KR20030073553A - Novel bioactive substance extracted from actinomycetes, method of extracting same, and pharmaceutical composition containing same - Google Patents

Abstract

PURPOSE: Provided is a physiological active material of Bisboralactin which exhibits strong toxicity with respect to cancer cells of leukemia, lung cancer, uterine carcinoma, melanoma, large intestine cancer and central nervous system cancer, and works as a multi-purpose anticancer agent. CONSTITUTION: A physiological active material is a compound expressed by the formula 1 where R1 and R3 being identical with or different from each other are hydrogen and methyl or ethyl, respectively, and R2 and R4 being identical with or different from each other are hydrogen, and methyl, ethyl, propyl or isopropyl, respectively. The compound is extracted by culturing the Actinomycetes of Streptomyces sp., isolating mycelium from the culture, and purifying it.

Description

Novel bioactive substance derived from actinomycetes, a method of extracting the same, and a pharmaceutical composition comprising the same.

The present invention relates to a novel bioactive substance extracted from cultured mycelium of Streptomyces sp. Actinomycetes and a method for extracting the same, and a pharmaceutical composition comprising the same.

It is widely known that actinomycetes contain various new biologically active substances, and industrial developments such as pharmaceuticals are being made using them. The present invention has also been made in connection with the development of new materials derived from actinomycetes as part of this research.

An object of the present invention is to provide a method for extracting a bioactive substance from actinomycetes and a novel bioactive substance obtained thereby.

Another object of the present invention is to provide a pharmaceutical composition having anticancer activity, comprising the novel bioactive substance obtained as described above.

In order to achieve the above object, the present invention provides a compound having the following structural formula.

Wherein R ¹ and R ³ may be the same or different from each other and are each hydrogen, methyl or ethyl, and R ² and R ⁴ may be the same or different from each other and are each hydrogen, methyl, ethyl, propyl or isopropyl, all asymmetric Isomers at carbon.

Method for obtaining a novel compound of formula 1 from actinomycetes, the actinomycetes of Streptomyces sp. Incubated in a liquid medium, mycelium is isolated from the culture and extracted with an organic solvent, the solvent is evaporated, The residues are partitioned according to polarity to give a polar organic concentrate, which is separated and purified by chromatography.

In addition, the present invention provides an anticancer composition comprising a novel compound of formula (1) in order to achieve the other object.

The novel compound of formula 1 obtained from actinomycetes of the Streptomyces sp. Was named Bisboraractin by the present inventors and is hereinafter referred to by the same name.

The method for extracting the novel compound bisboralactin according to the present invention is in detail,

(1) culturing the unclassified species of Streptomyces sp., An actinomycetes, in a liquid medium;

(2) filtering the culture to separate mycelium;

(3) extracting the mycelium with one or two or more mixtures of methanol, ethanol, chloroform, acetone and dichloromethane;

(4) evaporating and drying the crude extract obtained by extraction as described above under reduced pressure to obtain a residue;

(5) fractionating the residue according to polarity;

(6) evaporating to dry the polar material fractions under reduced pressure to obtain a polar organic concentrate; And

(7) separating and purifying the concentrate by chromatography.

The molecular structure of the separated material was determined by nuclear magnetic resonance (NMR) spectra, infrared and ultraviolet spectroscopic data, and high resolution mass spectrometry data. The assignment of hydrogen nuclear magnetic resonance ( ¹ H-NMR) and carbon nuclear magnetic resonance ( ¹³ C-NMR) signals in nuclear magnetic resonance spectra is correlation spectroscopy (COSY), total correlation spectroscopy (TOCSY), and gradient HSQC. multidimensional nuclear magnetic resonance experiments, such as gradient heteronuclear single quantum coherence and gradient heteronuclear multiple bond coherence (HMBC). By experiment.

Among the bisboraractins thus obtained, the chemical formula, physical properties, and spectroscopic characteristics of bisboraractin A are as follows.

(1) Molecular Formula: C ₂₁ H ₃₄ O ₆

(2) molecular weight: 382

(3) Appearance: Colorless, viscous semi-solid substance, which is generally stable at room temperature, and soluble in polar and medium polar organic solvents such as methanol, acetone, chloroform and ethyl acetate.

(4) Infrared absorption band (KBr): 2970, 2840, 1730, 1570, 1460, 1190 cm^-One

(5) Ultraviolet absorption band (MeOH): no maximum absorption at 200-400 nm

(7) ¹ H and ¹³ C-NMR designations: see Table 1 below.

In the following Table 1, ¹ H and ¹³ C-NMR spectra were measured at 500 MHz and 125 MHz in chloroform-d solvent, respectively. Chemical transitions were based on tetramethylsilane peak (0 ppm) for ¹ H and residual solvent peak (77.0 ppm) in chloroform-d for ¹³ C.

location Bisvoractin A ¹ H ¹³ C 1234 5 6789101112 13 1415161718192021 2.52 (1 H, m) 4.01 (1 H, dt, J = 6.8, 7.3 Hz) 1.93 (1 H, m); 1.55 (1 H, m) 1.98 (1 H, m); 1.49 (1 H, m) 3.83 (1 H, m) 1.76 (2 H, m) 4.90 (1 H, m) 2.50 (1 H, m) 4.02 (1 H, dt, J = 6.8, 7.3 Hz) 1.93 (1 H, m); 1.55 (1 H, m) 1.98 (1 H, m); 1.49 (1 H, m) 3.85 (1 H, m) 1.72 (2 H, m) 4.94 (1 H, m) 1.07 (3 H, d, J = 7.3 Hz) 1.61 (2 H, m) 0.87 (3 H, t, J = 6.8 Hz) 1.09 (3H, d, J = 6.8 Hz) 1.22 (3H, d J = 5.9 Hz) 174.2 s45.0 d79.8 d28.1 ^a t31.4 ^b t76.4 d39.9 t73.2 d174.5 s45.2 d80.0 d28.0 ^a t31.3 ^b t76.2 d42.2 t69.0 d12.6 q27.4 t9.3 q13.1 q20.4 q

^ab Carbon assignments can change

Bisboralactin A (C ₂₁ H ₃₄ O ₆ : 382), whose structure is determined in this way, is a macrocyclic dilactone in which two molecules of iso-fatty acid having a side chain of methyl or ethyl group are bonded to each other. It is classified as a systemic substance. The backbone of this material consists of an 18-membered dilactone in which the oxygen atom of carbon 1 is linked to carbon 16 and the oxygen atom of carbon 9 is linked to carbon 8. Here again, carbons 3 and 6 and carbon 11 and 14 are triple ring compounds having tetrahydrofuran groups linked by pentagonal ether rings by oxygen atoms, respectively.

Such fatty acid dilactones or similar organic substances with octagonal rings are very rarely found, whether natural or synthetic, particularly those having two pentagonal ether rings in the octagonal dilactone rings, such as the compounds of the present invention. No literature was found.

The novel compound bisboralactin A of the present invention shows strong toxicity against cancer cells such as leukemia, non-small cell lung cancer, uterine cancer, melanoma, colorectal cancer, central nervous system cancer, etc., as a result of physiological activity test, and developed as a multi-purpose anticancer agent. It is expected to be.

Hereinafter, the culture of the strain Streptomyces sp. To obtain the bisvoractin of the present invention, the extraction method of the bisvoractin from the culture, and the measurement of the physiological activity of the bisboractin will be described in detail through the examples. However, these Examples are only illustrative of the present invention, the present invention is not limited to these.

Example 1 Culture of Streptomyces Strain

Unclassified actinomycetes from Streptomyces isolated from sponges collected offshore in Jeju Island were cultured in a solid medium containing Petri dishes. The composition of the medium was 10 g of glucose, 2 g of tryptone, 1 g of yeast extract, 1 g of beef extract, and 20 g of agar. Incubated in the incubator for 10 days at 32 ℃. 10 ml of distilled water was added to the Petri dish, shaken, and filtered to obtain spore solutions.

4 L of sterilized medium was placed in a 7 L fermentor (Korea Fermentation Company). The composition of the medium was 10 g of glucose per liter of medium, 2 g of tryptone, 1 g of yeast extract, and 1 g of beef extract, and fixed at pH 7.0 at room temperature. 2 ml of the above spore solution was added to the liquid medium, followed by incubation with air at 32 ° C. for 7 days (2.5-3 kgF / Nm 3, 300 rpm).

Example 2: Separation and Purification Method of Bisvoractin A

Solid components were separated from the culture medium of Streptomyces sp. Obtained in Example 1 by using a tangential filtering system equipped with a 0.2 μm membrane. The extra mycelium was removed from the mycelium thus separated using a high-speed centrifuge (ultracentrifuge, 9000 rpm, 15 minutes). 51 g of the mycelium was added to a beaker, and 500 ml of methanol was added for 24 hours, followed by filtration to obtain a supernatant. After repeating this process twice, 500 ml of dichloromethane was added to obtain a supernatant in the same manner. Methanol and dichloromethane solution were combined and distilled under reduced pressure to obtain 5.74 g of a purple semi-solid crude extract.

The crude extract was partitioned according to the polarity using a fraction of 500 ml each of water and butanol. 1.49 g of the residue obtained by drying the butanol layer under reduced pressure was partitioned according to polarity using a fraction of hexane and 10% water / methanol. The water / methanol layer was dried under reduced pressure to obtain 0.95 g of a polar organic substance.

This polar organic material was divided into several fractions using reversed-phase vacuum flash chromatography. The column was a glass filter column 100 × 95 mm (inner diameter × length), the stationary phase was C ₁₈ semi-prepared silica for TLC, the eluent was started with 50% water / 50% methanol and increased by 10% methanol. After 100% methanol, the residue was washed with ethyl acetate and dichloromethane.

The eight fractions thus obtained contained significant metabolites in fractions 6 and 7 eluted with 100% methanol and 100% ethyl acetate, respectively, by toxicological experiments on hydrogen nuclear magnetic resonance spectroscopy and leukemia cancer cells. Confirmed.

Accordingly, the fractions 6 and 7 were combined to evaporate the solvent under reduced pressure to obtain 0.73 g of a residue, which was dissolved in 5% water / 95% methanol and then filtered (spartan filter; Aldrich) to remove insoluble matters. C _18, using the same elution fluid and the solution reverse phase semi-preparative high performance liquid chromatography column _{(C 18 reversed-phase semi-} preparative HPLC column) (YMC-ODS-A column, particle size 5 ㎛, 10 × 250 ㎜ (inner diameter X length), eluent: 5% water / 95% methanol, elution rate: 2 ml / min, refractive index detector) to obtain 87.8 mg of bisvoractin A as a colorless, viscous liquid substance at a holding time of 17 minutes. .

Example 3: Activity Determination Experiment of Bisvoractin A

The inhibitory activity (ED ₅₀ ) of the bisboralactin A on cancer cells of the present invention is MTT (3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyl tetrazolium bromide) reduction by intracellular reductase. It was measured by the method of measuring live cells according to the method using the sulforhodamine B (SRB).

Cancer cells used in this experiment were leukemia cell-line (K-562), lung cancer (non-small cell lung cancer cell-line: A-549), ovarian cancer cell-line (SK-OV-3), Melanoma cell-line (SK-MEL-2), colorectal cancer (colon cancer cell-line (HCT-15) and central nervous system tumor cell-line (XF-498) cells.

(1) MTT method

In the activity measurement, all samples were dissolved in an appropriate amount of dimethyl sulfoxide (DMSO) at each concentration and dispensed into 1.5 ml Eppendorf tubes. The cancer cells to be used in the experiment for cell culture were 37 ° C. and 5% CO in a flask having a surface area of 25 cm 2, using a RPMI 1640 medium containing 10 µg / ml of 10% FBS (Fetal Bovine Serum) and kanamycin (kanamycin). _The cells were cultured in an incubator maintained at ₂ . In measuring cytotoxicity, the cells were washed by centrifugation and then suspended in 2 × 10 ⁴ cells / ml in fresh culture.

The MTT assay was carried out as follows: 0.1 ml of cancer cell cultures which reached the log phase were inoculated into 96 well plates, and serial dilutions of the samples (boralactin A) were added 10 times each, and 0.1 ml were added to give final volume of 0.2 after ㎖ is presented, in 37 ℃, 5% CO ₂ incubator which is maintained and cultured for 4 days. After the incubation, 50 μl of MTT solution (1 mg / ml) dissolved in distilled water was added to each well and further incubated for 4 hours. The culture supernatant was removed and the formazone crystals formed by the reductase of living cells were dissolved in 0.15 ml of DMSO and measured with a microplate reader using an 540 nm optical filter. Cells that did not process the sample appeared dark purple, and when all cancer cells died, the result was an almost clear solution, and the number of living cells could be measured according to the absorbance.

As a result of the experiment, when measured by viable cell number measurement method according to MTT reduction by intracellular reductase, bisboralactin A of the present invention is 10 ^-23 ㎍ / ㎖ for leukemia cell-line (K-562) cells Inhibitory activity of (ED ₅₀ ) was shown.

(2) SRB method

Solid cancer cells to be used in the preservation experiments were cultured in an incubator maintained at 37 ° C. and 5% CO ₂ in a flask having a surface area of 25 cm 2 using RPMI 1640 medium containing 10% FBS. Cancer cells were cultured to reach log phase and then numbered and diluted to appropriate concentrations (1-2 × 10 ⁴ cells per well) and aliquoted into 96 well microtiter plates. After culturing the cells for 24 hours, the bisvoractin A dissolved in the medium was added to a well containing cancer cells, and cultured for 48 hours in an incubator maintained at 37 ° C. and 5% CO ₂ . After the incubation was completed, the culture medium fixed with cold TCA was fixed with 0.4% SRB dissolved in 1% acetic acid solution. 10 mM unbuffered tris salt was added to the culture and stirred to dissolve the fixed dye, and then absorbance was measured using a microplate reader using an 520 nm optical filter. In order to increase the accuracy of the measurement, all samples were tested in three multiples, and the concentration at which the absorbance reached 50% of the standard was defined as ED ₅₀ .

As a result, in the measurement method using SRB, the bisvoractin A of the present invention is non-small cell lung cancer cell-line (A-549), ovarian cancer cell-line (SK-OV-3), melanoma 0.002 and 0.001 for melanoma cell-line (SK-MEL-2), colon cancer cell-line (HCT-15) and central nervous system tumor cell-line (XF-498) cells, respectively. , 0.001, 0.002 and 0.002 μg / ml showed inhibitory activity (ED ₅₀ ).

As described above, the novel physiologically active substance boralactin obtained by extracting from the culture mycelium of actinomycetes of Streptomyces sp., As a result of physiological activity experiment, leukemia, non-small cell lung cancer, uterine cancer It is expected to be developed as a versatile anticancer agent by showing strong toxicity against cancer cells such as melanoma, colorectal cancer, and central nervous system cancer.

Claims (4)

A compound having the following structural formula. [Formula 1] Wherein R ¹ and R ³ may be the same or different from each other and are each hydrogen, methyl or ethyl, and R ² and R ⁴ may be the same or different from each other and are each hydrogen, methyl, ethyl, propyl or isopropyl, all asymmetric Isomers at carbon. Actinomycetes of Streptomyces sp. Were cultured in a liquid medium, mycelium was isolated from the culture, extracted with an organic solvent, the solvent was evaporated, and the residue was distributed according to polarity to polar organic concentrate. Obtaining, and separating and purifying the concentrate by chromatography. The method of claim 2, (1) culturing the unclassified species of Streptomyces sp., An actinomycetes, in a liquid medium; (2) filtering the culture to separate mycelium; (3) extracting the mycelium with one or two or more mixtures of methanol, ethanol, chloroform, acetone and dichloromethane; (4) evaporating and drying the crude extract obtained by extraction as described above under reduced pressure to obtain a residue; (5) fractionating the residue according to polarity; (6) evaporating to dry the polar material fractions under reduced pressure to obtain a polar organic concentrate; And (7) an extraction method comprising the step of separating and purifying the concentrate by chromatography. An anticancer agent composition comprising the compound of claim 1.