Secalonic Acid and Benzoic Acid Analogues Exhibiting Cyto toxicity against Cancer Cells Isolated from Claviceps yanagawaensis ()
1. Introduction
Members of the genus Claviceps, which includes parasitic fungi of mainly Poaceae and Cyperaceae families, produce ergot alkaloids in sclerotia (ergot) [1]. EAs (Ergot alkaloids) are the causative agent of ergotism, the main symptoms of which include convulsions and miscarriage [1]. Ergotism occurred frequently in medieval Europe and was feared as the “Fire of St. Anthony” [2]. In modern times, ergotism outbreaks were reported in India in 1975 (caused by C. fusiformis) and Ethiopia in 2001 (caused by C. purpurea) [3] [4]. Since the Middle Ages, ergotism outbreaks have caused significant health problems worldwide. Ergotism outbreaks have also affected Japan, and many miscarriages occurred in 1943 due to ingestion of bread made from bamboo grass seeds [5]. In livestock production, ergotism symptoms have also been reported in calves [6].
Our studies have focused on the genus Claviceps in Japan. Clavicepsyanagawaensis,a parasite of Zoysia japonica (family: Poaceae), has been isolated only in Japan [7] [8]. Bioactivity screening showed that a methanol extract of C. yanagawaensis cultured on rice was cytotoxic to HepG2 cells. Although ergot alkaloids are cytotoxic to cancer cells, we previously reported that C. yanagawaensis does not produce ergot alkaloids in artificial culture [9] [10]. To date, no detailed metabolite studies have been reported for C. yanagawaensis. Therefore, we screened for cytotoxic components other than EAs using methanol extracts of rice cultures of C. yanagawaensis. A new secalonic acid analogue, (-)-5-epi-F-7 (1), ergochrysin C (2), and a new benzoic acid analogue, dimethyl bigutol (3), were isolated, along with the known compounds 3,4-dihydroxy-5-(3-methyl-2-buten-1-yl)benzoic acid (4) and methyl veratrate (5) (Figure1 and FigureS1). In this paper, we report the structures of 1-3 and the cytotoxicity of 1 - 5 against five cancer cell lines (HepG2, HL60, HT29, PANC-1, and T98G) determined using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay.
2. Materials and Methods
2.1. Experimental Instruments
HPLC analysis was performed using an 1100 series HPLC system (binary pump: G1312A, auto sampler: A1329A, column compartment: G1316A, UV detector: G1314A, Agilent Technologies, Inc., Santa Clara, CA, USA) equipped with an
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Figure 1. Structures of compounds 1 - 6.
Inertsil ODS-3 column (3 μm, 2.1 × 150 mm) (GL Science Inc., Tokyo, Japan). Open column chromatography for compound isolation was performed using 100 g of Sephadex LH-20 (GE Healthcare, Chicago, IL, USA) in a 3.5 × 80 cm glass column. Preparative MPLC was performed on a Shimadzu LC-20AT and SPD-10AV instrument equipped with an ULTRA PACK ODS-S-50B column (50 μm, 26 × 300 mm) (Yamazen Corp., Osaka, Japan). Preparative HPLC was performed on a Shimadzu LC-20AT and SPD-10AV instrument equipped with a GL Science InertSustain C18 column (5 μm, 10 × 250 mm). NMR spectra were recorded on an ECA-600II instrument (1H: 600.17 MHz; 13C: 150.91 MHz) (JEOL, Tokyo, Japan). Chemical shifts for 1H- and 13C-NMR are given in parts per million (δ) relative to residual solvent signals ([δH 7.26]/[δC 77.0] for CDCl3, [δH 2.49]/[δC 39.5] for DMSO-d6 as internal standards). Mass spectra were measured on a JEOL JMS-T100LP or JMS-700. Circular dichroism (CD) spectra were recorded on a J-820 spectropolarimeter (Jasco). Liquid cultures were fermented in a Taitec Bio-Shaker BR-300LF (Taitec Corp., Saitama, Japan). Rice cultures were fermented in a Sanyo MIR-554 incubator (Sanyo Electric Co., Ltd., Osaka, Japan).
2.2. Material
The C. yanagawaensis MAFF 247556 isolate was obtained from ears of Zoysia japonica.
2.3. Fermentation
Clavicepsyanagawaensis was pre-cultured on potato dextrose agar (PDA, Nissui Pharmaceutical Co., Ltd., Tokyo, Japan). Fermentation was conducted using M102 medium (sucrose 30 g, malt extract 20 g, peptone 2 g, yeast extract 1 g, MgSO4-7H2O 0.5 g, KCl 0.5 g, KH2PO4 1.0 g, purified water 1 L, NaOH [pH 6.0]) and rice medium (rice: 750 g, tap water: 450 mL).
First, C. yanagawaensis was pre-cultured on PDA for 21 days at 25˚C, after which two pieces of cultured agar (1 × 2 cm2) were transferred into 500-mL Erlenmeyer flasks containing 200 mL of M102 medium and grown at 25˚C with shaking at 150 rpm for 7 days. Next, 10-mL aliquots of cultured M102 medium were transferred into 500-mL Roux flasks containing rice medium (each flask contained 150 g of medium as dry rice) and grown at 25˚C for 28 days.
2.4. Extraction and Isolation
Each rice culture of C. yanagawaensis was extracted with 2.5 L of methanol, and then the methanol extract was evaporated to dryness in a vacuum evaporator. The crude methanol extract (9.83 g) was suspended in 300 mL of water and extracted three times with an equal volume of ethyl acetate. The resulting ethyl acetate extract (2.1 g) was obtained by evaporation under vacuum, suspended in 300 mL of n-hexane, and extracted three times with an equal volume of acetonitrile. The solvent was removed under vacuum, and the acetonitrile extract (1461.5 mg) was separated into 11 fractions (A-K) by open column chromatography using Sephadex LH-20. The mobile phases were n-hexane:chloroform (1:4; 200 mL), chloroform:acetone (3:2; 200 mL and 1:4; 200 mL), acetone (200 mL), and MeOH (500 mL). Fraction E (136.4 mg) was purified by HPLC on an ODS column eluted with 50% acetonitrile to obtain 1 (3.5 mg) as a yellow amorphous powder. Fraction F (135.3 mg) was separated by MPLC on an ODS column eluted with 50% acetonitrile to obtain 7 fractions. Fraction F-5 (19.3 mg) was purified by HPLC on an ODS column eluted with 45% acetonitrile to obtain 2 (2.1 mg) as a yellow amorphous powder. Fraction C (77.5 mg) was purified by HPLC on an ODS column eluted with 50% acetonitrile to obtain 3 (27.3 mg) as a colorless oil. Fraction I (43.4 mg) was separated by MPLC on an ODS column eluted with 50% acetonitrile to obtain 7 fractions. Fraction I-5 (10.7 mg) was purified by HPLC on an ODS column eluted with 50% acetonitrile to obtain 3,4-dihydroxy-5-(3-methyl-2-buten-1-yl)benzoic acid (4; 7.9 mg) as a white amorphous powder [11]. Fraction B (162.6 mg) was separated by MPLC on an ODS column eluted with 50% acetonitrile to obtain 4 fractions. Fraction B-2 (34.6 mg) was purified by HPLC on an ODS column eluted with 50% acetonitrile to obtain methyl veratrate (5; 16.2 mg) as a white amorphous powder [12]. The absence of EAs in the extracts was confirmed by HPLC analysis (data not shown).
Compound 1: yellow amorphous powder.
−351.3 (c 0.1, CHCl3). UV (MeOH) λmax 204 nm (logε 5.57), 335 (logε 5.38). 1H- and 13C-NMR data, see Table 1. HR-ESI-MS (negative ion) m/z [M-H]− 653.1514 (calcd for C32H29O15: 653.1506).
Compound 2: yellow amorphous powder.
−222.02 (c 0.1, CHCl3). UV (MeOH) λmax 204 nm (logε 5.49), 335 (logε 5.23). 1H- and 13C-NMR data, see Table 1. HR-ESI-MS (negative ion) m/z [M-H]− 639.1328 (calcd for C31H27O15: 639.1350).
Compound 3: colorless oil. UV (MeOH) λmax 206 nm (logε 4.69), 273 (logε 0.67). 1H- and 13C-NMR data, see Table 2. HR-EI-MS (positive ion) m/z [M]+ 236.1413 (calcd for C14H20O3: 236.1412).
2.5. Cell Lines and Cell Culture
Human hepatoma cells (HepG2) were obtained from the National Institutes of Bio-medical Innovation, Health, and Nutrition (Osaka, Japan). Human promyelocytic leukemia cells (HL60), human pancreatic carcinoma cells (PANC-1), and human glioblastoma multiforme tumor cells (T98G) were obtained from Riken BioResource Research Center (Ibaraki, Japan). Human colon adenocarcinoma cells (HT29) were obtained from KAC Co., Ltd. (Kyoto, Japan). Cells were cultured in DMEM (Nacalai Tesque, Inc., Kyoto, Japan) or RPMI 1640 (Nacalai Tesque, Inc.) supplemented with 10% heat-inactivated fetal bovine serum (Merck, Darmstadt, Germany) and 1% Antibiotic-Antimycotic Mixed Stock Solution (Nacalai Tesque, Inc.) at 37˚C in a humid incubator containing ambient air supplemented with 5% CO2.
2.6. Cell Viability Assay
Cell viability was measured using an MTT Cell Count kit (Nacalai Tesque, Inc.) according to the manufacturer’s instructions. Briefly, cells were seeded in a 96-well plate at 5.0 × 103 cells per well; each experiment was performed with four replicates. After 48 h, the growth medium was replaced with fresh medium supplemented with test compounds (at concentrations ranging from 6.25 to 100 μM), and the plates were cultured for another 72 h. Next, 10 μL of MTT solution was added to each well, and the plates were incubated at 37˚C for 3 h and then treated with 100 μL/well of solubilization solution to dissolve the formazan crystals. The absorbance at 570 nm was measured using a microplate reader (As One Corp., Osaka, Japan). Cell viability is expressed as the relative percentage of absorbance in the experimental groups normalized to that of the negative control (no added compound) group; where appropriate, outliers were excluded based on identification using the Smirnov-Grubbs test.
2.7. Statistical Analysis
Statistical analysis was performed using the multcomp package in R, version 4.1.0 [13]. Where appropriate, values are expressed as mean ± standard deviation (SD). Comparisons to the respective negative controls were conducted using Dunnett’s multiple comparisons test. Values of p < 0.05 were considered statistically significant.
3. Results
3.1. Characterization of Isolated Compounds
Compound 1 {
−351.3 (c 0.1, CHCl3)} was obtained as a yellow amorphous powder solid. Compound 1 was revealed to have the molecular formula C32H30O15 [m/z 653.1514, [M-H]−, calcd 653.1506] based on HR-ESI-MS data. The 1H-NMR spectrum of 1 in CDCl3 (Table 1) showed four doublet aromatic protons [δH 7.57 (d, J = 8.7 Hz), 7.47 (d, J = 8.7 Hz), 6.68 (d, J = 8.7 Hz), 6.63 (d, J = 8.7 Hz)] and suggested the presence of two benzene rings with ortho coupling. The 1H-NMR spectrum also showed two doublet methyl protons [δH 1.22 (d, J = 6.6 Hz) and δH 1.17 (d, J = 5.8 Hz)], two oxygen-bearing methyl protons (δH 3.68 and 3.72), and two oxygen-bearing methine protons [δH 4.50 (d, J = 10.7 Hz) and δH 3.93 (d, J = 11.6 Hz)]. The 13C-NMR spectrum showed three ketone carbonyl (δC 198.9, 191.8, 187.1) and three sp3 quaternary carbons, four sp3 methyl carbons, two sp3 methylene carbons, two sp3 methine carbons, two oxygen-bearing sp3 methine carbons, and 16 sp2 carbons (estimated benzene rings, an olefin carbon, an oxygen-bearing olefin carbon or ester carbons).
Next, the planar structure of 1 was elucidated through detailed analyses of 2D NMR data, including 1H-1H correlation spectroscopy (COSY), heteronuclear single quantum coherence (HSQC), and heteronuclear multiple bond correlation (HMBC) spectra in CDCl3 (Figure 2 and Figures S2-S6). The 1H-1H COSY correlations for H-3 (δH 7.57)/H-4 (δH 6.68) and HMBC correlations from H-3 to
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Table 1. 1H- and 13C-NMR spectral data of compounds 1, 2, and F-7 (6) in CDCl3.
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Figure 2. Key 1H-1H COSY and HMBC correlations of compound 1.
C-1 (δC 160.6) and C-4a (δC 157.4), from H-4 to C-2 (δC 118.5), C-4a and C-9a (δC 106.6), and from OH-1 (δH 11.77) to C-1, C-2, and C-9a suggested a benzene ring with a hydroxy group at the 1-position and an oxygen attached at the 4a-position. The 1H-1H COSY correlations for H-11 (δH 1.22)/H-6 (δH 2.07), H-6 (δH 2.07)/H-7(δH 2.49, 2.94)/H-5 (δH 4.50) and HMBC correlations from H-7 to C-8 (δC 198.9) and C-8a (δC 71. 7) and from H-5 to C-10a (δC 89.4) suggested a cyclohexanone ring with a methyl group at the 6-position, a hydroxy group at the 5-position, and a carbonyl group at the 8-position. In addition, HMBC correlation from the H-5 methine and H-13 methyl to C-12 (δC 167.9) suggested partial structure A with a carboxymethyl group at the 10a-position (Figure 2).
Similarly, the 1H-1H COSY correlations for H-3’ (δH 7.47)/H-4’ (δH 6.63) and HMBC correlations from H-3’ to C-1’ (δC 159.2), from H-4’ to C-2’ (δC 117.4), C-4a’ (δC 158.3), C-9a’ (δC 106.9), and C-9’ (δC 187.1) and from OH-1’ (δH 11.73) to C-1’, C-2’, and C-9a’ suggested another benzene ring with a hydroxy group at the 1’-position and an oxygen attached at the 4a’-position. The 1H-1H COSY correlations for H-11’ (δH 1.17)/H-6’ (δH 2.42), H-6’/H-7’ (δH 2.32, 2.74), and H-5’ (δH 3.93) and HMBC correlations from H-7’ to C-8’ (δC 177.5) and C-8a’ (δC 101.5), from H-5’ to C-10a’ (δC 84.8), and from OH-8’ (δH 13.76) to C-7’ (δC 36.2), C-8’, and C-8a’ suggested a cyclohexene ring with a methyl group at the 6’-position and a hydroxy group at the 5’- and 8’-positions. In addition, HMBC correlation from the H-5’ methine and H-13’ methyl to C-12’ (δC 170.3) suggested partial structure B. The HMBC correlations from H-3 to C-2’ and from H3’ to C-2 suggested a 2-2’ bond between partial structures A and B.
These results and UV spectra (λmax 204, 264 and 335 nm) suggested that 1 is a secalonic acid analogue as a dimer of tetrahydroxanthone derivatives. The 1H- and 13C-NMR data indicated that 1 was similar to the secalonic acid analogue F-7 (6), except for the coupling constant at the 5-position (1: d, 10.7 Hz, F-7 (6): brs). Therefore, we considered 1 to be a stereoisomer of F-7 (6) (Figure 2) [14].
The configuration of 1 was also examined. The 5’-H/6’-H coupling constant of 1 was 11.6 Hz, similar to that of F-7 (6, 11.0 Hz), and these two hydrogens had a trans-diaxial configuration, but 5-H/6-H differed significantly from that of F-7 (6; brs) at 10.7 Hz. Thus, the 5-H/6-H configuration of 1 was suggested as trans, different from that of F-7 (6).
Next, we examined the configuration at the 10a- and 10a’-positions of 1. In the case of secalonic acids, it has been reported that the Cotton around 330 nm in the CD spectrum correlates with the configuration at the 10a- and 10a’-positions [14] [15] [16] [17] [18]. The CD spectrum of 1, which showed a negative Cotton around 333 nm, suggested that 1 has a configuration of 10aR and 10a’S, which differs from that of F-7 (6; 10aS, 10a’R) (Figure 3).
The configurations at positions 5, 6, and 10a and 5’, 6’, and 10a’ of 1 were estimated from the biosynthetic reaction of secalonic acids. The tetrahydroxanthone skeleton, the monomer unit of secalonic acids, is formed by spontaneous cyclization of the biosynthetic intermediate diphenylmethanone analogue A (FigureS7). During this spontaneous cyclization reaction, the carboxymethyl group at position 10a and the methyl group at position 11 are enantioselectively placed in the trans configuration due to 1,3-axial pseudoaxial interaction [19]
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Figure 3. CD spectrum of compound 1 in methanol.
[20]. Therefore, the configuration of 1 was 5S, 6R, 10aR, 5’S, 6’R, and 10a’S, and the structure of 1 was suggested to be (-)-5-epi-F-7 (Figure 1).
Compound 2 {
−222.02 (c 0.1, CHCl3)} was obtained as a yellow amorphous powder solid. Compound 2 was revealed to have the molecular formula C31H28O15 [m/z 639.1328, [M-H]−, calcd 639.1350] by HR-ESI-MS data. From the molecular formula and UV spectra (λmax 204 and 335 nm), 2 was estimated to be a secalonic acid analogue with a molecular weight lower by CH2 than compound 1. Comparing 1H- and 13C-NMR spectra in CDCl3 (Table 1, Figure 4 and Figures S8-S12) with those of 1, the 13-position methyl disappeared, and the carbon at the 8-position shifted downfield. Therefore, the structure of 2 was suggested to be a lactone ring formed between positions 10a and 8 of 1. This result was supported by detailed analysis of the 2D NMR spectra (Figure 4).
The configurations at 5-H/6-H and 5’-H/6’-H of 2 were trans-diaxial due to similarity to the coupling constants of 1. The CD spectra of 2 showed a negative Cotton around 333 nm (Figure 5). The configuration at the chiral center of 2 was estimated as 5S, 6R, 8R, 10aR, 5’S, 6’R, and 10a’S, and the structure of 2 was suggested as shown in Figure 1.
Compound 3 was obtained as a colorless oil. Compound 3 was revealed to have the molecular formula C14H20O3 [m/z 236.1413, [M]+, calcd. 236.1412] based on HR-EI-MS data. The 1H-NMR spectrum of 3 in DMSO-d6 (Table 2) showed two doublet aromatic protons [δH 6.81 (d, J = 1.4 Hz), 6.66 (d, J = 1.4 Hz)] and suggested the presence of a 1,3,4,5-tetrasubstituted benzene ring with meta coupling. One olefine proton (δH 5.18) suggested the presence of a trisubstituted olefinic moiety. Two oxygen-bearing methyl protons (δH 3.65 and 3.75) suggested two methoxy groups. The 1H-NMR spectrum also showed two broadened singlet methyl protons (δH 1.67 and δH 1.68), one oxygen-bearing methylene proton [δH 4.37 (d, J = 5.5 Hz)], and a doublet methylene-attached olefin [δH 3.23 (d, J = 7.6 Hz)]. The 13C-NMR spectrum showed two sp3 methyl carbons, two oxygen-bearing sp3 methyl carbons, one sp3 methylene carbon and one oxygen-bearing sp3 methylene carbon, and eight sp2 carbons (estimating a
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Figure 4. Key 1H-1H COSY and HMBC correlations of compound 2.
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Figure 5. CD spectra of compound 2 in methanol.
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Table 2. 1H- and 13C-NMR spectral data of compound 3 in DMSO-d6.
benzene ring and two olefin carbons).
The gross structure of 3 was elucidated through detailed analyses of 2D NMR data, including 1H-1H COSY, HSQC, and HMBC spectra in DMSO-d6 (Figure 6 and Figures S13-S17). The 1H-1H COSY correlations for OH-12 (δH 5.09)/H-12
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Figure 6. Key 1H-1H COSY and HMBC correlations of compound 3.
(δH 4.37) and HMBC correlations from H-4 (δH 6.81) to C-2 (δC 145.0), C-3 (δC 152.1), C-5 (δC 138.1), C-6 (δC 119.2), and C-12 (δC 62.9), from H-6 (δH 6.66) to C-2, C-4 (δC 108.9), and C-12, and from H-12 to C-4, C-5, and C-6 suggested a benzene ring with a hydroxy methyl group at the 5-position. The HMBC correlations from H-13 (δH 3.75) to C-3 and from H-14 (δH 3.65) to C-2 suggested methoxy groups at the 2- and 3-positions. The 1H-1H COSY correlations for H-7 (δH 3.23)/H-8 (δH 5.18) and HMBC correlations from H-11 (δH 1.68) to C-10 (δC 25.5) and from H-10 (δH 1.67), C-8 (δC 123.2), C-9 (δC 131.2), and C-11 (δC 17.6) suggested the presence of a dimethylallyl group. The HMBC correlations from H-7 (δH 3.23) to C-1 (δC 134.1), C-2, and C-6 and from H-6 to C-7 (δC 28.1) suggested that the dimethylallyl group was attached at the 1-position. Therefore, the structure of 3 was determined as a 3-methoxy derivative (Figure 1) of methyl bigutol isolated from the mycoparasite Verticilliumbiguttatum (Table S1) [21].
3.2. Effect of the Compounds on the Viability of Cancer Cell Lines
MTT assays were performed using HepG2, HL60, HT29, PANC-1, and T98G cell lines to investigate the cytotoxicity of the isolated compounds against cancer cells (Figure 7). Compound 1 decreased the viability of HepG2, HT29, PANC-1, and T98G at 100 μM, especially T98G at 12.5 μM in a concentration-dependent manner. Compound 2 slightly decreased the viability in HepG2 and PANC-1 cells at 100 μM. Compound 3 decreased the viability of HepG2, HL60, HT29, and PANC-1 at 100 μM, especially HL60 at 6.25 μM in a concentration-dependent manner. Compound 4 decreased the viability of HepG2, HL60, and PANC-1 cells at 100 μM, reducing the viability of PANC-1 cells to <50% relative to control cells. Compound 5 at 100 μM did not affect the viability of any of the cancer cell lines examined.
4. Discussion
In this study, secalonic acid analogues 1 and 2 and benzoic acid analogues 3 - 5 were isolated from methanol extracts of C. yanagawaensis. These compounds
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Figure 7. Effect of isolated compounds (1 - 5) and the methanol extracts (Ext.) on the viability of cancer cells, as assessed using the MTT assay. The methanol extract of C. yanagawaensis cultured on rice (Ext., 400 μg/mL) and Doxorubicin (DOX, 0.5 μM) were employed as a positive (cell death–inducing) control. Data are shown as the mean ± SD (n = 4). Comparisons with the negative control (no added compound) were performed using Dunnett’s multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001. Conditions without an asterisk exhibited no significant difference. (A) HepG2; (B) HL60; (C) HT29; (D) PANC-1; (E) T98G.
exhibited cytotoxicity against several cancer cell lines, including HepG2, HT29, PANC-1, and T98G cells.
Secalonic acids are structurally diverse, and polyketide synthases are involved in biosynthesis [19] [22]. Compound 1 has the same planar structure as F-7 (6) isolated from Aspergillusaculeatus MBT 102, but detailed analysis of NMR and CD spectra suggested that the configuration of compound 1 differs from that of F-7 (6). Secalonic acids with dimeric tetrahydroxanthone derivative structures are produced by fungi in the Aspergillus, Pyrenochaeta, and other genera, in addition to Claviceps. The monomers (blennolide A, B, [-]-blennolide A, B, and their derivatives with chiral carbons at C-5, C-6, and C-10a) constituting the dimer differ according to the genus of the producing fungi (FigureS18) [23]. Secalonic acids produced byAspergillus and Claviceps species commonly produce blennolide A (5S, 6S, 10aR) as a component, whereas Claviceps and Pyrenochaeta commonly produce (-)-blennolide B (5S, 6R, 10aS), Aspergillus produces blennolide B (5R, 6S, 10aR), and Pyrenochaeta produces (-)-blennolide A (5R, 6R, 10aS) [19] [20] [24]. Therefore, Aspergillus produces secalonic acid B; blennolide A dimer, secalonic acid D; blennolide B dimer and secalonic acid F; blennolide A and blennolide B dimer (FigureS18). In contrast, Claviceps produces secalonic acid A: (-)-blennolide B dimer, secalonic acid B; blennolide A dimer, secalonic acid C; blennolide A and (-)-blennolide B dimer (FigureS18). The conformation of 1 was found to be 5S, 6R, 8R, 10aR, 5’S, 6’R, and 10a’S, the same conformation as the dimer of (-)-blennolide B and its derivatives, in agreement with the abovementioned studies.
Compound 2 is similar to 1 and estimated to have the same 5S, 6R, 10aR, 5’S, 6’R, and 10a’S configuration based on NMR and CD spectra. Secalonic acid analogues with a lactone ring have been reported, including ergoflavin, ergochrysin A, and ergochrysin B, and secalonic acid analogues with a lactone ring and hydroxy group attached at the 8-position, as in 2, were isolated from Claviceps by Lünne et al. [17]. Lactone cyclization of ergoflavin, ergochrysin A, and ergochrysin B is predicted to proceed via the addition of H2O to the 8-8a olefin and condensation of the hydroxy group at the 8-position with the carboxymethyl group at the 10a-position (FigureS19) [25] [26]. In contrast, it is expected that the lactone ring of 2 cyclizes in a different manner and has a hydroxy group attached at the 8-position.
MTT assay results showed that 1 was cytotoxic to T98G cells, whereas 3 was cytotoxic to HL60 cells and 4 to PANC-1 cells (Figure 7). The secalonic acid F analogue F-7 (6) has been shown to induce apoptosis and inhibit microtubule formation in MDA-MB-231 human breast cancer cells [14]. Secalonic acid A inhibits topoisomerases I and II in CCF-STTG-1 glioma cells and induces apoptosis in Jurkat human T-cell leukemia-derived cells [17] [27]. In this study, secalonic acid (-)-5-epi-F-7 (1), isolated from C. yanagawaensis, was shown to be cytotoxic to cancer cells and therefore expected to exhibit similar activity.
5. Conclusion
Novel cytotoxic secalonic acid analogues (-)-5-epi-F-7 (1), ergochrysin C (2), and dimethyl bigutol (3) were isolated from methanol extracts of C. yanagawaensis rice cultures. These results highlight the potential of members of the genus Claviceps to produce bioactive substances. We plan to carry out further investigations of ergot alkaloids and other bioactive substances produced by C. yanagawaensis sclerotia to better characterize the possibility of poisoning in nature.
Fund
This work was supported by JSPS KAKENHI grant number 16K07238.
Supplementary
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Table S1. NMR data for compound 3 and methyl bigutol in CDCl3.
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Figure S1. HPLC chromatogram (210 nm) of methanol extract of C. yanagawaensis rice culture.
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Figure S5. 1H-1H COSY spectrum of 1 in CDCl3.
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Figure S7. Spontaneous heterocyclization of hydroxycyclohexenones stereochemically controlled by 1,3-pseudoaxial interaction.
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Figure S18. Structures of major secalonic acids and their monomers isolated from Claviceps and Aspergillus.
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Figure S19. Predicted scheme of the lactonization of ergoflavin, ergochrysin A, and ergochrysin B [25].