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A new antibacterial quinolone derivative from the endophytic fungus Aspergillus versicolor strain Eich.5.2.2
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A new antibacterial quinolone derivative from the endophytic fungus Aspergillus versicolor strain Eich.5.2.2 Sherif S. Ebadaa,b,*, Weaam Ebrahimc,* a
Department of Pharmacognosy, Faculty of Pharmacy, Ain Shams University, 11566 Abbassia, Cairo, Egypt Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Mu’tah University, 61710 Al-Karak, Jordan c Department of Pharmacognosy, Faculty of Pharmacy, Mansoura University, 35516 Mansoura, Egypt b
A R T I C L E
I N F O
Article History: Received 12 September 2019 Revised 23 November 2019 Accepted 9 December 2019 Available online xxx Edited by MG Kulkarni Keywords: Endophyte Quinolone Epimers Antibacterial activity
A B S T R A C T
Fine chromatographic fractionation of the EtOAc (ethyl acetate) extract of a solid rice culture of the endophytic fungus Aspergillus versicolor strain Eich.5.2.2 from the petals of flowers of the Egyptian water hyacinth Eichhornia crassipes revealed the isolation of a new quinolone derivative (1) along with its known isomer aniduquinolone A (2). Structure elucidation of the isolated compounds was unambiguously determined through HRESIMS (High-Resolution Electrospray Ionization Mass Spectrometry) and extensive 1D and 2D NMR (Nuclear Magnetic Resonance) spectroscopy. The relative and absolute configurations of the new compound are deduced through ROESY (Rotational Overhauser Effect SpectroscopY), optical rotation as well as biogenetic consideration. Interestingly, the epimers (1/2) altogether exhibited a significant antibacterial activity against the bacterium Staphylococcus aureus (ATCC700699) with a MIC (Minimum Inhibitory Concentration) of 0.4 mg/mL and fortunately with no cytotoxic effects highlighting a specificity of the antibacterial effect. © 2019 SAAB. Published by Elsevier B.V. All rights reserved.
1. Introduction Endophytes are those bacteria or fungi, that live inside healthy plant tissues and they do not cause any harmful effect to the host plant under normal conditions (Stone et al., 2000). These endophytes live in peace inside their hosts in the form of colonies and they are found either in inter- and/or intracellular spaces of host plants (Pimentel et al., 2011). The genus Aspergillus is one of the largest genera and comprises about 250 species (Geiser et al., 2007). Many species which belong to this genus have the ability to grow in key nutrient-poor environments (Lee et al., 2013). Furthermore, most of these species are known to be successfully grown in a wide range of temperatures, pHs and salinity (0 34%) (Meyer et al., 2011). Aspergillus versicolor is known to metabolise a plethora of unique new/bioactive secondary metabolites, which belong to terpenoids (Belofsky et al., 1998), polyketides (Lee et al., 2010) and peptides (Lee et al., 2011). Authors have previously isolated different fungal strains belonging to the genus Aspergillus from Egyptian Eichhornia crassipes but from different locations and different parts of the plant. The chemical investigation of these Aspergillus strains in either axenic, cocultures or OSMAC-modified cultures resulted in the isolation of many new and bioactive natural products. These fungal secondary metabolites belong to xanthones, dimeric-xanthones, diphenyl * Corresponding authors. E-mail addresses: [email protected], [email protected] (S.S. Ebada), [email protected] (W. Ebrahim).
ethers, alkaloids, lactones and isocoumarins (Ebrahim et al., 2016; Ebada et al., 2018; Abdelwahab et al., 2018). These results encouraged us to investigate more fungi from the same plant but from a different location in Egypt. As a part of our ongoing research aiming at recognizing new/bioactive fungal compounds from endophytes living inside the aquatic plant Eichhornia crassipes (Ebrahim et al., 2016; Ebada et al., 2018; Abdelwahab et al., 2018), an A. versicolor strain Eich.5.2.2 was isolated from the fresh healthy petals of flowers of this plant collected from the main stream of the River Nile near the location of El-Kanater El-Khayriah in Egypt, and fermented on solid rice medium. In this study, we report the structure elucidation of two epimeric quinolone derivatives (1 and 2). All isolated compounds were subjected to antibacterial and cytotoxicity (MTT) assays. 2. Materials and methods 2.1. General experimental procedure The following equipment were used in the present study; PerkinElmer-241 MC polarimeter (for optical rotation), HPLC-MS (High Pressure Liquid Chromatography-Mass Spectrometry) HP1100 Agilent Finnigan LCQ Deca XP Thermoquest (for mass spectra), FTHRMSOrbitrap (Thermo Finnigan) mass spectrometer [for HRESIMS (High Resolution Electrospray Ionization Mass Spectrometry)], Dionex Ultimate 3000 LC system (for analytical HPLC measurements) using ready-made separation columns (125 £ 4 mm, L £ ID), prefilled with Eurospher-10 C18 (Knauer, Germany), RP-HPLC (Reversed-Phase
https://doi.org/10.1016/j.sajb.2019.12.004 0254-6299/© 2019 SAAB. Published by Elsevier B.V. All rights reserved.
Please cite this article as: S.S. Ebada and W. Ebrahim, A new antibacterial quinolone derivative from the endophytic fungus Aspergillus versicolor strain Eich.5.2.2, South African Journal of Botany (2019), https://doi.org/10.1016/j.sajb.2019.12.004
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High Pressure Liquid Chromatography) system of LaChrom-Merk Hitachi (for HPLC separation), Merck MN silica gel 60 M (0.04 0.063 mm) or Sephadex LH-20 stationary phases (for routine column chromatography), Bruker AVANCE DMX 600 using deuterated NMR (Nuclear Magnetic Resonance) solvents (Sigma-Aldrich, Germany) [for 1D (1H and 13C NMR) and 2D NMR (chemical shifts in ppm)], TLC (Thin-Layer Chromatography) plates precoated with silica gel 60 F254 (Merck, Darmstadt, Germany) followed by detection under UV light at 254 and 365 nm wavelengths or after spraying with anisaldehyde and vanillin sulfuric acid spray reagents (for analytical purposes). Spectroscopic and deuterated grade solvents were utilized for spectroscopic and NMR measurements, respectively. All Solvents used during the experiments were predistilled before usage. ChiralpakÒ IA column with 250 mm £ 4.6 mm L £ ID, 5 mm P.S. (Daicel, Chemical Industries Ltd.) was used during chiral separation. 2.2. Fungal material The endophytic fungus A. versicolor strain Eich.5.2.2 was isolated from fresh healthy petals of flowers of E. crassipes, family Pontederiaceae, collected off the main stream of the River Nile near the location of El-Kanater El-Khayriah in Egypt (GPS coordinates: 30.191413, 31.129488) in May, 2014. Both authors authenticated the plant identity after confirmation by Prof. Dr. Ibrahim Mashaly; Professor of Taxonomy, Faculty of Science, Mansoura University, Egypt. A voucher specimen coded as SW436655 was deposited at authors’ labs at Department of Pharmacognosy, Faculty of Pharmacy, Ain Shams University, 11566 Abbassia, Cairo, Egypt (S. S. E.) and Department of Pharmacognosy, Faculty of Pharmacy, Mansoura University, 35516 Mansoura, Egypt (W. E.). The entire Fresh, healthy petals of flowers of E. crassipes were washed twice with sterilized distilled water, airdried within a laminar flow cabinet before they were surface sterilized using 70% ethanol for 2 min (twice) followed by rinsing twice with sterilized distilled water. Two minutes after surface sterilization, the entire petals were then air-dried of ethanol remains within a laminar flow cabinet and placed flat on surface of Petri dishes containing (malt-agar medium) (medium composition: 15 g/L malt extract, 15 g/ L agar in distilled water, pH 7.4 7.8) and incubated at room temperature (25 °C). Each surface of the petal was placed flat on the surface of malt-agar petri dish for 1 min and then removed from the surface of the Petri dish. These Petri dishes were used as a control to test the efficacy of surface sterilization. After that, the same entire fresh healthy petals of flowers, removed from control-petri dishes, were then cut aseptically into small pieces (ca. 1 cm in length). These small pieces were then placed in a Petri dish (malt-agar medium) containing an antibiotic to suppress bacterial growth (medium composition: 15 g/L malt extract, 15 g/Lagar, and 0.2 g/L chloramphenicol in distilled water, pH 7.4 7.8) and incubated at room temperature (25 °C). After several days, the hyphae growing from the pieces of the petals of flowers were transferred to fresh malt-agar plates containing the same medium, incubated again for 10 days. All Petri dishes including the control ones were cultivated at the same culture conditions. These experiments were done with at least ten replicates. The isolated pure endophytic fungus A. versicolor strain Eich.5.2.2 was obtained from the incised fresh healthy petals of flowers only in the experiments in which the control Petri dishes were free from any microbial growth. 2.3. Identification of fungal culture A. versicolor strain Eich.5.2.2 was identified according to a molecular biological protocol using DNA amplification and sequencing of the ITS (Internal Transcribed Spacer) region as previously reported (Ebada et al., 2018). In the field of identifications of sampled fungi, ITS region is known to be one of the markers of the fast and efficient identification of them. It is also known that ITS is the fastest-evolving
component of the rRNA cistron. Moreover, it is well-known that most mycologists in the field of fungal taxonomy and systematics use the ITS as the official barcode for fungi in order to determine species relationships. This is due to its widespread use, ease of amplification, and suitably large barcode gap. Furthermore, for species identification, the ITS can be used alone or in combination with other protein coding genes (Raja et al., 2017). An accession number KM216390 was given the fungal sequence after approval of the GenBank. The NCBI taxonomy database is the generic nomenclature and classification repository consisting of GenBank for the International Nucleotide Sequence Database Collaboration (INSDC). The ITS sequence fragment is submitted to the Basic Local Alignment Search Tool (BLAST) in GenBank to verify identity. This is regulated by certain rules, such as (1) verifying that all query sequences are representative of the entire ITS region; (2) verifying orientation and chimeric sequences through BLAST; and (3) carefully checking taxonomic annotations using only authenticated sequences (Raja et al., 2017). The fungal strain which is used in the present study was kept at authors’ labs, at Department of Pharmacognosy, Faculty of Pharmacy, Ain Shams University, 11566 Abbassia, Cairo, Egypt (S. S. E.) and Department of Pharmacognosy, Faculty of Pharmacy, Mansoura University, 35516 Mansoura, Egypt (W. E.). 2.4. Extraction and isolation The fermentation of the investigated fungal strain was achieved by static culturing at room temperature (25 °C) on solid rice medium in 1 L Erlenmeyer flask (1 L) (Ebada et al., 2018). The culture was extracted using EtOAc (ethyl acetate) (1 £ 200 mL), filtered and then the solvent was evaporated under reduced pressure using rotary evaporator. Crude EtOAC extract (109 mg) was subjected to vacuum liquid chromatography (VLC) using silica gel 60 and a gradient elution solvent system of n-hexane-ethyl acetate and dichloromethanemethanol, with an elution volume of 50 mL each, to yield 6 fractions (AV1-6). Fraction 5 (AV5, 14.0 mg), eluted with dichloromethane: methanol (DCM:MeOH; 1:4), was further purified by preparative HPLC using C18 reversed phase column and eluted with a gradient of H2O and methanol to afford 1/2 (1.4 mg). 2.5. Antibacterial assay The antibacterial test was performed using broth microdilution method. The MIC was determined, and the growth method was used for the inoculum (Ebada et al., 2018). The positive control in this assay was moxifloxacin (MIC = 4 mg/mL). The human-pathogenic bacterium Staphylococcus aureus (ATCC700699) was obtained by purchasing. 2.6. Cytotoxicity (MTT) assay A microplate 3-(4,5-dimethythiazole-2yl) 2,5-diphenyl-tetrazolium bromide (MTT) assay was used to test the cytotoxic activity of 1/2 against human epithelial colorectal adenocarcinoma (Caco-2) cell line (Elissawy et al., 2019). Each well was supplemented with 100 ml of 1/2, dissolved in 5% dimethyl sulfoxide (DMSO) and Roswell Park Memorial Institute (RPMI) tissue culture medium. Two aliquots of 100 ml tissue culture medium [minimal essential medium (MEM) + foetal bovine serum 9:1 ratio], 100 ml sterile 5% DMSO and RPMI tissue culture medium were contained in the control wells. The wells were washed with PBS (Phosphate Buffer Saline) after the required incubation period (24 h) at 37 °C and the cells were incubated with MTT solution (2 mg/ml) for 1 h at 37 °C at 100 ml per well. Supernatants were then excluded by decantation and each well was then treated with 100 ml of DMSO to dissolve the formazan crystals produced in viable metabolically active cells. The absorbance of collected elutes from the 8 wells of each experiment was measured at
Please cite this article as: S.S. Ebada and W. Ebrahim, A new antibacterial quinolone derivative from the endophytic fungus Aspergillus versicolor strain Eich.5.2.2, South African Journal of Botany (2019), https://doi.org/10.1016/j.sajb.2019.12.004
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Fig. 1. Chemical structures of 1 and 2.
Fig. 2. Key 1H
540 nm. Similar treatment was performed for control cells. The percentage of cytotoxicity was then determined as in (Elissawy et al., 2019). The cytotoxic methotrexate (IC50 = 0.7 mM) was used as a positive control in this assay. 2.7. Statistical analysis and software used The mathematical, non-linear regression (4-parameter model) was performed in cytotoxicity assay. The GraphPad Prism software was used in the statistical analysis, ChemDraw software was used to draw the chemical structures and MestreNova software was used for NMR processing. 3. Results and discussion Compound 1 (22S-aniduquinolone A, Fig. 1) was isolated as a white amorphous solid showing a clear single spot on TLC and in LRESIMS revealed pseudomolecular ion peaks at m/z 436.0 [M+H]+,
3
1
H COSY and HMBC correlations of 1 and 2.
458.1 [M+Na]+ and 434.4 [M-H] . The molecular formula of 1 was determined based on HRESIMS exhibiting pseudomolecular ion peaks at m/z 436.2116 [M+H]+ (calcd for C26H30NO5; m/z 436.2118) and m/z 458.1933 [M+Na]+ (calcd for C26H29NNaO5; m/z 458.1938) indicating the presence of thirteen degrees of unsaturation. However, the HPLC chromatogram and UV spectrum unravelled the presence of one coalescent peak with absorption maxima (λmax) at 211, 238, 279 and 324 nm which are characteristic for 4-phenyl-3,4-dihydroquinolone derivatives (Neff et al., 2012; An et al., 2013; Chen et al., 2014; Ebada et al., 2018). The 1H NMR spectrum measured in both aprotic (DMSO‑d6) and protic (MeOH-d4) deuterated NMR solvents (see supplemental material, Figure S4 and S5) distinguished that there are two sets of protons that are very close to that of aniduquinolone A rather than to aniduquinolones B or C (An et al., 2013). This notion confirmed the presence of an isomer of aniduquinolone A. Interestingly, 1H NMR spectrum clearly revealed the presence of two isomers in a ratio of 0.75:1 based on the integration of comparable protons. The 1H NMR
Table 1 1 H and 13C NMR data of 1 and 2. #
1 a
dH (multi, J value in Hz) 1-NH 2 3 4 5 6 7 8 9 10 11 12/16 13/15 14 17 18 19 20 21 22 23 24 25 26 27 4-OH a b c
dC
a,b
2 c
dH (multi, J value in Hz)
a
dH (multi, J value in Hz)
d
a,b C
10.29 (1H, br s) 3.61 (1H, s)
7.36 (1H, d, 8.3) 6.45 (1H, d, 8.3)
7.28 (2H, m) 7.32 (2H, m) 7.32 (1H, m) 6.86 (1H, d, 16.3) 6.31 (1H, d, 16.3) Hax: 2.04 (1H, m) Heq: 1.83 (1H, m) Hax: 1.83 (1H, m) Heq: 2.11 (1H, m) 4.58 (1H, br s) 1.77 (3H, s) Ha: 4.80 (1H, s) Hb: 5.07 (1H, s) 1.41 (3H, s) 3.54 (3H, s)
168.6, CO 85.8, CH 79.9, C 112.4, C 156.5, C 122.0, C 127.8, CH 107.8, CH 136.9, C 140.3, C 127.1, CH 129.5, CH 129.5, CH 121.9, CH 136.0, CH 84.7, C 39.3, CH2 31.8, CH2 82.5, CH 146.6, C 18.1, CH3 111.2, CH2 27.0, CH3 59.0, CH3
dHc (multi, J value in Hz) 10.30 (1H, br s)
3.61 (1H, d, 1.3)
3.61 (1H, s)
7.35 (1H, d, 8.3) 6.44 (1H, d, 8.3)
7.38 (1H, d, 8.3) 6.45 (1H, d, 8.3)
7.19 (2H, m) 7.34 (2H, m) 7.34 (2H, m) 6.74 (1H, d, 16.2) 6.29 (1H, d, 16.2)
7.28 (2H, m) 7.32 (2H, m) 7.32 (1H, m) 6.80 (1H, d, 16.2) 6.24 (1H, d, 16.2)
Hax: 1.99 (1H, m) Heq: 1.74 (1H, m) Hax: 1.74 (1H, m) Heq: 2.05 (1H, m) 4.37 (1H, br s)
Hax: 1.92 (1H, m) Heq: 1.83 (1H, m) Hax: 1.83 (1H, m) Heq: 2.04 (1H, m) 4.49 (1H, t, 6.9)
1.69 (3H, s) Ha: 4.76 (1H, s) Hb: 5.01 (1H, s) 1.31 (3H, s) 3.44 (3H, s) 3.95 (s)
1.73 (3H, s) Ha: 4.82 (1H, s) Hb: 5.01 (1H, s) 1.43 (3H, s) 3.55 (3H, s)
168.7, CO 86.0, CH 79.9, C 112.2, C 156.2, C 122.2, C 127.9, CH 107.8, CH 136.8, C 140.3, C 127.1, CH 129.6, CH 129.6, CH 122.2, CH 134.4, CH 84.8, C 38.7, CH2 31.6, CH2 83.4, CH 147.0, C 17.9, CH3 110.7, CH2 27.2, CH3 58.8, CH3
3.60 (1H, d, 1.3)
7.37 (1H, d, 8.3) 6.44 (1H, d, 8.3)
7.21 (2H, m) 7.34 (2H, m) 7.34 (2H, m) 6.65 (1H, d, 16.2) 6.24 (1H, d, 16.2) Hax: 1.91 (1H, m) Heq: 1.83 (1H, m) Hax: 1.74 (1H, m) Heq: 1.99 (1H, m) 4.32 (1H, t, 6.9) 1.67 (3H, s) Ha: 4.75 (1H, s) Hb: 4.96 (1H, s) 1.33 (3H, s) 3.44 (3H, s) 3.84 (s)
Measured in methanol-d4 (1H at 600 MHz and 13C at 150 MHz). Data was assigned and confirmed via gHMQC and gHMBC spectra. Measured in DMSO‑d6 (1H at 600 MHz).
Please cite this article as: S.S. Ebada and W. Ebrahim, A new antibacterial quinolone derivative from the endophytic fungus Aspergillus versicolor strain Eich.5.2.2, South African Journal of Botany (2019), https://doi.org/10.1016/j.sajb.2019.12.004
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Fig. 3. Key ROESY correlations of 1 and 2.
results in DMSO‑d6 for one of the two sets (Table 1) was closely related to aniduquinolone A (2) (An et al., 2013) with characteristic proton signals spread over a wide field range including an amide proton, a monosubstituted phenyl moiety (H-12 to H-16), a 1,2,3,4-tetrasubstituted aromatic unit (H-8 and H-9), trans-olefinic protons (H-17 and H-18) and terminal downfield double bond methylene (H2-25). In addition, two oxymethine protons (H-3 and H-22), an oxygenated methyl singlet (H3-27), signals indicative to two methylenes (H2-20 and H2-21) and two singlet methyls (H3-24 and H3-26) at the upfield region were also recognized on the same spectrum. Further confirmation to the suggested identity for one of the two compounds to be aniduquinolone A (2) was obtained through extensive 2D NMR spectral analyses such as 1H 1H COSY, HMBC and HMQC (see supplemental material, Figure S6-S9) which revealed (Fig. 2) key correlations unambiguously confirming the presence of the different structural fragments of aniduquinolone A namely, 4-phenyl-3,4-dihydroquinolone together with the terpenoid side-chain containing a tetrahydrofuran ring with a terminal allyl moiety. The relative configuration of 2 was defined on the basis of coupling constant (J-values) calculations, ROESY spectral analysis (Fig. 3) and by comparison with the reported relative and absolute configuration of aniduquinolone A (An et al., 2013) which revealed identical key ROESY correlations and J-values especially to those indicating the cofacial orientation of H-3 and the phenyl ring; 3-OMe on the opposite side in addition to E-geometry for the double bond at C-17/C-18 on the terpenoid portion of the molecule which was undoubtedly confirmed through the characteristic huge coupling constant values for both H-17 and H-18 (J = 16.2 Hz). In addition, key ROESY correlations of H-22 at dH 4.49 (t, J = 6.9 Hz) to two methyl singlets (H3-24 and H3-26) and from methyl group (H3-26) to H-17 and H-18. In conclusion, one set of the observed NMR data was unambiguously related to aniduquinolone A with tentatively similar relative and absolute configurations (An et al., 2013). Due to the close similarity of the second set of proton resonances to those observed for 1 together with revealing the same molecular formula from HRESIMS, the second set was suggested to be belonging to an isomer of 1. A careful comparison of 1D and 2D NMR data (Table 1) for both sets of proton peaks resulted in the identification of a second compound which is very similar to aniduquinolone A (2) showing similar main molecular features namely, 4-phenyl-3,4-dihydroquinolone and the terpenoid side-chain. Further investigation of the second compound was obtained through careful examination of 2D NMR spectra including 1H 1H COSY, HMBC and HMQC (Fig. 2) to unambiguously assign all proton and respective carbon resonances (Table 1). The main difference noticed between the two different compounds is the proton peak representing H-22 on the tetrahydrofuran ring of the terpenoid side-chain portion of the molecule which appeared at dH 4.58 (br s) and bound to a carbon resonance at dC 82.5 in compound (1) compared to a proton peak at dH 4.49 (t, J = 6.9 Hz) bond to a carbon peak at dC 83.4 in compound (2) which suggests
that the stereochemistry of C-22 is the sole difference between 1 and 2 explaining the close similarity in their NMR data. The relative configuration of 1 was determined by careful examination of ROESY spectrum which revealed similar key correlations (Fig. 3) to those exhibited by aniduquinolone A (2). The major difference in ROESY spectrum between 1 and 2 was the absence of the key ROESY correlations from H-22 to methyl groups (H3-24 and H3-26) in 1 unlike 2 while keeping the key ROESY correlations from H-8 at dH 7.36 (d, J = 8.3 Hz) to H-18 at dH 6.31 (d, J = 16.3 Hz) and also from methyl group (H3-26) at dH 1.41 (s) to H-17 at dH 6.86 (t, J = 16.3 Hz) and H-18. These findings suggested the inversion of the stereochemistry at C-22 in 1 compared to 2. Based on the comparison of the measured NMR spectral data of 1 in DMSO‑d6 and methanol-d4 (Table 1) to the reported data for aniduquinolone A (2) (An et al., 2013) including both its relative and absolute configurations, compound 1 was confirmed to be a 22-epimer of aniduquinolone A which was given a trivial name aniduquinolone D (22S-aniduquinolone A). Surprisingly and despite that 1 and 2 are epimers, all trials to separate them into individual components using different solvent systems by normal, reversed and chiral chromatography deemed impossible (see supplemental material, Figure S1c). Careful investigation of the literature revealed many similar cases in which also epimers were unseparable. These cases such as in Jiang et al. (2019), Senadeera et al. (2018) and (Li et al. 2008). Moreover, (Schurig 1998) described clearly possible six scenarios of peak coalescence that hindering chromatographic resolution of isomers in enantioselective chromatography. Compound 1/2 were tested for their antibacterial and cytotoxic activities. Interestingly, 1/2 exhibited a significant antibacterial activity against the human-pathogenic bacterium Staphylococcus aureus (ATCC700699) with a MIC of 0.4 mg/mL using moxifloxacin as a reference antibiotic (MIC = 4 mg/mL) with no cytotoxic activity. It is worthy to mention that aniduquinolone A did not show any antibacterial activity as described in the literature (An et al., 2013), which highlights that the antibacterial activity that is found in the present study is either exclusively due to the new compound (1) or the co-existence of it with compound (2) has a synergistic antibacterial action. 4. Conclusion A new quinolone derivative (22S-aniduquinolone A, 1) along with its known isomer aniduquinolone A (2) were obtained from the EtOAc (ethyl acetate) extract of a solid rice culture of the endophytic fungus Aspergillus versicolor strain Eich.5.2.2 derived from the flower petals of the Egyptian water hyacinth Eichhornia crassipes. Structure elucidation along with the relative and absolute configurations of the isolated compounds were unambiguously determined through extensive spectroscopic analysis. Interestingly, the epimers (1/2) altogether exhibited significant antibacterial activity against the bacterium Staphylococcus aureus (ATCC700699) and with no cytotoxic effects demonstrating a specificity of the antibacterial effect.
Please cite this article as: S.S. Ebada and W. Ebrahim, A new antibacterial quinolone derivative from the endophytic fungus Aspergillus versicolor strain Eich.5.2.2, South African Journal of Botany (2019), https://doi.org/10.1016/j.sajb.2019.12.004
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Declaration of Competing Interest The authors declare that there is no conflict of interest. Supplementary materials Supplementary material associated with this article can be found in the online version at doi:10.1016/j.sajb.2019.12.004. References ndi, A., Mu € ller, W.E., Fouad, M.A., Kamel, M.S., Liu, Z., Abdelwahab, M.F., Kurt an, T., Ma Ebrahim, W., Daletos, G., Proksch, P., 2018. Induced secondary metabolites from the endophytic fungus Aspergillus versicolor through bacterial co-culture and osmac approaches. Tetrahedron Letters 59, 2647–2652. An, C.-.Y., Li, X.-.M., Luo, H., Li, C.-.S., Wang, M.-.H., Xu, G.-.M., Wang, B.-.G., 2013. 4-Phenyl-3,4-dihydroquinolone derivatives from Aspergillus nidulans MA-143, an endophytic fungus isolated from the mangrove plant Rhizophora stylosa. Journal of Natural Products 76, 1896–1901. Belofsky, G.N., Jensen, P.R., Renner, M.K., Fenical, W., 1998. New cytotoxic sesquiterpenoid nitrobenzyl esters from a marine isolate of the fungus Aspergillus versicolor. Tetrahedron 54, 1715–1724. Chen, M., Shao, C.-.L., Meng, H., She, Z.-.G., Wang, C.-.Y., 2014. Anti-respiratory syncytial virus prenylated dihydroquinolone derivatives from the Gorgonian-derived fungus Aspergillus sp. XS-20090B15. Journal of Natural Products 77, 2720–2724. ndi, A., Kurtan, T., Kalscheuer, R., Ebada, S.S., El-Neketi, M., Ebrahim, W., Ma € ller, W.E., Proksch, P., 2018. Cytotoxic secondary metabolites from the endoMu phytic fungus Aspergillus versicolor KU258497. Phytochemistry Letters 24, 88–93. Ebrahim, W., El-Neketi, M., Lewald, L.-.I., Orfali, R.S., Lin, W., Rehberg, N., Kalscheuer, R., Daletos, G., Proksch, P., 2016. Metabolites from the fungal endopyhte Aspergillus austroafricanus in axenic culture and in fungal-bacterial mixed cultures. Journal of Natural Products 79, 914–922. Elissawy, A.M., Ebada, S.S., Ashour, M.L., El-Neketi, M., Ebrahim, W., Singab, A.B., 2019. New secondary metabolites from the mangrove-derived fungus Aspergillus sp. AV2. Phytochemistry Letters 29, 1–5.
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Geiser, D., Klich, M., Frisvad, J.C., Peterson, S., Varga, J., Samson, R.A., 2007. The current status of species recognition and identification in Aspergillus. Studies in Mycology 59, 1–10. Jiang, M.-.H., Hu, Y., Jiao, L., Dong, P., Yin, S., Zhao, Z.-.M., Yang, D.-.P., Wang, D.-.M., 2019. A new labdane-type diterpenoid from Leonurus japonicus. Journal Asian Natural Products Research 21, 627–632. Lee, Y.M., Kim, M.J., Li, H., Zhang, P., Bao, B., Lee, K.J., Jung, J.H., 2013. Marine-derived Aspergillus species as a source of bioactive secondary metabolites. Marine Biotechnology 15, 499–519. Lee, Y.M., Li, H., Hong, J., Cho, H.Y., Bae, K.S., Kim, M.A., Kim, D.-.K., Jung, J.H., 2010. Bioactive metabolites from the sponge-derived fungus Aspergillus versicolor. Archives of Pharmacal Research 33, 231–235. Lee, Y.-.M., Li, J., Zhang, P., Hong, J.-.K., Lee, C.-.O., Jung, J.-.H., 2011. A cytotoxic fellutamide analogue from the sponge-derived fungus Aspergillus versicolor. Bulletin of Korean Chemical Society 32, 3817–3820. Li, S.-.H., Niu, X.-.M., Zahn, S., Gershenzon, J., Weston, J., Schneider, B., 2008. Diastereomeric stilbene glucoside dimers from the bark of Norway spruce (Picea abies). Phytochemistry 69, 772–782. Meyer, V., Wu, B., Ram, A.F., 2011. Aspergillus as a multi-purpose cell factory: current status and perspectives. Biotechnology Letters 33, 469–476. Neff, S.A., Lee, S.U., Asami, Y., Ahn, J.S., Oh, H., Baltrusaitis, J., Gloer, J.B., Wicklow, D.T., 2012. Aflaquinolones A-G: Secondary metabolites from marine and fungicolous isolates of Aspergillus spp. Journal of Natural Products 75, 464–472. stica, Junior, M.R., Pastore, G.M., 2011. Pimentel, M.R., Molina, G., Dionísio, A.P., Maro The use of endophytes to obtain bioactive compounds and their application in biotransformation process. Biotechnology research international 2011. Raja, H.A., Miller, A.N., Pearce, C.J., Oberlies, N.H., 2017. Fungal identification using molecular tools: a primer for the natural products research community. Journal of natural products 80 (3), 756–770. Schurig, V., 1998. Peak coalescence phenomena in enantioselective chromatography. Chirality The Pharmacological, Biological, and Chemical Consequences of Molecular Asymmetry 10, 140–146. Senadeera, S.P., Lucantoni, L., Duffy, S., Avery, V.M., Carroll, A.R., 2018. Antiplasmodial b-triketone-flavanone hybrids from the flowers of the Australian tree Corymbia torelliana. Journal of Natural Products 81, 1588–1597. Stone, J.K., Bacon, C.W., White, Jr, J.F., 2000. An Overview of Endophytic Microbes: Endophytism Defined in Microbial endophytes. CRC Press, pp. 17–44.
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A new antibacterial quinolone derivative from the endophytic fungus Aspergillus versicolor strain Eich.5.2.2 Sherif S. Ebadaa,b,*, Weaam Ebrahimc,* a
Department of Pharmacognosy, Faculty of Pharmacy, Ain Shams University, 11566 Abbassia, Cairo, Egypt Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Mu’tah University, 61710 Al-Karak, Jordan c Department of Pharmacognosy, Faculty of Pharmacy, Mansoura University, 35516 Mansoura, Egypt b
A R T I C L E
I N F O
Article History: Received 12 September 2019 Revised 23 November 2019 Accepted 9 December 2019 Available online xxx Edited by MG Kulkarni Keywords: Endophyte Quinolone Epimers Antibacterial activity
A B S T R A C T
Fine chromatographic fractionation of the EtOAc (ethyl acetate) extract of a solid rice culture of the endophytic fungus Aspergillus versicolor strain Eich.5.2.2 from the petals of flowers of the Egyptian water hyacinth Eichhornia crassipes revealed the isolation of a new quinolone derivative (1) along with its known isomer aniduquinolone A (2). Structure elucidation of the isolated compounds was unambiguously determined through HRESIMS (High-Resolution Electrospray Ionization Mass Spectrometry) and extensive 1D and 2D NMR (Nuclear Magnetic Resonance) spectroscopy. The relative and absolute configurations of the new compound are deduced through ROESY (Rotational Overhauser Effect SpectroscopY), optical rotation as well as biogenetic consideration. Interestingly, the epimers (1/2) altogether exhibited a significant antibacterial activity against the bacterium Staphylococcus aureus (ATCC700699) with a MIC (Minimum Inhibitory Concentration) of 0.4 mg/mL and fortunately with no cytotoxic effects highlighting a specificity of the antibacterial effect. © 2019 SAAB. Published by Elsevier B.V. All rights reserved.
1. Introduction Endophytes are those bacteria or fungi, that live inside healthy plant tissues and they do not cause any harmful effect to the host plant under normal conditions (Stone et al., 2000). These endophytes live in peace inside their hosts in the form of colonies and they are found either in inter- and/or intracellular spaces of host plants (Pimentel et al., 2011). The genus Aspergillus is one of the largest genera and comprises about 250 species (Geiser et al., 2007). Many species which belong to this genus have the ability to grow in key nutrient-poor environments (Lee et al., 2013). Furthermore, most of these species are known to be successfully grown in a wide range of temperatures, pHs and salinity (0 34%) (Meyer et al., 2011). Aspergillus versicolor is known to metabolise a plethora of unique new/bioactive secondary metabolites, which belong to terpenoids (Belofsky et al., 1998), polyketides (Lee et al., 2010) and peptides (Lee et al., 2011). Authors have previously isolated different fungal strains belonging to the genus Aspergillus from Egyptian Eichhornia crassipes but from different locations and different parts of the plant. The chemical investigation of these Aspergillus strains in either axenic, cocultures or OSMAC-modified cultures resulted in the isolation of many new and bioactive natural products. These fungal secondary metabolites belong to xanthones, dimeric-xanthones, diphenyl * Corresponding authors. E-mail addresses: [email protected], [email protected] (S.S. Ebada), [email protected] (W. Ebrahim).
ethers, alkaloids, lactones and isocoumarins (Ebrahim et al., 2016; Ebada et al., 2018; Abdelwahab et al., 2018). These results encouraged us to investigate more fungi from the same plant but from a different location in Egypt. As a part of our ongoing research aiming at recognizing new/bioactive fungal compounds from endophytes living inside the aquatic plant Eichhornia crassipes (Ebrahim et al., 2016; Ebada et al., 2018; Abdelwahab et al., 2018), an A. versicolor strain Eich.5.2.2 was isolated from the fresh healthy petals of flowers of this plant collected from the main stream of the River Nile near the location of El-Kanater El-Khayriah in Egypt, and fermented on solid rice medium. In this study, we report the structure elucidation of two epimeric quinolone derivatives (1 and 2). All isolated compounds were subjected to antibacterial and cytotoxicity (MTT) assays. 2. Materials and methods 2.1. General experimental procedure The following equipment were used in the present study; PerkinElmer-241 MC polarimeter (for optical rotation), HPLC-MS (High Pressure Liquid Chromatography-Mass Spectrometry) HP1100 Agilent Finnigan LCQ Deca XP Thermoquest (for mass spectra), FTHRMSOrbitrap (Thermo Finnigan) mass spectrometer [for HRESIMS (High Resolution Electrospray Ionization Mass Spectrometry)], Dionex Ultimate 3000 LC system (for analytical HPLC measurements) using ready-made separation columns (125 £ 4 mm, L £ ID), prefilled with Eurospher-10 C18 (Knauer, Germany), RP-HPLC (Reversed-Phase
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High Pressure Liquid Chromatography) system of LaChrom-Merk Hitachi (for HPLC separation), Merck MN silica gel 60 M (0.04 0.063 mm) or Sephadex LH-20 stationary phases (for routine column chromatography), Bruker AVANCE DMX 600 using deuterated NMR (Nuclear Magnetic Resonance) solvents (Sigma-Aldrich, Germany) [for 1D (1H and 13C NMR) and 2D NMR (chemical shifts in ppm)], TLC (Thin-Layer Chromatography) plates precoated with silica gel 60 F254 (Merck, Darmstadt, Germany) followed by detection under UV light at 254 and 365 nm wavelengths or after spraying with anisaldehyde and vanillin sulfuric acid spray reagents (for analytical purposes). Spectroscopic and deuterated grade solvents were utilized for spectroscopic and NMR measurements, respectively. All Solvents used during the experiments were predistilled before usage. ChiralpakÒ IA column with 250 mm £ 4.6 mm L £ ID, 5 mm P.S. (Daicel, Chemical Industries Ltd.) was used during chiral separation. 2.2. Fungal material The endophytic fungus A. versicolor strain Eich.5.2.2 was isolated from fresh healthy petals of flowers of E. crassipes, family Pontederiaceae, collected off the main stream of the River Nile near the location of El-Kanater El-Khayriah in Egypt (GPS coordinates: 30.191413, 31.129488) in May, 2014. Both authors authenticated the plant identity after confirmation by Prof. Dr. Ibrahim Mashaly; Professor of Taxonomy, Faculty of Science, Mansoura University, Egypt. A voucher specimen coded as SW436655 was deposited at authors’ labs at Department of Pharmacognosy, Faculty of Pharmacy, Ain Shams University, 11566 Abbassia, Cairo, Egypt (S. S. E.) and Department of Pharmacognosy, Faculty of Pharmacy, Mansoura University, 35516 Mansoura, Egypt (W. E.). The entire Fresh, healthy petals of flowers of E. crassipes were washed twice with sterilized distilled water, airdried within a laminar flow cabinet before they were surface sterilized using 70% ethanol for 2 min (twice) followed by rinsing twice with sterilized distilled water. Two minutes after surface sterilization, the entire petals were then air-dried of ethanol remains within a laminar flow cabinet and placed flat on surface of Petri dishes containing (malt-agar medium) (medium composition: 15 g/L malt extract, 15 g/ L agar in distilled water, pH 7.4 7.8) and incubated at room temperature (25 °C). Each surface of the petal was placed flat on the surface of malt-agar petri dish for 1 min and then removed from the surface of the Petri dish. These Petri dishes were used as a control to test the efficacy of surface sterilization. After that, the same entire fresh healthy petals of flowers, removed from control-petri dishes, were then cut aseptically into small pieces (ca. 1 cm in length). These small pieces were then placed in a Petri dish (malt-agar medium) containing an antibiotic to suppress bacterial growth (medium composition: 15 g/L malt extract, 15 g/Lagar, and 0.2 g/L chloramphenicol in distilled water, pH 7.4 7.8) and incubated at room temperature (25 °C). After several days, the hyphae growing from the pieces of the petals of flowers were transferred to fresh malt-agar plates containing the same medium, incubated again for 10 days. All Petri dishes including the control ones were cultivated at the same culture conditions. These experiments were done with at least ten replicates. The isolated pure endophytic fungus A. versicolor strain Eich.5.2.2 was obtained from the incised fresh healthy petals of flowers only in the experiments in which the control Petri dishes were free from any microbial growth. 2.3. Identification of fungal culture A. versicolor strain Eich.5.2.2 was identified according to a molecular biological protocol using DNA amplification and sequencing of the ITS (Internal Transcribed Spacer) region as previously reported (Ebada et al., 2018). In the field of identifications of sampled fungi, ITS region is known to be one of the markers of the fast and efficient identification of them. It is also known that ITS is the fastest-evolving
component of the rRNA cistron. Moreover, it is well-known that most mycologists in the field of fungal taxonomy and systematics use the ITS as the official barcode for fungi in order to determine species relationships. This is due to its widespread use, ease of amplification, and suitably large barcode gap. Furthermore, for species identification, the ITS can be used alone or in combination with other protein coding genes (Raja et al., 2017). An accession number KM216390 was given the fungal sequence after approval of the GenBank. The NCBI taxonomy database is the generic nomenclature and classification repository consisting of GenBank for the International Nucleotide Sequence Database Collaboration (INSDC). The ITS sequence fragment is submitted to the Basic Local Alignment Search Tool (BLAST) in GenBank to verify identity. This is regulated by certain rules, such as (1) verifying that all query sequences are representative of the entire ITS region; (2) verifying orientation and chimeric sequences through BLAST; and (3) carefully checking taxonomic annotations using only authenticated sequences (Raja et al., 2017). The fungal strain which is used in the present study was kept at authors’ labs, at Department of Pharmacognosy, Faculty of Pharmacy, Ain Shams University, 11566 Abbassia, Cairo, Egypt (S. S. E.) and Department of Pharmacognosy, Faculty of Pharmacy, Mansoura University, 35516 Mansoura, Egypt (W. E.). 2.4. Extraction and isolation The fermentation of the investigated fungal strain was achieved by static culturing at room temperature (25 °C) on solid rice medium in 1 L Erlenmeyer flask (1 L) (Ebada et al., 2018). The culture was extracted using EtOAc (ethyl acetate) (1 £ 200 mL), filtered and then the solvent was evaporated under reduced pressure using rotary evaporator. Crude EtOAC extract (109 mg) was subjected to vacuum liquid chromatography (VLC) using silica gel 60 and a gradient elution solvent system of n-hexane-ethyl acetate and dichloromethanemethanol, with an elution volume of 50 mL each, to yield 6 fractions (AV1-6). Fraction 5 (AV5, 14.0 mg), eluted with dichloromethane: methanol (DCM:MeOH; 1:4), was further purified by preparative HPLC using C18 reversed phase column and eluted with a gradient of H2O and methanol to afford 1/2 (1.4 mg). 2.5. Antibacterial assay The antibacterial test was performed using broth microdilution method. The MIC was determined, and the growth method was used for the inoculum (Ebada et al., 2018). The positive control in this assay was moxifloxacin (MIC = 4 mg/mL). The human-pathogenic bacterium Staphylococcus aureus (ATCC700699) was obtained by purchasing. 2.6. Cytotoxicity (MTT) assay A microplate 3-(4,5-dimethythiazole-2yl) 2,5-diphenyl-tetrazolium bromide (MTT) assay was used to test the cytotoxic activity of 1/2 against human epithelial colorectal adenocarcinoma (Caco-2) cell line (Elissawy et al., 2019). Each well was supplemented with 100 ml of 1/2, dissolved in 5% dimethyl sulfoxide (DMSO) and Roswell Park Memorial Institute (RPMI) tissue culture medium. Two aliquots of 100 ml tissue culture medium [minimal essential medium (MEM) + foetal bovine serum 9:1 ratio], 100 ml sterile 5% DMSO and RPMI tissue culture medium were contained in the control wells. The wells were washed with PBS (Phosphate Buffer Saline) after the required incubation period (24 h) at 37 °C and the cells were incubated with MTT solution (2 mg/ml) for 1 h at 37 °C at 100 ml per well. Supernatants were then excluded by decantation and each well was then treated with 100 ml of DMSO to dissolve the formazan crystals produced in viable metabolically active cells. The absorbance of collected elutes from the 8 wells of each experiment was measured at
Please cite this article as: S.S. Ebada and W. Ebrahim, A new antibacterial quinolone derivative from the endophytic fungus Aspergillus versicolor strain Eich.5.2.2, South African Journal of Botany (2019), https://doi.org/10.1016/j.sajb.2019.12.004
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Fig. 1. Chemical structures of 1 and 2.
Fig. 2. Key 1H
540 nm. Similar treatment was performed for control cells. The percentage of cytotoxicity was then determined as in (Elissawy et al., 2019). The cytotoxic methotrexate (IC50 = 0.7 mM) was used as a positive control in this assay. 2.7. Statistical analysis and software used The mathematical, non-linear regression (4-parameter model) was performed in cytotoxicity assay. The GraphPad Prism software was used in the statistical analysis, ChemDraw software was used to draw the chemical structures and MestreNova software was used for NMR processing. 3. Results and discussion Compound 1 (22S-aniduquinolone A, Fig. 1) was isolated as a white amorphous solid showing a clear single spot on TLC and in LRESIMS revealed pseudomolecular ion peaks at m/z 436.0 [M+H]+,
3
1
H COSY and HMBC correlations of 1 and 2.
458.1 [M+Na]+ and 434.4 [M-H] . The molecular formula of 1 was determined based on HRESIMS exhibiting pseudomolecular ion peaks at m/z 436.2116 [M+H]+ (calcd for C26H30NO5; m/z 436.2118) and m/z 458.1933 [M+Na]+ (calcd for C26H29NNaO5; m/z 458.1938) indicating the presence of thirteen degrees of unsaturation. However, the HPLC chromatogram and UV spectrum unravelled the presence of one coalescent peak with absorption maxima (λmax) at 211, 238, 279 and 324 nm which are characteristic for 4-phenyl-3,4-dihydroquinolone derivatives (Neff et al., 2012; An et al., 2013; Chen et al., 2014; Ebada et al., 2018). The 1H NMR spectrum measured in both aprotic (DMSO‑d6) and protic (MeOH-d4) deuterated NMR solvents (see supplemental material, Figure S4 and S5) distinguished that there are two sets of protons that are very close to that of aniduquinolone A rather than to aniduquinolones B or C (An et al., 2013). This notion confirmed the presence of an isomer of aniduquinolone A. Interestingly, 1H NMR spectrum clearly revealed the presence of two isomers in a ratio of 0.75:1 based on the integration of comparable protons. The 1H NMR
Table 1 1 H and 13C NMR data of 1 and 2. #
1 a
dH (multi, J value in Hz) 1-NH 2 3 4 5 6 7 8 9 10 11 12/16 13/15 14 17 18 19 20 21 22 23 24 25 26 27 4-OH a b c
dC
a,b
2 c
dH (multi, J value in Hz)
a
dH (multi, J value in Hz)
d
a,b C
10.29 (1H, br s) 3.61 (1H, s)
7.36 (1H, d, 8.3) 6.45 (1H, d, 8.3)
7.28 (2H, m) 7.32 (2H, m) 7.32 (1H, m) 6.86 (1H, d, 16.3) 6.31 (1H, d, 16.3) Hax: 2.04 (1H, m) Heq: 1.83 (1H, m) Hax: 1.83 (1H, m) Heq: 2.11 (1H, m) 4.58 (1H, br s) 1.77 (3H, s) Ha: 4.80 (1H, s) Hb: 5.07 (1H, s) 1.41 (3H, s) 3.54 (3H, s)
168.6, CO 85.8, CH 79.9, C 112.4, C 156.5, C 122.0, C 127.8, CH 107.8, CH 136.9, C 140.3, C 127.1, CH 129.5, CH 129.5, CH 121.9, CH 136.0, CH 84.7, C 39.3, CH2 31.8, CH2 82.5, CH 146.6, C 18.1, CH3 111.2, CH2 27.0, CH3 59.0, CH3
dHc (multi, J value in Hz) 10.30 (1H, br s)
3.61 (1H, d, 1.3)
3.61 (1H, s)
7.35 (1H, d, 8.3) 6.44 (1H, d, 8.3)
7.38 (1H, d, 8.3) 6.45 (1H, d, 8.3)
7.19 (2H, m) 7.34 (2H, m) 7.34 (2H, m) 6.74 (1H, d, 16.2) 6.29 (1H, d, 16.2)
7.28 (2H, m) 7.32 (2H, m) 7.32 (1H, m) 6.80 (1H, d, 16.2) 6.24 (1H, d, 16.2)
Hax: 1.99 (1H, m) Heq: 1.74 (1H, m) Hax: 1.74 (1H, m) Heq: 2.05 (1H, m) 4.37 (1H, br s)
Hax: 1.92 (1H, m) Heq: 1.83 (1H, m) Hax: 1.83 (1H, m) Heq: 2.04 (1H, m) 4.49 (1H, t, 6.9)
1.69 (3H, s) Ha: 4.76 (1H, s) Hb: 5.01 (1H, s) 1.31 (3H, s) 3.44 (3H, s) 3.95 (s)
1.73 (3H, s) Ha: 4.82 (1H, s) Hb: 5.01 (1H, s) 1.43 (3H, s) 3.55 (3H, s)
168.7, CO 86.0, CH 79.9, C 112.2, C 156.2, C 122.2, C 127.9, CH 107.8, CH 136.8, C 140.3, C 127.1, CH 129.6, CH 129.6, CH 122.2, CH 134.4, CH 84.8, C 38.7, CH2 31.6, CH2 83.4, CH 147.0, C 17.9, CH3 110.7, CH2 27.2, CH3 58.8, CH3
3.60 (1H, d, 1.3)
7.37 (1H, d, 8.3) 6.44 (1H, d, 8.3)
7.21 (2H, m) 7.34 (2H, m) 7.34 (2H, m) 6.65 (1H, d, 16.2) 6.24 (1H, d, 16.2) Hax: 1.91 (1H, m) Heq: 1.83 (1H, m) Hax: 1.74 (1H, m) Heq: 1.99 (1H, m) 4.32 (1H, t, 6.9) 1.67 (3H, s) Ha: 4.75 (1H, s) Hb: 4.96 (1H, s) 1.33 (3H, s) 3.44 (3H, s) 3.84 (s)
Measured in methanol-d4 (1H at 600 MHz and 13C at 150 MHz). Data was assigned and confirmed via gHMQC and gHMBC spectra. Measured in DMSO‑d6 (1H at 600 MHz).
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Fig. 3. Key ROESY correlations of 1 and 2.
results in DMSO‑d6 for one of the two sets (Table 1) was closely related to aniduquinolone A (2) (An et al., 2013) with characteristic proton signals spread over a wide field range including an amide proton, a monosubstituted phenyl moiety (H-12 to H-16), a 1,2,3,4-tetrasubstituted aromatic unit (H-8 and H-9), trans-olefinic protons (H-17 and H-18) and terminal downfield double bond methylene (H2-25). In addition, two oxymethine protons (H-3 and H-22), an oxygenated methyl singlet (H3-27), signals indicative to two methylenes (H2-20 and H2-21) and two singlet methyls (H3-24 and H3-26) at the upfield region were also recognized on the same spectrum. Further confirmation to the suggested identity for one of the two compounds to be aniduquinolone A (2) was obtained through extensive 2D NMR spectral analyses such as 1H 1H COSY, HMBC and HMQC (see supplemental material, Figure S6-S9) which revealed (Fig. 2) key correlations unambiguously confirming the presence of the different structural fragments of aniduquinolone A namely, 4-phenyl-3,4-dihydroquinolone together with the terpenoid side-chain containing a tetrahydrofuran ring with a terminal allyl moiety. The relative configuration of 2 was defined on the basis of coupling constant (J-values) calculations, ROESY spectral analysis (Fig. 3) and by comparison with the reported relative and absolute configuration of aniduquinolone A (An et al., 2013) which revealed identical key ROESY correlations and J-values especially to those indicating the cofacial orientation of H-3 and the phenyl ring; 3-OMe on the opposite side in addition to E-geometry for the double bond at C-17/C-18 on the terpenoid portion of the molecule which was undoubtedly confirmed through the characteristic huge coupling constant values for both H-17 and H-18 (J = 16.2 Hz). In addition, key ROESY correlations of H-22 at dH 4.49 (t, J = 6.9 Hz) to two methyl singlets (H3-24 and H3-26) and from methyl group (H3-26) to H-17 and H-18. In conclusion, one set of the observed NMR data was unambiguously related to aniduquinolone A with tentatively similar relative and absolute configurations (An et al., 2013). Due to the close similarity of the second set of proton resonances to those observed for 1 together with revealing the same molecular formula from HRESIMS, the second set was suggested to be belonging to an isomer of 1. A careful comparison of 1D and 2D NMR data (Table 1) for both sets of proton peaks resulted in the identification of a second compound which is very similar to aniduquinolone A (2) showing similar main molecular features namely, 4-phenyl-3,4-dihydroquinolone and the terpenoid side-chain. Further investigation of the second compound was obtained through careful examination of 2D NMR spectra including 1H 1H COSY, HMBC and HMQC (Fig. 2) to unambiguously assign all proton and respective carbon resonances (Table 1). The main difference noticed between the two different compounds is the proton peak representing H-22 on the tetrahydrofuran ring of the terpenoid side-chain portion of the molecule which appeared at dH 4.58 (br s) and bound to a carbon resonance at dC 82.5 in compound (1) compared to a proton peak at dH 4.49 (t, J = 6.9 Hz) bond to a carbon peak at dC 83.4 in compound (2) which suggests
that the stereochemistry of C-22 is the sole difference between 1 and 2 explaining the close similarity in their NMR data. The relative configuration of 1 was determined by careful examination of ROESY spectrum which revealed similar key correlations (Fig. 3) to those exhibited by aniduquinolone A (2). The major difference in ROESY spectrum between 1 and 2 was the absence of the key ROESY correlations from H-22 to methyl groups (H3-24 and H3-26) in 1 unlike 2 while keeping the key ROESY correlations from H-8 at dH 7.36 (d, J = 8.3 Hz) to H-18 at dH 6.31 (d, J = 16.3 Hz) and also from methyl group (H3-26) at dH 1.41 (s) to H-17 at dH 6.86 (t, J = 16.3 Hz) and H-18. These findings suggested the inversion of the stereochemistry at C-22 in 1 compared to 2. Based on the comparison of the measured NMR spectral data of 1 in DMSO‑d6 and methanol-d4 (Table 1) to the reported data for aniduquinolone A (2) (An et al., 2013) including both its relative and absolute configurations, compound 1 was confirmed to be a 22-epimer of aniduquinolone A which was given a trivial name aniduquinolone D (22S-aniduquinolone A). Surprisingly and despite that 1 and 2 are epimers, all trials to separate them into individual components using different solvent systems by normal, reversed and chiral chromatography deemed impossible (see supplemental material, Figure S1c). Careful investigation of the literature revealed many similar cases in which also epimers were unseparable. These cases such as in Jiang et al. (2019), Senadeera et al. (2018) and (Li et al. 2008). Moreover, (Schurig 1998) described clearly possible six scenarios of peak coalescence that hindering chromatographic resolution of isomers in enantioselective chromatography. Compound 1/2 were tested for their antibacterial and cytotoxic activities. Interestingly, 1/2 exhibited a significant antibacterial activity against the human-pathogenic bacterium Staphylococcus aureus (ATCC700699) with a MIC of 0.4 mg/mL using moxifloxacin as a reference antibiotic (MIC = 4 mg/mL) with no cytotoxic activity. It is worthy to mention that aniduquinolone A did not show any antibacterial activity as described in the literature (An et al., 2013), which highlights that the antibacterial activity that is found in the present study is either exclusively due to the new compound (1) or the co-existence of it with compound (2) has a synergistic antibacterial action. 4. Conclusion A new quinolone derivative (22S-aniduquinolone A, 1) along with its known isomer aniduquinolone A (2) were obtained from the EtOAc (ethyl acetate) extract of a solid rice culture of the endophytic fungus Aspergillus versicolor strain Eich.5.2.2 derived from the flower petals of the Egyptian water hyacinth Eichhornia crassipes. Structure elucidation along with the relative and absolute configurations of the isolated compounds were unambiguously determined through extensive spectroscopic analysis. Interestingly, the epimers (1/2) altogether exhibited significant antibacterial activity against the bacterium Staphylococcus aureus (ATCC700699) and with no cytotoxic effects demonstrating a specificity of the antibacterial effect.
Please cite this article as: S.S. Ebada and W. Ebrahim, A new antibacterial quinolone derivative from the endophytic fungus Aspergillus versicolor strain Eich.5.2.2, South African Journal of Botany (2019), https://doi.org/10.1016/j.sajb.2019.12.004
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Please cite this article as: S.S. Ebada and W. Ebrahim, A new antibacterial quinolone derivative from the endophytic fungus Aspergillus versicolor strain Eich.5.2.2, South African Journal of Botany (2019), https://doi.org/10.1016/j.sajb.2019.12.004