2.4.5. Effects of Compounds **3** and **4** on Protein Expressions of Bax and Bcl-2 in A549 Cells

To determine whether compounds **3** and **4** could influence the expression of proteins related to A549 cells apoptosis, compounds **3** and **4** (6.25, 12.5, and 25 μM) were added to A549 cells. Figures 9 and 10 showed that the expression level of pro-apoptotic protein, bax was obviously higher with 25 μM treatment of compound **3** or **4** than with 12.5 or 6.25 μM treatment. On the contrary, the cells treated with 25 μM of compound **3** or **4** showed higher Bcl-2 (anti-apoptotic protein) expression than that treated with 12.5 or 6.25 μM. The results showed that compounds **3** and **4** suppressed the expression of Bcl-2 and increased bax expression.

**Figure 10.** Western blot analysis for Bcl-2 (**a**), Bax (**b**), pro-caspase 3 (**c**), and cleaved-caspase 3 (**d**) in each group. Treatment with epiremisporine B (**4**) significantly reduced the expression levels of Bcl-2 and pro-caspase 3, and increased the expression levels of Bax and cleaved-caspase 3. As-terisks indicate significant differences (\* *p* < 0.05 and \*\* *p* < 0.01) compared with the control group.

#### **3. Experimental Section**

#### *3.1. General Procedures*

Optical rotations were measured using a Jasco *P*-2000 polarimeter (Japan Spectroscopic Corporation, Tokyo, Japan) in CHCl3. Circular dichroism (CD) spectra were obtained on a J-715 spectropolarimeter (Jasco, Easton, MD, USA). Ultraviolet (UV) spectra were obtained on a Hitachi U-2800 Double Beam Spectrophotometer (Hitachi High-Technologies Corporation, Tokyo, Japan). Infrared (IR) spectra (neat or KBr) were recorded on a Shimadzu IRAffinity-1S FT-IR Spectrophotometer (Shimadzu Corporation, Kyoto, Japan). Nuclear magnetic resonance (NMR) spectra, including correlation spectroscopy (COSY), rotating frame nuclear Overhauser effect spectroscopy (ROESY), heteronuclear multiple-bond correlation (HMBC), and heteronuclear single-quantum coherence (HSQC) experiments, were acquired using a BRUKER AVIII-500 or a BRUKER AVIII-600 spectrometer (Bruker, Bremen, Germany), operating at 500 or 600 MHz (1H) and 125 or 150 MHz (13C), respectively, with chemical shifts given in the ppm (δ), using CDCl3 as an internal standard (peak at 7.263 ppm in 1H NMR and 77.0 ppm in 13C NMR spectrum). Electrospray ionization (ESI) and high-resolution electrospray ionization (HRESI)-mass spectra were recorded on a Bruker APEX II Mass Spectrometer (Bruker, Bremen, Germany). Silica gel [70–230 mesh (63–200 μm) and 230–400 mesh (40–63 μm), Merck] was used for column chromatography (CC). Silica gel 60 F-254 (Merck, Darmstadt, Germany) was used for thin-layer chromatography (TLC) and preparative thin-layer chromatography (PTLC).

#### *3.2. Fungal Material*

The fungal strain *Penicillium citrinum* BCRC 09F458 was isolated from waste water, which was collected from Hazailiao, Dongshi, Chiayi, Taiwan, in 2009. The fungal strain was identified as *Penicillium citrinum* (family Trichocomaceae) by the BCRC center, based on cultural and anamorphic data. The rDNA-ITS (internal transcribed spacer) region was used for further identification. After searching the GenBank database through BLAST

(nucleotide sequence comparison program), it was found to have a 100% similarity to *P. citrinum*. *P. citrinum* BCRC 09F458 was stored in the Biological Resources Collection and Research Center (BCRC) of the Food Industry Research and Development Institute (FIRDI).

## Cultivation and Preparation of the Fungal Strain

We kept *P. citrinum* BCRC 09F0458 on potato dextrose agar (PDA), and cultivated the strain on PDA at 25 ◦C for one week, and finally harvested the spores with sterile water. The spores (5 × 105) were seeded into 300 mL shake flasks containing 50 mL RGY medium (3% rice starch, 7% glycerol, 1.1% polypeptone, 3% soybean powder, 0.1% MgSO4, and 0.2% NaNO3), and cultivated with shaking (150 rpm) at 25 ◦C, for 3 days. After the mycelium enrichment step, an inoculum mixing 100 mL mycelium broth and 100 mL RGY medium was inoculated into plastic boxes (25 cm × 30 cm) containing 1.5 kg sterile rice and cultivated at 25 ◦C for producing rice, and the above mentioned RGY medium was added for maintaining the growth. After 21 days of cultivation, the rice was harvested, and used as a sample for further extraction.
