Integrating Activity-Guided Strategy and Fingerprint Analysis to Target Potent Cytotoxic Brefeldin A from a Fungal Library of the Medicinal Mangrove Acanthus ilicifolius

Abstract

Mangrove-associated fungi are rich sources of novel and bioactive compounds. A total of 102 fungal strains were isolated from the medicinal mangrove Acanthus ilicifolius collected from the South China Sea. Eighty-four independent culturable isolates were identified using a combination of morphological characteristics and internal transcribed spacer (ITS) sequence analyses, of which thirty-seven strains were selected for phylogenetic analysis. The identified fungi belonged to 22 genera within seven taxonomic orders of one phyla, of which four genera Verticillium, Neocosmospora, Valsa, and Pyrenochaeta were first isolated from mangroves. The cytotoxic activity of organic extracts from 55 identified fungi was evaluated against human lung cancer cell lines (A-549), human cervical carcinoma cell lines (HeLa), human hepatoma cells (HepG2), and human acute lymphoblastic leukemia cell lines (Jurkat). The crude extracts of 31 fungi (56.4%) displayed strong cytotoxicity at the concentration of 50 μg/mL. Furthermore, the fungus Penicillium sp. (HS-N-27) still showed strong cytotoxic activity at the concentration of 25 µg/mL. Integrating cytotoxic activity-guided strategy and fingerprint analysis, a well-known natural Golgi-disruptor and Arf-GEFs inhibitor, brefeldin A, was isolated from the target active strain HS-N-27. It displayed potential activity against A549, HeLa and HepG2 cell lines with the IC₅₀ values of 101.2, 171.9 and 239.1 nM, respectively. Therefore, combining activity-guided strategy with fingerprint analysis as a discovery tool will be implemented as a systematic strategy for quick discovery of active compounds.

Keywords: Acanthus ilicifolius; activity-guided strategy; cytotoxic activity; endophytic fungi; fungal metabolites.

Figure 1

The detailed flowchart of this study.

Figure 2

The distribution of each species in different parts.

Figure 3

Cytotoxicity of organic extracts of 55 identified fungi. (a) The cytotoxic activities of 55 identified fungi against A-549, HeLa, HepG2 and Jurkat tumour cell lines at the concentration of 50 μg/mL. (b) The cytotoxic activities of the active strains at the concentration of 25 μg/mL.

Figure 4

The neighbor-joining of phylogenetic tree of HS-N-27 fungi in Hypocreales order fungi. The values at each node represent the bootstrap values from 1000 replicates, and the scale bar = 0.01 substitutions per nucleotide.

Figure 5

Structures of isolated and identified compounds.