KR100768476B1 - Actinomycetes Streptomyces sp. Strain Producing Anticarcinogen Isolating Method Thereof Manufacturing Method of Semi-Solid Extract From The Same Semi-Solid Extract Resulting From The Same and Anti-cancer Agent composition Using The Same - Google Patents

Abstract

The present invention relates to actinomycetes Streptomyces sp. Strain producing anti-cancer substance, a method for isolation thereof, a method for preparing a semi-solid extract therefrom, a semi-solid extract derived therefrom, and an anticancer composition using the same, in particular, human leukemia cells Actinomycetes Streptomyces sp. KCCM-10664P strain, which produces anticancer substances, and crude extract anticancer active substances obtained from the cultures and the culture filtrates thereof according to the physiological activity test results. It has the effect of showing strong toxicity against the same cancer cells.

Anticancer substance, Streptomyces, Antibacterial

Description

Actinomycetes Streptomyces sp., Which produces anticancer substances, a method for isolation thereof, a method for preparing a semisolid extract therefrom, a semisolid extract derived therefrom, and an anticancer composition using the same {Actinomycetes Streptomyces sp. Strain Producing Anticarcinogen, Isolating Method Thereof, Manufacturing Method of Semi-Solid Extract From The Same, Semi-Solid Extract Resulting From The Same and Anti-cancer Agent composition Using The Same}

1 is a photograph of the actinomycetes Streptomyces genus KCCM-10664P strain cultured for 7 days in agar medium for separation of actinomycetes (ISP-4) according to a preferred embodiment of the present invention,

Figure 2 is a scanning electron micrograph showing the spores of the actinomycetes Streptomyces sp. KCCM-10664P strain cultured for 3 days in modified Bennett's medium according to an embodiment of the present invention,

Figure 3 is a phylogenetic tree based on the 16S rDNA gene of the actinomycetes Streptomyces genus KCCM-10664P strain according to an embodiment of the present invention.

The present invention relates to an actinomycetes Streptomyces sp. Strain, in particular the actinomycetes Streptomyces sp. Strain producing anticancer substances, a method for isolation thereof, a method for preparing a semi-solid extract therefrom, It relates to a semi-solid extract derived and an anticancer composition using the same. More specifically, it is a strain of Streptomyces spp. That produces an anticancer substance that exhibits anticancer activity against human leukemia cells.

Representative cancer treatments of modern medicine include surgical surgery, biotherapy, radiation therapy, and chemotherapy with chemotherapy. Chemotherapy is a method of inhibiting the proliferation of cancer cells by administering oral or injecting an anticancer agent, the advantage of chemotherapy is that the drug can reach any cancer in any part of the body, and can treat metastatic cancer, Chemotherapy is currently used as a standard therapy for treating metastatic cancer. Of course, chemotherapy can not cure cancer, but it plays an important role in relieving symptoms and extending lifespan.

To date, the most widely used anticancer agent among natural anticancer agents is taxol, which is used to treat breast and ovarian cancers. However, these chemotherapeutic agents have problems such as side effects and anticancer drug resistance, and despite many studies on cancer until now, due to the diversity of cancer itself and the diversification of pathogenesis, the side effects are low and the resistance can be overcome. No anticancer drugs have been developed.

Conventional terrestrial organisms have limitations in the development of new drug materials, so new microbial diversity must be secured for the development of new materials. The global marine bio market is expected to reach $ 3.3 billion in 2000 and $ 16.3 billion in 2010. In particular, the medical marine new material market is expected to grow at an annual rate of 18%, to $ 560,000 in 2010. . The ocean is a treasure trove of biodiversity, where 80% of the world's species live, and the potential for developing unused microbial resources, especially when incorporating advanced marine biotechnology. Moreover, only 1% of marine microorganisms can be cultured in a laboratory, and more than 99% remain in egg culture. As such, there are many unknown and diverse biological resources in the ocean, and since marine organisms reproduce in an environment different from that of terrestrial organisms, many compounds having high novel chemical structures have been found in the produced materials.

Among them, the strain Streptomyces genus produces a variety of secondary metabolites and its physiological activity is very diverse. In particular, about 2/3 of the 100,000 bioactive substances detected from microorganisms were found in Streptomyces spp., About 13% in bacteria and 23% in fungi. . Actinomycetes, including the genus Streptomyces, have become a very important microorganism from an industrial or medical point of view as a production bacterium of various bioactive substances. The present invention is also intended to isolate marine actinomycetes exhibiting anticancer activity as part of this study, and to use it for the treatment of leukemia.

The present invention has been made to solve the above problems, to provide a novel actinomycetes Streptomyces sp. KCCM-10664P strain that produces an anti-cancer substance for human leukemia cells. Through this, it is intended to provide an anticancer composition having anticancer activity against cancer cells such as leukemia in human body by separating crude extracting anticancer active material obtained from the culture and culture filtrate thereof.

In addition, according to the present invention, as well as actinomycetes Streptomyces genus strain producing an anticancer substance, a method for separating it, a method for preparing a semi-solid extract from it, a semi-solid extract derived therefrom and an anticancer composition using the same In order to use a variety of anticancer drugs in the future.

The present invention for achieving the above object is the actinomycetes Streptomyces sp. KCCM-10664P to produce anti-cancer material for human leukemia cells.

Accordingly, the anticancer composition comprising the actinomycetes Streptomyces genus KCCM-10664P as described above is also included in the scope of the present invention.

Another preferred embodiment for attaining another object of the present invention is a method for separating such actinomycetes Streptomyces genus KCCM-10664P. That is, collecting sedimentary soil samples from the deep sea; Heat treating the sample; Culturing the heat treated sample in modified Bennett's medium; And separating the strain by smearing the cultured sample. Isolation of actinomycetes Streptomyces sp. KCCM-10664P, which produces an antiangiogenic material for leukemia cells of the human body, characterized in that it comprises a. Way.

Embodiments for achieving another object of the present invention is a method for producing a semi-solid extract having anticancer activity by culturing the strain as described above. In particular, after culturing the actinomycetes Streptomyces sp. KCCM-10664P strain that produces an antiangiogenic material to human leukemia cells, the culture is evaporated to dryness in the liquid or under pressure, anticancer activity Method of producing a semi-solid crude extract having a culture and the culture of the KCCM-10664P strain, the supernatant from which the mycelium was removed from the culture by evaporation to dryness in liquid or under pressure, to prepare a semi-solid crude extract having anticancer activity How is it possible?

Of course, it is obvious that semi-solid crude extracts having anticancer activity and anticancer agent compositions comprising the same, which are produced by such a production method, also belong to another preferred embodiment of the present invention.

Hereinafter, a novel marine actinomycetes Streptomyces sp. KCCM-10664P strain producing anticancer substances according to the present invention, a separation method thereof, a method of preparing a semisolid extract therefrom, a semisolid extract derived therefrom and an anticancer composition using the same It will be described in detail.

First, the present invention relates to a strain of Streptomyces genus that produces an anticancer active substance having anticancer activity against human leukemia cells, and a microbial preparation using the strain. The present invention is characterized in that a new strain of actinomycetes Streptomyces sp. KCCM-10664P producing an anticancer substance against human leukemia cells.

The inventors of the present invention strain Streptomyces sp strains Streptomyces sp. It was named KORDI-3238, which was deposited with the Korea Microorganism Conservation Center (KCCM) on July 7, 2005 and was given accession number KCCM-10664P.

The strain Streptomyces genus according to the present invention is a strain isolated from the deep sea sediment, and the present inventors have comprehensively examined the morphological, culture and biochemical properties in order to identify marine actinomycetes producing such anti-cancer active substances. , 16S rDNA sequencing was confirmed by phylogenetic method.

As a result, the strain of the genus Streptomyces sp. According to the present invention was identified as a new strain having anticancer activity against cancer cells such as human leukemia cells. The mycological properties of these strains are preferably as follows. First, it was confirmed that the growth temperature of the strain is preferably in the range of 15 ℃ to 40 ℃.

Morphological characteristics and optical microscopy (Lechevalier, HN Bergey's Manual of Systematic Bacteriology. Vol. 4, p. 2344, 1989) and biochemical tests (MacFaddin, TF Biochemical tests for identification of medical bacteria. & Wilkins, Baltimore, p. 36-308, 1984). As a morphological characteristic, the substract mycelium of the genus Streptomyces sp. Is brown or purple, and a gray or yellow weight above it. Mycelia (aerial mycelium) is formed, the back color can be brown or purple.

In addition, the spore chain of the genus Streptomyces genus strain is spiral, spore surface is smooth, spores may be a circular shape having a size in the range of 0.5 ㎛ to 0.9 ㎛. Furthermore, using di (D) -glucose, di (D) -mannitol, di (D) -xylose, di (D) -galactose, di (D) -suscross and L (L) -rhamnose as carbon sources It is preferable that gelatin liquefaction ability, starch decomposition ability, nitrate reduction ability, and hydrogen sulfide generation ability are positive, and casein resolution is negative. In addition, the cell wall component may be KCCM-10664P of the actinomycete Streptomyces genus, which comprises glucose, ribose, and LL-diaminopimellic acid.

More specifically, the strains were identified based on phylogenetic analysis by 16S rDNA gene sequencing of the actinomycetes Streptomyces strain KCCM-10664P. The marine actinomycetes of the present invention belong to the genus Streptomyces in the phylogenetic position but all formed independent branches having a sequential difference of 1% or more from other Streptomyces strains. That is, the strain Streptomyces sp. Of the present invention is a new strain belonging to Streptomyces. Accordingly, the 16S rDNA gene of the actinomycetes Streptomyces sp. KCCM-10664P strain according to the present invention preferably comprises a DNA sequence of SEQ ID NO: 1, and the 16S rDNA gene is Streptomyces lavendulae . More preferably 97% homology with IFO 12789 ^T (D85116) is the KCCM-10664P genus Actinomycetes Streptomyces.

Accordingly, the anticancer composition comprising the actinomycetes Streptomyces genus KCCM-10664P as described above is also included in the scope of the present invention. Streptomyces sp. KCCM-10664P strain according to the present invention produces an anticancer substance having anticancer activity, in particular anticancer activity against human leukemia cells, preferably human leukemia cell-line K-562 and leukemia cell-line It is an anticancer composition for producing an anticancer active substance against HL-60 cells.

Another preferred embodiment for attaining another object of the present invention is a method for separating such actinomycetes Streptomyces genus KCCM-10664P. That is, collecting sedimentary soil samples from the deep sea; Heat treating the sample; Culturing the heat treated sample in modified Bennett's medium; And separating the strain by smearing the cultured sample. Isolation of actinomycetes Streptomyces sp. KCCM-10664P, which produces an antiangiogenic material for leukemia cells of the human body, characterized in that it comprises a. Way.

Here, the actinomycetes Streptomyces sp. KCCM-10664P strain according to the present invention can be cultured or grown in actinomycetes culture medium, and representative culture mediums include modified Bennett's, Starch-casein KNO ₃ , various There is an International Streptomyces Project (ISP) badge. Culture conditions are preferably cultured shaken for 3 to 7 days at 25 to 30 ℃. The medium may be any one used for culturing the strain of the genus Streptomyces, but is not limited thereto.

In particular, the modified Bennett medium is 0.1% yeast extract, 0.1% beef extract, 0.2% tryptone, 1% glucose, 1.5% agar, pH 7.2, Aging seawater: distilled water = 7: 3), ISP-2 (International Streptomyces Project-2, 3.74%, pH 7.2, Aging seawater: distilled water = 7: 3, Difco, USA), ISP-4 (International Streptomyces Project-4, 3.74%, pH 7.2, Aged seawater: distilled water = 7: 3), Starch-casein KNO ₃ , 1% soluble starch, 0.03% casein, 0.2% potassium nitrate (KNO ₃ ), 0.2% Potassium phosphate (K ₂ HPO ₄ ), 0.2% sodium chloride (NaCl), ferrous sulfate hydrate (FeSO ₄ · 7H ₂ O), magnesium sulfate hydrate (MgSO ₄ · 7H ₂ O) and calcium carbonate (CaCO ₃ ) It may be characterized by.

Embodiments for achieving another object of the present invention is a method for producing a semi-solid extract having anticancer activity by culturing the strain as described above. In particular, after culturing the actinomycetes Streptomyces sp. KCCM-10664P strain that produces an antiangiogenic material for human leukemia cells, the culture is evaporated to dryness in the liquid or under pressure, anticancer activity Method of preparing a semi-solid crude extract having a cultivation of actinomycetes Streptomyces sp. KCCM-10664P strain producing an antiangiogenic material for human leukemia cells, and then the mycelia from the culture The removed supernatant is evaporated to dryness in a liquid phase or under reduced pressure to prepare a semi-solid crude extract having anticancer activity.

Of course, it is obvious that semi-solid crude extracts having anticancer activity and anticancer agent compositions comprising the same, which are produced by such a production method, also belong to another preferred embodiment of the present invention.

In addition, the anticancer substance produced from the Streptomyces sp. KCCM-10664P strain according to the present invention can be secreted extracellularly. Therefore, the present invention can provide an anticancer composition comprising Streptomyces genus KCCM-10664P, a culture thereof, and a culture filtrate comprising at least one selected from the group consisting of active ingredients. The content of the active ingredient in the anticancer agent composition may be appropriately selected in consideration of the purpose and method of use, and may be, for example, 0.01 wt% or more. In addition, the anticancer composition may further include an additive commonly used.

The culture may be obtained by incubating Streptomyces genus KCCM-10664P in a solid agar medium or liquid culture medium for 3 to 14 days at 15 to 40 ℃, as a crude crude extract evaporated to dry liquid or reduced pressure under reduced pressure It may be included in the anticancer agent composition. The culture filtrate is a supernatant from which the mycelium is removed from the culture. The culture filtrate may be obtained by centrifugation or filtration. The culture filtrate may be included in the anticancer composition as a crude extract obtained by evaporation to dryness under liquid or reduced pressure. The anticancer agent composition of the present invention may further include a carrier according to the use purpose, purpose and method in addition to the above-mentioned effective ingredient. In addition, the anticancer composition may be used as a food additive and a medicament.

Hereinafter, one preferred embodiment of the present invention will be described in detail with reference to the accompanying drawings. The invention can be better understood by the following examples, which are intended for the purpose of illustration of the invention and are not intended to limit the scope of protection defined by the appended claims.

Example  1: Actinomycetes Genus Streptomyces KCCM Isolation of -10664P

First, we tried to isolate KCCM-10664P genus Actinomycetes Streptomyces according to the present invention. About 1 g of sample taken from deep sea sediment in Ayu Trough (01 ° 38 '355N, 132 ° 43' 591E) in the Western Pacific was placed in a vial filled with 20 ml of sterile seawater and sonified for 5 minutes. Then, in order to kill the general bacteria (bacteria), the heat treatment for 50 minutes in a 60 ℃ dry oven, and the heat-treated samples were sequentially diluted to 10 ^-1 , 10 ^-2 , 10 ^-3 .

Subsequently, the diluted sample was subjected to modified Bennett medium, that is, actinomycetes separation medium, that is, modified Bennett (0.1% yeast extract, 0.1% beef extract, 0.2% tryptone, 1%). Glucose, 1.5% agar, pH 7.2, aged seawater: distilled water = 7: 3), ISP-2 (International Streptomyces Project-2, 3.74%, pH 7.2, aged seawater: distilled water = 7: 3, Difco, USA), ISP-4 (International Streptomyces Project-4, 3.74%, pH 7.2, Aged seawater: distilled water = 7: 3) and Starch-casein KNO ₃ (1% soluble starch, 0.03% casein) casein), 0.2% potassium nitrate (KNO ₃ ), 0.2% potassium diphosphate (K ₂ HPO ₄ ) 0.2% sodium chloride (NaCl), traces of ferrous sulfate hydrate (FeSO ₄ · 7H ₂ O), traces of magnesium sulfate hydrate (MgSO ₄ · 7H ₂ O, trace calcium carbonate (CaCO ₃ ), 1.5% agar (agar), pH 7.2, aged seawater: distilled water = 7: 3) 0.1 ml of the medium was inoculated and plated sequentially.

Subsequently, after culturing for 14 days in a 30 ° C. incubator, when the actinomycetes colonies were observed, the strains separated by tertiary streaking in modified Bennett's medium for secondary separation were collected in colony form and under an optical microscope. Observations were made of 300 strains of the typical actinomycetes as primary pure separation and screening.

Example  2: Screening of Anticancer Strains

Strains showing anticancer activity were selected from Actinomycetes Streptomyces sp. KCCM-10664P isolated according to Example 1. Actinomycetes completely formed spores among the isolated strains were stored in 20% (w / v) glycerol and allowed to stand at room temperature for one day and then vortex and stored at -70 ℃. The cryopreserved actinomycetes were activated by culturing in a modified Bennett liquid medium for 7 days, and using leukemia cell-line K-562 and HL-60, MTT (3- [4,5-dimethylthiazo1) described below. 60 kinds of actinomycetes were selected by searching for primary anticancer activity according to -2-yl] -2,5-diphenyl tetrazolium bromide) method (see Example 7). Selected from Streptomyces sp. It was named KORDI-3238, and it was deposited with the Korea Microorganism Conservation Center (KCCM) under the accession number KCCM-10664P.

Example  3: Morphology, Physiology and Biochemical Properties of Strains

Actinomycetes Streptomyces spp. KCCM-10664P strain, which was selected in Example 1, was highly biochemically identified according to the microbial classification method, biochemical test method, and International Streptomyces Project (ISP). Morphological features were observed under light microscopy after incubation in modified Bennett medium and International Streptomyces project medium.

Streptomyces sp. KCCM-10664P strain according to the present invention was generally good growth in the medium, the growth temperature range was 15 ~ 40 ℃, the optimum growth temperature was 25 ~ 30 ℃. Substract mycelium was mainly brown or purple with gray or yellow aerial mycelium on it, and its backing was mainly brown or purple (see FIG. 1). The culture characteristics of the Streptomyces genus KCCM-10664P are summarized in Table 1 below.

Table 1: Culture Characteristics of KCCM-10664P in Streptomyces]

 badge  Growth  Aerial hyphae color  Substrate hyphae color  Back color  Water soluble pigment  Modified Bennet Agar Badge  Very good  Light yellow  purple  purple  purple  Tryptone-Yeast Extract Agar Medium (ISP-1)  good  Light gray  Dark yellow  Dark yellow  Light yellow  Yeast Extract- Malt Extract Agar Medium (ISP-2)  good  Light gray  Light brown  Dark brown  Light yellow  Inorganic Salt-Starch Agar Medium (ISP-4)  good  Light gray  purple  purple  none  Peptone-Yeast Extract-Iron Agar Medium (ISP-6)  usually  Light gray  Light yellow  Light yellow  none

In addition, the actinomycetes Streptomyces spp. KCCM-10664P strain cultured in the modified Bennett medium for 3 days was preliminarily fixed with 8% glutaaldehyde solution for one day, and the well-spore-formed site was cut and fixed for one day. After gold coating using the coating apparatus, spores were observed using a scanning electron microscope (Phillips model 515). As shown in Figure 2, the spore chain of the bacteriostatic strain according to the present invention was a spiral, the spore surface was smooth, did not have a specific protrusion, spores were circular size 0.5 ~ 0.9 ㎛, substrate mycelia are segmented It did not show form.

In addition, the carbon source utilization characteristics of the Streptomyces genus KCCM-10664P were investigated, and the results are shown in Table 2 below. That is, the actinomycetes according to the present invention are di (D) -glucose, di (D) -mannitol, di (D) -xylose, di (D) -galactose, di (D) -sucrose, L (L) in the medium. Most sugars, such as) -rhamnose, were used, but no di (D) -fructose and L (L) -arabinose were used.

[Table 2: Carbon Source Utilization Characteristics of KCCM-10664P in Streptomyces]

 Carbon source  Utilization  Di (D) -glucose  positivity    Di (D) -mannitol  positivity    D (D) -Fructose  voice    D (D) -Xylose  positivity    D (D) -Sucrose  positivity    Dee (D) -galactose  positivity    L (L) -Arabinos  voice   L (L)-Rhamnos  positivity

Furthermore, the physiological characteristics of the Streptomyces genus KCCM-10664P were investigated, and the results are shown in Table 3 below. In other words, actinomycetes according to the present invention was gelatin liquefaction ability, starch decomposition ability, nitrate reduction ability, hydrogen sulfide generating ability was positive, but the casein resolution was negative, melanin was not produced in ISP-6 and ISP-7 medium.

Table 3: Physiological Characteristics of KCCM-10664P in Streptomyces

 Item  characteristic  Melanin production ability  voice  Nitrate Reducing Ability  positivity  Starch resolution  positivity  Gelatin liquefaction  positivity  Casein resolution  voice  Hydrogen sulfide generating ability  positivity

In addition, the cell wall component characteristics of the Streptomyces genus KCCM-10664P were investigated. Cell wall component analysis was performed by analyzing the sugars of diaminopimellic acid and the total hydrolysates of whole cells in the peptide glycan of the cell wall according to the method of Staneck and Roberts and the analysis method using TLC. . As a result of the cell wall degradation assay, glucose and ribose were detected as shown in Table 4 below. In the analysis of amino acids, LL-diaminopimellic acid was detected.

Table 4: Properties of Cell Wall Components of Streptomyces genus KCCM-10664P

 Item  ingredient  Diaminopimellic acid (DAP)    LL-diaminopimellic acid    Glycine    voice   Party    Glucose, ribose

According to these results, the classification method according to cell wall characteristics (Goodfellow, M. and DE Minnikin. Chemical method in bacterial systematics. P. 131-143. Academic Press. New York, 1985; Lechevalier, HN Bergey's Manual of Systematic Bacteriology. 4, p. 2344, 1989), Streptomyces sp. KCCM-10664P strain according to the present invention can be classified as actinomycetes having a cell-wall type I (type I).

Example  4: 16S of strain rRNA  Gene Sequencing and Genealogy

In this example, 16S rRNA gene sequences were analyzed to identify the phylogenetic location of KCCM-10664P in Streptomyces. First, the cells were suspended and cultured in modified Bennett medium for 3 days for DNA extraction, and the cells were separated by centrifugation. Genomic DNA extraction was performed using AccuPrep ^™ Genomic DNA extraction kit (Bioneer), and amplification was performed by PCR reaction. 16S rRNA gene fragments were cloned using pGEM-T easy vector (Promega).

After extracting the plasmid from the transformed E. coli, the nucleotide sequence (1461 bp) was analyzed using Termination Sequencing Ready Reaction kit (Perkin Elmer) and ABI 377 genetic analyser (Perkin Elmer), and NCBI BLAST program was used. Homology was confirmed. As a result, the Streptomyces genus KCCM-10664P strain according to the present invention is actinomycetes Streptomyces Ravendule ( Streptomyces lavendulae ) 97% homology with IFO 12789 ^T (D85116), and more than 1% nucleotide sequence variation with all of the existing strains of Streptomyces in 16S rDNA homology.

Based on this, the nucleotide sequences of the strains of Streptomyces sp. Already analyzed were taken from the gene bank, and the phylogenetic tree was drawn by the neighbor-joining method using the MEGA 2.1 package program, which is shown in FIG. 3. All. Thereafter, in order to evaluate the topology of the phylogenetic tree, the bootstrap was repeatedly performed 1000 times using Streptosporangium roseum DSM 43021 ^T (X89947) as an out group. Streptomyces sp. KCCM-10664P strain according to the present invention belonged to Streptomyces in the phylogenetic tree, but formed a separate branch having a difference in sequence of more than 1% with all other Streptomyces strains. That is, by the phylogenetic analysis, the strain KCCM-10664P of the genus Streptomyces according to the present invention was confirmed to be a new strain belonging to the genus Streptomyces.

As a result, the morphological, culture, and biochemical characteristics of Streptomyces sp. KCCM-10664P strain producing anti-cancer active substances were comprehensively reviewed, and phylogenetic classification was performed using 16S rDNA sequencing and MEGA program. As a result, the inventors named the strain KORDI-3238 of Streptomyces genus, and deposited it on July 7, 2005 at the Korea Microorganism Conservation Center, an internationally recognized deposit institution for patent microorganisms under the Budapest Treaty (Accession No. KCCM- 10664P).

Example  5: Genus Streptomyces KCCM Culture of -10664P Strains

Streptomyces sp. KCCM-10664P strain according to the above-described embodiment was activated by incubating in a modified Bennett's medium for 7 days, and then activated at 27 ° C. and 150 rpm in a flask (× 3) containing 100 ml of the same medium. Seed culture was carried out for 4 days. The seeded strains were inoculated again in 30 ml portions of 3 l Fernbach flask (× 10) containing 0.8 L of the same medium, followed by shaking culture for 7 days under the same conditions. As a result, it was confirmed that the growth temperature of the strain is preferably within the range of 15 ℃ to 40 ℃.

Example  6: Extraction of anticancer agent composition

Mycelium was removed from the culture medium of Streptomyces sp. KCCM-10664P strain obtained in Example 5 using a high-speed centrifuge (9000 rpm, 15 minutes). The culture filtrate thus separated was again filtered through a 0.2 μm membrane filter to completely remove extra mycelium. The culture filtrate thus obtained was concentrated by evaporation under reduced pressure to about 1 L to obtain a semi-solid crude extract having anticancer activity.

Example  7: Crude extraction  Anticancer activity measurement of anticancer agent composition

Inhibitory activity (IC ₅₀ ) on the semi-solid extract obtained in Example 6 from the Streptomyces sp. KCCM-10664P strain according to the present invention, MTT (3- [4,5-dimethylthiazo1-) by intracellular reductase 2-yl] -2,5-diphenyl tetrazolium bromide) was measured by viable cell count measurement method. The cancer cells used in this experiment were leukemia cell-line K-562 and HL-60.

When measuring the activity, the sample was dissolved in an appropriate amount of dimethyl sulfoxide (DMSO) at each concentration and dispensed into 1.5 ml Eppendorf tubes. The cancer cells used in the experiment for cell culture were 37 ° C. and 5% CO in a flask having a surface area of 25 cm 3 using RPMI 1640 medium containing 20 μg / ml of 10% FBS (Fetal Bovine Serum) and kanamycin (kanamycin). _The cells were cultured in an incubator maintained at ₂ . In measuring cytotoxicity, cells were washed by centrifugation and suspended in 2 × 10 ⁴ cells / ml in fresh culture.

The MTT assay was performed as follows: 0.1 ml of cancer cell cultures which reached the log phase were inoculated into 96 well plates, and the samples were serially diluted 10-fold and 0.1 ml were added to make the final volume 0.2 ml. , 37 ° C., and incubated for 4 days in an incubator maintained at 5% CO ₂ . 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.

The cells that did not process the sample appeared dark purple, and when all the cancer cells died, the result was an almost clear solution, and the number of living cells could be measured according to the absorbance. In order to increase the accuracy of the measurement, all samples were tested in three-fold multiples, and the concentration at which the absorbance reached 50% of the standard was defined as IC ₅₀ . As a result, the anti-solid crude anti-cancer active substance according to the present invention is leukemia cell-line K-562 and HL- which are leukemia cells of the human body, as measured by the method of measuring viable cell number according to MTT reduction by intracellular reductase. The inhibitory activity (IC ₅₀ ) of 49.6 and 0.01 μg / ml was shown for 60, respectively.

As described above, according to the present invention can provide a novel actinomycetes Streptomyces sp. KCCM-10664P strain producing an anticancer substance for human leukemia cells. Through this, the crude extract anticancer active material obtained from the culture and the culture filtrate thereof has the effect of showing strong toxicity against cancer cells such as leukemia of the human body as a result of the physiological activity experiment.

According to the present invention, as well as the actinomycetes Streptomyces genus strain producing an anticancer substance, a method for separating it, a method for preparing a semi-solid extract from it, a semi-solid extract derived therefrom and an anticancer composition using the same In addition, it can be developed in various ways for anticancer drugs in the future.

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Claims (16)

Actinomycetes Streptomyces sp. Streptomyces sp. KCCM-10664P, which produces an anticancer substance against human leukemia cells, wherein the 16S rDNA gene comprises a DNA sequence of SEQ ID NO: 1. The actinomycete Streptomyces genus KCCM-10664P according to claim 1, wherein the growth temperature is in the range of 15 ° C to 40 ° C. The genus Streptomyces genus of claim 1, wherein the substract mycelium is brown or purple, and gray or yellow aerial mycelium is formed thereon, and the back color is brown or purple. KCCM-10664P. The actinomycetes Streptomyces genus KCCM-10664P according to claim 1, wherein the spore chain is helical, the spore surface is smooth, and the spores have a circular shape and have a size in the range of 0.5 µm to 0.9 µm. The method of claim 1, wherein di (D) -glucose, di (D) -mannitol, di (D) -xylose, di (D) -galactose, di (D) -suscross and L (L) -rhamnose Actinomycetes Streptomyces genus KCCM-10664P, characterized in that used as a carbon source. The actinomycetes Streptomyces genus KCCM-10664P according to claim 1, wherein the gelatin liquefaction ability, starch decomposition ability, nitrate reduction ability, hydrogen sulfide generating ability is positive, and casein resolution is negative. The actinomycetes Streptomyces genus KCCM-10664P according to claim 1, wherein the cell wall component comprises glucose, ribose and LL-diaminopimellic acid. delete The actinomycetes Streptomyces genus KCCM-10664P according to claim 1, wherein the 16S rDNA gene has a homology of 97% with Streptomyces lavendulae IFO 12789 ^T (D85116). An anticancer agent composition comprising the actinomycetes Streptomyces genus KCCM-10664P according to claim 1. In a method for isolating actinomycetes Streptomyces sp. Microorganisms producing an antiangiogenic substance to human leukemia cells, Collecting sediment samples from deep sea; Heat treating the sample; Culturing the heat treated sample in modified Bennett's medium; And Smearing the cultured sample to separate strains; and The modified Bennett medium is 0.1% yeast extract (yeast extract), 0.1% beef extract (beef extract), 0.2% tryptone, 1% glucose (glucose), 1.5% agar (agar, pH 7.2, aged seawater) Distilled water = 7: 3), ISP-2 (International Streptomyces Project-2, 3.74%, pH 7.2, Aged seawater: Distilled water = 7: 3, Difco, USA), ISP-4 (International Streptomyces Project-4, 3.74%) , pH 7.2, Aged seawater: distilled water = 7: 3), Starch-casein KNO ₃ , 1% soluble starch, 0.03% casein, 0.2% potassium nitrate (KNO ₃ ), 0.2% diphosphate Potassium (K ₂ HPO ₄ ), 0.2% sodium chloride (NaCl), ferrous sulfate hydrate (FeSO ₄ · 7H ₂ O), magnesium sulfate hydrate (MgSO ₄ · 7H ₂ O) and calcium carbonate (CaCO ₃ ) Separation method of actinomycetes Streptomyces genus KCCM-10664P. The method of claim 11, wherein the actinomycetes Streptomyces genus KCCM-10664P is 16S rDNA gene, characterized in that the DNA sequence of SEQ ID NO: 1 separation method of Streptomyces genus KCCM-10664P. To produce an antiangiogenic material for human leukemia cells, the 16S rDNA gene is cultured actinomycetes Streptomyces sp. KCCM-10664P strain containing the DNA sequence of SEQ ID NO: 1, and then Method of producing a semi-solid crude extract having an anticancer activity by evaporating to dry the culture in the liquid phase. To produce an antiangiogenic material for human leukemia cells, the 16S rDNA gene is cultured actinomycetes Streptomyces sp. KCCM-10664P strain containing the DNA sequence of SEQ ID NO: 1, and then A method of preparing a semi-solid crude extract having anticancer activity by evaporating and drying the supernatant from which the mycelium is removed from the culture in the liquid phase. 16. rDNA gene prepared by the method according to claim 13 or 14, characterized in that the 16S rDNA gene comprises Actinomycetes Streptomyces sp. KCCM-10664P comprising the DNA sequence of SEQ ID NO: Semi-solid crude extract having anticancer activity. An anticancer agent composition comprising the semi-solid crude extract according to claim 15.

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