Bioprospection of endophytic fungi Paraphaeosphaeria sporulosa and Cochliobolus eragrostidis isolated from Paepalanthus planifolius (Eriocaulaceae) and epigenetic study of Aspergillus terreus

Author(s)

Marcelo Rodrigues de Amorim

• Document type: Doctoral Thesis
• Press: Araraquara. 2020-01-29.
• Institution: Universidade Estadual Paulista (Unesp). Instituto de Química. Araraquara
• Defense date: 2019-12-17
• Advisor: Lourdes Campaner dos Santos

Abstract

Microorganisms are considered a promising source of substances with biological potential and high biosynthetic capacity to produce new substances with application in various areas such as agriculture, medicine and food. Endophytic fungi colonize the interior of plants and have shown great importance in the production of bioactive secondary metabolites. Thus, this work was conceived aiming the bioprospection of extracts produced from endophytic fungi associated with Paepalanthus planifolius, a Cerrado plant species, and the epigenetic evaluation in Aspergillus terreus, isolated from Sonora Desert plant species, to obtain new substances. The isolated endophytic fungi of P. planifolius (15 strains) were cultivated in small scale in potato and dextrose liquid medium (PDB) at 25 oC, under static mode to obtain the respective EtOAc crude extracts. Antimicrobial and cytotoxic activities were evaluated and the initial bioprospection led to the selection of two endophytic fungi identified as Paraphaeosphaeria sporulosa and Cochliobolus eragrostidis. These were cultivated in different liquid and solid media (reduced scale) to again evaluate the biological activity of the extracts and select the most promising ones for isolation and structural determination/elucidation of the secondary metabolites on larger scale. From the cultivation of P. sporulosa in the solid medium of rice, six new substances, sporulosaldeins A-F (1-6) were isolated, with good antifungal activity (MIC <100 µg/mL) against Candida spp. From the extract obtained from the P. sporulosa PDB liquid medium, four substances were isolated, three unpublished 7-(hydroxymethyl)-8-methoxy-3-methylisoquinoline-6-ol (7), 8-methoxy-3,7-dimethylisoquinoline-6-ol (8) and N-(6-hydroxy-8-methoxy-3-methylisoquinoline-7-yl) methyl acetamide (9), and the known chaetoquadrin F (10). From the cultivation of C. eragrostidis in PDB liquid medium, seven substances were identified: phomalactone (11), musacin D (12), musacin E (13), sapinofuranone A (14), cycle (L-valyl-L-tryptophan) (15), indole-3-carboxylic acid (16) and indole-3-acetic acid (17). Metabolite 11 showed promising cytotoxic activity for tumor cells (IC50 = 5.3 - 8.8 µg/mL) and was responsible for the biological activity presented by the extract, while the other substances (12-17) did not show 50% inhibition at the highest concentration evaluated (> 30 µg/mL). The A. terreus cultivation in PDB liquid medium with SAHA epigenetic modifier induced the production of secondary metabolites observed by HPLC. This endophyte was grown on a larger scale in the presence and absence of SAHA resulting in the isolation of two new 3-pyrone-coumarin metabolites, 18 and 19, and a new isocoumarin, 3,4-dihydro-3-methyl-6,7,8-trihydroxy-1H-2-benzopyran-1-one (20), together with known compounds such as terrein (21), 6-hydroxymellein (22), orthosporin (23), 6,7-dimethoxymellein (24), 6-methoxymellein (25), farnesol (26), 3,4-dihydro-3-methyl-5,6,8-trihydroxy-1H-2-benzopyran-1-one (27) and ethyl 3-methylorsellinate (28). Additionally, compounds 18 and 19 were subjected to acetylation and methylation reactions providing the semi-synthetic derivatives 18a, 18b and 19a that helped in the structural elucidation of 18 and 19. The absolute configuration of substances 5, 6, 18, 19 and 20 was determined by Eletronic Circular Dichroism. (AU)

• FAPESP's process: 15/11058-5 - Chemical and biological prospecting of endophytic fungi associated with Paepalanthus planifolius (Eriocaulaceae)
• Grantee: Marcelo Rodrigues de Amorim
• Support Opportunities: Scholarships in Brazil - Doctorate