Introduction

Anthraquinones and their derivatives comprise a large group of natural quinoid compounds. The chemical diversity of these compounds and their biological properties have attracted the attention of the pharmaceutical, textile, dyeing, and food colorant industries. Anthraquinones exhibit a wide range of biological activities including antibacterial, antifungal, antiviral, insecticidal, and anticancer [1, 2]. Anthraquinones are widespread in fungi, especially endophytic fungi [3,4,5,6].

Insect pathogenic fungi are known to be potential sources of novel bioactive substances; however, only a few species of ant pathogens have been chemically explored [7, 8]. As part of our ongoing program on bioactive compounds from insect pathogenic fungi, we selected an ant pathogen (Cordyceps morakotii, strain BCC 56811) for chemical investigation, since the crude extract exhibited antibacterial activity against Bacillus cereus (IC50 3.13 µg ml−1) and HPLC and 1H NMR spectra showed profiles suggestive of novel compounds. Nine anthraquinone compounds were isolated, including new morakotins A–E (1–5), lunatin (6) [9], rheoemodin (7) [10], YM187781 (8) [11], and bislunatin (9) [12] (Fig. 1), together with (1′S)-6-(1-hydroxypentyl)-4-methoxypyran-2-one [13], 9,11-dehydroergosterol peroxide [14], and cerevisterol [15]. We report here the isolation, structure elucidation, and biological activities of these compounds.

Fig. 1
figure 1

Chemical structures of compounds 1–9

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Results and discussion

Morakotin A (1) was obtained as an orange solid. The molecular formula was established as C16H12O7 by HRESIMS, in combination with 13C NMR spectroscopy. The UV spectrum showed the typical absorption pattern of an anthraquinone chromophore at 233, 286, 320, and 443 nm. The IR spectrum exhibited major absorption bands for hydroxyl (3261 cm−1), non-chelated quinone carboxyl (1622 cm−1), and chelated quinone carbonyl (1606 cm−1). The 1H and 13C NMR spectra indicated the presence of two D2O exchangeable protons, one singlet aromatic proton, two meta-coupling aromatic protons, two methoxy groups, five oxygenated quaternary carbons, and two carbonyl groups (Table 1). The downfield hydroxyl protons at δH 12.17 and 12.28 indicated H-bonding with a carbonyl, which corresponded to the low absorption frequency of a chelated quinone carbonyl group at 1606 cm−1 in the IR spectrum. The HMBC spectrum showed correlations from H-4 to C-2/C-3/C-4a/C-9a/C-10, H-5 to C-7/C-10, H-7 to C-5/C-8, 2-OCH3 to C-2, and 3-OCH3 to C-3. These data revealed the structure of morakotin A as depicted in Fig. 1. The correlations from 3-OCH3 to H-4 and 2-OCH3 in NOESY spectrum also supported the position of the methoxy groups at C-2 and C-3. Therefore, morakotin A (1) was determined to be 1,6,8-trihydroxy-2,3-dimethoxyanthracene-9,10-dione.

Table 1 NMR spectroscopic data for compounds 1–3 (500 MHz for 1H and 125] MHz for 13C)
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The UV and IR spectra of morakotin B (2) were almost identical to those of morakotin A (1). The 1H NMR spectra of these two compounds were also similar except for the presence of one more methoxy signal in morakotin B (2). HRESIMS gave the molecular formula C17H14O7, 14 amu higher than that of morakotin A (1), which indicated that morakotin B (2) has one additional methoxy group. The HMBC correlation from these methoxy protons (δH 3.93) to C-6 (Fig. 2) led to the deduction that the hydroxyl group at C-6 in morakotin A (1) is replaced by a methoxy group in morakotin B (2). This assignment was further confirmed by the correlations from 6-OCH3 to H-5 and H-7 in the NOESY spectrum. Consequently, morakotin B (2) was identified as 1,8-dihydroxy-2,3,6-trimethoxyanthracene-9,10-dione.

Fig. 2
figure 2

Selected HMBC, and NOESY correlations of compounds 2 and 5

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The presence of 15 carbon signals in the 13C NMR spectrum and the quasimolecular ion peak in the HRESIMS (m/z 601.0632 [M−H]−, calcd. for C30H17O14 601.0624) led to the conclusion that morakotin C (3) was a symmetrical anthraquinone dimer. When compared with that of the known co-metabolite bislunatin (9) [12], the 1H NMR spectrum of morakotin C (3) showed the absence of one of the meta-coupled aromatic protons and the presence of one more carbonyl chelated hydroxyl group (δH 13.04) per monomeric unit. The IR spectrum exhibited only a chelated quinone carbonyl band at 1598 cm−1. In combination with the molecular formula, which indicated the presence of two more oxygen atoms than that of bislunatin, the planar structure of morakotin C (3) was established as shown in Fig. 1. The HMBC correlations from H-2 to C-4/C-9a, H-7 to C-5/C-8a, 1-OH to C-2/C-1/C-9a, 5-OH to C-5/C-6/C-10a, 8-OH to C-7/C-8/C-8a, and 6-OCH3 to C-6 also supported the structural features of morakotin C (3) as described above. Morakotin C (3) was, thus, determined as 1,1′,3,3′,5,5′,8,8′-octahydroxy-6,6′-dimethoxy-4,4′-bianthracene-9,9′,10,10′-tetraone.

The HRESIMS data established the molecular formula of morakotin D (4) as C29H16O12. The UV and IR spectra were similar to those of the known co-metabolite bislunatin (9) [11]. The NMR spectroscopic data revealed the presence of four hydroxyl groups, two pairs of meta-coupling aromatic protons, two singlet aromatic protons, one methoxy group, and four carbonyl groups (Table 2), indicating an unsymmetrical dimeric anthraquinone. The correlations from H-2 to C-4/C-9a, H-5 to C-7/C-10/C-8a, H-7 to C-5/C-8a, 1-OH to C-1/C-2/C-9a, 8-OH to C-7/C-8/C-8a, and OCH3 to C-6 in the HMBC spectrum and the correlations from methoxy protons to H-5 and H-7 in the NOESY spectrum established one of the monomer units as the known co-metabolite lunatin (6) [9]. The other part was found to resemble that of the known co-metabolite rheoemodin (7) [10], which is joined to another unit at C-4 and C-4′ positions, as deduced by the HMBC correlations from H-2′ to C-4′ /C-9a′, H-5′ to C-7′/C-10′/C-8a′, H-7′ to C-5′/C-8a′, 1′-OH to C-1′/C-2′/C-9a′, 8′-OH to C-7′/C-8′/C-8a′. The chemical shifts at C-4 and C-4′ were also consistent with those of other 4,4′ coupled bianthraquinone derivatives [11, 12]. Therefore, morakotin D (4) was identified as 1,1′,3,3′,6′,8,8′-heptahydroxy-6-methoxy-4,4′-bianthracene-9,9′,10,10′-tetraone.

Table 2 NMR spectroscopic data for compounds 4 and 5 (500 MHz for 1H and 125 MHz for 13C)
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Morakotin E (5) gave the same molecular formula to that of morakotin D (4), i.e., C29H16O12, as deduced from HRESIMS. Comparison of the 1H and 13C NMR spectra between these two compounds revealed the significant shift of two singlet aromatic protons (from δH 6.68, 6.69 in compound 4 to 7.28, 7.29 in compound 5) and two quaternary carbons (from δC 123.6, 124.0 in compound 4 to 113.8, 113.9 in compound 5). The HMBC correlations from H-4 to C-2/C-9a/C-10, H-5 to C-7/C-10, H-7 to C-5/C-8a, 1-OH to C-1/C-9a, 8-OH to C-7/C-8, 6-OCH3 to C-6, H-4′ to C-2′/C-9a′/C-10′, H-5′ to C-8a′/C-10′, H-7′ to C-5′ and the NOESY correlations from 6-OCH3 to H-5 and H-7 led to the deduction that Morakotin E (5) has the same monomer units as those of morakotin D (4) with the connection at C-2 and C-2′. The chemical shifts at the junction (C-2 and C-2′) were in agreement with those obtained from other 2,2′ coupled bianthraquinone compounds [16]. Morakotin E (5) was, thus, determined to be 1,1′,3,3′,6′,8,8′-heptahydroxy-6-methoxy-2,2′-bianthracene-9,9′,10,10′-tetraone.

The absolute configurations of bianthraquinone compounds were determined by application of the exciton chirality method [17,18,19,20]. For morakotin C (3), CD coupling was found with strong negative first and positive second Cotton effect due to the long axis of the anthraquinone moieties, indicating the two chromophores were twisted in an anticlockwise manner. Therefore, the absolute configuration was suggested to be R. The opposite CD cotton curve, positive first and negative second Cotton effect, was observed for the known co-metabolite bislunatin (9), which showed that the anthraquinone chromophores were twisted in a clockwise manner; however, the optical rotation value was low \(([\alpha]^{25}_{\mathrm{D}} + 44, c \,{\mathrm{0.61}} \,{\mathrm{dioxane}})\) when compared with that reported in the literature (\([\alpha]^{26}_{\mathrm{D}} \)+1690, c 0.25, EtOH) [12], suggesting the non-equivalent mixture of atropisomers slightly rich in S-isomer. Morakotins D (4), E (5), and compound 8 exhibited low values of optical rotations and weak CD signals, which suggested that these compounds were also non-equivalent mixtures of atropisomers.

The seven known compounds were identified as lunatin (6) [9], rheoemodin (7) [10], YM187781 (8) [11], bislunatin (9) [12], (1′S)-6-(1-hydroxypentyl)-4-methoxypyran-2-one [13], 9,11-dehydroergosterol peroxide [14], and cerevisterol [15] by analysis of their spectroscopic data and their physical properties, which were identical to those reported in the literature data.

Compounds 3, 4, and 6–9 were assayed for antimicrobial activity against various bacterial and fungal strains (Mycobacterium tuberculosis H37Ra, B. cereus, Staphylococcus aureus, Acinetobacter baumannii, Pseudomonas aeruginosa, and Candia albicans) and cytotoxicity against mammalian cancer cell lines (MCF-7, and NCI-H187) and noncancerous Vero cells (Table 3). Compounds 1, 2, and 5 were not tested owing to insufficient sample. Compounds 3, 7, 8, and 9 exhibited weak to moderate anti-TB and antifungal activities. For antibacterial activity, compounds 4 and 8 were active against both B. cereus and S. aureus (MIC 3.13–25 µg ml−1), whereas compounds 3 and 9 were active only against B. cereus (MIC 12.5 and 3.13 µg ml−1, respectively). Only compound 7 was active against A. baumannii (MIC 12.5 µg ml−1). All compounds showed cytotocixity against all tested cell-lines (IC50 5.37–47.97 µg ml−1), except that compound 4 was inactive against Vero cells.

Table 3 Biological activities of compounds 3, 4 and 6–9
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Materials and methods

General experimental procedures

Melting points were measured using a Metler MP90 melting point apparatus and are uncorrected. Optical rotation measurements were conducted by using a JASCO P-1030 digital polarimeter. UV and FT-IR spectra were recorded on an Analytic Jena Spekol 1200 spectrophotometer and a Bruker Alpha spectrometer. The CD spectra were recorded on a JASCO J-180 spectropolarimeter. NMR spectra were recorded on Bruker Advance III 400 and Bruker Advance 500 spectrometers. ESITOF MS data were obtained on a Bruker micrOTOF mass spectrometer. Column chromatography was performed on silica gel 60 (70–230 Mesh ASTM, Merck). HPLC experiments were performed using Dionex-Ultimate 3000 series equipped with a binary pump, an autosampler and a diode array detector.

Fungal material

The fungus used in this study was isolated from an ant (Hymenoptera), collected from the evergreen forest in Chiang Mai Province, Thailand. The organism was deposited in the BIOTEC Culture Collection (BCC) as BCC 56811 on 16 November 2012. This fungus was identified as C. morakotii of the Class Sordariomycetes, order Hypocreales, family Cordycipitaceae, on the basis of morphology, by Miss Kanoksri Tasanathai, BIOTEC, and was confirmed by the molecular identification method using the rDNA internal transcribed spacer region (ITS rDNA: GenBank accession number MH828333), analyzed by Dr. Nattawut Boonyuen, BIOTEC.

Fermentation, extraction, and isolation

C. morakotii BCC 56811 was cultured on potato dextrose agar at 25 °C for 30 days. The agar was cut into pieces (1 × 1 cm) and inoculated into 8 × 250-ml Erlenmeyer flasks containing 25 ml of potato dextrose broth (PDB medium contained potato starch 4.0 g and dextrose 20.0 g in 1.0 l distilled water). After incubation at 25 °C for 7 days on a rotary shaker (200 rpm), each primary culture was transferred into a 1-l Erlenmeyer flask containing 250 ml of the same medium and incubated under the same conditions for 7 days. Each 25 ml portion of the secondary culture was transferred into 80 × 1-l Erlenmeyer flasks containing 250 ml of PDB and was statically incubated at 25 °C for 53 days.

After filtration of the culture, the cells were macerated in MeOH (1 l) for 3 days, and then in CH2Cl2 (1 l) for 3 days. The MeOH and CH2Cl2 extracts were combined and evaporated under reduced pressure. Water (800 ml) was added and the mixture was extracted with hexanes (3 × 800 ml), followed by EtOAc (3 × 800 ml). Trituration of the dark brown solid (3.05 g) obtained from the EtOAc extract with MeOH followed by filtration gave a dark red solid (1.35 g), which was further purified by preparative HPLC using a reverse phase column (SunFire C18 OBD, 10 μm, 19 × 150 mm, step gradient elution with 40−70% MeCN/H2O) to furnish compounds 2 (0.6 mg), 3 (38.3 mg), 4 (6.6 mg), 6 (25.4 mg), 7 (3.9 mg), 8 (18.9 mg), and 9 (17.2 mg). The filtrate (1.69 g) was fractionated using Sephadex LH 20 (4.0 × 36 cm) and eluted with 100% MeOH to provide 8 fractions (A1–A8). Compounds 6 (49.6 mg) and 7 (43.4 mg) were obtained from fractions A5 and A6, respectively. Further purification of fraction A2 by preparative HPLC (step gradient elution with 20−100% MeCN/H2O) afforded 6-(1-hydroxypentyl)-4-methoxypyran-2-one (7.9 mg). Fraction A3 was subjected to preparative HPLC (step gradient elution with 70−100% MeCN/H2O) to give compounds 2 (1.3 mg) and 9 (0.5 mg). Compounds 1 (0.6 mg) and 6 (2.1 mg) were obtained from fraction A4 after purification by preparative HPLC (step gradient elution with 45−75% MeCN/H2O). Purification of fraction A7 by preparative HPLC (step gradient elution with 55−75% MeCN/H2O) yielded compound 8 (0.7 mg) and 9 (0.3 mg). Fraction A8 was purified by preparative HPLC (step gradient elution with 40−80% MeCN/H2O) to afford compounds 4 (5.4 mg), 5 (1.4 mg), 8 (1.6 mg), and 9 (0.3 mg).

The hexanes extract of the mycelium (19.49 g) was subjected to silica gel column chromatography (7.3 × 22.5 cm), using 2% MeOH/CH2Cl2 as an eluent, to give six fractions (B1–B6). Compound 2 (3.3 mg) and cerevisterol (63.9 mg) were obtained from fraction B2 and B6, respectively, after trituration with MeOH followed by filtration. Fraction B4 was fractionated by Sephadex LH 20 (4.5 × 60 cm) and eluted with 100% MeOH to provide 4 subfractions (B4-1–B4-4). Further purification of subfraction B4-2 by preparative HPLC (step gradient elution with 55−75% MeCN/H2O) yielded 9,11-dehydroergosterol peroxide (12.6 mg). Trituration of subfraction B4-4 with MeOH followed by filtration furnished compound 6 (25.0 mg) as an orange solid. Consecutive purification of fraction B5 by Sephadex LH 20, eluted with 100% MeOH, followed by preparative HPLC (step gradient elution with 35−70% MeCN/H2O) gave compound 7 (1.8 mg) and 9 (0.9 mg).

The culture broth was extracted with EtOAc (3 × 20 l) and evaporated to dryness, leaving a brown gum (7.69 g). The crude extract was consecutively purified by Sephadex LH 20, eluted with 100% MeOH, followed by preparative HPLC to provide compounds 3 (16.0 mg), 6 (9.2 mg), and 7 (13.1 mg).

Morakotin A (1): Orange solid; UV (MeOH) λmax (log ε) 233 (3.96), 286 (4.13), 320 (3.85), 443 (3.79) nm; IR (ATR) νmax 3261, 2924, 1622, 1606, 1571, 1452, 1400, 1364, 1325, 1274, 1167, 1136 cm-1; 1H and 13C NMR spectroscopic data in acetone-d6, Table 1; HRMS (ESITOF) m/z 339.0480 [M + Na]+ (calcd. for C16H12O7+Na, 339.0475).

Morakotin B (2): Orange solid; UV (MeOH) λmax (log ε) 233 (3.78), 283 (3.91), 319 (3.63), 439 (3.65) nm; IR (ATR) νmax 3451, 2925, 1627, 1604, 1461, 1415, 1368, 1270, 1186, 1164, 1137 cm-1; 1H and 13C NMR spectroscopic data in DMSO-d6, Table 1; HRMS (ESITOF) m/z 353.0616 [M + Na]+ (calcd. for C17H14O7+Na, 353.0632).

Morakotin C (3): Dark red solid; m.p. (dec.) 200.0–202.0°C; \([\alpha]^{25}_{\mathrm{D}}\)−184 (c 0.22, dioxane); UV (dioxane) λmax (log ε) 271 (4.24), 284 (4.25), 312 (4.29), 487 (4.27), 504 (4.28), 539 (4.20) nm; CD (dioxane) [θ] (nm) 0 (276), −161200 (264), 0 (255), +19300 (251), 0 (249); IR (ATR) νmax 3434, 2924, 1598, 1466, 1403, 1307, 1271, 1227 cm-1; 1H and 13C NMR spectroscopic data in DMSO-d6, Table 1; HRMS (ESITOF) m/z 601.0632 [M − H]− (calcd. for C30H17O14, 601.0624).

Morakotin D (4): Red solid; \([\alpha]^{25}_{\mathrm{D}}\)+11 (c 0.19, dioxane); UV (dioxane) λmax (log ε) 272 (3.99), 298 (4.14), 457 (3.98), 485 (3.93) nm; IR (ATR) νmax 3407, 2924, 1622, 1601, 1461, 1380, 1240 cm-1; 1H and 13C NMR spectroscopic data in DMSO-d6, Table 2; HRMS (ESITOF) m/z 579.0531 [M + Na]+ (calcd. for C29H16O12+Na, 579.0534).

Morakotin E (5): Orange solid; \([\alpha]^{25}_{\mathrm{D}}\)−22 (c 0.16, dioxane); UV (dioxane) λmax (log ε) 262 (4.07), 295 (4.37), 321 (4.07), 451 (4.09) nm; IR (ATR) νmax 3363, 2924, 1620, 1597, 1440, 1383, 1313, 1253 cm-1; 1H and 13C NMR spectroscopic data in DMSO-d6, Table 2; HRMS (ESITOF) m/z 555.0561 [M − H]− (calcd. for C29H15O12, 555.0569).

Biological assays

Growth inhibitory activity against M. tuberculosis H37Ra and cytotoxicity to Vero cells (African green monkey kidney fibroblast, ATCC CCL-81) was performed by using the green fluorescent protein (GFP) based method [21, 22]. The resazurin microplate assay was used to evaluate anti-B. cereus (ATCC 11778), anti-C. albicans (ATCC 90028) and cytotoxicity against MCF-7 cells (human breast cancer, ATCC HTC-22) and NCI-H187 cells (human small-cell lung cancer, ATCC CRL-5804) [23]. Antibacterial activity against S. aureus (ATCC 29213), A. baumannii (ATCC 19606) combined with PAβN, and P. aeruginosa (K 2733) was evaluated by using the standard protocols published by the Clinical and Laboratory Standard Institute [24, 25].