Dibenzo-α-pyrones from the endophytic fungus Alternaria sp. Samif01: isolation, structure elucidation, and their antibacterial and antioxidant activities

Abstract The EtOAc extract of the liquid fermentation of Alternaria sp. Samif01, an endophytic fungus obtained from Salvia miltiorrhiza Bunge, showed antibacterial activity against several tested bacterial pathogens. Fractionation of this extract led to the isolation of seven dibenzo-α-pyrones (1–7), including one new compound, 2-acetoxy-2-epi-altenuene (1) and one new natural product, 3-epi-dihydroaltenuene A (2). The structures of the new metabolites were elucidated by comprehensive analysis of the spectroscopic data including (1D, 2D) NMR and HRESIMS, while the absolute configuration of 1 was determined by TDDFT–ECD computation. Altenuisol (5), 4-hydroxyalternariol-9-methyl ether (6), and alternariol (7) showed inhibitory activities against the tested bacteria with minimum inhibitory concentration values in the range of 86.7–364.7 μM. A preliminary structure–antibacterial activity relationship was discussed. In addition, compounds 2, 5 and 6 displayed promising antioxidant effects using DPPH and hydroxyl radical assays. The cytotoxicity of the isolated compounds was evaluated as well. The endophytic fungus Alternaria sp. Samif01 isolated from Salvia miltiorrhiza Bunge produced seven bioactive dibenzo-α-pyrones, including one new compound (1), whose absolute configuration was determined by ECD calculation.


Introduction
Endophytic fungi derived from plant and other sources continue to be important producers of bioactive metabolites with diverse types of structures (Aly et al. 2011;Zhao et al. 2011;Newman & Cragg 2015), ever since the first report on taxol production by an endophytic fungus Taxomyces andreanae in 1993 (Stierle et al. 1993). These metabolites were reported to have a broad range of bioactivities, such as cytotoxic/antitumor (Chen et al. 2016), antibacterial (Radić & Štrukelj 2012) and antifungal activities (Kumar & Kaushik 2012).
In our course to search for bioactive fungal metabolites derived from plant endophytes Huang et al. 2009;Meng et al. 2012;Shan et al. 2014), an endophytic fungus Alternaria sp. Samif01 isolated from the medicinal plant Salvia miltiorrhiza Bunge displayed promising antibacterial activities against the tested bacterial pathogens (MIC 100-150 μg/ mL) (Lou, Fu, Luo et al. 2013). The Alternaria fungi are producers of many types of secondary metabolites, such as macrosporin dimers , triterpenoid saponins (Mu et al. 2015), isocoumarins ) and in particular dibenzo-α-pyrones (Lou, Fu, Peng et al. 2013;Xu et al. 2015), in which one phenolic ring was connected to a six-membered carbon ring through a lactone moiety to form a tricyclic skeleton. Dibenzo-α-pyrones were reported to have a broad range of bioactivities ) such as cytotoxic, antioxidant and antimicrobial. In this study, bio-guided fractionation of the fungal extract has afforded the isolation and identification of seven dibenzo-α-pyrones, including two new natural products (1-2). Among them, compounds 5-7 displayed antibacterial activities, while 2, 5 and 6 showed promising antioxidant effects. Herein, we described the isolation, structural elucidation and bioactivities of these metabolites.

Structure elucidation
Fractionation of the fungal extract over silica gel and LH-20, and subsequent purification by semi-preparative HPLC resulted in the isolation and identification of seven dibenzo-α-pyrone derivatives (1-7) ( Figure 1).
The NMR data of 1 were similar to those of 4 (Bradburn et al. 1994;Jiao et al. 2006), except that the presence of an additional acetoxyl group in 1. A noticeable downfield shift of H-2 (δ H 5.28, dd) in 1 was observed when compared to that in 4, suggesting that the acetoxyl group was located at C-2. This was corroborated by the HMBC correlation seen from H-2 to the carbonyl (δ C 172.2) of the acetoxyl group. Thus, the planar structure of 1 was established as the 2-acetoxy derivative of 4. This conclusion was secured by analysis of the 2D NMR spectra ( 1 H-1 H COSY, HMQC, HMBC and NOESY) (Figures S1 and S2, see Supplementary material).
The relative configuration was established by analysis of the coupling constants (J) and NOEs. The small vicinal J value (5.6 Hz) between H-2 (δ H 5.28) and H-3 (δ H 3.97) indicating their cis relationship was consistent with the NOESY correlation observed between them. The correlation between H-2, H-3 and H-4b (δ H 2.11), indicated that these protons were oriented to the same face of the cyclohexene ring, while the correlation observed between 11-Me (δ H 1.55) and H-4a (δ H 2.40) suggested that they were directed to the opposite face ( Figure S2). Thus, the relative configuration was established. Interestingly, (±)-altenuene-2-acetoxy esters, having a same gross structure as 1, but with a different configuration (i.e. H-2, and H-3 was trans), were recently reported from the endophytic fungus A. alternata derived from Camellia sinensis .
The absolute configuration was determined by TDDFT-ECD computation (Bringmann et al. 2009). First, an arbitrarily chosen structure of 1 (2R, 3S, 4aS-) was subjected to random conformational search using SYBYL-X, which generated 13 conformers within 0-10 kcal/mol. Then, these conformers were optimised at B3LYP/6-31G (d) level and the solvent (MeOH) effect was taken into consideration using the CPCM solvent model, which resulted in the identification of six low-energy conformers above 1% (Conf. A-F, Figure S3). Interestingly, in the dominant conformers, i.e. Conf. A (32.9%) and Conf. B (31.9%), 9-OMe was oriented to the left side of the aromatic ring; on the contrary, 9-OMe directed to the right in other low-energy conformers. The ECD spectra for these conformers were, respectively, calculated at TD-DFT B3LYP/6-31+G (d) level using CPCM model (solvent = MeOH), and the calculated ECD spectrum for compound 1 was then generated by average of each conformer's ECD data according to their Boltzmann populations. The experimental ECD spectrum displayed positive Cotton effect at 280 nm and negative at 233 nm. The calculated ECD spectrum was in good agreement with that of the experimental one (Figure 2), thus the absolute configuration was elucidated as 2R, 3S, 4aS.
Compound 2 was isolated as a yellow amorphous solid. Its molecular formula was determined to be C 15 H 18 O 6 by HRESIMS, which is the same as dihydroaltenuene A (Jiao et al. 2006). The NMR data of 2 (Table S1) were similar to those of dihydroaltenuene A, however, the coupling constants between H-3 and H 2 -4 were significantly different (4.4, 11.0 Hz, in 2; vs. 2.3, 3.0 Hz, in dihydroaltenuene A). This indicated that H-3 was axial in 2 rather than equatorial as found in dihydroaltenuene A. This was corroborated by NOESY experiment, as correlations were seen between H-3 (δ H 3.56, m), H-4 eq (δ H 2.24, dd) and H 3 -11 (δ H 1.22, s) ( Figure S4). Thus, compound 2 was identified as 3-epi-dihydroaltenuene A. Interestingly, this compound had been obtained previously as a synthetic product by hydrogenation of isoaltenuene (Altemöller & Podlech 2009). And the NMR data of 2 matched those of the synthetic product. Hence, compound 2 was a new natural product.
Compound 4 was identified as (+)-2-epi-altenuene (4) by comparison of the NMR, MS and [α] D data with the literature (Bradburn et al. 1994;Jiao et al. 2006). Previously, the absolute configuration of 2-epi-altenuene (4) was proposed only from a biogenetic consideration by analogy to isoaltenuene (Jiao et al. 2006). Compound 4 was closely related to compound 1, and they only differed at C-2 for having a different substituent. The great similarity between the CD spectra of 1 and 4 ( Figure S5) indicates that they share a same absolute configuration. This result provided further evidence to support the absolute configuration of 4.

Biological activities
Altenuisol was reported to show inhibition against Staphylococcus aureus (Kim et al. 2014), while alternariol was active against Bacillus Subtilis ) and S. aureus (Lai et al. 2013). In the present study, the antibacterial effects of the isolated compounds were tested against six pathogenic bacteria including Agrobacterium tumefaciens, B. subtilis, Pseudomonas lachrymans, Ralstonia solanacearum, Staphylococcus hemolyticus and Xanthomonas vesicatorya (Table 1). Among them, altenuisol (5), 4-hydroxyalternariol-9-methyl ether (6) and alternariol (7) showed inhibition against all the tested bacteria with minimum inhibitory concentration (MIC) values in the range of 86.7-364.7 μM, and IC 50 values of 7.3-150.3 μM. Among the tested bacteria, P. lachrymans, the causal agent of bacterial angular leaf spot of cucumber, was the most sensitive to the active compounds. Interestingly, it was reported that (+)-2-epi-altenuene (4) was inactive against S. aureus, B. subtilis and Escherichia coli at 100 μg/disc, while altenuene (3) showed antibacterial activity against S. aureus and B. subtilis (Jiao et al. 2006). A preliminary structure-activity relationship (SAR) revealed that a tricyclic aromatic core spanning from C-1 to C10b, i.e. 6H-benzo[c]chromen-6-one, was essential to the antibacterial activity, as this was present in all the active compounds (5-7), while absent in those inactive ones (1-4). However, no significant differences were found between the antibacterial activities of the active compounds. More derivatives should be evaluated to obtain a clear SAR.
The antioxidant activities of these metabolites were tested using DPPH and hydroxyl radical scavenging assays (Table 2). Compounds 2, 5 and 6 showed scavenging ability on hydroxyl radical, with EC 50 values of 267.1, 261.5 and 68.3 μM, respectively. In addition, altenuisol (5) displayed scavenging activity against DPPH radical with EC 50 of 474.5 μM. The other tested compounds were inactive.
Dibenzo-α-pyrones from the Alternaria fungi were reported to be cytotoxic (Lou, Fu, Peng et al. 2013). We also tested the cytotoxicity of compounds 1, 2, 4-6 against five human cancer cell lines (HCT-116, HepG2, BGC-823, NCI-H1650 and A2780), however, none of them showed potent activity (IC 50 > 10 μM). This was not so surprising for 1, 2 and 4, which only had one phenyl group in the structure, as such type of compounds was reported to be non-cytotoxic against the L5178Y mouse lymphoma cells (Aly et al. 2008) and against the human tumour  . Similarly, compound 6 was reported to be inactive against the mouse lymphoma cells (Aly et al. 2008). In contrast, altenuisol (5) was reported to cytotoxic towards HeLa cells (Pero et al. 1973).

General experimental procedure
Please refer to the Supplementary Material online for more on the "General experimental procedure" section.

Fungal source
The endophytic fungus Alternaria sp. Samif01 was isolated from the inner tissue of the fresh root of S. miltiorrhiza Bunge collected in Beijing Medicinal Plant Garden in July 2011 and identified by morphological identification and sequencing of the fungal ITS region (Lou, Fu, Luo et al. 2013). A BLAST search in Genbank indicated that this fungus (GenBank accession No.: KC878695) showed a 100% similarity to that of Alternaria sp. YLN4 KC139492 in their ITS region. A voucher specimen was deposited in the Department of Plant Pathology, China Agricultural university.

Fermentation
The fungus was cultured on PDA (potato dextrose agar) medium for 7 days at 25 °C, and then a slice of agar containing fungal hyphae was used to inoculate the PDB (potato dextrose broth) (150 mL) in a 250-mL Erlenmeyer flask. The cultivation was performed in a rotatory shaker for another 5 days at 150 rpm and 25 °C to produce the seed culture. A scale-up fermentation was carried out using 50 1 L-Erlenmeyer flasks (each containing 500 mL PDB medium and 1 mL seed culture) under shaking condition at 150 rpm and 25 °C for 20 days.

ECD calculation for 1
An arbitrarily chosen structure of 1 (2R, 3S, 4aS-) was submitted to random conformational search with the MMFF94s force field using SYBYL-X 2.0 software package. The resulting conformers (0-10 kcal/mol) were further optimised using the density functional theory (DFT) method at B3LYP/6-31G(d) level with CPCM for MeOH and frequency was calculated at the same level of theory. TDDFT-ECD calculation of the low-energy conformers (≥1%) without imaginary frequencies was performed at B3LYP/6-31+G(d) level with CPCM for MeOH. ECD spectrum of different conformer was simulated using SpecDis with a half-bandwidth of 0.3 eV, and the Boltzmann-averaged ECD spectrum was generated according to the Boltzmann distributions of each conformer. All calculations were performed with Gaussian 09 program package.

Antibacterial assay
The antibacterial activities of the isolated compounds were evaluated against the plant/ human pathogenic bacteria, including two Gram-positive (B. subtilis ATCC 11562 and S. hemolyticus ATCC 29970), and four Gram-negative (R. solanacearum ATCC11696, X. vesicatoria ATCC 11633, A. tumefaciens ATCC 11158 and P. syringae pv. lachrymans ATCC 11,921). Streptomycin sulphate was used as the positive control. MICs and median inhibitory concentrations (IC 50 ) of the tested substances were determined in sterile 96-well plates by the modified broth dilution colorimetric assay .

DPPH radical scavenging assay
DPPH radical scavenging activity was determined based on the reduction of 1,1-diphenyl-2-picrylhydrazyl (DPPH) as reported previously . Butylated hydroxytoluene (BHT) was used as the positive control. For details, see the Supplementary material.

Hydroxyl radical scavenging assay
The hydroxyl radical scavenging activity was determined as described previously with some modifications (Li et al. 2012). Ascorbic acid was used as the positive control. For details, see the Supplementary material.

Disclosure statement
All the authors declare no conflict of interest.