Abstract
Aiming to identify new sources of bioactive secondary metabolites, we isolated 82 endophytic fungi from stems and barks of the native Brazilian tree Caesalpinia echinata Lam. (Fabaceae). We tested their ethyl acetate extracts in several in vitro assays. The organic extracts from three isolates showed antibacterial activity against Staphylococcus aureus and Escherichia coli [minimal inhibitory concentration (MIC) 32-64 μg/mL]. One isolate inhibited the growth of Salmonella typhimurium (MIC 64 μg/mL) and two isolates inhibited the growth of Klebsiella oxytoca (MIC 64 μg/mL), Candida albicans and Candida tropicalis (MIC 64-128 μg/mL). Fourteen extracts at a concentration of 20 μg/mL showed antitumour activities against human breast cancer and human renal cancer cells, while two isolates showed anti-tumour activities against human melanoma cancer cells. Six extracts were able to reduce the proliferation of human peripheral blood mononuclear cells, indicating some degree of selective toxicity. Four isolates were able to inhibit Leishmania (Leishmania) amazonensis and one isolate inhibited Trypanosoma cruzi by at least 40% at 20 μg/mL. The trypanocidal extract obtained from Fusarium sp. [KF611679] culture was subjected to bioguided fractionation, which revealed beauvericin as the compound responsible for the observed toxicity of Fusarium sp. to T. cruzi. This depsipeptide showed a half maximal inhibitory concentration of 1.9 μg/mL (2.43 μM) in a T. cruzi cellular culture assay.
Highlights
Aiming to identify new sources of bioactive secondary metabolites, we isolated 82 endophytic fungi from stems and barks of the native Brazilian tree Caesalpinia echinata Lam. (Fabaceae)
Based on the results of their ITS1-5.8S-ITS4 partial sequences, these isolates were submitted to the GenBank to obtain their accession numbers and the closest related species were achieved by BLAST analysis
ALL: allopurinol tested at 20 μg/mL; AMB: amphotericin B tested at 0.02 μg/mL; BNZ: benznidazole tested at 1.0 μg/mL = 3.8 μM; DEX: dexamethasone tested at 20 μg/mL; ETO: etoposide tested at 1.6 μg/mL in tumour cell lineages and at 20 μg/mL in human peripheral blood mononuclear cell (PBMC); LA: amastigotes forms of Leishmania (Leishmania) amazonensis; MCF-7: human breast cancer; NT: not tested; TC: amastigote and trypomastigote forms of Trypanosoma cruzi; TK-10: human renal cancer; UACC-62: human melanoma cancer; WC: working code; -: inactive; ity against the host cell used in the T. cruzi assay, showing an IC50 of 5 μg/mL (6.38 μM)
Summary
All fungal ITS-rDNA sequences obtained in this work were deposited in the GenBank with accessions KF611676-KF611689 (Table I). The solution was injected into a semi-preparative reverse phase high-performance liquid chromatographic (RP-HPLC) column [250 mm × 4.6 mm internal dimension (i.d.)], 5 μm particle diameter) using a Shimadzu chromatograph (Shimadzu Corp, Japan) equipped with a LC6AD pump and manual injection valve (RheodyneTM 7125, Rheodyne Co, USA) using a fixed 1.000 μL sample loop and a dual-wavelength detector (SPD M10A) controlled by LCsolution software v.1.25 (Shimadzu Corp). Liquid chromatography-diode-array detection-mass spectrometry (LC-DAD-MS) analysis of Fr-5 was performed in a Thermo Surveyor Plus (Thermo Fisher Scientific, USA) chromatograph equipped with a Finnigan Surveyer PDA Plus diode-array detector and C18 column (Atlantis C18, Waters, USA) (3 μm particle diameter, 150 mm × 2.1 mm i.d.). The LC-DAD-MS was conducted in a gradient system using a mixture of water (A) and MeOH (B) with 0.1% formic acid, 1%B-100%B for 13 min, 100%B for 4 min, 100%B-1%B for 0.5 min, 1%B for 11.5 min
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