Secondary metabolites with antimicrobial, anticancer and antiviral effects isolated from Fusarium oxysporum yp9b strain.

Abstract

The invention relates to secondary metabolites produced by and isolated from the Fusarium oxysporum YP9B strain. These metabolites These are compounds with potent antimicrobial, antibacterial, anticancer, and / or antiviral effects, and are potential pharmaceutical active ingredients.

Description

1 TARIFF ANTIMICROBIAL ISOLATED FROM FUSARIUM OXYSPORUM YP9B STRAIN, SECONDARY METABOLITE WITH ANTICANCER AND ANTIVIRAL EFFECTS Technical Field to Which the Invention Relates The invention relates to the secondary bacteria produced by Fusarium oxysporum YP9B strain and isolated from it. It is related to metabolites. These metabolites have strong antimicrobial, antibacterial, and anticancer properties. and / or antiviral compounds, which are potential drug active ingredients. State of the Art Relating to the Invention (Prior Art) In the pharmaceutical industry, antimicrobial agents developed by microorganisms are increasing every day. A new drug with strong antimicrobial, antiviral, and anticancer effects to address the resistance problem. The development of precursor materials is of great importance. For the last 50 years, various diseases... Numerous potential drugs / drug precursors effective against these agents are being discovered, and these 15 These substances are made available for therapeutic use as a result of further research. Secondary metabolites are produced by the organism but are not necessary for the organism's growth. These are compounds that are not essential and are only present in limited taxonomic groups. Compounds are commonly found, especially in plants and microorganisms. Microbial 20 Secondary metabolites possess a variety of biological activities and are used in hormones, antibiotics, toxins, It functions as an anti-migraine agent, an anticancer agent, or an insecticide. Microbial secondary metabolites are currently used mostly in agriculture, industry, and healthcare. It is used in this field. It is one of the topics that has been given considerable attention in recent years. Ecological farming uses natural metabolites that increase the productivity and resilience of plants and soil. Its use has come to the forefront. In industry, secondary metabolites are a more stable product. It is preferred because it generates less energy and requires less power. 2 Many bioactive secondary metabolites are produced by fungi, the most prominent of which are... The most important of these are antimicrobial metabolites released into the external environment. The most important of the secondary metabolites are... Obtaining it in high yield and at the lowest cost is commercially important and for therapeutic purposes. It finds wide use in applications. Drugs against pathogenic microorganisms. Increased resistance and the presence of side effects from these chemical drugs have led to the development of new, naturally derived 5 This necessitates research into antimicrobial substances. Plants, bacteria, and fungi are known as the most common therapeutic sources, and these Many studies are being conducted on organisms. However, the production of these substances depends on pH. Temperature is largely influenced by parameters such as culture medium content, 10 optimum for the maximum efficiency of production of materials that may have economic value The existence of certain conditions is of great importance. Naturally derived medicines, anti-inflammatory drugs. It is quite important in research and many of the methods currently used in clinical practice. Antibacterial drugs are semi-synthetic, natural, or designed from prototypes of natural products. It is used. 15 Liquids or extracts obtained from microbial ferments are processed using the agar well method. Various antibiotics can be easily discovered by measuring bacterial inhibition zones. Bacterial Research into new antibacterial agents with few side effects due to the prevalence of resistance. It is among the important targets in the health sector. Fungi are of various types. It stands out as a source of new chemical compounds with biological activity. Fungal volatile metabolites, mycotoxins, nonribosomal peptides, polyketides, terpenes, a wide biodiversity for the production of secondary compounds such as diketopiperazines and alkaloids It is known to have Fusarium oxysporum, which infects approximately 150 plant species. It is an important species of phytopathogenic fungus with a wide host range. Fusarium sp. 25 The secondary metabolites secreted by these species vary from species to species. In the study by Wang et al. (2011), 9 different secondary strains were obtained from Fusarium oxysporum strains. Metabolites were isolated and their cytotoxic activities were determined using the MTT method for three different types of metabolites. Tested against cancer cell lines (PC-3, PANC-1, and A549). Extracted The efficacy of Beauvericin against PC-3, PANC-1 and A549 cell lines was measured with IC50 values ​​of 49.5±3.8, 30, respectively. The reported values ​​were 47.2±2.9 and 10.4±1.6 μM. Beauvericin is also similar to methicillin. resistant Staphylococcus aureus (MIC=3.125 μg / mL) and Bacillus subtilis (MIC=3.125 3 It has also been reported to have antibacterial activity against strains (μg / mL). Nascimento et al., In their study (2012), they found that Fusarium oxysporum SS46 isolate was cultured in Czapek liquid medium and Ethyl acetate extract of filtrate obtained from culture in solid rice medium and F. oxysporum Hexane extracts of SS50 isolate were used in vitro against three cancer cell lines (HCT-8, MDA-MB435 and SF295 have been reported to have anticancer effects. In the current technique, a 5 In another study, Sondergaard et al. (2016), 14 different Fusarium species were studied. Extracts were fractionated by HPLC, revealing the red pigments aurofusarin and bicaverine. Its presence was monitored and tested against Lactobacillus acidophilus at 8 micrograms and Bifidobacterium It has been reported to have antibacterial activity against breve at 64 micrograms. However, Broad-spectrum antimicrobial and anticancer drugs related to existing Fusarium oxysporum strains. 10 and strains that produce secondary metabolites or metabolites exhibiting antiviral activity It is not available. Due to the challenges and inadequacies of existing solutions regarding the issue, in the relevant technical field An improvement has been deemed necessary. The invention in question represents a 15% improvement over the prior art. isolated from Fusarium oxysporum YP9B strain, where the disadvantages were eliminated secondary metabolites with antimicrobial, anticancer and antiviral effects and their use as drugs It is related to its usage. Brief Description and Objectives of the Invention 20 In the present invention, a secondary metabolite isolated from the Fusarium oxysporum YP9B strain and Its antimicrobial, anticancer, and antiviral effects are explained. The aforementioned The secondary metabolite is Compound-5, which has the GGI-4B fraction, and is formulated as follows in Formula IV: It is shown that the GGI-4B fraction is pure and consists of a single substance. Formula IV 4 One purpose of the invention is to develop pharmaceutical active ingredients with antimicrobial, anticancer and / or antiviral effects. The goal is to obtain the materials. Definitions of the Figures Illustrating the Invention Figure 1: Extraction scheme of Fusarium oxysporum YP9B isolate. 5 Figure 2: LC-MS spectrum of compound GGI-4B. Figure 3: IR spectrum of compound GGI-4B. Figure 4: Fractions obtained from Fusarium oxysporum YP9B isolate (GGI 62, 3, 4, 6) and 4B) Cytotoxic effect on the VERO cell line. Figure 5: Secondary (GGI 3, 4, 62, and 10) obtained from Fusarium oxysporum YP9B isolate. 4B) Antiviral activities of metabolites on HSV type-1. Figure 6: Secondary metabolites obtained from Fusarium oxysporum YP9B isolate. Anticancer activity on MCF-7, PC-3 and A549 cell lines. Detailed Description of the Invention The present invention describes an antimicrobial agent isolated from Fusarium oxysporum YP9B strain. It is related to a secondary metabolite with anticancer and antiviral effects. The Fusarium used in the invention oxysporum YP9B; Fungi Kingdom, Ascomycota phylum, Pezizomycotina Subphylum, Sordariomycetes Class, Hypocreomycetidae Subclass, Hypocreales Order, Nectriaceae 20 It is a mycelial microfungus belonging to the family and genus Fusarium. When chemical and physical conditions are changed, the fungus Fusarium oxysporum YP9B... capable of producing numerous partially different antimicrobial / antiviral / anticancer-effective metabolites It was found to be a specific strain. 25 The metabolites it naturally produces in the culture medium have effective antimicrobial activity. However, when ethyl acetate extractions are performed, the metabolites it produces... Partial or complete purification is possible from F. oxysporum YP9B. The diagram showing the synthesis of the metabolites is given in Figure 1. In Figure 1, 30 metabolites are obtained in stages Fr. 1-4. Isolation of compounds GGI-3 (Compounds 2a, 2b, 2c) and GGI-4 (Compounds 2a, 3a, 3b), Fr. 4-8 Isolation of GGI-4B (Compound 5) in phase Fr. 9-28, and GGI-62 in phase Fr. Isolation of compounds (Compound 1) and GGI-6 (Compounds 4a, 4b, 4c) is achieved. This The conditions in the extraction scheme are as follows: • Fr. 1-4 : n-Hexane:Chloroform (100:0 50:50) • Fr. 4-8 : Chloroform:Ethylacetate (100:0 50:50) 5 • Fr. 9-28 : Ethylacetate:Methanol (100:0 0:100) • Fr. 29-30 : Methanol (100) In one application of the invention, the secondary metabolites (compounds) that are the subject of the invention are obtained. Fusarium oxysporum YP9B strain from tomato root soil, Dikloran Rose Bengal 10 isolated using Chloramphenicol (DRBC) and Potato Dextrose Agar (PDA) media The isolated strain was then cultured both in traditional (various culture media) and other culture media. (Magnified macroscopic and microscopic views and spore shapes) as well as molecular (Fusarium nuclear rDNA from ITS1-5.8S-ITS2 intragenic gene) using methods It has been identified as Fusarium oxysporum. The reproduction and growth of the Fusarium oxysporum YP9B strain... The optimal incubation period for high levels of secondary metabolite production is approximately 15 days. The ideal temperature for potatoes is between 20-28°C, and the most suitable growing medium is... It was determined that the medium was dextrose agar (PDA) for the Fusarium oxysporum YP9B strain. PDA agar (5-10 L) was prepared for the production of high amounts of secondary metabolites and 26 The cultures were incubated in an incubator at °C for 10 days to allow them to grow. The resulting cultures were then transferred to 2-liter containers. Harvested in an Erlenmeyer flask and approximately twice (1:2) ethyl acetate added, then left at room temperature for 2 days. Extraction was performed by shaking at 120 rpm at a certain temperature. After extraction, ethyl... For the purification and characterization of extracellular metabolites by removing the acetate portion. It has been used. The invention describes ethyl 25, coded as GGI and GGI-B, produced at two different times and under different conditions. different fractions obtained from acetate extracts (hexane, chloroform, ethyl acetate and methanol) has strong antimicrobial activities against a group of microorganisms that are human pathogens. It has been determined that the other fractions (chloroform, ethyl acetate and excluding hexane) exist. methanol) against a human pathogenic microorganism that has been absolutely tested It has been observed to possess antimicrobial activity. 30 6 Raw filtrate of PDA liquid and solid agar culture of Fusarium oxysporum YP9B (its lysate) has potent antimicrobial properties against a range of human pathogen culture collection strains. Preliminary studies have determined that it exhibits activity. In this invention, Fusarium oxysporum YP9B was inoculated at three points on the PDA substrate, and these inoculations lasted 8 days and 26 weeks. Produced at °C, extracted with ethyl acetate for 24 hours, and fractionated with different solvents. 5 and coded as GGI. The resulting fractions are primarily antimicrobial. Their activities were tested using the agar well diffusion method and they showed strong antimicrobial properties. activity was observed (Table 1). Table 1. Fractions of GGI group ethyl acetate extract of F. oxysporum YP9B. Antimicrobial activities (Test quantity: 50 µL). FN Fraction Stock Kon. Antimicrobial efficacy determined by the agar well method. Gram (-) Gram (+) ARB+ Yeast Fungi Hexane µg / mL Ec Yp Pa Sa Ef Bc Ms Ca Ct Sc 1 GGI-1 12000 - - - - - - 6 - - - Control - - - - - - - - - - Chloroform Ec Yp Pa Sa Ef Bc Ms Ca Ct Sc 2 GGI-2 235000 >30 >30 >30 >30 20 >30* 40 >30 >30 >40 3 GGI-3 74 000 >30 >30 >30 >30 20 >30* 20 >30 >30 >40 Control - - - - - - - - - - Ethyl acetate Ec Yp Pa Sa Ef Bc Ms Ca Ct Sc 4 GGI-4 35 600 16 16 20 8 8 10* 40 16 * 15 14* GGI-5 70 000 15 15 18 10 10 12* 26 18* 15 16 Control - - 6 - - - - 6 - - Methanol Ec Yp Pa Sa Ef Bc Ms Ca Ct Sc 6 GGI-6 73 500 8 - 6 28 25 22 45 15 15 - 7 GGI-61 52 300 - - 6 24 20 24 30 22 20 20 8 GGI-62*** 128000 6 6 6 14 12 18 28 8 8 - 9 GGI-63 64 000 6 6 10 16 16 24 22 12 12 10 GGI-64 58 000 6 6 6 8 6 10 12 12 12 8 11 GGI-65 16 000 6 6 6 8 6 12 12 14 14 14 12 GGI-66 38 000 6 6 6 8 6 14 12 14 15 15 13 GGI-67 132000 6 6 6 10 6 17 12 12 12 12 14 GGI-68 14 000 6 6 8 8 6 - - 12 12 12 GGI-7 14 000 6 6 6 6 - - - 10 10 10 Control 6 6 - - 6 - - 6 6 6 In the second repetition of the study, the culture conditions were slightly changed (28 °C and 10 days). incubation) tested, coded as “GGI-B”, in the first hexane fraction obtained (GGI-B1) showed no antimicrobial activity; 15 in the second chloroform fractions obtained. (GGI-B2 and GGI-B3) are all fractions in increasing proportion depending on the fraction order. They have low levels of antimicrobial activity against microorganisms 7 The final fraction was separated with methanol, resulting in a total of 10 different fractions. and strong antimicrobial activity has been determined (Table 2). Table 2. Fractions of GGI-B group ethyl acetate extract of F. oxysporum YP9B. Antimicrobial activities (Test quantity 50 µL). 5 FN Fraction Stock Cons. Antimicrobial activity (mm) was measured on agar plates. g / mL Gram (-) Gram (+) ARB+ Maya stains. Hekzan Ec Yp Pa Sa Ef Bc Ms Ca Ct Sc 1 GGI-B1 64300 - 6 - - - 6 10 - - Chloroform Ec Yp Pa Sa Ef Bc Ms Ca Ct Sc 2 GGI-B2 278400 8 8 8 6 6 6 3 GGI-B3 11800 10 10 10 10 12 6 8 8 8 Ethyl acetate Ec Yp Pa Sa Ef Bc Ms Ca Ct Sc 4 GGI-B4 7600 18 15 30 16 26 20 26 16 16 15 GGI-B5 48300 22 18 26 20 26 20 20 26 20 26 20 Methanol Ec Yp Pa Sa Ef Bc Ms Ca Ct Sc 6 GGI-B6 65400 8 - - 20 25 18 >30 20 20 - 7 GGI-B61 2400 6 10 - 12 16 15 28 12 10 10 8 GGI- B62 5200 8 8 8 12 10 12 20 10 - 9 GGI- B63 8400 6 8 10 10 8 14 35 10 10 8 GGI- B64 46600 6 6 10 10 8 14 15 10 12 8 11 GGI- B65 24300 6 6 6 20 18 16 10 10 10 10 12 GGI- B66 21600 15 8 6 15 8 15 24 15 16 15 13 GGI- B67 10800 8 8 6 12 - 20 24 15 16 15 14 GGI- B68 27500 8 6 10 15 10 20 15 15 16 15 GGI-B7 7600 6 6 6 12 6 20 15 15 16 15 The invention involves 4 from the first culture and 1 from the second culture, both of which have the best antimicrobial activity. Five fractions were completely and partially purified, totaling [number] fractions. Of the five purified fractions... Two of them (compounds GGI-62 and GGI-4B) consist of a single pure substance, while the other three are fractions. It has been determined that each of them is a compound consisting of three separate substances. 10 GGI-3 (2a, 2b, 2c) from the chloroform fraction; GGI-4 from the ethyl acetate fraction (2a, 3a, 3b); GGI-6 (4a, 4b, 4c) and GGI-62 from the methanol fraction and from the ethyl acetate fraction GGI-4B compounds were detected using chemical methods (NMR, FT-IR, LC-MS / MS, IR, etc.) were characterized. All of the characterized fractions were resistant to at least one group of bacteria (15). They have been found to possess strong antibacterial activity. Five fractions, partially and completely purified, are pathogenic to humans. tested against microorganisms, at least one or more disease-causing agents 8 minimal inhibition (MIC) and minimal bactericidal concentration against microorganisms They demonstrated bacteriostatic or bactericidal activity using (MBK) methods. Antimicrobial properties of crude and purified ethyl acetate extracts were found (Table 3). The activities of 10 different bacteria and 3 different yeasts totaled 13 different species. Tested against microorganisms (Table 3). At least 5 microorganisms were required for the production of each microorganism. Suitable culture media were used, and both the crude extract and the purified and obtained The compounds obtained were found to possess strong antimicrobial activities. When considering their overall antimicrobial activity, all of the selected fractions It has been observed to have high antimicrobial activity at low concentrations. The purified compound, the subject of the invention, has a potent efficacy against all tested microorganisms. It has antimicrobial activity, with minimal inhibitory concentration (MIC) values ​​of 0.8- It was determined to be in the range of 12.5 µg / ml. Table 3. MIC and MBK of purified fractions of F. oxysporum YP9B metabolites. values Fraction No Stock Cons. Minimal Inhibitory Concentration Values ​​(MIC) (µg) Gram (-) Gram (+) ARB+ Yeast Fungus µg / ml Ec Yp Kp Pa Sa Ef Sm Lc Bc Ms Ca Ct Sc GGI-4B 2000 1.6 1.6 1.6 1.6 0.8 1.6 6.3 12.5 0.8 3.1 3.1 3.1 6.3 Kont. - - - - - - - - - - - - - - Minimal Bakterisidal Konsantrasyon Değerleri (MBK) Gram (-) Gram (+) ARB+ Maya Mantarı Ec Yp Kp Pa Sa Ef Sm Lc Bc Ms Ca Ct Sc GGI-4B 2000 3.1 3.1 3.1 3.1 6.3 12.5 12.5 25 - - 12.5 12.5 12.5 Kontrol - - - - - - - - - - - - (Ec: Escherichia coli ATCC 25922, Yp: Yersinia pseudotuberculosis ATCC 911, Kp: Klepsiella 15 pneumonia ATCC 13883, Pa: Pseudomonas aeruginosa ATCC27853, Sa: Staphylococcus aureus ATCC 25923, Ef: Enterococcus faecalis ATCC 29212, Sm Streptococcus mutances RSKK07038, Lactobacillus casei RSK591, Bc: Bacillus cereus 702 Rome, Mycobacterium smegmatis ATCC607, Ca: Candida albicans ATCC 60193, Ct: Candida tropicalis ATCC13803, Sc: Saccharomyces cerevisiae RSKK 251, (-): aktivite yok) 20 GGI-4B is a potent antimicrobial agent (active agent) against 13 different microorganisms tested. (substance) and very low (MIC values ​​0.8-12.5 µg / ml and MBC 3.1-25 µg / ml 9 It was observed that it has high efficacy at concentrations (values). Most sensitive The microorganisms were identified as Gram-negative bacteria. The extracellular metabolite GGI-4B (Compound 5) was tested in the yeast (Candida albicans, 5). C. tropicalis and Saccharomyces cerevisiae fungi at a concentration of 12.5 µg / ml It has been determined to have a lethal effect. The GGI-4B fraction of extracellular metabolites was tested from Bacillus bacteria. Lethal to all bacteria tested except Mycobacterium cereus and Mycobacterium smegmatis. (MBC) They have been determined to have activity. The secondary metabolites that are the subject of this invention were isolated from Fusarium oxysporum YP9B. microfungus; - It can grow in solid and liquid culture media, 15 - It can grow in a wide pH range (pH; 4.5-7.2), - It can reproduce at temperatures of 15-30 °C. - It can reproduce in the presence of various carbon and nitrogen sources. - It has a high tolerance to salt and sugar. - Better results in a liquid medium with agitation at 120 rpm rotation. is reproducing, - It can reproduce in both dark and bright environments and produce different substances. - It is an important biological material that can produce different compounds depending on the culture medium. The secondary metabolites that are the subject of this invention are 25, whose chemical structures and properties are given below. Compounds are 1, 2a, 2b, 2c, 3a, 3b, 4a, 4b, 4c and 5. Compound 5 (GGI-4B): 2-oxo-8-azatricyclo[9.3.1.13,7]-hexadeca-1(15),3(16),4,6,11,13- hexane-10-on Formula IV Melting point (°C): 125-130°C Rf: 0.78 (Ethyl acetate) FT-IR (ATR, cm-1): 3355 (-NH voltage band), 2926, 2855 (-CH voltage band), 1673 (=CO voltage band), 1588, 1450 (C=C voltage band), 1282, 1233 (-CO voltage band), 998, 769, 692. LC-Q-TOF MS, 5:10 Calculated values ​​for C14H11NO2 were 225.24, measured values ​​were 226.82 [M+H]+, 247.21 [M+Na-H]+, and 250.98. [M+Na+2H]+. The 1H, 13C, APT NMR spectrum values ​​for compound GGI-4B are given below: 1H-NMR (300MHz, Acetone-d6): δ: 6.98 (2H, bd, J= 2.40), 6.76 (4H, d, J= 8.8), 7.12 (5H, t, J=7.8), 6.96-6.72 (6H, dd, J= 8.8, 8.16), 7.48 (2I H, dd, J= 1.76, 2.06), 7.56, (4I H, d, J= 15 7.68), 7.33 (5I H, t, J= 7.35), 7.05-7.08 (6I H, dd, J=0.88, 8.0), 4.22 (2'' H, s), 8.60 (C6H5- NH, bs, 1H). 13C-NMR (75MHZ, Acetone-d6): δ: 159.22 (1C,-C-), 115.30 (2C, -CH-), 140.05 (3C, -C), 118.18 (4C, -CH-), 131.15 (5H, -CH-), 116.57 (6H, -CH-), 159.42 (1' C, -C-), 121.83 (2I 20 C,-CH-), 138.51 (3IC, -C-), 122.39, (4'C,-CH-), 131.48 (5'C, -CH-), 121.74 (6'C, -CH), 196.89 (1''C, C=O), 45.03 (2''C, -CH2). 11 Five fractions isolated and purified from Fusarium oxysporum YP9B were used to treat African green VERO cell line, which is a kidney epithelial cell of monkeys (Cercopithecus aetiops). Their cytotoxic activities were tested, and the results are given in Table 4 and Figure 4. The cytotoxic activities of the 5 purified fractions against the VERO cell line were investigated. Table 4. Cytotoxic effects of fractions obtained from F. oxysporum YP9B on VERO cells. effect. Fraction Control Cell Fraction Concentration (µM) and Viable Cell Count 40 20 10 5 2.5 1.25 0.625 GGI-4B 100 - 9.9306 8.2092 44.7893 65.9744 72.4708 87.2145 High concentrations of purified GGI-4B compound (GGI-4B ≥2.5 µM) in VERO It is cytotoxic to the cell line, but not cytotoxic at low concentrations. 10 shown According to the results obtained, the fraction GGI-4B is present in dilutions of 1.25 (µM) and below. It has been determined that it has no cytotoxic activity against VERO cell lines. The antiviral activity of the fraction in question is due to its ability to lyse VERO cells with HSV type-1 DNA. It was determined using the virus. HSV type-1 is a virus that lyses VERO cells. VERO 15 The number of surviving cells in the cell line is higher than that under virus control. High concentrations were determined as antiviral activity values ​​(Table 5, Figure 5). The fraction studied was GGI-4B at dilutions of ≤ 1.25 µM (a total of six-fold dilution). Because it is effective, it has been determined to be usable in antiviral activity tests. Table 5. Antiviral effects of fractions obtained from F. oxysporum YP9B on HSV type-1. effect. Fractional Cell Virus Acyclovir Substances in Antiviral Activity Tests concentrations studied (µM) 2.5 1.25 0.625 0.312 0.156 0.078 0.039 GGI-4B 100 33.697 92.828 - - 37.362 43.462 37.110 36.558 35.517 23.698 The secondary metabolite (GGI-4B) of Fusarium oxysporum YP9B has anti-cancer activity. breast cancer cell line (MCF-7), prostate cancer cell line (PC-3), and lung cancer 12 Tested using cell line (A549). Compound GGI-4B was tested in three cell lines. It has been determined to have anticancer activity against the cancer cell lines (MCF-7, PC-3 and A549). (Table 5, Figure 5). MCF-7, a breast cancer cell line of fraction GGI-4B, is a prostate cancer cell line. Anticancer activity against PC-3 and the lung cancer cell line A549 was observed. It has been determined that they possess it. Table 6. Fractions obtained from F. oxysporum YP9B in three different cancer cell lines. on anticancer activity values ​​(IC50 values). Fractions Cancer Cell Lines and IC50 (µM) ± SD Values ​​(µM) MCF-7 PC-3 A549 GGI-4B 790.33± 32.56 917.06± 84.66 905.35± 82.24 Doxorubicin 0.053± 0.004 0.09± 0.014 17.75 ± 5.61 (MCF-7; Breast cancer cell line; PC-3; Prostate cancer cell line; A549; Lung cancer cell line) PDA agar for secondary metabolite production of Fusarium oxysporum. YP9B isolate. culture conditions were used. After being produced in an incubator at 28°C for 7-10 days, solid medium was used. harvested. The culture media, along with the culture material, were broken up in sterile flasks and mixed in a 1:1 ratio. Ethyl acetate was added. The Erlenmeyer flasks were incubated at 28°C, 120 rpm, with shaking for 24 and 48 hours. It was shaken. After extraction, the extract was filtered through sterile filter paper before use. It was kept at 4ºC for the specified time. Obtained from the Refik Saydam Culture Collection to determine their antibacterial activity. detected Escherichia coli ATCC25922, Klebsiella pneumonia ATCC13883, Yersinia 20 pseudotuberculosis ATCC911, Pseudomonas aeruginosa ATCC27853, Staphylococcus aureus ATCC25923, Enterococcus faecalis ATCC29212, Bacillus cereus 702 Rome, Bacillus subtilis ATCC6633, Streptococcus mutans RSK, Lactobacillus acidophilus RSKK06029, Lactobacillus casei RSK591 and Mycobacterium smegmatis ATCC607 etc. Strains were used. Candida albicans ATCC 60193, 25 was used to determine its antifungal activity. Yeasts Candida tropicalis ATCC 13803 and Saccharomyces cerevisiae RSKK 251 Mueller Hinton agar-liquid (MHB, MHA) was used to test bacteria in the study. Brain Heart Infusion agar-liquid (BHIB, BHIA) media and lactobacilli for M. smegmatis. 13 MRS agar for fungi and potato dextrose agar and malt extract liquid (PDA, MEB) for other fungi. Culture media were used. Extracts and fractions were obtained by agar well diffusion method. Tested against microorganisms for antimicrobial activity. Strong antimicrobial. Ethyl acetate extracts of secondary metabolites, whose activity was determined, were fractionated. purified. The antimicrobial 5 of the fully or partially purified GGI-4B fraction. Efficacy doses Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBK) values ​​were used in the test. For this purpose, each different group was tested again. Mueller Hinton (MHB), Brain Heart Infusion (BHI), and Malt are suitable for microorganisms. MIC values ​​were determined by dilution method (96) using extract (MEB) liquid culture media. MBC values ​​were determined in well microplates and then by culture method. 10 The invention relates to approximately 5 g of ethyl acetate produced at two separate times from the F. oxysporum YP9B strain. The extract was obtained using column chromatography with hexane, chloroform, ethyl acetate, methanol, etc. Purification was performed using different solvents and at different gradients. The collected materials... The fractions were subjected to Thin Layer Chromatography (TLC normal phase silica gel 60 F254), 15 The process was carried out in different solvent environments. A cabinet was used to control thin-layer decomposition. A 254 nm UV lamp located inside was used. UV control of inactive compounds. For this, acid is sprayed onto the ITK plate, followed by a burning process on a hot plate. NMR spectra were obtained from the fractions that were treated and thought to be pure. CDCl3 and Adeton-d6 were used as solvents during the process. 20 After fractionation by column chromatography, the active extract was separated from the normal extract. or repeated analysis with isocrotic mobile phase system by reverse phase (C8 or C18) HPLC method Chromatographic analysis of phenolic or other compounds was performed using the previously developed method. The study was conducted using a Shimadzu brand HPLC instrument. A Zorbax 25 was used as the analytical column. Eclipse XDB-C8 (150 mm × 4.6 mm, 5µm) or similar columns were used. Isocrotic mobile. The phase was adjusted to have a suitable gradient, and repeated HPLC was run. Column oven. The temperature was set to 40 °C. The HPLC system has an existing photodiode array detector. HPLC analysis was performed using a wavelength of 280 nm. 14 The isolated active natural metabolite was analyzed using 400 MHz NMR (1H, 13C, APT / DEPT and 2D NMR). methods), UV, FT-IR and mass spectroscopy (such as EI, LC-TOF, LC / MS-MS, GC-MS) It was characterized by its methods. 1H and COSY NMR spectra are relative to the TMS peak, while 13C and APT spectra are relative to acetone-d6 5. The values ​​were adjusted according to the solvent peaks of (δ 206.7, 30.7 ppm) and CDCl3 (δ 77.0 ppm). The spectra were obtained using the electron spray (ES) method. Column chromatography (CC) and thin-layer chromatography (TLC) were used for isolation. In column chromatography (CC), the normal phase uses 230-400 mesh silica gel, while the thin layer is 10 In the chromatography (TC) of the thin layer, normal phase silica gel 60 F254 was used. A 254 nm UV lamp located inside the cabinet was used to monitor separations, or ITK acid spraying onto the plate and subsequent burning process on a hot plate. It has been implemented. Gas Chromatography and Gas Chromatography Mass Spectrometry (GC and GC / MS) In these studies, the fatty acids obtained were converted into fatty acid methyl esters, thus altering their composition. It has been analyzed. The electron catcher detector used in the mass spectrometer was captured. The ionization of ions is 70 eV. The device used has a film thickness of 60 m × 0.25 mm id. A capillary column (0.25 µm) was used. The flow rate of helium used as the carrier gas was 1 20 The injection rate is mL / min. Injection was performed in splitless mode at 230 °C. From the prepared samples... The resulting volatile components were placed in the column at 60 °C for 2 minutes, then incrementally increased by 3 °C. It was run at temperatures up to 240 °C. The structure of volatile components in each sample was determined by comparing retention indices (RI) of 25 Retention indices were attempted to be determined for fatty acid methyl ester components. Spectral data for all volatile components were determined using (C4-C24) standards. Library of mass spectra of the instrument (NIST and Wiley 7NL) (Adams, 2004), standards (FAMEs; C4-C24) and compared with sources in the literature. Analyzed twice. The peak areas of the samples were calculated using correlation factors. 30 Preparation of fatty acid methyl esters; Preparation of Fatty Acid Methyl Esters (FAMEs) First, 5 mL of 5% NaOH solution is added to 100 mg of sample and incubated at 60-70 °C. The samples were left to stand for 3-4 hours. 5 mL of methanol was then added to the cooled samples in an ice bath. The samples were placed in the test tubes and BBr3 was added drop by drop. The test tubes were incubated in a water bath at 100 °C for 6 hours. The samples were heated for an hour and then cooled. 3 mL of water was added to each sample, which was then divided into 2 mL portions. It was extracted twice with hexane. The resulting hexane phases were dried with Na2SO4, The samples were filtered, and fatty acid methyl esters (FAMEs) were obtained from the samples after the organic solvents were evaporated. It has been obtained. 15 16 REFERENCES Wang, QX, Li, SF, Zhao, F., Dai, HQ, Li, CS, Ding, R., Gao, H., Zhang, LX, Liu, H. (2011). Chemical constituents from endophytic fungus Fusarium oxysporum. Fitoterapia. 82: 777-781. Sondergaard, T.E., Fredborg, M., Christensen, A.M.O., Damsgaard, S.K., Kramer, N.F., Giese, H. and Sørensen, J.L. (2016). Fast Screening of Antibacterial Compounds from Fusaria. Toxins, 8:355 (1-9). doi:10.3390 / toxins8120355. do Nascimento, A.M., Conti, R., Turatti, I.C.C., Cavalcanti, B.C., Costa-Lotufo, L.V., 10 Pessoa, C., de Moraes, M.O., Manfrim, V., Toledo, J.S., Cruz, A.K., Pupo, M.T. (2012). Bioactive extracts and chemical constituents of two endophytic strains of Fusarium oxysporum. Revista Brasileira de Farmacognosia Brazilian Journal of Pharmacognosy 22(6): 1276-1281. ISSN 0102-695X http: / / dx.doi.org / 10.1590 / S0102695X2012005000106 15 25

Claims

1 REQUESTS 1. Fusarium oxysporum, with the following chemical structure shown as Formula IV. Antimicrobial secondary metabolite 5 obtained from the isolate of strain YP9B. Formula IV 2. 2-oxo-8-azatricyclo[9.3.1.13,7]-hexadeca-1(15),3(16),4,6,11,13-hexaen-10-one (Compound 5) is a secondary metabolite according to Claim 1, with the chemical formula.

3. It is a secondary metabolite according to claim 2 and its characteristic is; 2-oxo-8-azatricyclo[9.3.1.13,7]-15 Formula IV of hexadeca-1(15),3(16),4,6,11,13-hexaene-10-one (Compound 5) It has a chemical structure.

4. It is a secondary metabolite according to claim 2 and its characteristic is; 2-oxo-8-azatricyclo[9.3.1.13,7]- Melting point of hexadeca-1(15),3(16),4,6,11,13-hexane-10-one compound (Compound 5) 20 its temperature should be in the range of 125-130 °C.

5. It is a secondary metabolite according to claim 2 and its characteristic is; 2-oxo-8-azatricyclo[9.3.1.13,7]- Retention of hexadeca-1(15),3(16),4,6,11,13-hexaene-10-on compound (Compound 5) The factor is Rf: 0.78 (ethyl acetate). 25 2 6. It is a secondary metabolite according to claim 2 and its characteristic is; 2-oxo-8-azatricyclo[9.3.1.13,7]- infrared (IR) of hexadeca-1(15),3(16),4,6,11,13-hexane-10-one compound (Compound 5) FT-IR spectrum (ATR, cm-1): 3355 (-NH voltage band), 2926, 2855 (-CH voltage band), 1673 (=CO voltage band), 1588, 1450 (C=C voltage band), 1282, It includes peaks 1233 (-CO voltage band), 998, 769, and 692. 5 7. It is a secondary metabolite according to claim 2 and its characteristic is; 2-oxo-8-azatricyclo[9.3.1.13,7]- liquid of hexadeca-1(15),3(16),4,6,11,13-hexane-10-one compound (Compound 5) Chromatography-time-of-flight mass spectrum calculated for C14H11NO2 The measured peaks were 225.24, 226.82 [M+H]+, 247.21 [M+Na-H]+, and 250.98 [M+Na+2H]+. It includes.

8. It is a secondary metabolite according to claim 2 and its characteristic is; 2-oxo-8-azatricyclo[9.3.1.13,7]- 1H-NMR of hexadeca-1(15),3(16),4,6,11,13-hexane-10-one compound (Compound 5) 1H-NMR of the spectrum (300MHz, Acetone-d6): δ: 6.98 (2H, bd, J= 2.40), 6.76 (4H, 15 d, J= 8.8 ), 7.12 (5H, t, J=7.8), 6.96-6.72 (6H, dd, J= 8.8, 8.16), 7.48 (2I H, dd, J= 1.76, 2.06), 7.56, (4I H, d, J= 7.68), 7.33 (5I H, t, J= 7.35), 7.05-7.08 (6I H, dd, It includes peaks of J=0.88, 8.0), 4.22 (2'' H, s), 8.60 (C6H5-NH, bs, 1H).

9. According to claim 2, it is a secondary metabolite with the characteristic of; 2-oxo-8-20 azatricyclo[9.3.1.13,7]-hexadeca-1(15),3(16),4,6,11,13-hexaen-10-on compound (Compound 5) 13C-NMR spectrum of 13C-NMR(75MHz, Acetone-d6): δ: 159.22 (1C,- C-), 115.30 (2C, -CH-), 140.05 (3C, -C), 118.18 (4C, -CH-), 131.15 (5H, -CH-), 116.57 (6H, -CH-), 159.42 (1'C, -C-), 121.83 (2IC, -CH-), 138.51 (3IC, -C-), 122.39, (4'C,-CH-), 131.48 (5'C, -CH-), 121.74 (6'C, -CH), 196.89 (1''C, C=O), 45.03 (2'' 25 It contains C, -CH2) peaks.

10. It is a secondary metabolite according to claim 1 or 2, and its characteristic is that it is effective against microorganisms. minimal inhibitory concentration (MIC) values ​​between 0.8-12.5 µg / ml The minimum bactericidal concentration (MBC) is between 3.1-25 µg / ml. It is within the range. 3 11. According to claim 10, it is a secondary metabolite and its characteristic is antimicrobial activity. The microorganisms it exhibited were Escherichia coli and Yersinia pseudotuberculosis. Klepsiella pneumonia, Pseudomonas aeruginosa, Staphylococcus aureus, Enterococcus faecalis, Streptococcus mutances, Lactobacillus casei, Bacillus cereus, Mycobacterium smegmatis, Candida albicans, Candida tropicalis and / or 5 It is Saccharomyces cerevisiae.

12. According to claim 11, it is a secondary metabolite with the characteristic of having a concentration of 12.5 µg / ml. Compound 5 is effective against Candida albicans, C. tropicalis and Saccharomyces cerevisiae yeast. It has a fungicidal effect. 10 13. According to claim 2, it is a secondary metabolite and its characteristic is; Cercopithecus aetiops kidney. It has cytotoxic activity against the VERO cell line, which is an epithelial cell.

14. It is a secondary metabolite against claim 13, characterized by its presence in ≥2.5 µM of compound 5. Cercopithecus aetiops versus the VERO cell line, which is a renal epithelial cell line. It has cytotoxic activity.