Isoshamixanthone: a new pyrano xanthone from endophytic Aspergillus sp. ASCLA and absolute configuration of epiisoshamixanthone

Abstract Isoshamixanthone (1), a new stereoisomeric pyrano xanthone together with the previously known fungal metabolites, epiisoshamixanthone (2), sterigmatocystin (3), arugosin C (4), norlichexanthone (5), diorcinol (6), ergosterol and methyllinoleate, were obtained from the endophytic fungal strain Aspergillus sp. ASCLA isolated from leaf tissues of the medicinal plant Callistemon subulatus. The chemical structure of the new xanthone (1) was elucidated by extensive 1D, 2D NMR, and ESI HR mass measurements, and by comparison with literature data. The constitutions and absolute configurations of 1 and epiisoshamixanthone (2) were additionally confirmed by X-ray crystallography. Compounds 1,2 were evaluated for their potential anticancer activity using the human cervix carcinoma cell line (KB-3-1). The antimicrobial activities of the fungal extract and compounds 1,2 were studied using a panel of pathogenic microorganisms as well. Graphical Abstract


Introduction
Fungal endophytes colonizing internal tissues of healthy medicinal plants represent one of the largest and important components of plant micro-ecosystems, however, they are still the least-explored sources of novel small-molecule natural products (Strobel and Daisy 2003;Arnold 2007;Kusari and Spiteller 2011;Wanigasekara et al. 2013;Xu et al. 2013). There is a great biological diversity of endophytic fungi, occurring naturally in the temperate regions, tropical rainforests, and hot deserts, where about 300,000 terrestrial host-plant species are distributed (Arnold 2007;Jia et al. 2016). It has been estimated that over one million endophytic fungal species occur in Nature (Faeth and Fagan 2002). Although the interactions between endophytes with their hosts are not yet fully understood in most cases, many endophytes produce bioactive natural products, which are mostly responsible for the protection of hosts from herbivores, plant pathogens, and abiotic stressors such as drought (Clay and Holah 1999).
The bioactive compounds produced by endophytic fungi are very important to increase the adaptability of both endophytic fungi and their host plants, such as the tolerances to biotic and abiotic stress (Jia et al. 2016). These compounds can be exploited and applied as important medicinal resources (Zhang et al. 2006;Fir akov a et al. 2007;Rodriguez et al. 2009). Particularly, up to one hundred anticancer compounds were reported from endophytic fungi in the period between 1990 and 2010 (Kharwar et al. 2011) along with antiviral potency (Hawas et al. 2016;Laatsch 2017).
In the context of our ongoing studies concerning the discovery of new bioactive metabolites, the endophytic fungal strain Aspergillus sp. ASCLA cultivated on modified rice solid medium, followed by work-up of the obtained organic extract and separation using different chromatographic techniques afforded isoshamixanthone (1) and epiisoshamixanthone (2) (Figure 1). The known metabolites sterigmatocystin (3), arugosin C (4), norlichexanthone (5), diorcinol (6), ergosterol, and methyllinoleate were identified as well. The chemical structure of the new xanthone (1) was elucidated by 1D-and 2D-NMR, and ESI HR mass measurement. The constitutions and absolute configurations of 1 and 2 were unequivocally confirmed by X-ray crystallography.

Isolation and maintenance of the producing strain
The fungal strain Aspergillus sp. ASCLA was isolated from a healthy leaf tissue of the medicinal plant Callistemon subulatus according to our previous work (Mahmoud et al. 2018). The pure strain's colonies were cultivated on potato-dextrose agar (PDA) and maintained at the Microbial Chemistry Department, National Research Centre, Giza-Egypt.

Phenotypic and genotypic characteristics
The growth of the fungal strain ASCLA produces colonies of white hyphae, which turned into black with the formation of conidia. Based on phylogenetic analysis of its 18S rRNA gene sequence ( Figure S2), the fungal strain ASCLA has been confirmed to be belonged to the genus Aspergillus. The aligned sequence of ASCLA has been recorded in GenBank database (accession no. MH665645, https://www.ncbi.nlm.nih. gov/nuccore/MH665645).

Structural elucidation
Chemical screening of the fungal extract was initially performed by thin-layer chromatography (TLC), displaying several bands with a wide range of polarities. Staining of un-polar compounds without any UV absorbance using anisaldehyde/sulfuric acid gave a broad range of colours (yellow-violet), while UV-absorbing bands stained yellow or brown. Visible yellow bands (including compounds 1 and 2) turned dark blue upon spraying with anisaldehyde/sulfuric acid and heating. Isolation of the metabolites produced by the strain was carried out using a series of chromatographic techniques (see the experimental section). The physico-chemical properties of isoshamixanthone (1) and epiisoshamixanthone (2) are listed in Table S1 and experimental section.
Compound 1, a moderately polar yellow solid, appeared on TLC as yellow spot that changed to dark blue on spraying with anisaldehyde/sulfuric acid and heating. As it did not show any colour change with aqueous sodium hydroxide, but turned brown with conc. sulfuric acid, a peri-hydroxy xanthone moiety, present in linear or angular form (Kondo et al. 1998;Shaaban 2004;Wang et al. 2018) was likely. The molecular formula of 1 was determined as C 25 H 26 O 5 by ESI-HR-MS (Table S1), indicating the presence of thirteen double bond equivalents (DBE).
The 1 H-NMR spectrum together with HSQC data (Table S2) exhibited a singlet at d H 12.92 being for a peri-hydroxyl group (1-OH), and four signals corresponding to methyl groups attached to sp 2 -carbons of olefinic and/ or aromatic residues. Signals of two ocoupled sp 2 -methine protons at d H 7.40 (H-3) and 6.79 (H-4), indicated the presence of a 1,2,3,4-tetrasubsituted aromatic moiety. According to the 13 C/DEPT NMR spectra, compound 1 gives rise to 25 carbon signals classified into four methyl groups (d C 25.8-17.4 [C-20-22,25]), three methylene groups, one of them being sp 2 hybridized (112.3 [C-24]) six methine carbons, among them four being sp 2 hybridized, one oxygenated sp 3 methine carbon (63.2 [C-16]), and a non-oxygenated sp 3 methine carbon (d C 45.0 [C-15]). The remaining twelve quaternary carbons are sp 2 hybridized, among them the xanthone carbonyl (184.5, C-10) and four oxygenated carbons (d C 158.7-149.3 C-1,7,12,13]. Based on the evidence mentioned above, and 13 DBE deduced from the molecular formula, compound 1 is belonging to angular tetracyclic pyrano-xanthone skeleton, bearing aromatic-attached methyl and prenyl groups, along with an aliphatic-bound isopropenyl side chain. Further confirmation for the planar structure of 1 were deduced by HMBC and H,H COSY experiments shown in Figure S3. Based on the presented chromatographic behavior, NMR spectroscopic (Table S2 and Figure S3) and mass spectrometric analyses and searching in different data bases, structure 1 was predominantly concluded as isoshamixanthone (1) or epiisoshamixanthone (2) (Xu et al. 2013;Chexal et al. 1975). However, a comparison of the NMR data (Table S2) of both compounds, revealed the presence of a clear difference in the resonating shifts (d H & d C ), multiplicities and coupling constants at the chiral carbons (C-15 and C-16) in ring D (Table S2 and Figure S3), such that the multiplicity of H-15 was established as quartet (J 3.4 Hz) in 1, meanwhile it is doublet triplet (J 12.0, 5.5 Hz) for compound 2. Additionally, H-16 displayed a ddt multiplicity of J 5.5, 3.3 and 1.6 Hz (the coupling with 1.6 Hz represents a long-range coupling to one H-14 according to the W-mechanism) in 1, while the same proton H-16 in 2 showed a td multiplicity of J 3.8 and 1.4 Hz. Furthermore, the 1 H NMR of attached isopropenyl group at C-15 showed a downfield shifting with Dd 0.14-0.26 ppm in 1 than 2, reflecting the orientation nucleophilic influence of OH-16 on the latter in both compounds. Accordingly, compounds 1 and 2 were deduced as two diastereomers at C-15 and C-16 and compound 1 represents a novel xanthone, which we assigned herein as isoshamixanthone (1). Shamixanthone (7) and epishamixanthone (8), representing pyrano-xanthone isomers of 1 and 2, respectively, were reported previously from Aspergillus variecolor (Chexal et al. 1974;Chexal et al. 1975) and Aspergillus rugulosus (Ishida et al. 1976), respectively. The absolute configuration of 7 was assigned by application of the Horeau asymmetric synthesis (Chexal et al. 1975), while that for 8 was deduced by Xray analysis (Ishida et al. 1976;Fukuyama et al. 1978). A comparison of the chemical shifts and multiplicities of the chiral centers H-15 and H-16 in 2 and those for the constitutional isomer 8 with literatures displayed strong close similarities (Chexal et al. 1975;Ishida et al. 1976). By a similar manner, compound 1 showed high similarity in chemical shifts ( 1 H/ 13 C) and multiplicities at H 2 -14, H-15, H-16 and OH-16 to those reported for shamixanthone (7) (Chexal et al. 1974). According to literature, the configurations of both C-15 and C-16 atoms in 8 are R (Ishida et al. 1976;Fukuyama et al. 1978), while C-15 and C-16 in 7 were assigned as S and R, respectively (Chexal et al. 1974).
The absolute configurations of isoshamixanthone (1) and epiisoshamixanthone (2) were unambiguously assigned by X-ray structure determination. Single crystals of both compounds were grown from dichloromethane: methanol (1:3) solution. On the basis of anomalous dispersion, the chiral carbons C-15 and C-16 in isoshamixanthone (1) were assigned as (R) and (S) configuration, respectively ( Figure 2) opposing those reported for shamixanthone (7). The reported relative configuration of epiisoshamixanthone (2) stated that both chiral centers C-15 and C-16 have the same configurations as (R) or (S), while herein both stereocenters were absolutely confirmed to have (R) configuration ( Figure 2).

Biological activities
Based on agar diffusion testing method (Mahmoud et al. 2018), the crude extract of the fungus ASCLA (40 mg/ml) exhibited moderate-high activity against Staphylococcus aurous, Pseudomonas aeruginosa, Candida albicans, Saccharomyces cerevisiae, and Aspergillus niger; while it showed low activity against Bacillus subtilis DSMZ 704 and Pseudomonas agarici (Table S3). Both xanthone diastereomers 1 and 2 exerted closely related moderate-high activities (11-16 mm,  DSMZ 1605, S. warneri DSMZ 20036 and Aspergillus niger, in comparison with gentamycin as reference. Examination of the in vitro cytotoxic activity of the fungal extract and compounds 1 and 2 against the human cervix carcinoma cell line KB-3-1 and its multi-drug resistant subclone (KB-V1) reported no significant cytotoxicity in comparison with (þ) griseofulvin.

General experimental details
Column chromatography was carried out on silica gel (0.06-0.2 mm, Merck, Darmstadt, Germany). Gel filtration was carried out on Sephadex LH-20 (GE Healthcare, Uppsala, Sweden). Preparative TLC (0.5 mm thick) and analytical TLC was performed on Merck pre-coated silica gel 60 PF 254 þ 366 plates (Merck, Darmstadt, Germany). R f values of the bioactive compounds and visualization of their chromatograms was carried out under UV light (254 and 366 nm) and further by spraying with anisaldehyde/sulfuric acid followed by heating. High Resolution ESI-MS was done on a Micromass AC-TOF micro mass spectrometer (Micromass, Agilent Technologies 1200 series, Waldbronn, Germany). Optical rotations were measured on a P-1020 polarimeter (JASCO, Tokyo, Japan). 1 D NMR and 2 D (COSY, HMQC, HMBC, NOESY) NMR spectra were recorded on an Avance 500 MHz spectrometer (Bruker, Rheinstetten, Germany) at 500 MHz ( 1 H) and 125 MHz ( 13 C) at 298 K using the residual solvent peaks as a reference. X-ray crystallographic data was collected on a Rigaku Supernova diffractometer (Rigaku Innovative Technologies, Auburn Hills, MI, USA).

Isolation of the producing organism
The host medicinal plant Callistemon subulatus was collected in February 2017 from Orman Botanic Garden, Giza region, Egypt, and identified by Department of pharmacognosy, Faculty of Pharmacy, Helwan University. A voucher reference specimen (no. CS2017) of the plant was authenticated at the Herbarium of the National Research Centre, Egypt. The plant leaves were washed three times with sterilized water to remove a loosely attached bacteria, debris and salts. The outer surface was sterilized with 70% ethanol then by sterilized water (Debbab et al. 2009). Thereafter, the plant leaves were cut into small pieces using a sterile scalpel to reach the inner tissue surface. The cut pieces were plated on Potato-Dextrose Agar and incubated at 30 C for 3-6 weeks. The selected colonies have been deposited in the Microbial Chemistry Department, National Research Centre (NRC), Egypt.

DNA isolation and 18S rRNA gene sequencing
Analyses for genomic DNA of ASCLA have been carried out using bead beatingextracted DNA using spin column purification by ABT DNA mini extraction kit (Applied Biotechnology Co. Ltd, Egypt). The fungal spores were suspended in 200 ul of distilled water containing zirconia silica beads, then homogenised using vortex. Consequently, the mixture was incubated at 100 C for 15 min. Furthermore, the lysis buffer and proteinase K were added to the sample under investigation; and DNA extraction was preceded according to manufacturer instructions. The genomic DNA was further investigated using fungi-specific primer set ITS1 (5 0 -TCCGTAGGTGAACCTGCGG-3 0 )/ITS4 (5 0 -TCCTCCGCTTATTGATATGC-3 0 ). The following amplification profile was used: an initial denaturation step at 95 C for 5 min was followed by 35 amplification cycles of 55 C for 30 sec., 72 C for 90 sec. and a final extension step at 72 C for 3 min. The PCR product was detected and visualized by (UV) fluorescence after ethidium bromide staining. The 18S rRNA gene sequence was aligned using BLAST (blastn) available at NCBI database (National Centre Biotechnology Information, http://www.ncbi.nlm.nih. govThe phylogenetic tree was constructed using neighbor-joining tree method using the software MEGA7 (Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets) (Kumar et al. 2016).

Large-scale fermentation and isolation
The spore suspension of the fungal strain ASCLA was inoculated into 100 mL of yeast extract/malt extract broth (g/L): malt extract 10 g; yeast extract 4 g; glucose 4 g and cultivated at 30 C for 3 days as seed culture. 5 mL of seed culture were used to inoculate 1 L Erlenmeyer flasks (5 flasks) under aseptic conditions each containing modified rice medium composition (100 g/commercial rice; 150 mL distilled water containing 0.5% peptone) and applied to incubation at 30 C for 14 days. After harvesting, the cultures were combined and socked in methanol for 3 hrs. The methanol extract was separated from rice by filtration under vacuum. After filtration, the water/methanol fraction was concentrated in vacuo, and the afforded water residue was re-extracted by ethyl acetate and obtained ethyl acetate-extract was finally concentrated in vacuo to dryness affording 4.7 g of a reddish-brown crude extract.
For further details see the CIF files attached in the Supporting Information. CCDC 1858754 for isoshamixanthone (1) and CCDC 1858755 for epiisoshamixanthone (2) contain the supplementary crystallographic data for this paper. These data can be obtained free of charge via http://www.ccdc.cam.ac.uk/conts/retrieving.html.

Antimicrobial activity assay
Antimicrobial activity testing of the bacterial crude extract and compounds 1, 2 were carried out against a set of microorganisms using paper-disk diffusion assay (Bauer et al. 1966) with some modifications according to our previous work (Mahmoud et al. 2018).

Cytotoxicity assay
Methodology of the cytotoxic assaying of the fungal extract and compounds 1, 2 against the human cervix carcinoma cell line KB-3-1 and its multi-drug resistant subclone (KB-V1) were carried out according to our previous work (Hamed et al. 2017).

Conclusion
Isoshamixanthone (1), a new diastereomer of the known metabolite epiisoshamixanthone (2), was obtained from the endophytic fungus Aspergillus sp. ASCLA isolated from Callistemon subulatus. Its chemical structure was established on the basis of HR-ESI-MS, 1D and 2D NMR data. The crystal structure as well as absolute configuration of 1 was derived from X-ray crystallography. The absolute configuration and crystal structure of epiisoshamixanthone (2) was reported herein as well to the first time based on X-ray single crystal analysis. Antimicrobial and cytotoxic activities of the fungal extract and the diastereomeric xanthones 1, 2 were studied as well, displaying a closely related antimicrobial potency.