KR20110045763A - Aspergillus sp. KMD 901 strain, diketopiperazines isolated therefrom, and a composition for the prevention of the treatment of cancer comprising the same - Google Patents

Abstract

PURPOSE: A novel compound isolated from Aspergillus sp. KMD901 is provided to be used as a anticancer agent. CONSTITUTION: An Aspergillus sp. strain(deposit number KCCM 11047P) is derived from marine deposit soil. A composition for preventing or treating cancer contains Aspergillus sp. strain, culture material of the strain, dried material or concentrate of the culture material, organic solvent extract which is obtained from the culture using ethyl acetate, alcohol of C1-C4, methylene chloride, ether, or chloroform, or fraction. A composition for preventing or treating cancer contains acetylaranotin of chemical formula 1, acetylapoaranotin of chemical formula 2, dehydro-deoxyapoaranotin of chemical formula 3 or pharmaceutically acceptable salt thereof as an active ingredient.

Description

Aspergillus WMD 901 strain, a diketopiperazine compound isolated therefrom, and a composition for preventing or treating cancer containing the same {Aspergillus sp. KMD 901 strain, diketopiperazines isolated therefrom, and a composition for the prevention of the treatment of cancer comprising the same}

Aspergillus fungus KMD 901 from Aspergillus sp. KMD 901), a novel compound isolated therefrom, Dehydro-deoxyapoaranotin, Acetylaranotin, and Acetylapoaranotin, and their use in inhibiting cancer cell growth It is about. Fungi isolated from the marine sediment and secondary metabolites derived therefrom can be usefully used as candidates for anticancer drugs.

The development of anticancer drugs is the core of life science-related technology development research, and cancer is competing for the top two causes of death not only in Korea but also in the United States and Japan. In particular, it accounts for more than half of the death rates of large hospitals. Biotechnology Laboratories, 3: 55, 1995; Yeong-Gi-Sup, Seong-Gun, Cancer Chemotherapy, Seokwang Seolim, 351, 1991). As part of efforts to conquer these cancers, many advances have been made in the diagnostic methods for early detection of various cancers. Surgery, radiation therapy, development of anticancer drugs, and biological methods have also been developed in the treatment of cancer. In the case of anticancer drugs, there have been many attempts using various kinds of cancer cells, but until now, there is no anticancer drug as a true therapeutic agent, and only drugs for the purpose of supporting life extension for a short period of time have been developed.

The anti-cancer agents mainly used in the clinic are substances that exhibit cytotoxicity by reacting with nucleic acid synthesis inhibitors such as 5-fluorouracil and citrarabine or nucleic acids such as alkylated drugs, mitomycin, cisplatin, procarbazine, etc. Can be classified. However, these anticancer drugs have many problems because they have various side effects, and there is an urgent need for the development of new cancer treatments that solve these problems.

In recent years, international trends in chemotherapy have shown that conventional methods of anti-cancer therapies include their inhibitors or replaceable compounds that target DNA biosynthesis, DNA conjugates, topoisomerase, spindles and hormones. Recently, the target has been transferred to signal transduction (EGF), angiogenesis suppression (MMPI's), cell cycle (CDK's), apoptosis (bcl2), immunotherapy, gene therapy, and monoclonal antibodies. have.

In addition, natural and organic synthesis methods have been used mainly as new anticancer drugs. Attempts have been made to separate components with anticancer effects from natural products through efficacy evaluations using several cancer cell lines and animal tumor models. Recent examples include the development of Taxol and Campotesine.

Taxol is a unique mechanism of cancer cytotoxicity through anti-spinning activity and has a strong effect on breast and ovarian cancer (PB Schiff et al. J. Supramolec. Struct. 8: 328, 1978; EK Rowinsky et al. Cancer Res. 48: 4093, 1988; N. Kumar, J. Biol. Chem. 256: 10435, 1981; FA Holmes et al. J. Natl. Cancer Invest. 83: 1797, 1991). In addition, campotethecin is an inhibitor of topoisomerase I, which showed a very strong effect in experimental tumors but showed severe cystitis in the clinic (M.E. Wall et al. J. Am. Chem. Soc. 88: 3888, 1966). However, many derivatives, such as CPT-11 and SN38, have been developed with campotesin as a leader (T. Kunimoto et al. Cancer Res. 47: 5944, 1987; N. Kaneda et al. Cancer Res. 50: 1721, 1990; S. Negoro et al. J. Natl. Cancer Inst. 83: 1164, 1991). Phase II clinical trials are currently underway for CPT-11, including lung cancer (M. Fukuoka et al. J. Clin. Oncol. 110: 16, 1992), leukemia and lymphoma (R. Ohno et al. J. Clin. Oncol). 8: 1907, 1990; MR Boyd et al. Agent Chemother. 31: 1238, 1987). However, a single anticancer agent does not show anticancer activity against all cancers, and the anticancer activity of each cancer is different, so the development of a new anticancer agent is continuously needed.

On the other hand, marine life is known to inhabit hundreds of thousands of species, which account for 80% of the world's animals, and new living organisms that do not exist at all on land have been reported for future drug development. In particular, marine microorganisms are recognized as highly efficient biological resources for the acquisition of new compounds, which are one of the most important factors in drug development.

Unlike plant or animal resources, where mass cultivation or aquaculture is essential, the desired extract or compound can be obtained using a relatively rapid and cost-effective mass culturing method for microorganisms. Salinosporamide A (Feling, RH et al. Angew. Chem Intl. Ed. 42, 355-357, 2003) and Porboxazole A (Searie, PA & Molinski, TFJ Am. Chem. Soc. 117, 8126-8131, 1995) is a representative drug derived from marine microorganisms. For salinosporamide A, a substance derived from bacteria isolated from marine sediments is under clinical research for the treatment of myeloma. Sol-A is a compound isolated from bacteria separated from the sponges and has a function of inhibiting cell division, and a function of inhibiting the cell cycle at a very low concentration.

Representative components exhibiting anticancer or cancer cytotoxic efficacy derived from Aspergillus sp. Include fumagillin (Lu, J. et al. J. Med. Chem. 49 (19), 5645-5648 , 2006), Tropolactone (Cueto, M. et al. Phytochemistry 67 (16), 1826-1831, 2006), Asterriquinone (Kaji, A. et al. Anticancer Res. 17 ( 5A), 3675-3679, 1997), Aspergillazine (Capon, RJ et al. Org. Biomol. Chem. 3 (1), 123-129, 2005), Aspyridone (Bergmann, S. et al. Nat. Chem. Biol. 3 (4), 213-217, 2007), and tryprostatin (Zhao, S. et al. J. Med. Chem. 45 (8), 1559 -1562, 2002).

More than 180 species of fungal strains of the genus Aspergillus sp. Have been reported since it was first classified by Italian priest and biologist Micheli, P. A in 1729 (Lubertozzi, D. Biotechnol. Adv. 27, 53-75, 2009). Many fungi, which play an important role in food and fermentation industry, are included in the Aspergillus, and since amylase and protease activities are strong, they have been used for brewing alcoholic beverages and intestines since ancient times. It is also used in the production of drugs and enzymes such as diaastase, glucoamylase and protease (Bennett, JWJ Biotechnol. 66 (2-3): 101-107, 1998).

Aspergillus aurise ; Aspergllus oryzae is a fungus that has long been used for fermentation (Machida, M. et al. DNA Research) as a representative strain belonging to the genus Aspergillus sp. , 15, 173-183, 2008), Aspergillus granules ( A. sojae ) are used for the preparation of soy sauce (Chang, PK Appl. Microbiol. Biotechnol. 76, 977-984, 2007) ( A.niger ) is used for organic acid fermentation (Withers, JM et.al. Biotechnol. Bioeng. 59 (4): 407-418, 1998), and Aspergillus wenti (A. wentii) Used for processing (Manabe, M. et. Al. In Toxigenic Fungi. 4-14, 1984), Aspergillus fungi are fungi essential to the fermentation industry.

On the other hand, Aspergillus pumigatus , Aspergillus flavus ( A. flavus ) and A.niger can damage the parenchyma of the lung, forming fungal gall (aspergilloma), which can cause aspergillosis in humans (Latg ㅹ, JP Clin. Microbiol. Rev. 310-350, 1999).

On the other hand, acetyl aranotin is Neuss, N. et al. Was first reported (Neuss, N. et al. Tetrahedron lett. 42, 4467-4471, 1968), and subsequently isolated from Aspergillus terreus strains and is widely known as a plant growth inhibitor. (Kamata, S. et al. Agric. Biol. Chem. 47 (11), 2637-2638, 1983). In addition, the antiviral effect on the respiratory virus Coxsackie A-21 by Murdock, KC (Murdock, KCJ Med. Chem. 17, 827-835, 1974) and the inhibition of platelet aggregation (Ihara, Tetsuo et al. JP 02300129, 1990), but have not been reported to inhibit the growth of cancer cells. In addition, in the case of acetylapoaranotin of the present invention, a compound having an antibiotic effect has been studied for its chemical properties (Nagarajan, RJ Am. Chem. Soc. 95 (22), 7212-7222, 1973). Inhibition of platelet aggregation has been reported (Ihara, Tetsuo et al. JP 02300130, 1990), but no action on cancer cells has been reported.

The present inventors completed the present invention by confirming that Aspergillus sp. Strain (KMD 901) isolated from marine sedimentary soils, and compounds isolated therefrom, are effective in inhibiting growth of various types of cancer cells.

Thus, one example of the present invention provides Aspergillus sp. Strain (Accession No. KFCC 11450P) isolated from marine sediment.

Another example provides the novel compound Dehydro-deoxyapoaranotin of Formula 3 isolated from the Aspergillus strain.

<Formula 3>

Another example is at least one selected from the group consisting of the genus Aspergillus strain, the culture of the strain, the dried product of the culture, the concentrate of the culture, the organic solvent extract of the culture, and the alcohol / water fraction of the extract It provides a composition for the prevention or treatment of cancer comprising as an active ingredient

Another example provides a composition for the prophylaxis or treatment of cancer containing the diketopiperazine compound of Formula 1-3 isolated from the Aspergillus strain (aranotin derivative) as an active ingredient.

<Formula 1>

<Formula 2>

<Formula 3>

Another example is the genus Aspergillus strain, the culture of the strain, the dried product of the culture, the concentrate of the culture, the organic solvent extract of the culture, the alcohol / water fraction of the extract, the diketopy of formula 1-3 It provides a functional food for the prevention or amelioration of cancer comprising a perrazine (aranotin derivative) compound and at least one member selected from the group consisting of pharmaceutically acceptable salts of the compound.

Another example provides a method of preparing a compound of Formula 1 to 3 from the Aspergillus strain.

The present inventors prepared liquid extracts of Asperigillus sp. KMD 901 strain isolated from marine sediments and prepared extracts using ethyl acetate, from which a new compound, dihydro-deoxyaporanotin ( Compounds of three diketopiperazine families, including acetylaranotin and acetylapoaranotin, including dehydro-deoxyapoaranotin were isolated to identify the structure of the compound. The present invention was completed by evaluating their cancer cell growth inhibitory activity.

First, a novel Aspergillu s sp. Strain (KMD 901) isolated from marine sediments is provided.

The present inventors collected a marine sediment with a piston core having an inner diameter of 5 cm x 1 m in length at a depth of 1,198 m at a position of 130 degrees 0 minutes and 37 degrees 3.4 minutes in the vicinity of Ulleungdo in the East Sea, and dried it in the shade to prepare a marine sediment sample. It was. The dried product was suspended in physiological saline, diluted 1/10, and then inoculated in a Czapek-Dox solid medium, and a single strain produced while incubating at room temperature was inoculated again in a Czapek-Dox solid medium. Pure strains were isolated and selected. The isolated KMD 901 strains had a yellow colony with a downy colony in the Czapek-Dox solid medium (see FIG. 1), 5.8S rRNA, 18S rRNA and 28S rRNA gene sequences of SEQ ID NO: 1. It is characterized in that the secretion of the aranotine derivatives of the formula 1 to 3 as a secondary metabolite. The strain KMD 901 was deposited on June 23, 2009 at the Korea Microbial Conservation Center, and was given accession number KCCM 11047P.

As a result of analyzing the nucleotide sequences of the 5.8S rRNA, 18S rRNA, and 28S rRNA genes of the KMD 901 strain, it showed 98.8% homology with Aspergillus versicolor NRRL 227. Secretion and cancer cell growth inhibitory activity of aranotine derivatives such as compounds have not been reported at all in Aspergillus versicolor.

The base sequences of 5.8S rRNA genes, 18S rRNA genes, 28S rRNA genes of the KMD 901 strain are as follows (SEQ ID NO: 1):

In addition, the Aspergillus sp. KMD 901 strain is first cultured in a solid medium, and then a colony generated therein is inoculated in a liquid medium, thereby providing a method of bulk liquid culture.

In addition, the Aspergillus sp. KMD 901 strain, the culture of the strain, the dried product of the culture, the concentrate of the culture, the organic solvent extract of the culture, and the alcohol / water fraction of the extract Provided is a composition for preventing or treating cancer, which contains at least one selected from the group as an active ingredient.

Aspergillus sp. It was confirmed that there is a substance having cancer cell growth inhibitory activity in the secondary metabolite of the strain KMD 901 (see Test Examples <1> to <3>). These useful secondary metabolites have been produced from about 4 days after culture. Therefore, cultures of Aspergillus sp. KMD 901 strain as well as Aspergillus sp. KMD 901 strain can be used as an active ingredient of the composition for preventing or treating cancer.

In order to provide optimal conditions for the production of useful secondary metabolites, the culture medium is preferably saccharose medium. In the present invention, saccharose medium refers to a medium having a saccharose content of 50% by weight or more based on the total amount of ingredients excluding water, for example, Czapek-Dox medium (Saccharose 30 g, Sodium Nitrate 3.0 g, Dipotassium Phophate). 1.0 g, Magnesium Sulfate 0.5 g, Potassium Chloride 0.5 g, and Ferrous Sulfate 0.01 g as the main ingredient). The saccharose medium form may be both a solid medium and a liquid medium, and preferably, may be a liquid medium.

The culture period is preferably about 4 days or more, preferably 4 to 20 days, more preferably 6 to 15 days active production of effective secondary metabolites.

In one embodiment of the present invention, the culture may be obtained by incubating 4 to 10 days, preferably 6 to 8 days in 20 to 30 ml of saccharose liquid medium per 2-3 KMD 901 strain colonies.

In the present invention, the dry matter and the concentrate mean that obtained by drying or concentrating the culture in a conventional manner.

The organic solvent extract is obtained by extracting a culture of the KMD 901 strain with at least one organic solvent selected from the group consisting of ethyl acetate, alcohol having 1 to 4 carbon atoms (preferably butanol), methylene chloride, ether, chloroform, and the like. It may be an extract. The extraction may be replaced by a method using an extraction apparatus such as supercritical extraction, high pressure extraction or ultrasonic extraction, or by using a celite or polystyrene or polyamide adsorption resin, but is not limited thereto. At the time of extraction, the solvent is preferably extracted by adding 1 to 3 times the volume of the culture solution, and more preferably, by adding about 2 volumes. Extraction at room temperature is preferred, but is not limited thereto. The number of extraction is preferably 1 to 5 times, and more preferably three times of extraction, but is not limited thereto. Drying under reduced pressure preferably uses a rotary vacuum evaporator, but is not limited thereto. When drying under reduced pressure, the temperature is preferably 20 to 40 ° C, more preferably 30 ° C, but is not limited thereto.

The alcohol / water fraction of the extract may be a fraction obtained by fractionating the organic solvent extract using an alcohol / water mixed solution having 1 to 4 carbon atoms. The alcohol / water mixed solution having 1 to 4 carbon atoms may be about 70 to 90% (v / v) alcohol / water mixed solution having 1 to 4 carbon atoms, preferably 70 to 90% (v / v) methanol / water mixed solution. Can be. In another example, the fraction is obtained by fractionating the 70-90% (v / v) alcohol / water mixed solution having 1 to 4 carbon atoms, preferably 70-90% (v / v) methanol / water mixed solution. Small fraction obtained by further fractionation of the fraction into 40 to 60% (v / v) alcohol / water mixed solution having 1 to 4 carbon atoms, preferably 40 to 60% (v / v) methanol / water mixed solution. Can be.

The fractions and / or small fractions may contain useful secondary metabolites, including the compounds of Formulas 1 to 3, to exhibit excellent cancer cell growth inhibition effects.

In addition, acetyl aranotin (Acetylaranotin) of formula (I), Acetylapoara notin of formula (2), and a novel compound dehydro-deoxyapoaranotin (Dehydro-deoxyapoaranotin) isolated from the KMD 901 strain ) Is provided. All of these compounds were shown to have excellent cancer cell growth inhibitory effect. Thus, there is provided a composition for the prevention or treatment of cancer containing the compound of formula 1 to 3 and their pharmaceutically acceptable salts as an active ingredient.

<Formula 1>

<Formula 2>

<Formula 3>

Acetylaranotin of Formula 1, Acetylapoaranotin of Formula 2, and a novel compound dehydro-deoxyapoaranotin of Formula 3 are used as the active ingredient. It can be used by itself or in the form of a salt. The salt is preferably an acid addition salt formed by free acid, which is a pharmaceutically acceptable salt. Organic acids and inorganic acids may be used as the free acid. The organic acid may be any pharmaceutically acceptable organic acid, for example citric acid, acetic acid, lactic acid, tartaric acid, maleic acid, fumaric acid, formic acid, propionic acid, oxalic acid, trifluoroacetic acid, benzoic acid, gluconic acid, metasulfonic acid, glycolic acid, Succinic acid, 4-toluenesulfonic acid, glutanoic acid, aspartic acid, and the like, but is not limited thereto. In addition, the inorganic acid may be any pharmaceutically acceptable inorganic acid, for example, hydrochloric acid, bromic acid, sulfuric acid, phosphoric acid and the like, but is not limited thereto.

The Aspergillus KMD 901 strain, and / or the compound isolated therefrom, has the advantage of being cytotoxic to a variety of cancers, which can be used for a wide range of cancer treatments. Therefore, the composition for preventing or treating cancer has excellent prevention, treatment, and improvement effect against all kinds of cancers, preferably all solid cancers such as gastric cancer, liver cancer, colon cancer, lung cancer, breast cancer, and prostate cancer.

The cancer cytotoxicity of these compounds was AGS (stomach cancer, ATCC cell code- CRL-1739 ^TM ), HepG2 (liver cancer, HB-8065 ^TM ), HCT116 (colon cancer) received from American Type Culture Collection (ATCC, Manassas, VA, USA) , CCL-247 ^™ ), A549 (lung cancer, CCL-185 ^™ ) and MCF-7 (breast cancer, HTB-22 ^™ ) cancer cell lines. As a result, Acetylaranotin showed significant cancer cell growth inhibitory effect on lung cancer (A549), while Acetylapoaranotin showed significant cancer cell growth on lung cancer (A549), breast cancer (MCF-7) and gastric cancer (AGS). Growth inhibitory effect was shown, dehydro-deoxyapoaranotin (Dehydro-deoxyapoaranotin) was confirmed that the cancer cell growth inhibitory effect on lung cancer (A549). Therefore, it can be seen that the composition of the present invention can be very preferably used for the treatment of lung cancer, breast cancer or stomach cancer. In addition, the present invention was confirmed that the three single compounds cause apoptosis of colorectal cancer cells (apoptosis).

Aspergillus KMD 901 strain in the composition, the strain culture, the dried product of the culture, the concentrate, the organic solvent extract of the culture, the fraction of the extract, or the content of the compound of formula 1 to 3 may be a symptom of symptoms, symptoms It can be appropriately adjusted according to the degree of progress, the condition of the patient, and the like, for example, 0.0001 to 99.9% by weight, preferably 0.001 to 50% by weight based on the total weight of the composition, but is not limited thereto. The content ratio is a value based on the dry amount of the solvent, in the case of cultures, extracts and fractions.

The composition may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions, and oral preparations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like according to conventional methods It may be used in the form of a dosage form, an external preparation, a suppository, or a sterile injectable solution.

In formulating the composition, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations may include at least one or more excipients and / or lubricants. Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups, and various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories, and the like.

The preferred dosage of the composition depends on the condition and weight of the patient, the severity of the disease, the form of the drug, the route of administration and the duration, but may be appropriately selected by those skilled in the art. For a more preferable effect, the dosage of the composition of the present invention may be about 1 to 200 mg / kg per day as an active ingredient, for example, 1 day when the active ingredient is the extract, culture, dried product or concentrate. 10 to 200 mg / kg on the basis of, if the active ingredient is a compound of formula 1 to 3 or a pharmaceutically acceptable salt thereof may be 1 to 10 mg / kg per day. The administration may be carried out once a day or divided into several doses. The compositions of the present invention can be administered by various routes to an animal, preferably a mammal, including a human. All modes of administration can be envisaged, for example, by oral, intravenous, intramuscular, subcutaneous injection or the like. The pharmaceutical dosage form of the composition of the present invention may be used in the form of a pharmaceutically acceptable salt of the active ingredient, and may be used alone or in combination with other pharmaceutically active compounds as well as in a suitable collection.

In addition, the Aspergillus KMD 901 strain, the culture of the strain, the dried product of the culture, the concentrate of the culture, the organic solvent extract of the culture, the alcohol / water fraction of the extract, Diketope of formulas 1 to 3 Provided is a functional food for preventing and / or ameliorating cancer comprising a perrazine compound (aranotin derivative) and at least one member selected from the group consisting of pharmaceutically acceptable salts of the compound.

The health functional food of the present invention may be used in various foods, beverages, gums, teas, health functional supplements, food additives, and the like for improving diabetes, and may be used in the form of powders, granules, tablets, capsules, or beverages.

At this time, the content of the Aspergillus KMD 901 strain in the functional food can be appropriately adjusted according to the type, function, efficacy, etc. of the food, for example, about 0.00001 to 100% by weight of the total food weight, preferably 0.0001 to 50% by weight, more preferably 0.001 to 10% by weight, more preferably 0.01 to 1% by weight, but is not limited thereto.

In another example, a method of preparing by separating and purifying the compounds of Formulas 1 to 3 from the KMD 901 strain is provided. The method may include the following steps:

1) extracting the Aspergillus KMD 901 strain by adding at least one organic solvent selected from the group consisting of ethyl acetate, alcohol having 1 to 4 carbon atoms, methylene chloride, ether and chloroform to the culture culture liquid; And

2) obtaining a fraction by column chromatography of the obtained extract using an alcohol / water mixed solution having 1 to 4 carbon atoms of 70 to 90% (v / v);

3) column chromatography of the fractions with an alcohol / water mixed solution of 40 to 60% (v / v) of 1 to 4 carbon atoms.

In the above production method, the liquid culture of step 1) is a saccharose liquid medium such as Czapek-Dox liquid medium (Saccharose 30 g, Sodium Nitrate 3.0 g, Dipotassium Phophate 1.0 g, Magnesium Sulfate 0.5 g, Potassium Chloride). 0.5 g and 0.01 g of Ferrous Sulfate as the main ingredient), and is preferably inoculated at a ratio of 20 to 30 ml of liquid medium per 2-3 strain colonies.

When extracting the organic solvent of the culture solution, the solvent is preferably extracted by adding 1 to 3 times the volume of the culture solution, and more preferably, about 2 volumes of the extract is added. Extraction at room temperature is preferred, but is not limited thereto. The number of extraction is preferably 1 to 5 times, and more preferably three times of extraction, but is not limited thereto. Drying under reduced pressure preferably uses a rotary vacuum evaporator, but is not limited thereto. When drying under reduced pressure, the temperature is preferably 20 to 40 ° C, more preferably 30 ° C, but is not limited thereto.

Column chromatography of step 2) or step 3) is performed by column chromatography using a filler selected from the group consisting of silica gel, Sephadex, RP-18, RP-8, RP-4, polyamide, Toyopearl and XAD resin. It may be photography. Column chromatography can be carried out several times by selecting the appropriate filler as necessary, and chloroform (CHCl ₃ ) -alcohol (eg methanol), ethyl acetate-alcohol (eg methanol), methylene chloride as a solvent. (dicloromethane) -alcohol (eg methanol), alcohol (eg methanol) -water or acetonitrile-water may be used, but is not limited thereto.

In an embodiment of the present invention, elution conditions were water and methanol, starting with 20% methanol / water to increase the methanol content by 20%, and 100% methanol was used as the final solvent. As a result of analyzing the main components of the fractions obtained by high performance liquid chromatography, three compounds were identified in the 80% methanol / water fraction. In addition to 50% methanol / water isocratic condition and an eluent gun at a flow rate of 4 ml per minute, the fraction was purified by reverse phase liquid chromatography using a RP-18 column of 10 mm x 250 mm and the retention time was 10 minutes. Acetylaranotin represented by the formula (1) and dihydro-dioxyaporanotin represented by the formula (3) at the retention time 20 minutes with acetylapoaranotin represented by the formula (2) at the retention time 12 minutes (Dehydro-deoxyapoaranotin) was obtained respectively.

The separated compounds were analyzed by MS and nuclear spectroscopy to identify chemical structures. Acetylaranotin was an amorphous white powder having a molecular weight of 504 and a molecular formula of C ₂₂ H ₂₀ N ₂ O ₈ S ₂ , and acetylaphores. Aranotin (Acetylapoaranotin) is an amorphous white powder with a molecular weight of 488 and a molecular formula of C ₂₂ H ₂₀ N ₂ O ₇ S ₂ , Dehydro-deoxyapoaranotin has a molecular weight of 428 and a molecular formula of C ₂₀ H _16. It was confirmed that it was an amorphous light yellow powdery substance which was N ₂ O ₅ S ₂ .

As described above, the present invention has been developed a liquid culture method of Aspergillus sp. KMD 901 fungi, a novel compound, dehydro-deoxyapoaranotin and acetylaranotin (Acetylaranotin), Acetylapoaranotin was isolated and purified. These compounds showed a strong cytotoxic effect on cancer cells and three single compounds in colon cancer cells caused natural death. In addition, in vivo anti-cancer activity targeting acetylapoaranotin, the most toxic drug, showed tumor growth inhibition effect. Therefore, liquid culture fluid extracts of Aspergillus sp. KMD 901 fungus or acetylaranotin and acetylapoaranotin (including the novel compound Dehydro-deoxyapoaranotin) Acetylapoaranotin) or a pharmaceutical composition containing the pharmaceutically acceptable salt thereof can be usefully used as a candidate for anticancer drug development.

Aspergillus sp. KMD 901 fungi strains of the present invention, the culture extract and the compound produced by liquid culture method Acetylaranotin, Acetylapoaranotin, Dihydro-deoxyapoara Notine (Dehydro-deoxyapoaranotin) may be usefully used in the manufacture of a pharmaceutical composition for treating cancer.

Hereinafter, the present invention will be described in detail by the following examples.

However, the following examples illustrate the present invention in detail, and the content of the present invention is not limited by the following examples.

Example 1 Screening and Screening of Mold Strains

Marine sediments were collected at a depth of 1,198 m at a depth of 1,198 m at a location of 130 degrees 0 minutes and 37 degrees 3.4 minutes longitude near Ulleungdo, and then dried in the shade to prepare marine sediment samples. The prepared sample was suspended in physiological saline and diluted to 1/10 (w / v), followed by Czapek-Dox solid medium (30 g Saccharose, 3 g Sodium Nitrate, 1 g Dipotassium Phosphate, 0.5 g Magnesium Sulfate). , 0.5 g Potassium Chloride, 0.01 g Ferrous Sulfate, 18 g Agar in 1 L of DI water) was inoculated in 100 ml and then cultured at 25 ℃ for 7 days to isolate a single strain to secure 20 strains. . Two kinds of sterile water were added to each of the 20 strain samples thus obtained, diluted to 1/10 (w / v), inoculated in 100 μl amount on the solid medium, and cultured for 7 days. It was.

Among them, the fungal strains to be used for cultivation were visually separated from the solid medium into a single species by selecting the strains having morphological and color differences with the naked eye, and then the Czapek-Dox liquid medium (per 1 liter of water). , 30 g Saccharose, 3 g Sodium Nitrate, 1 g Dipotassium Phosphate, 0.5 g Magnesium Sulfate, 0.5 g Potassium Chloride, 0.01 g Ferrous Sulfate Incubated for 5-7 days. Species analysis of the obtained strains was determined by analyzing nucleotide sequences of 5.8S, 18S, and 28S rRNA genes by separating chromosomal DNA. As a result, the strains named KMD 901 were sequence numbers of 5.8S, 18S, and 28S rRNA genes. As shown in Figure 1, it was confirmed that the homology of 98.8% with Aspergillus versicolor NRRL 227.

In the case of KMD 901 strain, 25 ml of the culture solution was extracted with ethyl acetate organic solvent, and dried under reduced pressure, and analyzed by analytical chromatography. there was. In addition, the cytotoxicity test against cancer cells against this small amount of extract showed strong cancer cytotoxicity, and KMD 901 was selected as an anticancer microbial strain.

KMD 901 strain is a Czapek-Dox solid medium (30 g Saccharose, 3 g Sodium Nitrate, 1 g Dipotassium Phosphate, 0.5 g Magnesium Sulfate, 0.5 g Potassium Chloride, 0.01 g Ferrous Sulfate, 18 g Agar in 1 L of DI In the water), the fluffy central point was found to form a yellow colony (see FIG. 1). The strain KMD 901 was deposited on June 23, 2009 at the Korea Microbial Conservation Center, and was given accession number KCCM 11047P.

Example 2 Aspergillus genus Aspergillus  sp.) Liquid Mass Culture of Strain KMD 901

       The inventors of the present invention are based on the fungus strain KMD 901 (2 to 3 colonies) Chapek dox (Czapek) consisting of 30 g Saccharose, 3 g Sodium Nitrate, 1 g Dipotassium Phosphate, 0.5 g Magnesium Sulfate, 0.5 g Potassium Chloride, 0.01 g Ferrous Sulfate -Dox) After inoculation into 25 ml of liquid medium, it was incubated for 7 days at 200 rpm and 25 ℃ shaking incubator. Thereafter, 500 ml of Chapek-Dox liquid medium was inoculated with 10 ml of the 25 ml culture medium in a 1 liter Erlenmeyer flask for incubation for 8 days.

In addition, continuous high-speed liquid chromatography was used to analyze the production of secondary metabolites from the liquid culture. Analysis conditions were as follows:

Instrument: Agilent 1100 LC / MS system;

Elution rate: 1 mL / min;

Eluent: increasing the composition of acetonitrile for 30 minutes from 10% acetonitrile / water to 100% acetonitrile;

Column: Phenomenex Luna 5u C18 (2) 4.6 × 150 mm;

Detector: DAD UV Detector (254 nm)

The analysis results of the high performance liquid chromatography under the above conditions are shown in FIG. 2. In the analysis of the high performance liquid chromatography, the extract was injected with 10 μL at a concentration of 1 mg / mL, and the effluent increasing the content of acetonitrile for 30 minutes from acetonitrile: water 1: 9 volume ratio to 100% acetonitrile. Solvent conditions were used and a C18 reversed phase column (4.6 mm × 150 mm, particle size 5 μm) was used.

As a result of analysis up to 15 days of culture, as shown in FIG. 2, the liquid culture medium of the fungal strain KMD 901 was acetylaranotin (Formula 1), which had a peak of 17.9 minutes, and acetylaporanotin (18.6 minutes). Acetylapoaranotin (Formula 2), Dehydro-deoxyapoaranotin (Formula 3), the peak of retention time 19.0 minutes.

Analysis of the liquid culture solution of KMD 901 every other day from the second day of culture showed that the compounds began to be produced from day 4 to day 15 of the culture.

Example 3 Separation and Purification Method of Compounds

After taking 25 ml of the culture solution incubated for 8 days obtained in Example 2, an organic solvent layer obtained by adding the same amount of ethyl acetate was dried under reduced pressure to prepare an ethyl acetate extract. The obtained extract was dried under reduced pressure at 40 ° C. using a rotary vacuum evaporator, and the extract was separated into five fractions using reverse phase column chromatography. Elution conditions start with 20% methanol / water using water and methanol as eluents to increase the methanol content by 20% (v / v) and 100% (v / v) methanol as the final outflow solvent. In the same manner as in the high performance liquid chromatography method of <Example 2>, reverse phase column chromatography was performed. Analysis of the principal components of the fractions obtained therefrom revealed three principal component peaks in <Example 2> in an 80% (v / v) methanol / water fraction (see 5d in FIGS. 5a to 5e). In the main component analysis, each fraction was injected with 10 μL at a concentration of 1 mg / mL, and the effluent solvent condition was increased for 30 minutes from acetonitrile: water 1: 9 volume ratio to 100% acetonitrile for 30 minutes. A C18 reversed phase column (4.6 mm x 150 mm, particle size 5 μm) was used.

The 80% methanol / water fractions were purified by reverse phase liquid chromatography using a 10 mm × 250 mm RP-18 column under isocratic conditions of 50% methanol / water and eluting at a flow rate of 4 ml per minute. Acetylaranotin of Formula 1 at time 10 minutes and Acetylapoaranotin of Formula 2 at retention time 12 minutes Dehydro-deoxyapoaranotin of Formula 3 at retention time 20 minutes ) Were respectively obtained.

Example 4 Identification of Compounds

The inventors determined the chemical structures of the three compounds in <Example 3> by using the instrumental analysis as in <Formula 1>, <Formula 2> and <Formula 3>. The chemical structure was analyzed by analyzing the following MS data and nuclear magnetic spectrometer data. The hydrogen and carbon nuclear magnetic spectrometer spectra were measured at 500 MHz and 125 MHz, respectively.

Acetylaranotin of Formula 1 is dimethyl sulfoxide- d ₆ Measured in (DMSO- d ₆ ) solvent and the chemical shift values of ¹ H and ¹³ C NMR for the structure of Acetylaranotin are as follows;

¹ H-NMR (500 MHz, DMSO-d ₆ ) δ: 1.90 (3H, s), 3.22 (1H, br ddd, J = 18.0, 1.5, 1.5 Hz), 3.84 (1H, br ddd, J = 18.0, 2.5, 1.5 Hz), 4.59 (1H, dd, J = 8.0, 2.0 Hz), 5.01 (1H, br ddt, J = 8.5, 2.0, 1.5 Hz), 5.42 (1H, ddd, J = 8.5, 2.5, 2.0 Hz), 6.45 (1H, dd, J = 8.0, 2.5 Hz), 6.82 (1H, br ddd, J = 2.5, 2.0, 1.5 Hz), ¹³ C-NMR (125 MHz, DMSO-d ₆ ) δ: 21.2 (s), 34.1 (s), 62.9 (s), 69.9 (s), 76.4 (s), 105.4 (s), 114.0 (s), 139.2 (s), 141.6 (s), 162.3 (s), 169.7 (s).

<Formula 1>

The chemical shift values of ¹ H and ¹³ C NMR for the structure of Acetylapoaranotin of Formula 2 are as follows;

¹ H-NMR (500 MHz, CDCl ₃ ) δ: 2.03 (3H, s), 2.15 (3H, s), 2.88 (1H, br dd, J = 18.5, 1.5 Hz), 2.99 (1H, ddd, J = 18.0, 2.0, 2.0 Hz), 3.81 (1H, dm, J = 18.5 Hz), 4.02 (1H, br ddd, J = 18.0, 2.5, 1.5 Hz), 4.61 (1H, dd, J = 8.5, 2.0 Hz) , 5.00 (1H, br dm, J = 13.2 Hz), 5.10 (1H, br dddd, J = 8.5, 2.0, 2.0, 1.5 Hz), 5.56 (1H, br dm, J = 13.2 Hz), 5.70 (1H, ddd, J = 8.5, 2.0, 2.0 Hz), 5.96-5.99 (2H, m ^a ), 6.05 (1H, br dm, J = 13.2 Hz), 6.31 (1H, dd, J = 8.5, 2.0 Hz), 6.61 (1H, broad doublet, J = 2.5, 2.0, 2.0 Hz), ¹³ C-NMR (125 MHz, CDCl ₃ ) δ: 21.0 (s), 21.3 (s), 34.5 (s), 36.1 (s), 62.9 (s), 64.5 (s), 69.8 (s), 73.9 (s), 75.9 (s), 78.2 (s), 105.3 (s), 113.4 (s), 119.9 (s), 124.5 (s), 127.8 (s), 132.2 (s), 139.2 (s), 141.2 (s), 162.2 (s), 163.1 (s), 170.0 (s), 170.5 (s). ^a Two proton peaks overlap.

<Formula 2>

Chemical shift values of ¹ H and ¹³ C NMR for the structure of the novel compound Dehydro-deoxyapoaranotin of Formula 3 are as follows.

¹ H-NMR (500 MHz, DMSO-d ₆ ) δ: 1.95 (3H, s), 3.34 (1H, br ddd, J = 18.5, 1.5, 1.5 Hz), 3.54 (1H, d, J = 18.5 Hz) , 3.91 (1H, br ddd, J = 18.5, 2.0, 1.5 Hz), 4.10 (1H, d, J = 18.5 Hz), 4.64 (1H, dd, J = 8.0, 2.0 Hz), 5.05 (1H, br ddt , J = 8.5, 2.0, 1.5 Hz), 5.53 (1H, ddd, J = 8.5, 2.0, 2.0 Hz), 6.48 (1H, dd, J = 8.0, 2.5 Hz), 6.86 (1H, br dt, J = 2.0, 2.0 Hz), 7.23 (1H, ddd, J = 7.5, 7.5, 1.0 Hz), 7.37 (1H, br dd, J = 7.5, 7.5 Hz), 7.45 (1H, br d, J = 7.5 Hz), 7.80 (1H, broad doublet, J = 7.5 Hz), ¹³ C-NMR (125 MHz, DMSO-d ₆ ) δ: 21.2 (s), 34.1 (s), 35.3 (s), 63.2 (s), 70.0 ( s), 76.2 (s), 76.9 (s), 105.4 (s), 114.3 (s), 115.1 (s), 126.0 (s), 126.3 (s), 129.0 (s), 129.3 (s), 138.2 ( s), 139.2 (s), 142.0 (s), 161.0 (s), 162.1 (s), 170.0 (s).

<Formula 3>

Test Example 1 Cytotoxic Effects of Compounds on Cancer Cells

Cytotoxicity evaluation of the isolated compounds on cancer cells using in vitro MTT [3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltrazdium bromide] assay to measure the amount of live cancer cells Was performed. The cancer cytotoxicity of the compounds was determined by AGS (Stomach Cancer, ATCC Cell Code- CRL-1739 ^TM ), HepG2 (Liver Cancer, HB-8065 ^TM ), HCT116 (Colon Cancer,) from the American Type Culture Collection (ATCC, Manassas, VA, USA). CCL-247 ^™ ), A549 (lung cancer, CCL-185 ^™ ), and MCF-7 (breast cancer, HTB-22 ^™ ) cancer cell lines.

Gastric cancer (AGS), liver cancer (HepG2), colorectal cancer (HCT-116), lung cancer (A549) and breast cancer (MCF-7) cell lines were added to a 96-well plate by 1 × 10 ⁴ cells, respectively. Was added to a total of 100 microliters and cultured in a CO ₂ incubator for 24 hours at 37 ℃. Stomach cancer cells, RPMI 1640 medium (main ingredient 25 mM HEPES, L-Glutamine, Hyclone SH30255.01; gastric cancer, lung cancer, breast cancer), DMEM / HIGH GLUCOSE (main ingredient 4.00 mM L-Glutamine, 4500 mg / L Glucose, Sodium pyruvate) , Hyclone SH30243.01; liver cancer), MEM / EBSS (main component 2.00 mM L-Glutamine, Earle's Balanced salts, Hyclone SH30024.01; colorectal cancer) medium. Then, after removing the existing medium, the compound was mixed with the existing medium as described above and replaced and incubated at 37 ℃. After 24 hours, 10 microliters of MTT solution was added thereto, and the cells were incubated at 37 ° C. for 1 hour, and the absorbance at 450 nm was measured for the amount of formazone formed by reductase in living cells, and the IC ₅₀ value was measured. The obtained results are shown in Table 1 below.

Cytotoxic Effects of Acetylaranotin, Acetylapoaranotin and Dehydro-deoxyapoaranotin on Five Cancer Cell Lines in Vitro IC ₅₀ (μM) Cancer cell line HCT116 AGS A549 MCF-7 HepG2 Acetylanranotin (1) 21.2 ± 1.90 32 ± 1.23 23 ± 1.96 36 ± 0.84 > 80 Acetylapoaranotine (2) 13.8 ± 1.59 12 ± 0.22 2 ± 1.08 10 ± 0.43 48.4 ± 2.16 Dihydro-Deoxyapoaranotine (3) 52 ± 1.47 29 ± 0.92 23 ± 0.48 31 ± 0.63 56 ± 1.28

As shown in Table 1, it can be seen that the isolated compounds have a high cytotoxic effect on various cancer cell lines.

Test Example 2 Effect of Compounds on Cell Natural Death against Colorectal Cancer Cells

In order to test the effect of apoptosis on colon cancer cells of the three isolated compounds, Western blotting was performed to measure the expression rate of proteins affecting apoptosis.

Colorectal cancer cells cultured in MEM / EBSS (main component 2.00 mM L-Glutamine, Earle's Balanced salts, Hyclone SH30024.01) medium were added to 2 mL of the MEM / EBSS medium by 5 × 10 ⁵ cells in a 6-well plate. CO ₂ during time Cultured in the incubator. After 24 hours, three single compounds were administered at 2 weight and 1/2 weight concentrations based on the 50% concentration of each compound inhibited in colorectal cancer cells. Treated during. Thereafter, colon cancer cells were collected and the protein was extracted using Bio-Rad's Protein assay reagent. 30 μg of these proteins were run on 4-10% gradient acrylamide gel and transferred to PVDF membrane (Polyvinylidene fluoride membrane, Bio-Rad). The expression rate of one poly-ATP ribose polymerase [Poly-ATP ribose polymerase (PARP)] was observed. The western blotting results are shown in FIG. 3.

As shown in FIG. 3, all three compounds were observed to decrease the expression rate of pap (PARP), which is one of proteins affecting apoptosis of cancer cells in a concentration-dependent manner. As a result, it can be seen that the compounds exhibit an effect of reducing cancer cell proliferation by reducing PAP, a protein that affects cell proliferation.

< Test Example  3> Of acetylapoaranotine in vivo  Anticancer activity measurement

BALB / c nude mice weighing 18-22 g in order to evaluate the in vivo tumor growth inhibitory activity of Acetylapoaranotin, which causes cytotoxicity at the lowest concentration among the three compounds according to the present invention Experimental animals), and HCT116 (colon cancer cell) cell line was injected into the right shoulder by 1 × 10 ⁶ cells in each rat, and when the tumor size was about 100 mm ³ , four randomly negative controls, experimental group 1 (acetyl) were used. Apoaranotin 5 mg / kg administration group) and Experimental Group 2 (acetylapoaranotin 20 mg / kg administration group) were divided into three groups. Thereafter, twice a week for three weeks, a sample of a certain concentration was dissolved in a solution of purified water: Tween 80: ethanol in a volume ratio of 95: 2.5: 2.5 and then orally administered. The tumor size was measured each time the sample was dosed.

Tumor size was calculated as 0.5 × width × (vertical) ² , and the length of the tumor was used as the short diameter and the length of the length of the tumor. The measurement results are shown in FIG. 4. As can be seen in Figure 4, the tumor size reduction effect is observed immediately after administration, compared to the negative control within 23 days 15.2% in experimental group 1 (acetyl apo alanotine 5 mg / kg), experimental group 2 (acetyl apo aranotin 20 mg / kg) showed a tumor growth inhibitory effect of 16.1% (*; P <0.2, **; P <0.10, ***; P <0.05).

That is, the Acetylapoaranotin compound isolated from Aspergillus sp. Strain KMD 901 was found to stably inhibit tumor growth immediately after administration (about 2 days) when compared to the control group.

As discussed above, Aspergillus sp. Strain KMD 901, and the compounds acetylaranotin, acetylapoaranotin, and the novel compound dehydro-deoxyapo, isolated therefrom Aranotin (Dehydro-deoxyapoaranotin) can all be usefully used as a composition for preventing and / or treating cancer.

1 is a photograph showing the morphology of the Aspergillus sp. KMD 901 fungi strain isolated from marine sediment in Czapek-Dox solid medium,

Figure 2 is a chromatographic observation of peak production of secondary metabolites through high-performance liquid chromatography after 8 days of incubation of the strain,

3 is a photograph evaluating the effect of the three compounds on the reduction of the expression rate of pap (PARP) by concentration by Western blotting method,

Figure 4 is a graph measuring the effect of acetylapo aranotin on the size growth of HCT116 colon cancer cells,

Figures 5a to 5e shows the results of the principal component analysis of 80% (v / v) methanol / water fraction of Aspergillus sp. KMD 901 strain culture, 5a is 20% methanol / water fraction, 5b is 40% Methanol / water fraction, 5c is 60% methanol / water fraction, 5d is 80% methanol / water fraction, and 5e is 100% methanol fraction.

<110> Korea Institute of Science and Technology <120> Aspergillus sp. KMD 901 strain, diketopiperazines isolated          therefrom, and a composition for the prevention of the treatment          of cancer comprising the same <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 567 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence of 5.8S, 18S, and 28S rRNA genes of          Aspergillus KMD 901 strain <400> 1 tcctccgctt attgatatgc ttaagttcag cgggtatccc tacctgatcc gaggtcaacc 60 tgaagaaaaa tggttggacg tcggctggcg cccggccggc cctaaatcga gcgggtgaca 120 aagccccata cgctcgagga ccggacacgg tgccgccgct gcctttcggg cccgtccccc 180 gggggggacg acgacccaac acacaagccg ggcttgatgg gcagcaatga cgctcggaca 240 ggcatgcccc ccggaatgcc agggggcgca atgtgcgttc aaagactcga tgattcactg 300 aattctgcaa ttcacattac ttatcgcagt tcgctgcgtt cttcatcgat gccggaacca 360 agagatccat tgttgaaagt tttgactgat tttatattca gactcagact gcatcactct 420 caggcatgaa gttcagtagt ccccggcggc tcgcccccga gagggctccc cgccgaagca 480 acagtgttag gtagtcacgg gtgggaggtt gggcgcccgg aggcagcccg cactcagtaa 540 tgatccttcc gcaggttcac ctacgga 567  

Claims (10)

The accession number KCCM 11047P Aspergillus (Aspergillus sp.) strains. Dehydro-deoxyapoaranotin of Formula 3: <Formula 3>

Aspergillus sp. Strain of Accession No. KFCC 11450P, Culture of said strain, Dried product of the culture, Concentrate of said culture, At least one organic solvent extract selected from the group consisting of ethyl acetate, alcohol having 1 to 4 carbon atoms, methylene chloride, ether and chloroform, and Fractions obtained by distilling the extract using at least one solvent selected from the group consisting of alcohols and mixed solvents of 1 to 4 carbon atoms A composition for the prevention or treatment of cancer containing at least one selected from the group consisting of active ingredients. 4. The method of claim 3 wherein the culture is accession number KFCC 11450P Aspergillus (Aspergillus of sp.) A composition for preventing or treating cancer, which is obtained by shaking culture for 4-20 days at 150-250 rpm and 22-28 ° C in saccharose liquid medium. The composition for preventing or treating cancer according to claim 3, wherein the fraction is a fraction obtained by distilling a 70-90% (v / v) C1-C4 alcohol / water mixed solution with a solvent. The composition for preventing or treating cancer according to claim 3, wherein the fraction is a fraction obtained by fractionating a 70-90% (v / v) methanol / water mixed solution with a solvent. A group consisting of Acetylaranotin of Formula 1, Acetylapoaranotin of Formula 2, Dehydro-deoxyapoaranotin of Formula 3, and pharmaceutically acceptable salts thereof A composition for preventing or treating cancer containing at least one selected from the group as an active ingredient: <Formula 1>

<Formula 2>

<Formula 3>

The composition according to any one of claims 3 to 7, wherein the cancer is gastric cancer, liver cancer, colon cancer, lung cancer, breast cancer or prostate cancer. Aspergillus sp. Strain of Accession No. KFCC 11450P, Culture of said strain, Dried product of the culture, Concentrate of said culture, At least one organic solvent extract selected from the group consisting of ethyl acetate, alcohol having 1 to 4 carbon atoms, methylene chloride, ether and chloroform, Fractions fractionated using at least one solvent selected from the group consisting of a mixed solvent of alcohol and water having 1 to 4 carbon atoms of the extract, Acetylaranotin of Formula 1, Acetylapoaranotin of Formula 2, Dehydro-deoxyapoaranotin of Formula 3, and Pharmaceutically acceptable salts of compounds of Formula 1-3 Functional food for the prevention or amelioration of cancer containing one or more selected from the group consisting of: <Formula 1>

<Formula 2>

<Formula 3>

1) At least one organic solvent selected from the group consisting of ethyl acetate, alcohol having 1 to 4 carbon atoms, methylene chloride, ether and chloroform in a culture medium in which the Aspergillus sp. Strain of accession number KFCC 11450P is liquid cultured Extracting by adding; And 2) obtaining a fraction by column chromatography of the obtained extract using an alcohol / water mixed solution having 1 to 4 carbon atoms of 70 to 90% (v / v); 3) column chromatography of the fractions with an alcohol / water mixed solution of 40 to 60% (v / v) of 1 to 4 carbon atoms A method for preparing acetylaranotin of Formula 1, acetylapoaranotin of Formula 2, or dehydro-deoxyapoaranotin of Formula 3, comprising: <Formula 1>

<Formula 2>

<Formula 3>

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