*2.4. Biological Studies*

#### 2.4.1. Inhibitory Activities on Neutrophil Pro-Inflammatory Responses

The anti-inflammatory effects of the isolated compounds from *Penicillium citrinum* were evaluated by their ability to suppress formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP)-induced O2 •– generation by human neutrophils. The anti-inflammatory activity data are shown in Table 2. The clinically used anti-inflammatory agent, ibuprofen, was used as the positive control. From the results of our anti-inflammatory tests, epiremisporine D (**2**), epiremisporine E (**3**), epiremisporine B (**4**), and penicitrinone A (**5**) exhibited inhibition (IC50 values ≤ 8.28 μM) of superoxide anion generation by human neutrophils, in response to fMLP. Thus, our study suggests *Penicillium citrinum* and its isolated compounds (especially **2**, **3**, **4**, and **5**) could be further developed as potential candidates for the treatment or prevention of various inflammatory diseases.

**Table 2.** Inhibitory effects of compounds **1**–**7** from *Penicillium citrinum* on superoxide anion generation by human neutrophils, in response to fMLP.


<sup>a</sup> Concentration necessary for 50% inhibition (IC50). <sup>b</sup> Ibuprofen (a fMLP receptor antagonist) was used as a positive control. Results are presented as average <sup>±</sup> SEM (*<sup>n</sup>* = 3). Values are expressed as average <sup>±</sup> SEM (*<sup>n</sup>* = 3). <sup>c</sup> *<sup>p</sup>* < 0.05; <sup>d</sup> *<sup>p</sup>* < 0.01; <sup>e</sup> *<sup>p</sup>* < 0.001 compared with the control.

#### 2.4.2. Cytotoxic Effects and Selectivity of Compounds **1**–**7**

In this study, the cytotoxic activities of seven compounds (**1**–**7**) against A549 (human lung carcinoma) and HT-29 (human colon carcinoma) cells were studied; shown in Table 3. Among the isolated compounds, compounds **3**, **4**, and **5** exhibited potent cytotoxic activities with IC50 values of 43.82 ± 6.33, 32.29 ± 4.83, and 49.15 ± 6.47 μM against A549 cells, respectively. In addition, compound **4** exhibited cytotoxic activities with an IC50 value of 50.88 ± 2.29 μM against HT-29 cells. According to the data in Table 2, compounds **3** and **5** showed selective cytotoxicity against A549 cancer cells. Among the chromone derivatives (**1**–**4**), epiremisporine B (**4**) (with 2 -hydroxy group) exhibited a more effective cytotoxic activity than its analogue, epiremisporines C–E (**1**–**3**) (without 2 -hydroxy group) against the A549 and HT-29 cells. New compound, epiremisporines E (**3**) (with 2 β-methoxyl group) exhibited a stronger anticancer activity than its analogues, epiremisporines C and D (**1** and **2**) (with 2 α-methoxyl group) against A549 cells.


**Table 3.** Cytotoxic effects of compounds **1**–**7** against A549 and HT-29 cells.

<sup>a</sup> The IC50 values were calculated from the slope of dose-response curves (SigmaPlot). Values are expressed as mean ± SEM (*<sup>n</sup>* = 3). <sup>c</sup> *<sup>p</sup>* < 0.05; <sup>d</sup> *<sup>p</sup>* < 0.01 compared with the control. <sup>b</sup> 5-Fluorouracil (5-FU) was used as a positive control.
