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

**Cui-Fang Wang 1,2, Jie Ma 1,2, Qian-Qian Jing 1, Xi-Zhen Cao 1, Lu Chen 1, Rong Chao 1, Ji-Yong Zheng 2, Chang-Lun Shao 1,2,\*, Xiao-Xi He 1,\* and Mei-Yan Wei 1,\***


**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*; endophytic fungi; fungal metabolites; cytotoxic activity; activityguided strategy
