Cytotoxic and antibacterial depsidones from the endophytic fungus Chaetomium brasiliense isolated from Thai rice

Abstract Four new depsidones, mollicellins V-Y (1-4), together with eight known depsidones (5-12) were isolated from the endophytic fungus, Chaetomium brasiliense, detached from stems of Thai rice. Their structures were determined by extensive spectroscopic methods. Mollicellins X, H, and F (3, 8 and 10) showed potent cytotoxicity against the human oral epidermoid carcinoma (KB) cell line, and mollicellin F (10) also showed a potent cytotoxicity against the human hepatocellular carcinoma (HepG2) cell line. Besides, mollicellin B (11) exhibited cytotoxicity against the colorectal adenocarcinoma (HT-29) cell line. Moreover, most of the isolated depsidones displayed potent antibacterial activity against Gram-positive bacteria, Bacillus cereus and Bacillus subtilis, and several of them showed moderate activity against Methicillin-resistant Staphylococcus aureus (MRSA) and clinical isolates of S. aureus. In addition, a few of them also showed moderate activity against a Gram-negative bacteria Pseudomonas aeruginosa. Graphical Abstract


Introduction
The Chaetomium genus belongs to the Chaetomiaceae family. It is widely distributed in soil, air, water, paper, plants and animals (Pinheiro et al. 2019;Tantapakul et al. 2020). It has been reported to produce various types of bioactive metabolites such as cytochalasin, depsidone, xanthone, sterigmatocystin, chromone, xanthoquinodin and dimeric epidithiodiketopiperazine (ETP) alkaloid. Many of these have shown bioactivities such as antimalarial, antibacterial, and antifungal, as well as cytotoxicity towards the properties of cancer cell lines (Li et al. 2008;Khumkomkhet et al. 2009;Zhang et al. 2013;Ouyang et al. 2018;Tantapakul et al. 2020;Zhao et al. 2021). Among bioactive compounds, depsidone is one of the groups that have attracted attention by natural product chemists, due to their biological activities and pharmaceutical applications (Li et al. 2008;Khumkomkhet et al. 2009;Ouyang et al. 2018;Zhao et al. 2021). Although depsidones have been known to be present in lichens (Elix et al. 1997;Rezanka and Guschina 1999;Elix et al. 2000;Pejin et al. 2012;Stojanovic et al. 2012;Bui et al. 2020;Nguyen et al. 2020), many of them have also been found in other sources such as fungi: Chaetomium brasiliense (Li et al. 2008;Khumkomkhet et al. 2009;Zhao et al. 2021), Preussia aurantiaca (Poch and Gloer 1991), Emericella unguis (Kawahara et al. 1988), Lasiodiplodia theobromae (Umeokoli et al. 2019), an endophytic fungus BCC 8616 (Pittayakhajonwut et al. 2006), a mangrove endophytic fungus Aspergillus sp. GXNU-A9 (Hao et al. 2020), a mushroom Pilobolus heterosporus (Rajachan et al. 2014) and leaves of Garcinia plants (Ito et al. 2001;Lannang et al. 2018). Our previous investigation on C. brasiliense revealed ten cytotoxic depsidones, including four new mollicellins K À N and six known mollicellins B, C, E, F, H and J (Khumkomkhet et al. 2009). Therefore, further search for new bioactive depsidones from C. brasiliense, an endophytic fungus isolated from Thai rice was our focus. Based on our primary screening of this fungus, it was found that the nhexane and EtOAc extracts from the cultured biomass showed cytotoxicity against five cancer cell lines: Human oral epidermoid carcinoma (KB), Human cervical carcinoma (Hela), Human hepatocellular carcinoma (HepG2), Colorectal adenocarcinoma (HT-29) and Human breast adenocarcinoma (MCF-7) with IC 50 values in the range of 4.31-39.08 mg/mL. Moreover, these crude extracts exhibited antibacterial activity against Gram-positive bacteria (Bacillus cereus, Bacillus subtilis and Staphylococcus aureus) with MICs in the range of 5-640 mg/mL and Gram-negative bacteria (Pseudomonas aeruginosa) with MICs in the range of 160-1,280 mg/mL. Consequently, the isolation, structural elucidation and bioactivities evaluation of isolated metabolites of crude n-hexane and EtOAc extracts from C. brasiliense are described.

Results and discussion
Chromatographic separation of n-hexane and EtOAc extracts of air-dried biomass of C. brasiliense yielded four new depsidones, mollicellins V-Y (1-4), eight known depsidones (5-12) (Figure 1), and an ergosterol. Structures of the known compounds were identified by physical and spectroscopic data (IR, 1 H and 13 C NMR, 2 D NMR) and by comparison of the data obtained with published values to be mollicellins R, C, E, H, N, F, B and M (5-12) (Khumkomkhet et al. 2009;Zhao et al. 2021) and an ergosterol (Martinez et al. 2015). The structure elucidation of four new depsidones together with their antibacterial and cytotoxicity activities are presented as follows.
Compound 3 was obtained as a white amorphous solid, and its molecular formula, C 22 H 21 ClO 8 , was deduced from HRESI-TOF-MS m/z 471.0752 [M þ Na] þ and 473.0755 [M þ 2 þ Na] þ in the ratio of 3:1, as well as 13 C NMR data, indicating a chlorine atom in the molecule and possessing 12 degrees of unsaturation. The IR spectrum of 3 showed absorption bands as in mollicellin W (2). The 1 H and 13 C NMR spectral data of 3 (Table S1) were similar to those of 2, except for the presence of a chlorine atom at C-2 (d C 122.0), which was determined by comparing its 13 C NMR data with those reported for known isolates mollicellins E (7), F (10), and M (12) (Khumkomkhet et al. 2009). The HMBC spectrum showed correlations of H-1 0 (d H 2.59) to C-2, and C-11a, and a hydroxyl proton at C-3 (d H 3.52) to C-3, C-2 and C-4 revealing a substituted chlorine at C-2 ( Figure S1). Thus, compound 3 was identified as a new depsidone and has been named mollicellin X.
All isolated depsidones, 1-12, were evaluated for their cytotoxicity and antibacterial activities and the results are shown in Tables S2 and S3   at C-4 show cytotoxicity toward most of the cell lines tested. In contrast, mollicellin Y (4), having a hydroxyl methyl group at C-4, was not cytotoxic towards any cell lines, except a weak activity against the HT-29 cell (IC 50 value of 80.47 mM). Therefore, an aldehyde group at C-4 in the depsidone core should be crucial to the cytotoxic activity. However, structure activity relationship of depsidones (2, 6, 9 and 11) and their chlorinated depsidones pairs (3, 7, 10 and 12) remains unclear. In addition, mollicellins V, W, R, C, H and M (1, 2, 5, 6, 8 and 12) exhibited weak antibacterial activity against a Gram-negative bacteria, P. aeruginosa, with MIC values in the range of 64-128 mg/mL. Moreover, mollicellins V, W and C-F (1, 2 and 6-10) exhibited strong antibacterial activity against Gram-positive bacteria B. cereus and B. subtilis with MIC values in the range of 2-8 mg/mL, which are close to the standard drug kanamycin (MIC value of 2 mg/mL). They also exhibited activity against S. aureus ATCC 25923, with MIC values in the range of 16-64 mg/mL. Mollicellins X, Y, C, H and B (3, 4, 6, 8 and 11) showed moderate antibacterial activity against MRSA ATCC 33591, MRSA ATCC 33592, and MRSA ATCC 43300 with MIC values in the range of 32-128 mg/mL, which are close to a standard drug, oxacillin (MIC value of 32-128 mg/mL). In addition, mollicellins C and B (6 and 11) displayed moderate antibacterial activity against clinical isolates S. aureus (SA1-3), with MIC values in the range of 32-128 mg/mL. Similarly for cytotoxic activity, an aldehyde group at C-4 in the depsidone core should be significant in antibacterial activity against all Gram-positive bacteria. In addition, a chlorine atom at C-2 of the depsidones has no or less effect than the antibacterial activity of their unchlorinated depsidone pairs. Overall, the complete lactone ring in the depsidone structure plays an important role in both cytotoxic and antibacterial activities (Zhang et al. 2014).

General experimental procedures
Melting points were determined on a Gallenkamp SANYO melting point apparatus. UV spectra were measured by an Agilent 8453 UV-visible spectrophotometer. IR spectra were taken on a Bruker Tenser 27 spectrophotometer. NMR spectra were recorded in CDCl 3 and CD 3 OD on a Varian Mercury Plus 400 spectrometer. HR-ESI-TOF-MS data were recorded on a Micromass Q-TOF-2 spectrometer. Column chromatography was carried out on Merck silica gel 60 (230 À 400 mesh). TLC was performed with precoated Merck silica gel 60 PF 254 on aluminium sheets. Preparative TLC was carried out on silica gel 60 PF 254 (0.5 mm, Merck) plates.

Fungal material
The fungus Chaetomium brasiliense Bat. & Pontual was isolated from stems of Thai rice (Pathum Thani 80) (Leewijit et al. 2016) and a voucher specimen No. PT302 was deposited at the Faculty of Agricultural Technology, King Mongkut's Institute of Technology. It was confirmed by a molecular phylogenetic study as having nuclear ribosomal DNA (rDNA) in the regions of the internal transcribed spacer (ITS), at the position of ITS-5.85-ITS2 in primers ITS1 and ITS 4. The fungus was cultured in conical flasks (200 mL each, 75 flasks) with potato dextrose broth (50 mL/flask) and incubated in a standing condition at 30 C for 4 weeks. The culture broth was filtered to give a wet biomass which was then air-dried at room temperature.

Biological activity procedures
Cytotoxicity and Antibacterial assays are provided in Supplementary Material.

Conclusion
Phytochemical investigation on biomass of an endophytic fungus C. brasiliense isolated from Thai rice led to the isolation of four new depsidones, mollicellins V-Y (1-4), along with eight known depsidones namely mollicellins R, C, E, H, N, F, B and M (5-12). Most of the isolated depsidones exhibited cytotoxicity towards five cancer cell lines (KB, Hela, HepG2, HT-29 and MCF-7) and Vero cell lines. In addition, they also displayed potent antibacterial activity against Gram-positive bacteria, B. cereus and B. subtilis and several of them showed moderate antibacterial activity against MRSA. These finding support that the fungus C. brasiliense is an alternative source for bioactive depsidones.
Innovation in Chemistry (PERCH-CIC), Ministry of Higher Education, Science, Research and Innovation is gratefully acknowledged. We would like to thank Assoc. Prof. Sophon Boonlue, Department of Microbiology, Faculty of Science, Khon Kaen University, Thailand for providing some bacterial cultures and laboratory facilities.

Disclosure statement
No potential conflict of interest was reported by the authors.