*2.2. Biological Activity*

In previous report, some alkaloids from marine-derived fungus showed pro-angiogenic activities in a zebrafish model [8]. We are also committed to find more marine natural products with angiogenesis related activity. In the present study, all isolated compounds were tested for the pro-angiogenic activities in a vatalanib (PTK787) induced vascular injury zebrafish model (Table S1). Compounds **5** and **7** (at concentrations of 30, 70 and 120 μg/mL) significantly promoted the angiogenesis, compounds **2** and **10** (70 and 120 μg/mL) also

had effects, and compounds **4** (120 μg/mL) exhibited moderate effects (Figure 4). Compared to compound **7**, compounds **8** and **9** were inactive with respect to pro-angiogenesis, indicating that phenolic hydroxyl group is necessary for pro-angiogenic activity. All compounds were also evaluated for anti-inflammatory effects in CuSO4-induced zebrafish inflammation model (Table S1). Compound **11** (30, 70, and 120 μg/mL) displayed potent anti-inflammatory activity, and compounds **7**, **8**, and **10** (70, and 120 μg/mL) had moderate effects (Figure 5). Compound **7** showed better anti-inflammatory activity than **8**, while **9** was ineffective, suggesting the phenolic hydroxyl group and the epoxide oxygen are important in the anti-inflammatory activity. Meanwhile, compared to compound **11**, compounds **12**–**14** displayed no anti-inflammatory activity, indicating that both phenolic and alcohol-hydroxyl groups are necessary for anti-inflammatory activity. In addition, all compounds were tested for cytotoxicity against human liver carcinoma cells HepG2 by MTT method (Table S1) [20], and compound **6** exhibit cytotoxicity with an IC50 value of 30 μg/mL (Figure S1). The pro-angiogenic, anti-inflammatory activities in zebrafish and cytotoxicity against HepG2 cells of these compounds were reported here for the first time.

**Figure 4.** Results of pro-angiogenesis activities. (**A**) Typical images of intersomitic vessels (ISV) in transgenic fluorescent zebrafish (Tg (vegfr2: GFP)) treated with PTK787 and different concentrations (30, 70 and 120 μg/mL) of compounds **2**, **4, 5**, **7**, and **10**, using ginsenoside Rg1 (120 μg/mL) as a positive control. (**B**) Quantitative analysis of the ISV index (number of intact vessels \* 1+number of defective vessels \* 0.5) in zebrafish treated with compounds **2**, **4**, **5**, **7**, and **10**. Data represented as mean ± SEM. ## *p* < 0.01 compared to the control group; \*\* *p* < 0.01 compared to the PTK787 group.

**Figure 5.** Results of anti-inflammatory activities. (**A**) Typical images on inflammatory sites in CuSO4-induced transgenic macrophages fluorescent of compounds **7**, **8**, **10**, and **11**, using ibuprofen (10 μM) as a positive control. (**B**) Quantitative analysis of the number of fluorescent macrophages. The data are represented as the mean ± SEM. ## *p* < 0.01 compared to the control group; \* *p* < 0.05 and \*\* *p* < 0.01 compared to the CuSO4 group.

#### **3. Materials and Methods**

#### *3.1. General Experimental Procedures*

Optical rotations were measured on a JASCO P-2000 digital polarimeter (JASCO, Tokyo, Japan). UV spectra were performed on an Eppendorf BioSpectrometer Basic photometer. IR spectra were recorded on a JASCO FT/IR-4600 spectrometer in KBr discs. CD data were obtained on a JASCO J-810 spectropolarimeter. NMR spectra were collected using a JEOL JNM-ECP 600 spectrometer (JEOL, Tokyo, Japan). HRESIMS data were acquired on an Agilent 6210 ESI/TOF mass spectrometer (Agilent, Santa Clara, CA, USA). Analytical high performance liquid chromatography (HPLC) system (Waters, Milford, MA, USA) consisted of Waters e2695, UV Detector 2489, and software Empower using a C18 column (Diamonsil C18(2), 250 × 4.6 mm, 5 μM). Semipreparative HPLC was operated on the same system using a C18 column (Cosmosil 5C18-MS-II, 250 × 10 mm, 5 μM). Vacuum-liquid chromatography (VLC) used silica gel H (Qingdao Marine Chemical Factory, Qingdao, China). Thin layer chromatography (TLC) and column chromatography were performed

on plates pre-coated with silica gel GF254 (10–40 μm) and Sephadex LH-20 (GE Healthcare Biosciences, Uppsala, Sweden), respectively.

## *3.2. Fungal Material*

The fungus Y32-2 was isolated from the seawater sample collected from a depth of about 30 m in the Indian Ocean (88◦59-51-- E, 2◦59-54-- S) in 2013. It was identified as *Aspergillus austroafricanus* (GenBank access No. MK267449) by rDNA amplification and sequence analysis of the ITS region. The producing strain was prepared on potato dextrose agar medium stored at 4 ◦C.

#### *3.3. Fermentation and Extraction*

The fungus was cultured in 500 mL Erlenmeyer flasks with fermentation media containing 80 g of rice and 120 mL of sea water at 28 ◦C for 40 days. The whole fermented material was extracted exhaustively with EtOAc. Then the EtOAc extract was dried under reduced pressure to obtain residue (30.1 g).

#### *3.4. Purification and Identification*

The EtOAc extract was subjected to silica gel chromatography with a vacuum liquid chromatography (VLC) column, using a stepwise gradient solvent system of petroleum ether (PE)-CH2Cl2 (7:3, 3:7 and 0:1), then of CH2Cl2-MeOH (99:1, 49:1, 19:1, 9:1, 4:1, 1:1, and 0:1) to obtain thirteen primary fractions (Fr.1–Fr.13). Fr.6–Fr.11 were individually subjected to Sephadex LH-20 column (120 × 2 cm) chromatography with CH2Cl2-MeOH (1:1) as mobile phase, and then fractions were purified separately by semipreparative HPLC column (Cosmosil 5C18-MS-II, 250 × 10 mm, 5 μM) using different gradients of MeOH in H2O. Fr.6 (3.5 g) afforded **6** (70% MeOH-H2O, *v*/*v*; *t*R = 23.5 min; 12.4 mg), **8** (60% MeOH-H2O, *v*/*v*; *t*R = 20.5 min; 91.2 mg), **9** (60% MeOH-H2O, *v*/*v*; *t*R = 21.9 min; 12.8 mg), **13** (65% MeOH-H2O, *v*/*v*; *t*R = 25.9 min; 8.6 mg), **14** (60% MeOH-H2O, *v*/*v*; *t*R = 24.8 min; 4.7 mg). Fr.7 (2.7 g) afforded **4** (40% MeOH-H2O, *v*/*v*; *t*R = 18.5 min; 4.4 mg). Fr.8 (1.5 g) afforded **1** (70% MeOH-H2O, *v*/*v*; *t*R = 10.4 min, 14.4min; 6.5 mg), **7** (60% MeOH-H2O, *v*/*v*; *t*R = 16.9 min; 21.4 mg), **11** (60% MeOH-H2O, *v*/*v*; *t*R = 22.8 min; 5.6 mg), **12** (65% MeOH-H2O, *v*/*v*; *t*R = 26.0 min; 14.3 mg). Fr.9 (0.5 g) afforded **3** (65% MeOH-H2O, *v*/*v*; *t*R = 28.5 min; 4.5 mg), 10 (60% MeOH-H2O, *v*/*v*; *t*R = 22.5 min; 5.5 mg). Fr.10 (1.1 g) afforded **2** (60% MeOH-H2O, *v*/*v*; *t*R = 15.0 min; 5.1 mg). Fr.11 (1.3 g) afforded **5** (50% MeOH-H2O, *v*/*v*; *t*R = 14.5 min; 3.0 mg).

*Di-6-hydroxydeoxybrevianamide E* (**1**)*:* Yellow amorphous powder; [α]<sup>20</sup> D +24 (*c* 0.1, MeOH); UV (MeOH) λmax 216, 299 nm; IR (KBr) νmax 3460, 2973, 2925, 1667, 1440, 1306, 1192, 1108, 1001, 920, 809 cm<sup>−</sup>1; 1H and 13C NMR data, see Table 1; HRESIMS *m*/*z* 731.3559 [M − H]− (calcd. for C42 H48 N6O6, 731.3557).

*Dinotoamide J* (**2**)*:* Yellow amorphous powder; [α]<sup>20</sup> D +22 (*c* 0.1, MeOH); UV (MeOH) λmax 210, 226 and 295 nm; IR (KBr) νmax 3447, 1646, 1442, 1186, 1105, 618 cm<sup>−</sup>1; 1H and 13C NMR data, see Table 1; HRESIMS *m*/*z* 763.3440 [M − H]− (calcd. for C42 H48 N6O8, 763.3456).

*Asperpteridinate A:* Yellow amorphous powder; [α]<sup>20</sup> D +63 (*c* 0.1, MeOH); UV (MeOH) λmax 218, 239, 300, 334 nm; 1H and 13C NMR data, see Table 2; IR (KBr) νmax 3465, 1633, 1263, 1192, 1105, 615 cm<sup>−</sup>1; HRESIMS *m*/*z* 465.1018 [M + Na]+ (calcd. for C20 H18 N4O8, 465.1023).

#### *3.5. ECD Computational Calculation*

The conformational analyses were carried out by random searching in the Sybyl-X 2.0 using the MMFF94S force field with an energy cutoff of 5.0 kcal/mol [21]. Subsequently, the conformers were re-optimized using DFT at the PBE0-D3/def2-SVP level in MeOH using the polarizable conductor calculation model (SMD) by the GAUSSIAN 09 program [22]. The energies, oscillator strengths, and rotational strengths (velocity) of the first 30 electronic excitations were calculated using the TDDFT methodology at the CAM-B3LYP-D3/def2- SVP level in MeOH. The ECD spectra were simulated by the overlapping Gaussian function (half the bandwidth at 1/e peak height, sigma = 0.30 for all) [23]. To ge<sup>t</sup> the final spectra, the

simulated spectra of the conformers were averaged according to the Boltzmann distribution theory and their relative Gibbs free energy (ΔG).

## *3.6. Bioassay Protocols*

#### 3.6.1. Cell Culture and Cytotoxicity Assay

According to previous report [24], The HepG2 cells were cultured with DMEM medium, pH 7.0, supplemented with 10% FBS and 1% antibiotics (10,000 IU mL−<sup>1</sup> of penicillin and 10 mg mL−<sup>1</sup> of streptomycin), and the culture flasks were incubated under a humidified atmosphere of 37 ◦C and 5% CO2. The cytotoxic activities of all compounds against HepG2 cells in vitro were determined by modified MTT assays as described previously [21]. Cells were seeded into a 96-well plate at a density 5 × 10<sup>4</sup> per well. After overnight incubation, the cells were treated with the chemicals for 24 h, and 10 μL MTT (5 mg/mL) was added to each well at 37 ◦C for 4 h, then 100 μL lysis buffer was added for the cell lysis. The OD value of each sample was detected at 560 nm using a microplate reader. The experiments were carried out in triplicate.

## 3.6.2. Zebrafish Maintenance

The zebrafish (*Danio rerio*) strains used in this assay were the AB wild-type, Tg (vegfr2-GFP) and Tg (zlyz-EGFP) transgenic lines [25,26]. They were maintained at 28.0 ◦C ± 0.5 ◦C in an automatic circulating tank system with light-dark cycle (14 h:10 h). The healthy adult zebrafish were placed in a breeding tank in the evening, and mated in the next morning. The fertilized eggs were collected, disinfected with methylene blue solution, and then raised in clean culture water including 5.0 mM NaCl, 0.17 mM KCl, 0.4 mM CaCl2, and 0.16 mM MgSO4 in a light-operated incubator.
