Cytotoxic and Antimicrobial Compounds from the Marine-Derived Fungus, Penicillium Species

The organic extract of liquid cultures of the marine-derived Penicillium sp. was investigated. Fractionation of the extracts of the fungus led to the purification and identification of two new compounds, penicillatides A (1) and B (2), together with the previously reported cyclo(R-Pro–S-Phe) (3) and cyclo(R-Pro–R-Phe) (4). The structures of compounds 1–4 were assigned by extensive interpretation of their NMR and high-resolution mass spectrometry (HRMS). The antiproliferative and cytotoxic activities of the compounds against three human cancer cell lines as well as their antimicrobial activity against several pathogens were evaluated. Compounds 2–4 displayed variable cytotoxic and antimicrobial activities.


Introduction
Members of the genus Penicillium are among the most investigated fungi by natural products chemists and are considered a major source for drug discovery. Prominent fungal-derived drugs include the antibiotic penicillin, which was obtained from Penicillium chrysogenum, and the antifungal griseofulvin from Penicillium griseofulvum. Nowadays, the genus Penicillium still represents a major producer of secondary metabolites with diverse bioactivities, as reported in many recent reviews [1][2][3][4][5].
In the last decade, a significantly increased interest in secondary metabolites from marine microbes has been reported [2]. 2,5-Diketopiperazines (2,5-DKPs) are obtained from different organisms, including marine microbes. They represent an important group of cyclic dipeptides with diverse structures and significant biological activities [2]. Since the number of reported compounds with significant biological properties is increasing, several reviews covering the structural determinations, biological activities, and proposed biosynthetic pathways of this class of compounds have been reported [6][7][8]. Consequently, marine fungi appear to be a promising source of these interesting dipeptides. Several members of the 2,5-DKP class displayed cytotoxic, anti-inflammatory, and antimicrobial activities [9][10][11][12].
Our growing interest in identifying secondary metabolites from marine microbes resulted in the identification of several compounds with different bioactivities [13][14][15][16][17]. The organic extract of the fungus Penicillium sp. gave two new compounds, named penicillatides A and B (1 and 2), together with cyclo(D-Pro-L-Phe) {cyclo(R-Pro-S-Phe)} (3) [18] and cyclo(R-Pro-R-Phe) (4) [19] (Figure 1). Structural determinations of the compounds were supported by interpretation of their spectroscopic data as well as comparison of available NMR data in the literature. In this paper, the structural determinations and the cytotoxic and antimicrobial activities of the compounds are reported.
In addition, 2-4 were evaluated for their antimicrobial activity against several microbes: S. aureus, Vibrio anguillarum, and C. albicans. Compounds 2-4 showed significant activity against V. anguillarum, with inhibition zones of 20, 24, and 25 mm, respectively. Similarly, compounds 2-4 showed moderate activity against both S. aureus and C. albicans, with inhibition zones between 10 and 19 mm (Table 3). The results in Table 3 clearly show that compounds 2-4 were selective against HCT-116 without any activity against HepG2. In comparison to 3 and 4, the hydroxylation of C-6 (in 2) potentiated the cytotoxic activity against HCT-116. In the antimicrobial screen, 2-4 were almost identically active against S. aureus and C. albicans, suggesting no effect for the OH at C-6. In addition, 2 and 3 were more active than 2 against V. anguillarum and showed similar activity to the positive control ciprofloxacin.
In addition, 2-4 were evaluated for their antimicrobial activity against several microbes: S. aureus, Vibrio anguillarum, and C. albicans. Compounds 2-4 showed significant activity against V. anguillarum, with inhibition zones of 20, 24, and 25 mm, respectively. Similarly, compounds 2-4 showed moderate activity against both S. aureus and C. albicans, with inhibition zones between 10 and 19 mm (Table 3). The results in Table 3 clearly show that compounds 2-4 were selective against HCT-116 without any activity against HepG2. In comparison to 3 and 4, the hydroxylation of C-6 (in 2) potentiated the cytotoxic activity against HCT-116. In the antimicrobial screen, 2-4 were almost identically active against S. aureus and C. albicans, suggesting no effect for the OH at C-6. In addition, 2 and 3 were more active than 2 against V. anguillarum and showed similar activity to the positive control ciprofloxacin.

Biological Materials
The marine-derived fungus Penicillium sp. was isolated from the Red Sea tunicate Didemnum sp., and the fungus was identified as previously described [13].

Culture Condition and Extraction
Large-scale culture of the fungus Penicillium sp. was carried out in 20 flasks (each 2 L). Each flask contained 500 mL of Sabouraud Dextrose (HiMedia Laboratories, Vadhani Ind. Est., LBS Marg, Mumbai, India) Broth (SDB) liquid medium. The prepared liquid cultures were shaken on an orbital shaker at 28 • C continuously for 14 days. After 2 weeks of shaking and incubation, the cultures were filtered using clean gauze to separate the formed fungal mycelia from the broth. The culture broth from each flask was extracted with EtOAc (3 × 300 mL). The mycelia formed during the shaking were lyophilized and extracted with MeOH. The ethyl acetate and methanolic extracts were combined and evaporated under vacuum, and the resulting extracts were used for fractionation and purification of the compounds.

Determination of Configuration of the Amino Acids in 1 and 2
About 0.5 mg each of compounds 1 and 2 was heated separately in 1 mL of 6 N HCl at 100 • C for 16 h, followed by removal of the excess HCl under vacuum. To each dry hydrolysate, 200 µL of 1% solution of FDAA [21] in acetone and 40 µL of 1.0 M NaHCO 3 were added. The reaction mixture was heated at 45 • C for 1.5 h, cooled, and acidified with 20 µL of 2.0 M HCl. Similarly, standard amino acids (D and L) of leucine and proline were derivatized separately. The derivatized standard amino acids and hydrolysates of 1 and 2 were subjected to HPLC on Nova-Pak C18 reverse-phase column (150 × 3.9 mm i.d., 4 mm particle size; Waters, Milford, MA, USA) using the following gradient program. Solvent A was a 50 mM triethylamine-phosphate buffer (pH 3.5) containing 25% (v/v) MeOH, and solvent B was the same buffer containing 70% MeOH. The mobile phase was a linear gradient from 0 to 100% B (100 to 0% A) in 40 min, at a flow rate of 0.65 mL/min at 25 • C. The eluted peaks were monitored at 340 nm. The retention times for FDAA derivatives of standards and compounds 1 and 2 were as follows: (L)-leucine (t R 27.2 min), (D)-leucine (t R 36.0 min), (L)-proline (t R 15.4 min), (D)-proline (t R 19.1 min), compound 1 (t R 27.2 min), and compound 2 (t R 15.4 min). The cytotoxicity of compounds 2-4 against 3 tumorous cell lines-colorectal carcinoma, breast cancer, and hepatocellular carcinoma-were evaluated using sulforhodamine assay [23]. Briefly, before adding the compounds, the cells were grown in 96-well plates for 24 h. After adding the compounds, incubation of the cells was carried out for another 48 h. The IC 50 values of the compounds were obtained from the log dose-response curve. The reported IC 50 values were obtained from the means of 3 experiments.

Antibacterial Evaluation of the Compounds
A disc diffusion assay was used to determine the antimicrobial activity of the compounds [24] with replication (n = 3). Staphylococcus aureus, Vibrio anguillarum, and Candida albicans served as target models for bacteria and fungi. A total of 100 µg of each compound was loaded onto 6-mm sterile circular filter-paper discs. The paper discs were left to air-dry. The dried paper discs were placed onto nutrient agar plates that had already been inoculated with a lawn of target microorganisms. After 24 h of incubation, the antimicrobial activity of the compounds was calculated.

Conclusions
Investigation of the marine-derived fungus Penicillium sp. gave two new compounds, penicillatides A and B (1 and 2), together with the known compounds cyclo(R-Pro-S-Phe) (3) and cyclo(R-Pro-R-Phe) (4). The structures of 1-4 were assigned by interpretation of their spectroscopic data by NMR and high-resolution mass spectroscopy (HRMS). Compounds 2 and 3 displayed significant and selective activity against HCT-116, with IC 50 of 6.0 and 9.57 µg/mL, respectively, while they were inactive against HepG2 and MCF-7. These results suggest a selective effect of 2 and 3 against HCT-116. Also, 2-4 showed potent antimicrobial activity against V. anguillarum, with inhibition zones of 20, 24, and 25 mm, respectively. On the other hand, 2-4 were moderately active against S. aureus and C. albicans.