Cytotoxic secondary metabolites from mangrove-rhizosphere-associated fungus Emericella sp. strain SWR1718

1 Introduction

Fungi represent a ubiquitous group of eukaryotes that are currently consisting of seven principal phyla: Chytridiomycota, Ascomycota, Basidiomycota, Microsporidia, Glomeromycota, Neocallimastigomycota and Blastocladiomycota (Simões et al., 2013). There is no doubt that fungi are a very essential soil component which play the roles of both decomposers and plant symbionts (Liu et al., 2015). Fungi have been a burgeoning source for several new and bioactive natural products (Suryanarayanan et al., 2012). Particularly, fungal strains that can withstand environments having high salt content proved to biosynthesize unique biomolecules (Ebada and Ebrahim, 2020). Being one of the unique ecosystems which are dramatically threatened, mangroves are coastal biotopes which are considered as an important source of terrestrial organic matter. This is because they harbor a broad spectrum of microbes especially fungi (Simões et al., 2015). Although, several researchers have attempted to study these ecosystems from the microbiological point of view, yet few reports explored their fungal communities (Simões et al., 2015). In fact, a huge proportion of the mangrove ecosystem total biomass is consisting of microbes that encourage natural product researchers to investigate the mangrove-related microbial community especially the fungal content of these ecosystems (Alongi, 1988). Unfortunately, the reports on fungi in the soil zone that is located in the vicinity of the roots (mangrove rhizospheres) are very scanty and reports which are found are mostly based on culture-dependent investigations (Simões et al., 2015).

Emericella is one of the mangrove-associated genera which is known to produce several interesting metabolites featuring complex structural stereochemistry such as cyclopeptides (Oh et al., 2007; Malmstrøm, 1999), diketopiperazines (Xu et al., 2013), alkaloids (Zhang et al., 2011), polyketides (Pang et al., 2018a; Wei et al., 2005), merosesqui- and sesterterpenoids (Chen et al., 2019; Pang et al., 2018b; He et al., 2017). Either the extracts and/or pure metabolites isolated from the genus Emericella revealed a vast array of bioactivities such as antiviral (Zhang et al., 2011), antibacterial (He et al., 2017), antileishmanial (Alves et al., 2018) and insecticidal activities (Abraham et al., 2015).

During our current research targeting the isolation and the identification of new and/or bioactive fungal secondary metabolites from mangrove-associated fungi (Ebrahim et al., 2012a, 2012b, 2013; Elissawy et al., 2019; Moussa et al., 2016; Umeokoli et al., 2019), we have isolated a mangrove-rhizosphere-associated fungus Emericella sp. strain SWR1718. This fungus was derived from the rhizosphere region of Avicennia marina (Forssk.) Vierh. (Acanthaceae) gathered at Jeddah coastline in Saudi Arabia. The phytobiological investigation for methanol extract prepared from liquid Wickerham medium of mangrove-rhizosphere-associated fungus Emericella sp. strain SWR1718 resulted in isolation of one unreported natural product, emericelactone E (1) (Fig. 1) along with six known metabolites belonging to various chemical classes namely; asperthecin (2) (Frisvad and Samson, 2004), nidulol (3) (Fujita et al., 1984), adenine (4) (Cao et al., 2019), adenosine (5), 3′-deoxyadenosine (6) and 3′-deoxy-5′-acetyladenosine (7) (Kawahara et al., 1992) (Fig. 1).

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Fig. 1

Chemical structures of compounds (1–7).

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In this study, we report isolation and structure elucidation of 1 including its relative and absolute configurations along with the results of assessing antiproliferative activity of isolated compounds against three different human tumor cell lines. HTB-176 cells are human lymphoblasts isolated from lymphoma. SW-620 cells are human colon cancer cells isolated from colon adenocarcinoma. HT-29 cells are human colorectal cancer cells isolated from colorectal adenocarcinoma.

2 Experimental

3 Results and discussion

Emericelactone E (1) was recovered as a colourless oil that revealed pseudomolecular ion peaks in LRESIMS at m/z 457.6 [M+H]⁺ and at m/z 455.5 [M−H]⁻. Its molecular formula was ascertained to be C₂₇H₃₆O₆ following its HRESIMS measurement that unravelled a pseudomolecular peak at m/z 457.2522 [M+H]⁺ (calcd for C₂₇H₃₇O₆, 457.2590). The determined molecular formula undoubtedly indicated the existence of ten degrees of unsaturation. Its ¹³C NMR data (Table 1) disclosed the presence of 27 different resonances distinguished into nine quaternary carbon including two ketocarbonyl carbons (δ_C 217.7 and δ_C 212.4), two carboxyl carbons (δ_C 174.5 and δ_C 171.1), one olefinic carbon (δ_C 138.5) and four aliphatic carbons (δ_C 85.7, δ_C 70.0, δ_C 59.9 and δ_C 49.1). In addition, ¹³C NMR spectrum of 1 revealed five olefinic carbons at δ_C 136.1, δ_C 132.2, δ_C 132.1, δ_C 131.5 and δ_C 130.2 together with one oxygenated methine carbons at δ_C 90.4 and three aliphatic methine carbons (δ_C 58.9, δ_C 45.4 and δ_C 39.6). The remaining nine carbon resonances were all designated for methyl groups (δ_C 25.0, δ_C 23.6, δ_C 20.8, δ_C 18.9, δ_C 17.2, δ_C 12.1, δ_C 11.8, δ_C 11.7 and δ_C 6.1). Compound 1 exhibited a maximal absorption (λ_max) at 288.6 nm in its UV spectrum. By exploring the reported literature and by comparison to the obtained data, compound 1 was suggested to be a related derivative to emericelactones A-D, polyketide fungal metabolites featuring a characteristic tetramethyloxabicyclo[2.2.1]heptanes-1,3-dione moiety (Pang et al., 2018a). The ¹H NMR data of 1 (Table 1) further supported its proposed chemical class of fungal metabolites via revealing five olefinic protons at δ_H 6.58 (dd, J = 14.7, 11.1 Hz), δ_H 6.30 (dd, J = 15.8, 10.5 Hz), δ_H 6.17 (dd, J = 14.7, 10.5 Hz), δ_H 6.05 (dd, J = 11.1, 1.5 Hz) and δ_H 5.55 (d, J = 15.8 Hz), three aliphatic methines at δ_H 2.73 (dq, J = 11.8, 7.0 Hz), δ_H 2.67 (dq, J = 11.8, 7.0 Hz) and δ_H 1.94 (t, J = 11.8 Hz) along with an oxygenated singlet methine proton (δ_H 4.69). The ¹H NMR data of 1 also disclosed the presence of six singlet methyl resonances at δ_H 2.01, δ_H 1.37, δ_H 1.26, δ_H 1.19, δ_H 1.16 and δ_H 1.11 together with three doublet methyl protons at δ_H 1.77 (d, J = 1.5 Hz), δ_H 0.96 (d, J = 7.0 Hz) and δ_H 0.92 (d, J = 7.0 Hz). Further confirmation of the unambiguous structure elucidation of 1 was achieved through the results obtained from extensive 2D NMR spectroscopic analyses (Fig. 2, Table 2) including ¹H–¹H COSY, HMBC, HMQC and NOESY experiments. The ¹H–¹H COSY spectrum (Fig. 2) displayed the existence of a triene moiety conjugating over C-7 to C-12 with an additional methyl group at C-12. The presence of this substructure was assured by the COSY spin system from H-7 to H-11 in addition to key HMBC correlations (Fig. 2, Table 2) from Me-23 at δ_H 1.77 (d, J = 1.5 Hz) to three carbons at δ_C 138.5 (C-12), δ_C 130.2 (C-11) and δ_C 58.9 (C-13). Further COSY correlations (Fig. 2, Table 2) were recognized featuring another spin system extending over Me-18/H-17/H-13/H-14/Me-24 together with key HMBC correlations from H-13 at δ_H 1.94 (t, J = 11.8 Hz) to C-15 (δ_C 217.7) and C-16 (δ_C 85.7), from H-14 at δ_H 2.67 (dq, J = 11.8, 7.0 Hz) to C-16 and C-17 (δ_C 39.6), from H-17 at δ_H 2.73 (dq, J = 11.8, 7.0 Hz) to C-12, C-14 (δ_C 45.4) and C-15, from Me-18 at δ_H 0.92 (d, J = 7.0 Hz) to C-13 and C-16, from Me-24 at δ_H 0.96 (d, J = 7.0 Hz) to C-13 and C-15 and from Me-25 at δ_H 1.16 (s) to C-15 and C-17 that altogether indicated the probable existence of a trimethylcyclopentan-1-one moiety with one oxygenated aliphatic quaternary carbon at C-16. By comparing spectroscopic data of 1 with the reported literature (Pang et al., 2018a), it turned out to be a close derivative to emericelactone B but with a higher molecular weight by 42 amu. Moreover, the methyl singlet at δ_H 2.01/δ_C 20.8 that was correlated via HMBC spectrum (Fig. 2) with a carboxyl carbon at δ_C 171.1 indicates the presence of an additional acetyl group in 1 explaining its higher molecular weight compared to emericelactone B. Based on NOESY correlations (Fig. 2, Table 2) along with the coupling constant values, all the three double bonds in (1) were distinguished all to be in E-configurations resembling their comparable positions in emericelactone B. The relative configuration of the tetramethyloxabicyclo[2.2.1]-heptanes-1,3-dione unit was recognized to be (2S*, 5R*, 6S*) similar to those reported for ukulactones A-C (Kaifuchi et al., 2015; Mori et al., 2011) together with emericelactones A-D (Pang et al., 2018a) as well by comparing their NMR data of the respective moiety. In addition, NOESY spectrum of 1 supported the suggested relative configuration by cross correlations of H-5 with H-8 and Me-22; and Me-19 with H-7. The relative configuration of trimethylcyclopentan-1-one moiety was determined based on NOE cross correlations of H-14 and H-17 with Me-25, H-13 with Me-18 and Me-24 indicating that H-14, H-17 and Me-25 similarly oriented, while H-13, Me-18 and Me-24 oriented toward the opposite side of the structure.

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Fig. 2

Key ¹H–¹H COSY, HMBC and NOESY correlations of compound 1.

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Interestingly, the presence of a single hydroxylated carbon at C-16 suggested that it is the only possible position to be acetylated supported by its more downfield carbon resonance (δ_C 85.7) compared to its corresponding carbon in the non-acetylated congener, emericelactone B (δ_C 75.9) (Pang et al., 2018a). Another evidence strengthened the placement of acetyl group at C-16 is the NOE interaction figured out between Me-27 with H-13 and Me-18. By comparing 1D/2D NMR data of 1 and emericelactone B which obviously revealed close values along with their biosynthetic similarities and their close optical rotation values, the absolute configuration of 1 was deduced to be as 2S,5R,6S,13S,14R,16S,17S. As a conclusion, compound 1 was determined to be 16-O-acetylemericelactone B which was given a trivial name emericelactone E.

It is noteworthy mentioning that emericelactone E (1) was carefully detected in the original HPLC chromatogram of total methanol extract of Emericella sp. strain SWR1718 in this study. Its identity was confirmed by means of its UV spectrum and LCMS to assure that it is a genuine natural product. In addition, its deacetylated ancestor, emericelactone B, cannot be traced in the same extract.

After collecting NMR data and performing biological assays on the freshly isolated compound 1, a trial was done in order to elucidate the absolute configuration by ECD. An ECD spectrum was obtained (please see supplementary material, Fig. S12) indicating that compound 1 was decomposed either upon storage or by the effect of deuterated DMSO used for NMR measurement.

Based on careful search in the reported literature, a plausible biosynthetic pathway for emericelactone (1) (Fig. 3) was deduced. It was proposed to be emerging from a polyketide biosynthetic cascade resembling those reported for structurally related fungal metabolites such as ukulactones (Kaifuchi et al., 2015; Mori et al., 2011), prugosenes (Lang et al., 2007) and wortmannilactones (Liu et al., 2018).

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Fig. 3

A plausible biosynthetic pathway of emericelactone E (1).

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The cytotoxic activities of compounds (1–7) were tested for against three human cancer cell lines using the MTT assay. The obtained results (Table 3) disclosed that most of these compounds possess moderate antiproliferative activities against the tested cell lines. Amongst the tested compounds, nidulol (3) revealed moderate cytotoxic activity against the three tested cell lines HTB-176 (lymphoma), SW-620 (colon) and HT-29 (colorectal adenocarcinoma) with IC₅₀ values of 18.6, 15.7 and 36.9 µM, respectively, compared to paclitaxel as a standard cytotoxic agent (IC₅₀ of 18.7–23.3 µM). Interestingly, the isolated compounds have not exhibited any remarkable toxicity against CCD-18 (normal colon) cell line.

4 Conclusion

Seven secondary metabolites including one new compound, emericelactone E (1), were identified from a crude methanol extract of mangrove-rhizosphere-associated fungus Emericella sp. strain SWR1718. Moreover, we demonstrate a plausible biosynthesis of a unique class of compounds to which emericelactone E belongs for the first time in our study. Interestingly, nidulol (3) revealed moderate cytotoxic activity against the three tested cell lines HTB-176 (lymphoma), SW-620 (colon) and HT-29 (colorectal adenocarcinoma) with IC₅₀ values of 18.6, 15.7 and 36.9 µM, respectively, compared to paclitaxel as a standard cytotoxic agent (IC₅₀ of 18.7–23.3 µM).