. Paterson, Nature). 387: 238, 1997) metabolites were investigated for antioxidant activity. Mucobacterium has recently gained global attention as a new source of bioactive substances, which have been isolated from mycobacterial bacteria such as myxoclin-A, epochilon, and amphotericin.

From this, the present inventors found excellent antioxidant activity in the culture solution of the bacterial bacterium Angiococcus disciformis , and isolated and purified this active body to confirm that it is myxochelin A. The present invention has been completed by providing a novel use of the myxochelin-A compound as an antioxidant and an anticancer agent by showing excellent growth inhibitory activity against various cancer cell lines of origin.

It is an object of the present invention to provide new uses for the use of myxochelin-A compounds as antioxidants and anticancer agents.

1 shows a ¹ H nuclear magnetic resonance spectrum of myxochelin A,

2 shows the ¹³ C nuclear magnetic resonance spectrum of myxoclin-A,

3 shows the infrared absorption spectrum of myxoclin-A,

4 shows the ultraviolet absorption spectrum of myxocellin-A.

In order to achieve the above object, the present invention provides a pharmaceutical composition for antioxidant using a myxochelin-A compound represented by the following formula (1 ) as an active ingredient.

The present invention also provides an anticancer pharmaceutical composition comprising the myxocellin-A compound as an active ingredient.

<Formula 1>

Hereinafter, the present invention will be described in detail.

Myxocelin-A is an slime bacteria isolated from the soil, Angiococcus disciformis , especially Angiococcus discipformis JW357 (Accession Number: ATCC 33172) (B. Kunze, N. Bedorf, W. Kohl , G. Hoffle and H. Reichenbach. J. Antibiotics , 42:14, 1989).

The method of isolating and purifying a myxochelin-A compound from the culture solution of angiococcus difficormis strain, for example

1) culturing the Angiococcus difficormis strain in the presence of the adsorptive resin and centrifuging the culture to separate the cells and the adsorbent resin;

2) extracting the active material by adding an organic solvent, preferably a mixed solvent of acetone and methanol to the separated cells and adsorptive resin, and then filter the filter medium with a filter paper to remove the cells and the adsorptive resin; And

3) extracting the crude extract of the liquid phase obtained by concentrating the obtained filtrate under reduced pressure with an organic solvent, for example ethyl acetate, to obtain an active fraction of the liquid phase; The active fractions are mixed with a solvent system, e.g. silica gel medium pressure liquid chromatography with dichloromethane / acetone / methanol (90: 5: 5) mixed solvent and a lower alcohol or organic solvent (preferably ethylacetate or dichloromethane). Final purification by sequentially performing a preparative HPLC (Recycling preparative HPLC) using a) to obtain a pure single material.

Slime bacteria which can produce them in mikso chelrin These are the addition OKO kusu dish formate Miss J mixer Socorro kusu glass tooth (Myxococcus xanthus), stigmasterol telra brother is thiazol car (Stigmatella aurantiaca) (W. Trowitzsch- kienast, B. Kunze, H. Irschik, V. Wray, H. Reichenbach and G. Hoffle, The 9th DECHEMA-Jahrestagung der Biotechnologen , Berlin, 1991; B. Silakowski, B. Kunze et al . Eur. J. Biochem. 267: 6476, 2000)

The myxochelin-A compound may be isolated from the mucin bacterial culture as described above or artificially prepared by chemical synthesis or semisynthesis by a conventional method.

The myxochelin-A compound of the present invention is a free radical 1,1-diphenyl-2-picrylhydrazyl (1,1-diphenyl-2-picrylhydrazyl, DPPH) measuring the antioxidant activity (MS Blois Nature , 181: 1199, 1958) shows about 2 to 4 times higher antioxidant activity than known synthetic antioxidants (BHA) and natural antioxidants (tocopherol).

In addition, the myxochelin-A compound of the present invention shows excellent anticancer activity against cancer cell lines of skin cancer (Melanoma) and central nervous system (CNS) cancer.

Based on the above results, the present invention provides a pharmaceutical composition for antioxidants having myxocellin-A as an active ingredient.

The pharmaceutical composition contains an antimycoclin-A compound having excellent antioxidant activity as an active ingredient, such as stroke, Parkinson's disease, brain diseases, ischemia, arteriosclerosis, skin diseases, digestive diseases, inflammation, rheumatism, autoimmune diseases, etc. It can be usefully used to prevent and treat various diseases and aging.

The present invention also provides an anticancer pharmaceutical composition comprising the myxocellin-A compound as an active ingredient.

The antimicrobial and anticancer pharmaceutical composition comprising the myxoclin-A of the present invention can be administered orally or parenterally during clinical administration and can be used in the form of a general pharmaceutical preparation.

That is, the pharmaceutical composition of the present invention may be administered in various oral and parenteral dosage forms during actual clinical administration, and when formulated, diluents such as fillers, extenders, binders, humectants, disintegrating agents, surfactants, etc. that are commonly used, or Formulated using excipients. Solid form preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, which form at least one excipient such as starch, calcium carbonate, sucrose (Sucrose) or lactose (Lactose), gelatin, etc. are mixed and prepared. In addition to simple excipients, lubricants such as magnesium, stearate and talc are also used. Oral liquid preparations include suspensions, solutions, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As a suppository base, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The pharmaceutical compositions of the invention can be administered via several routes including oral, transdermal, subcutaneous, intravenous or intramuscular.

A typical daily dosage of the myxochelin-A compound of the present invention is 10-200 mg in adults, preferably administered once or several times depending on symptoms. However, it is to be understood that the actual dosage of the active ingredient should be determined in light of several relevant factors such as the route of administration, the age, sex, weight of the patient and the severity of the patient, and thus the dosage should in any way be within the scope of the invention. It is not intended to limit.

Hereinafter, the present invention will be described in more detail with reference to Examples.

However, these examples are only for illustrating the present description, and the present invention is not limited only to these examples.

Example 1 Isolation and Purification of Myxochelin-A Compounds from Angiococcus Disymformis

The inventors of the present invention first obtained soil harvested from vineyards in Gyeongsan, Gyeongsangbuk-do to isolate mucus bacteria (S. Yamanaka, A. Kawaguchi and K. Komagata, J. Gen. Appl.Microbiol . 33: 247, 1987). Along with various kinds of mycobacterial bacteria, the mycobacterial strains producing the myxochelin-A compound of the present invention were isolated and named JW357. As a result of investigating the morphology of the strain JW357 and the growth state and physiological characteristics of various media, this strain was previously reported by Guntz et al. As a strain producing a myxochelin-A compound. (B. Kunze, N. Bedorf, W. Kohl, G. Hoffle and H. Reichenbach, J. Antibiotics 42:14, 1989). The Angiococcus difficormis strain was cultured according to the method established by Guntz et al., But the present inventors incubated rotation shaking (150 rpm) for 5 days using a 2 L culture flask instead of a large capacity fermenter.

The culture medium obtained by culturing in the above procedure was centrifuged to separate the cells and the adsorbent resin (Ambarlite XAD-16 [Amberlite XAD-16], Aldrich Chemical Co., Ltd.). 3 liters of acetone and 1 liter of methanol were added to the cells and the adsorption resins obtained above, and left at room temperature for 3 days. The filtrate thus obtained was concentrated under reduced pressure to obtain a crude crude extract. Then, the crude extract was suspended in 300 ml of water, extracted three times with 300 ml of ethyl acetate, and the extract was concentrated under reduced pressure to give 571 mg of an active fraction of a liquid. The active fraction was subjected to silica gel medium pressure liquid chromatography (silica gel 60, 230 to 400 mesh, diameter 20 mm × height 300 mm) to obtain an active fraction of 100 mg in the dichlormethane-acetone-methanol (90: 5: 5) fraction. Got. The liquid material obtained above was finally purified by recycling preparative HPLC equipped with a ZIGEL GS-310 column (GAIGEL GS-310, diameter 25 mm × height 500 mm) using methanol as a solvent, and 30 mg. A pure homogeneous substance was obtained.

Example 2 Structure Identification of Myxochelin-A Compound

As a result of various instrumental analysis experiments, the physicochemical properties of the pure purified compound of Example 1 were investigated as follows.

(1) Appearance of substance: Brown oil phase

(2) Molecular weight: 404

(3) Molecular formula: C ₂₀ H ₂₄ N ₂ O ₇

(4) Mass spectrometry spectrum (EI-MS, m / z ): 404 [M] ⁺

(5) Luminous intensity: [α] _D ²⁵ -25.8 ^。 ( c 0.61, MeOH)

(6) infrared absorption spectrum [υ _max (film) cm ⁻¹ ]: 3352 (br), 2939, 2863, 1638, 1586, 1545, 1262 ( FIG. 3 )

(7) UV absorption spectrum [λ _max ^MeOH nm (logε)]: 210 (4.97), 249 (4.54), 313 (4.13) ( FIG. 4 )

(8) Solubility: Soluble in methanol, acetone, ethyl acetate, dichloromethane. Insoluble in water and heptane.

(9) Color reaction: positive for vanillin-sulfuric acid solution and FeCl ₃ solution, negative for ninhydrin and Dragendorff solution.

(10) Silica gel thin layer chromatography (TLC): Rf = 0.58 (dichloromethane: methanol = 9: 1)

(11) ¹ H nuclear magnetic resonance spectrum (500 M Hz, acetone- d ₆ ):

δ 8.18 (1H, brt), 7.79 (1H, brd), 7.29 (1H, d, J = 8.5 Hz), 7.21 (1H, d, J = 8.5 Hz), 6.95 (2H, dd, J = 8.5, 2.0 Hz), 6.69 (2H, t, J = 8.5 Hz), 4.19 (1H, m), 3.66 (1H, d, J = 5.0 Hz), 3.43 (1H, m), 1.70 (1H, m) , 1.69 (1H, m), 1.51 (1H, m) ( FIG. 1 )

(12) ¹³ C nuclear magnetic resonance spectra (125 M Hz, acetone- d ₆ ):

171.2 (s), 171.1 (s), 150.9 (s), 150.8 (s), 147.4 (s), 147.2 (s), 118.8 (d), 118.7 (d), 118.6 (d), 118.5 (d), 117.6 (d), 117.3 (d), 115.3 (s), 115.2 (s), 64.5 (t), 52.3 (d), 39.7 (t), 31.0 (t), 29.6 (t), 23.9 (t) ( 2 )

From the results of the above physicochemical analysis and the following experimental results, the pure and purified compounds of the present invention were identified as myxochelin-A.

First, as a result of mass spectrometry, ¹³ C nuclear magnetic resonance spectra, and DEPT (Distortionless Enhancement by Polarization Transfer) spectra, the molecular formula of the compound was determined to be C ₂₀ H ₂₄ N ₂ O ₇ . ^{1 H- 1 H COSY (Correlation Spectroscopy} ) spectra, ¹ H- ¹³ C COSY spectral and HMBC (Heteronuclear Multiple Bond Coherence) analysis result of the spectrum, the skeleton portion of the structure other than the amide portion was estimated, ¹ H nuclear magnetic resonance spectrum And IR spectra suggest the presence of amide bonds in the molecule. Based on these findings and the literature review based on the fact that mucus bacteria are producers, the compounds of the present invention were isolated from mycobacteria by iron co-chelating compounds (H. Reichbach et al.). Kunze, N. Bedorf, W. Kohl, G. Hoffle and H. Reichenbach., J. Antibiotics , 42 : 14, 1989).

Example 3 Investigation of Antioxidant Activity of Myxoclin-A Compounds

Antioxidant activity exhibited by the myxocelin-A compound was investigated by measuring the antioxidant activity using the free radical DPPH (MS Blois., Nature, 181: 1199, 1958; BS Yun. J. Antibiotics , 53: 114, 2000).

First, 20 μl of sample compounds for each concentration dissolved in dimethyl sulfoxide (DMSO) were added to 980 μl of 150 μM DPPH ethanol solution. The sample was used as a control group of the myxoclin-A of the present invention, and BHA (Butylated hydroxyanisole) and tocopherol, which are known as antioxidants, were used as a control group. Each sample mixture was mixed with a vortex and left at room temperature for 20 minutes, after which absorbance was measured at 517 nm and the results are shown in Table 1 below. The ED ₅₀ value represents the amount of sample (μg / ml) required to reduce the absorbance value of the control by 50%.

compound Antioxidant activity (ED _50, μg / ml) Tocopherol 19.2 BHA 9.3 Myxochelin-A 4.9

As shown in Table 1 , the antioxidative activity of the myxochelin-A compound of the present invention was about 2 times higher than that of BHA, a synthetic antioxidant, and about 4 times higher than tocopherol, which is well known as a natural antioxidant. Thus, the myxochelin-A compounds of the present invention with good antioxidant power are useful as antioxidants.

Example 4 Investigation of Anticancer Activity of Myxoclin-A Compounds

The anticancer activity of various myocarcinoma cells of the myxoclin-A compound was examined by SRB assay (P. Skehan, et al ., J. Natl. Cancer Insti. , 82: 1107, 1990). The cancer cells used were skin cancer (Melanoma) cell lines MALME-3M, SK-MEL-2, SK-MEL-28, SK-MEL-5, SNB-19, M14, M19-MEL, LOXIMVI and the central nervous system cancer cell line U251, SNB-75, SNB-78, and XF-498, all of which were available from the US National Cancer Institute.

The cancer cell line was cultured using RPMI 1640 medium (Life Sciences Inc.) containing 10% fetal bovine serum, and the cultured cells were maintained by dispensing once or twice a week. The cancer cells thus cultured were dispensed into a 96-well plate at a constant concentration, and after one day, the comparative base absorbance of the cells was confirmed. At this time, the plate having the same cell number as the sample treatment as the required Tz (Time / Zero) plate was fixed with 10% TCA. Tz plates were washed with tap water after 1 hour at 4 ° C. Sample-treated plates were fixed with 100 μl of 10% TCA per well after 48 hours, and then tap water after 4 hours at 4 ° C. Washed with. The washed plates were dried at room temperature and stained with 100 μl of 0.4% SRB solution per well, and after 30 minutes, washed with 1% acetic acid solution and dried at room temperature. 100 μl of trisma base (pH 10.5) was added thereto to dissolve again, and then absorbance was measured at 520 nm using an ELISA reader, and the concentration of a compound that inhibits the growth of cancer cells by 50%, namely IC ₅₀ ( Μg / ml) and the results are shown in Table 2 below. At this time, cisplatin (Cisplatin, Sigma) and carboplatin (Carboplatin, Sigma) were used as a comparison group.

Anticancer activity against cancer cells Cancer cell line Anticancer activity (IC _50, μg / ml) Myxochelin-A Cisplatin Carboplatin U251 1.53 0.15 4.00 SNB-75 1.84 0.26 4.03 SNB-78 2.36 0.28 4.03 XF-498 1.81 0.22 4.76 MALME-3M 1.20 - - SK-MEL-2 1.27 0.88 5.27 SK-MEL-28 1.57 0.80 12.01 SK-MEL-5 1.22 0.31 3.44 SNB-19 1.52 0.22 3.88 M14 1.15 0.26 5.12 M19-MEL 1.24 0.33 9.81 LOXIMVI 1.38 - -

As shown in Table 2 , the myxochelin-A compound of the present invention showed excellent anticancer activity of inhibiting its growth against 12 cancer cells.

As described above, the myxochelin-A compound of the present invention can be produced by a cultivation method commonly used for culturing microorganisms, and industrial extraction and purification are not required. In addition to antioxidant activity, it shows excellent anticancer activity against various human cancer cells, so it can be usefully used as an effective antioxidant and anticancer agent.

Claims (5)

delete delete An anticancer pharmaceutical composition comprising the myxochelin-A compound represented by Formula 1 as an active ingredient. Formula 1 The method of claim 3, The pharmaceutical composition, characterized in that the myxochelin-A compound is isolated from the mycobacteria or chemically synthesized or semisynthesized. The method of claim 4, wherein 1) culturing the angiococcus disciformis strain, which is a mucus bacterium, in the presence of the adsorbent resin, and centrifuging the culture medium to separate the cells and the adsorbent resin; 2) adding a mixed solvent of acetone and methanol to the separated cells and adsorptive resin to extract the active material, and then filtering with a filter paper to remove the cells and the adsorptive resin; 3) extracting the crude extract of the liquid phase obtained by concentrating the obtained filtrate under reduced pressure with an organic solvent to obtain an active fraction of the liquid phase; 4) Pure separation of myxochelin-A from the active fraction by sequentially performing silica gel medium pressure liquid chromatography using a mixed solvent system and recycling preparative HPLC using a lower alcohol or an organic solvent. Pharmaceutical composition, characterized in that.