Anti-Cancer and Anti-Inflammatory Activities of Three New Chromone Derivatives from the Marine-Derived Penicillium citrinum

Three new and uncommon chromone analogs, epiremisporine F (1), epiremisporine G (2), and epiremisporine H (3), were isolated from marine-origin Penicillium citrinum. Among the isolated compounds, compounds 2–3 remarkably suppressed fMLP-induced superoxide anion generation by human neutrophils, with IC50 values of 31.68 ± 2.53, and 33.52 ± 0.42 μM, respectively. Compound 3 exhibited cytotoxic activities against human colon carcinoma (HT-29) and non-small lung cancer cell (A549) with IC50 values of 21.17 ± 4.89 and 31.43 ± 3.01 μM, respectively, and Western blot assay confirmed that compound 3 obviously induced apoptosis of HT-29 cells, via Bcl-2, Bax, and caspase 3 signaling cascades.

Human neutrophils play a significant role in host defenses against pathogen invasion and are the main acute inflammatory mediators [9,10]. After different stimuli, activated neutrophils produce a series of cytotoxins, such as superoxide anion (O 2 •-), granule proteases, and bioactive lipids [9,11,12]. Neutrophilic superoxide generation has been linked to many types of inflammation. An inadequately triggered oxidative burst may cause lipid peroxidation, tissue injury, and inflammatory diseases [13].
The main treatment strategies for cancer patients include chemotherapy, operations, and radiotherapy. However, in patients with metastatic cancer, many anti-cancer drugs show limited effects; thus, the development of more effective therapeutic drugs is urgently needed [14].
Undoubtedly, natural products are favorable drug candidates because they are easy to obtain and comparatively safe. Furthermore, natural compounds have been found to be useful to ameliorate the adverse effects of chemotherapeutic agents. Recently, the notion Undoubtedly, natural products are favorable drug candidates because they are easy to obtain and comparatively safe. Furthermore, natural compounds have been found to be useful to ameliorate the adverse effects of chemotherapeutic agents. Recently, the notion that natural products are an ideal resource for identifying anti-cancer therapeutics has grown globally [15][16][17].
Previously, we reported three new rare chromone analogues, epiremisporine C, epiremisporine D, and epiremisporine E [8]. In this study, we carried out the isolation and structure elucidation of three new compounds, epiremisporine F (1), epiremisporine G (2), and epiremisporine H (3), from the ethanol extract of Penicillium citrinum. In addition, we investigated the inhibitory effects of these compounds on superoxide anion generation by fMLP-activated human neutrophils. Moreover, the cytotoxicities of the isolated compounds against two cancer cell lines, colon cancer HT-29 and lung cancer A549, were also examined.

Fermentation, Extraction, and Isolation
In this study, the marine-derived fungal strain Penicillium citrinum (BCRC 09F0458) was cultured in solid-state culturing conditions, so as to abound the variability of the fungal secondary metabolites. Chromatographic isolation and purification of the n-BuOHsoluble fraction of an EtOH extract of Penicillium citrinum on a silica gel column and preparative thin-layer chromatography (TLC) obtained three new compounds (1-3) ( Figure  1).

Structural Elucidation
Compound 1 was isolated as a yellowish amorphous powder. Its molecular formula, C31H26O12, was determined on the basis of the positive HR-ESI-MS ion at m/z 613.13294 [M + Na] + (calcd. 613.13219) and supported by the 1 H and 13 C NMR data. The IR spectrum showed the presence of hydroxyl (3410 cm −1 ), ester carbonyl (1741 cm −1 ), and conjugated carbonyl (1657 cm −1 ) groups. The 1 H and 13 C NMR data of 1 showed the presence of two hydroxy groups, two methyl groups, three methoxy groups, two pairs of meta-coupling aromatic protons, two methylene protons, and three methine protons. The signals at δ 12.19 and 12.42 exhibited two chelated hydroxyl groups with the carbonyl group. Comparison of the 1 H and 13 C NMR data of 1 with those of epiremisporine C [8] suggested that their structures were closely related, except that the 2′β-methoxy group of 1 replaced the 2′α-methoxy group of epiremisporine C. This was supported by both HMBC correlations between OMe-2′ (δH 3.11) and C-2′ (δC 107.9), and the ROESY correlations between OMe-2′ (δH 3.11) and Hβ-4′ (δH 2.87). The relative configuration of 1 was confirmed by the basis of ROESY experiments. The ROESY cross-peaks between H-3/H-4, H-3/H-3′, H-3/Hα-4′,

Inhibitory Activities on Neutrophil Pro-Inflammatory Responses
The anti-inflammatory activities of the isolates from Penicillium citrinum were evaluated by their ability to inhibit formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP)induced O 2 •generation by human neutrophils. The anti-inflammatory activity data are shown in Table 2. The clinically used anti-inflammatory agent, ibuprofen [20][21][22][23], was used as the positive control. From the results of our anti-inflammatory tests, epiremisporine G (2) and epiremisporine H (3) exhibited inhibition (IC 50 values ≤ 33.52 µM) of superoxide anion release by human neutrophils, in response to fMLP. Among the chromone derivatives, epiremisporine H (3) (with 4α-methyl and 2 β-methoxy groups) exhibited more effective anti-inflammatory activity than its analogues, epiremisporine C (with 4α-hydrogen and 2 α-methoxy group) [8] and epiremisporine F (with 4α-hydrogen and 2 β-methoxy group). In addition, epiremisporine B (with 11-hydroxyl group) [8] exhibited stronger antiinflammatory activity than epiremisporine G (2) (with 11-methoxy group). Therefore, our study suggests Penicillium citrinum and its isolated compounds (2, 3, and epiremisporine B) could be further discovered as promising candidates for the therapy or prevention of various inflammatory diseases.

New Compound 3 Inhibited Proliferation of HT-29 Cells
Epiremisporine H (3) exhibited the most potent cytotoxicity, with an IC 50 value of 21.17 ± 4.89 µM against the HT-29 cell line. Compound 3 was selectively tested for clonogenic assay as it is a new compound and possesses cytotoxic activity against HT-29. The effect of compound 3 on colony formation of HT-29 cells was tested by using the clonogenic assay ( Figure 11). The HT-29 cell colonies were visualized as blue discs, through crystal violet staining. It was clearly observed that compound 3 (12.5 µM) significantly reduced the colony formation of HT-29 cells. Moreover, compound 3 almost completely inhibited the colony formation at 25 µM. a The IC50 values were calculated from the slope of dose-response curves (SigmaPlot). Values are expressed as mean ± SEM (n = 3). c p < 0.05; d p < 0.01; e p < 0.001 compared with the control. b 5-Fluorouracil (5-FU) was used as a positive control.

New Compound 3 Inhibited Proliferation of HT-29 Cells
Epiremisporine H (3) exhibited the most potent cytotoxicity, with an IC50 value of 21.17 ± 4.89 μM against the HT-29 cell line. Compound 3 was selectively tested for clonogenic assay as it is a new compound and possesses cytotoxic activity against HT-29. The effect of compound 3 on colony formation of HT-29 cells was tested by using the clonogenic assay (Figure 11). The HT-29 cell colonies were visualized as blue discs, through crystal violet staining. It was clearly observed that compound 3 (12.5 μM) significantly reduced the colony formation of HT-29 cells. Moreover, compound 3 almost completely inhibited the colony formation at 25 μM.

Effects of Epiremisporine F (3) on Protein Expressions of Pro-caspase 3 and Cleaved Caspase 3 in HT-29 and A549 Cells
Caspase 3 activation is a hallmark of apoptosis. Caspase 3 activation involves the cleavage of pro-caspase 3 (the inactive precursor form of caspase 3), leading to the formation of cleaved caspase 3 (which is the active caspase 3). Upon apoptosis, the procaspase 3 would decrease and the cleaved caspase 3 would increase accordingly [27][28][29]. We further investigated whether epiremisporine F (3) was able to influence these enzymatic activities of caspase 3. The results show that compound 3 suppressed pro-caspase 3 and increased the cleaved caspase 3 (Figures 12 and 13). Furthermore, compound 3 markedly induced apoptosis of HT-29 and A549 cells through caspase-3-dependent pathways.

Effects of Compound 3 on Protein Expressions of Bax and Bcl-2 in HT-29 and A549 Cells
To determine whether compound 3 could influence the expression of proteins related to HT-29 and A549 cells apoptosis, compound 3 (6.25, 12.5, 25, and 50 μM) was added to HT-29 and A549 cells. Figures 12 and 13 show that the expression level of pro-apoptotic protein bax was obviously higher with 50 μM treatment of compound 3 than with 6.25 or 12.5 μM treatment. On the contrary, the cells treated with 6.25 or 12.5 μM of compound 3

Effects of Epiremisporine F (3) on Protein Expressions of Pro-caspase 3 and Cleaved Caspase 3 in HT-29 and A549 Cells
Caspase 3 activation is a hallmark of apoptosis. Caspase 3 activation involves the cleavage of pro-caspase 3 (the inactive precursor form of caspase 3), leading to the formation of cleaved caspase 3 (which is the active caspase 3). Upon apoptosis, the pro-caspase 3 would decrease and the cleaved caspase 3 would increase accordingly [27][28][29]. We further investigated whether epiremisporine F (3) was able to influence these enzymatic activities of caspase 3. The results show that compound 3 suppressed pro-caspase 3 and increased the cleaved caspase 3 (Figures 12 and 13). Furthermore, compound 3 markedly induced apoptosis of HT-29 and A549 cells through caspase-3-dependent pathways.

Effects of Compound 3 on Protein Expressions of Bax and Bcl-2 in HT-29 and A549 Cells
To determine whether compound 3 could influence the expression of proteins related to HT-29 and A549 cells apoptosis, compound 3 (6.25, 12.5, 25, and 50 µM) was added to HT-29 and A549 cells. Figures 12 and 13 show that the expression level of pro-apoptotic protein bax was obviously higher with 50 µM treatment of compound 3 than with 6.25 or 12.5 µM treatment. On the contrary, the cells treated with 6.25 or 12.5 µM of compound 3 showed higher Bcl-2 (anti-apoptotic protein) expression than that treated with 50 µM. The results show that compound 3 suppressed the expression of Bcl-2 and increased bax expression.
showed higher Bcl-2 (anti-apoptotic protein) expression than that treated with 50 μM. The results show that compound 3 suppressed the expression of Bcl-2 and increased bax expression.   showed higher Bcl-2 (anti-apoptotic protein) expression than that treated with 50 μM. The results show that compound 3 suppressed the expression of Bcl-2 and increased bax expression.

Fungal Material
The fungal strain Penicillium citrinum BCRC 09F458 was isolated from wastewater, 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 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
P. citrinum BCRC 09F0458 was maintained on potato dextrose agar (PDA) and the strain was cultured on PDA at 25 • C for 7 days. The spores were seeded into 300 ml shake flasks containing 50 ml RGY (3% rice starch, 7% glycerol, 1.1% polypeptone, 3% soybean powder, 0.1% MgSO 4 , and 0.2% NaNO 3 ), 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.

Biological Assay
The anti-inflammatory effects of the isolated compounds from Penicillium citrinum were evaluated by suppressing fMLP-induced O 2 •generation by human neutrophils. In addition, anti-cancer activity was evaluated by cytotoxicity assay and Western blot analysis.

Preparation of Human Neutrophils
These studies were performed according with the code of ethics of the world medical association for (declaration of Helsinki) experiments involving humans, and all protocols were in compliance with the Institutional Review Board (IRB) of National Yang Ming University (protocol code YM106033E-2 and date of approval: 10 April 2019). Human neutrophils from the venous blood [11] of healthy, adult volunteers (20-35 years old) were isolated using a standard method of dextran sedimentation, prior to centrifugation in a Ficoll Hypaque gradient and hypotonic lysis of erythrocytes, as previously described [30]. Purified neutrophils containing >98% viable cells, as determined by the trypan blue exclu-sion method, were resuspended in HBSS buffer at pH 7.4 and were maintained at 4 • C, prior to use [31].

Measurement of O 2 •-Generation
The assay for detection of O 2 •generation was based on the SOD-inhibitable reduction of ferricytochrome c [32]. In short, neutrophils (1 × 10 6 cells/mL) pretreated with the various test agents (50 and 5 µM) at 37 • C for 5 min were stimulated with fMLP (1 µmol/L) in the presence of ferricytochrome c (0.5 mg/mL). Extracellular O 2 •production was evaluated with a UV spectrophotometer at 550 nm (Hitachi U-3010, Tokyo, Japan). The percentage of superoxide inhibition of the test compound was calculated as the percentage of inhibition = {(control − resting) − (compound − resting)}/(control − resting) × 100. The software SigmaPlot was used for determining the IC 50 values [31].

Cells and Culture Medium
HT-29 (human colon carcinoma) and A549 (human lung carcinoma) cells were kindly provided by Prof. Y. Su and Prof. T. M. Hu, respectively, of National Yang Ming Chiao Tung University, Taipei, Taiwan.
All cell lines were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, 100 µg/mL streptomycin, 2 µM L-glutamine, and 1 mM sodium pyruvate. The cells were incubated in an atmosphere of 37 • C and 5% CO 2 and passaged twice a week. Cells were stored in liquid nitrogen at −155 • C. After the cells were thawed, the experiment was completed before 30 generations. The purpose was to minimize experimental errors. The compound stock solution was stored in DMSO at a concentration of 10 mM and stored at −20 • C, and finally melted immediately before use.

Cytotoxicity Assay
The cell viability was conducted by the MTT assay method, as previously described [33]. The optical density at 570 nm was measured by ELISA plate reader (µ Quant) and the IC 50 value was calculated. The optical density of formazan formed in control (untreated) cells was taken as 100% viability.

Clonogenic Assay
The clonogenic assay was determined by the reference method with a slight modification [34]. In this assay, HT-29 cells were seeded in 6-well plates with 3 × 10 3 cells per well and incubated for 24 hours. The cells were then treated with the indicated concentrations of compound 3, and cultured for 14 days. The cells were washed three times using PBS and fixed using 99% methanol for 30 min. After washing three times with distilled water, the cells were stained using 0.2% crystal violet dye for 15 min and rinsed with distilled water to wash away the excess dye. The visible colonies were compared with the control samples and photographed using a standard camera under natural light.

Statistical Analysis
All results are presented as mean ± SEM. Statistical analysis was executed by using Student's t-test. A probability of 0.05 or less was considered to be statistically significant. Microsoft Excel 2019 was used for the statistical and graphical assessment. All experiments were executed at least 3 times.

Conclusions
Three novel compounds (1-3) were isolated and identified from Penicillium citrinum. The structures of these compounds were established on the basis of spectroscopic data. Reactive oxygen species (ROS) (e.g., superoxide anion (O 2 •− ), hydrogen peroxide) produced by human neutrophils contribute to the pathogenesis of inflammatory diseases. Among the isolated compounds, compounds 2 and 3 could inhibit fMLP-induced O 2 •− generation, with IC 50 values ≤ 33.52 µM. These isolated compounds are worth further research, as promising new leads for developing anti-inflammatory agents. Furthermore, compound 3 markedly induced apoptosis of HT-29 cells through the mitochondrial-and caspase-3-dependent pathways ( Figure 14). This suggests that compound 3 is worth further investigation and might be developed as a candidate for the treatment or prevention of colon cancer. biomolecular imager (GE Healthcare, Woburn, MA, USA). The band densities were quantified using the ImageJ software (NIH, Bethesda, MD, USA).

Statistical Analysis
All results are presented as mean ± SEM. Statistical analysis was executed by using Student's t-test. A probability of 0.05 or less was considered to be statistically significant. Microsoft Excel 2019 was used for the statistical and graphical assessment. All experiments were executed at least 3 times.

Conclusions
Three novel compounds (1-3) were isolated and identified from Penicillium citrinum. The structures of these compounds were established on the basis of spectroscopic data. Reactive oxygen species (ROS) (e.g., superoxide anion (O2 •− ), hydrogen peroxide) produced by human neutrophils contribute to the pathogenesis of inflammatory diseases. Among the isolated compounds, compounds 2 and 3 could inhibit fMLP-induced O2 •− generation, with IC50 values ≤ 33.52 μM. These isolated compounds are worth further research, as promising new leads for developing anti-inflammatory agents. Furthermore, compound 3 markedly induced apoptosis of HT-29 cells through the mitochondrial-and caspase-3-dependent pathways ( Figure 14). This suggests that compound 3 is worth further investigation and might be developed as a candidate for the treatment or prevention of colon cancer.   Institutional Review Board Statement: All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Institutional Review Board (IRB) of National Yang-Ming University (protocol code YM106033E-2 and date of approval: 10th April 2019).

Informed Consent Statement:
Informed consent was obtained from all subjects involved in the study.

Data Availability Statement:
The data presented in this study are available in the main text and the supplementary materials of this article.