*4.6. Cytotoxic Assay*

The cytotoxic activity was evaluated against human lung cancer cell line (A-549), human cervical carcinoma cells (HeLa), human hepatoma cells (HepG2) and Jurkat tumor cell lines by the MTT method, with adriamycin as a positive control [35]. The organic extracts, and adriamycin (the positive control) were dissolved in DMSO with the concentration of 50 μg/mL, 25 μg/mL, and 1 μM, respectively for bioassay.

#### *4.7. Extraction and Isolation of Compounds*

The organic extract of *Penicillium* sp. (HS-N-27) showed strong cytotoxic activity. The organic extract of the *Penicillium* sp. (HS-N-27) was subjected to silica gel column chromatography (CC) and eluted by a gradient of petroleum ether (PE)/ethyl acetate (EA) and then EA/MeOH to generate nine fractions (Fr. 1–9). All the fractions were further evaluated for cytotoxic activity. The Fr. 5 showed strong cytotoxic activity. Combined with fingerprint analysis,-the composition of Fr. 5 is relatively simple (Figure S5); it was further purified by semipreparative HPLC (MeOH−H2O, 80%; 2 mL/min) to obtain **1** (BFA) (19.0 mg).

The organic extract of the *Aspergillus flavus* (HS-N-06) was subjected to silica gel vacuum liquid chromatography (VLC) and eluted by a gradient of PE/EA and then EA/MeOH to afford four subfractions (Fr. 1−Fr. 4). Fr. 3 was separated by ODS CC (MeOH−H2O, 30–50%) to afford **2** (17.0 mg).

The organic extract of the *A. aculeatus* (HS-Y-23) was subjected to silica gel vacuum liquid chromatography (VLC) and eluted by a gradient of PE/EA and then EA/MeOH to afford seven subfractions (Fr. 1−Fr. 7). Fr. 4 was separated by ODS CC (MeOH−H2O, 30–100%) and then purified by semipreparative HPLC (MeOH−H2O, 70%; 2 mL/min) to afford **3** (7.0 mg).

The organic extract of the *Trichoderma harzianum* (HS-N-04) was subjected to silica gel column chromatography (CC) and eluted by a gradient of PE/EA and then EA/MeOH to generate six fractions (Fr. 1–6). Fr. 2 was further purified by using CC to generate five fractions (Fr. 2-1–2-5). Fr.2-2 was separated by normal phase silica gel column chromatography and purified by semi preparative HPLC (MeOH−H2O, 85%; 2 mL/min) to obtain **4** (10.0 mg). Fr.2-4 was separated by normal phase silica gel column chromatography and semipreparative HPLC to obtain **5** (2.4 mg). Fr.4 was separated into six fractions by Sephadex LH-20 eluting with MeOH gel column. Fr.4-2 was purified by semipreparative HPLC (MeOH−H2O, 70%; 2 mL/min) to yield **6** (5.3 mg). Fr.4-4 was purified by semipreparative HPLC (MeOH−H2O, 76%; 2 mL/min) to obtain **7** (5.0 mg).
