Terrenolide S, a new antileishmanial butenolide from the endophytic fungus Aspergillus terreus

Abstract Terrenolide S, a new butenolide derivative (6), together with six known compounds: (22E,24R)-stigmasta-5,7,22-trien-3-β-ol (1), stigmast-4-ene-3-one (2), stigmasta-4,6,8(14),22-tetraen-3-one (3), terretonin A (4), terretonin (5) and butyrolactone VI (7) have been isolated from the endophytic fungus Aspergillus terreus isolated from the roots of Carthamus lanatus (Asteraceae). Their structures were established by extensive spectroscopic analyses (1D, 2D NMR and HRESIMS), as well as optical rotation measurement and comparison with literature data. Compound 1 displayed a potent activity towards methicillin-resistant Staphylococcus aureus (MRSA) and Cryptococcus neoformans with IC50 values of 2.29 and 10.68 µM, respectively. Moreover, 1, 2 and 6 exhibited antileishmanial activity towards Leishmania donovani with IC50 values of 11.24, 15.32 and 27.27 µM, respectively and IC90 values of 14.68, 40.56 and 167.03 µM, respectively. Graphical Abstract


Introduction
Endophytic fungi living asymptomatically within plant tissues have been found in virtually all plant species (Saikkonen et al. 1998;Bacon & White 2000). Recently, attention has been focused on the biology and chemistry of the endophytic micro-organisms (Geris dos Santos & Rodrigues-Fo 2003). They became a hunting ground for novel drug leads (Strobel & Daisy 2003;larsen et al. 2005). The genus Aspergillus represents a diverse group of fungi, which are amongst the most abundant fungi in the world (Krijgsheld et al. 2012). Aspergillus is a filamentous, cosmopolitan and ubiquitous fungus commonly found in soil, plant debris and indoor air environment. This genus includes over 185 species and is famous for the production of bioactive secondary metabolites (e.g. antibiotic, mycotoxin, antifungal, β-glucuronidase inhibitor, cytotoxic compounds, etc.) (Balajee 2009;Deng et al. 2013;Haroon et al. 2013;Ibrahim et al. 2015). Aspergillus terreus is ubiquitous fungus isolated from both marine and terrestrial environments in tropical or sub-tropical areas (Balajee 2009;Parvatkar et al. 2009). It produces a variety of secondary metabolites that are economically significant, such as the antihypercholesterolemic drug lovastatin and several other metabolites, including sulochrin and terrein which have antibiotic activities (Macedo et al. 2004). In our continued search for biologically active metabolites from natural sources, we have identified a new butenolide derivative: terrenolide S (6), together with six known compounds (1-5 and 7) from the endophytic fungus A. terreus isolated from the roots of Carthamus lanatus (Figure 1(A)). Their chemical structures were identified by one-and two-dimensional NMR and HRESIMS analyses. In addition, their antimicrobial, antileishmanial, antimalarial and cytotoxic activities have been evaluated.
The fungus was cultured on rice solid medium. The metabolites from the rice culture have been extracted with EtoAc. The latter was concentrated and partitioned between n-hexane and 90% MeoH. The total 90% MeoH extract was subjected to vacuum liquid chromatography (VlC), silica gel, sephadex lH-20 and RP-18 column chromatography to yield a new (6) and six known compounds (1-5 and 7).
Compound 1 displayed potent activity against MRSA with an IC 50 value of 2.29 µM and good antifungal activity against Cryptococcus neoformans with an IC 50 value of 10.68 µM compared to ciprofloxacin (IC 50 0.21 µM) and amphotericin B (IC 50 0.37 µM), respectively, as well as it showed weak activity towards Staphylococcus aureus (IC 50 28.54 µM). The resting compounds 2-7 did not show any significant activity.

Isolation of the fungal material, identification, cultivation and isolation of metabolites
A. terreus was isolated from the internal tissue of the healthy roots of Carthamus lanatus l.
(Asteraceae) that was collected from the wildly growing plant at Al-Azhar university campus in February 2013. The strain was identified from the observed morphological features of the fungus by light microscopy (CX31RBSF, olympus) (Watanabe 2002 (AST No. Feb 2013). The fungus was grown on rice solid medium at room temperature. The metabolites from the rice culture were extracted with EtoAc at room temperature and concentrated to afford a residue. The latter was partitioned between n-hexane and 90% MeoH. The total 90% MeoH extract (11.8 g) was subjected to VlC using n-hexane, CHCl 3 , EtoAc and MeoH to yield four fractions. Fraction AS-2 (2.9 g) was subjected to VlC, silica gel and RP-18 column to obtain compounds 1-7. The details of the cultivation and isolation of metabolites have been mentioned in Supplementary materials.

Antimicrobial, antileishmanial, antimalarial and cytotoxicity assays
The antimicrobial activity of the isolated compounds 1-7 was assessed as described previously (Ibrahim et al. 2012;Al-Musayeib et al. 2014). Also, they were evaluated for their antileishmanial and antimalarial activities, as well as their cytotoxicity towards SK-MEl, KB, BT-549, SK-oV-3, llC-PK 11 and VERo cell lines (Borenfreund et al. 1990;El-Shanawany et al. 2011;Al-Musayeib et al. 2014). The details of the assays are mentioned in the Supplementary material.

Conclusion
A new butenolide derivative (6) along with six known compounds (1-5 and 7) have been isolated from the endophytic fungus A. terreus. Their structures were determined on the basis of extensive spectroscopic analyses. Compound 1 displayed potent activity against MRSA and C. neoformans. Moreover, 1, 2 and 6 exhibited antileishmanial activity towards L. donovani.

Supplementary material
A detailed experimental section, NMR spectral data of compounds 1-5 and 7, and NMR spectra of 6 are available as Supplementary material.