Original Articles
A new asteltoxin analog with insecticidal activity from Pochonia suchlasporia TAMA 87
2020 年 45 巻 2 号 p. 81-85
詳細
2020 年 45 巻 2 号 p. 81-85
A new asteltoxin analog, named asteltoxin H (1), was isolated by the solid-state fermentation of the fungus Pochonia suchlasporia var. suchlasporia TAMA 87. The chemical structure of 1 was deduced by spectroscopic methods, including 1D and 2D NMR, HRESIMS, and UV-Vis analyses. Compound 1 showed insecticidal activity against prepupae of the blowfly, Lucilia sericata, with an LD50 value of 0.94 µg/mg prepupal body weight.
During the last century, agrochemicals have helped increase food production.1) However, worldwide surveys have reported that these agrochemicals have an adverse effect on the environment and non-target organisms.2,3) Therefore, considerable research has been recently focused on determining new active compounds that are safer for the environment and human health.
Microorganisms are among the promising sources of useful natural products that are relatively safe for agricultural application. Microorganisms have been extensively screened for the production of bioactive secondary metabolites that can be used as antimicrobials, herbicides, and insecticides.4) While screening Pochonia suchlasporia var. suchlasporia TAMA 87 (hereinafter described as P. suchlasporia TAMA 87) for bioactive compounds, we found that the solid-state fermentation (SSF) culture of this fungus produced a novel polyhydroxylated pyrrolizidine alkaloid, pochonicine.5) This compound showed the potential inhibition of GlcNAcases from various organisms, including plants, mammals, fungi, and insects.5) Since GlcNAcase inhibition might affect insect growth, the MeOH extract of the SSF culture of P. suchlasporia TAMA 87 was examined for its effect on insect growth.
Our preliminary study revealed that the MeOH extract of the SSF culture of P. suchlasporia TAMA 87 showed insecticidal activity against the blowfly, Lucilia sericata. Insecticidal assay of the fractions obtained by partitioning the MeOH extract between EtOAc and water indicated that most of the insecticidal activity was recovered in the EtOAc-soluble fraction but not in the pochonicine-containing water-soluble fraction. Hence, the insecticidal activity was attributed to compound(s) other than pochonicine.
In this work, we purified the MeOH extract of the SSF culture of P. suchlasporia TAMA 87 by solvent extraction, silica gel chromatography, and preparative HPLC to obtain active compound 1. Extensive spectroscopic analysis and comparison with previously reported data showed that 1 has a structure analogous to that of asteltoxin6) and has, therefore, been named asteltoxin H (Fig. 1). The insecticidal activity of 1 was evaluated against the blowfly, L. sericata.
Column chromatography was performed on silica gel having particle sizes of 150–425 µm (Wakogel C-100, FUJIFILM Wako Pure Chemical Co., Osaka, Japan). Optical activity was measured with a P-2200 polarimeter (JASCO, Tokyo, Japan). Analytical HPLC was done using an Inertsil® ODS-3 column (φ4.6×250 mm, 5 µm) (GL Sciences, Tokyo, Japan) and a HITACHI system (Hitachi, Tokyo, Japan) equipped with an L-2300 column oven, L-7455 diode array detector, and an L-2130 pump. The solvent used was 55% MeOH, the flow rate was 1.0 mL/min, and the injection volume was 5 µL. Preparative HPLC was carried out using an Inertsil® ODS-3 column (φ20×250 mm, 5 µm) (GL Sciences, Tokyo, Japan) and a Waters system (Nihon Waters, Tokyo, Japan) equipped with a Waters 616 pump, a Waters 600 controller, a Waters 486 detector set at 254 nm, a Waters CHM column oven, and a HITACHI D-2500 integrator (Hitachi, Tokyo, Japan). The solvent used was 65% MeOH, the flow rate was 10 mL/min, and the injection volume was 1.8 mL. UV spectra were obtained using a UV-Vis 2550 spectrophotometer (Shimadzu, Tokyo, Japan). NMR spectra were recorded on a Varian NMR System 600 spectrometer (Varian, Palo Alto, CA, USA) operating at 600 MHz for 1H and 150 MHz for 13C NMR. The solvent used was deuterated chloroform. HRESIMS spectra were measured using a micrOTOF mass spectrometer (Bruker Daltonics GmbH, Bremen, Germany). All other chemicals were obtained commercially.
2. Fungal strain and insectsThe fungal strain P. suchlasporia TAMA 87 was isolated from a soil sample in Machida, Tokyo.5) The blowfly, L. sericata, was obtained from the Japan Maggot Company (Okayama, Japan) and reared in laboratory conditions at room temperature. The rearing plastic cup, containing beef liver and blowfly eggs, was placed onto a 5 cm-thick layer of sawdust. When the larvae reached the prepupal stage (4–5 days after hatching), they stopped feeding and dropped into the sawdust. The prepupae were then collected and used for the insecticidal assay.
3. Fermentation and isolationThe P. suchlasporia TAMA 87 culture was grown on a yeast extract agar slant. An aliquot of the stock culture was sub-cultured in a Petri dish with a diameter of 9 cm that contained a malt yeast agar peptone (MYA-P) medium consisting of 1% (w/v) glucose, 1% (w/v) malt extract (Becton, Dickinson and Company, Sparks, MD, USA), 0.1% (w/v) yeast extract (Nacalai Tesque Inc., Kyoto, Japan), 0.1% (w/v) Hipolypepton (Nihon Seiyaku, Tokyo, Japan), and 2% (w/v) agar (Nacalai Tesque Inc., Kyoto, Japan). After eight days of incubation at 22°C, discs 7 mm in diameter were cut from the growing edge of the fungal colony. Six such fungal discs were inoculated into six different Erlenmeyer flasks (200 mL), each containing an autoclaved rolled barley-based solid medium consisting of 9 g of rolled barley (Kyowa Seibaku Co., Kanagawa, Japan), 1 g of peeled oats (DoggyMan H.A Co., Ltd., Osaka, Japan), 10 mL of water, 20 mg of yeast extract (Nacalai Tesque Inc., Kyoto, Japan), 10 mg of sodium L-tartrate dihydrate, and 10 mg of KH2PO4. Fermentation was performed for 22 days in static conditions at 22°C. The fungal culture was extracted by adding 50 mL of MeOH to each flask, shaking the flasks well, and keeping them overnight at room temperature. The mixture was filtered and concentrated in vacuo to give an MeOH extract (2922 mg). The MeOH extract was partitioned between EtOAc and water. The EtOAc-soluble fraction was concentrated in vacuo to yield an EtOAc extract (550 mg), which was subsequently purified by silica gel column chromatography (φ2.0×26.15 cm) using hexane/EtOAc (90 : 10, 50 : 50, 30 : 70, 30 : 70, 30 : 70, 10 : 90 (v/v)) and EtOAc/MeOH (98 : 2, 70 : 30, 0 : 100 (v/v)) to afford nine fractions (F1–F9). F3 (45 mg), which was eluted by the 30 : 70 (v/v) hexane/EtOAc mixture, was further purified by preparative HPLC (stationary phase: Inertsil® ODS-3 column (φ20×250 mm, 5 µm); solvent: 55% MeOH; flow rate: 10 mL/min) to obtain 1 (6.1 mg).
Compound 1: yellowish amorphous solid; [α]D19+15.4 (c 0.55, MeOH); UV λmax (MeOH) nm (log ε): 378 (4.46), 283 (4.45), 212 (3.95); 1H and 13C NMR are shown in Table 1; HRESIMS m/z: 425.1933 [M+Na]+ (Calcd. for C23H30NaO6: 425.1935). The spectral data of 1 are given in the Supplemental Figs. S1–S8.
| Position | Compound 1a) | Asteltoxinb) | ||
|---|---|---|---|---|
| δC | δH (mult., J, int.) | δC | δH (mult., J, int.) | |
| 1 | 11.4 | 1.06 (t, 7.5, 3H) | 11.5 | 1.05 (t, 7.5, 3H) |
| 2 | 21.7 | 1.56 (m, 2H) | 21.8 | 1.56 (m, 2H) |
| 3 | 89.8 | 4.31 (dd, 8.1, 4.8, 1H) | 89.9 | 4.31 (dd, 8.0, 5.0, 1H) |
| 4-OH | 1.65 (brs, 1H) | —c) | ||
| 4 | 81.1 | 81.2 | ||
| 5 | 62.3 | 62.4 | ||
| 6 | 111.8 | 5.29 (s, 3H) | 111.9 | 5.29 (s, 3H) |
| 7-OH | 1.79 (d, 4.4, 1H) | —c) | ||
| 7 | 78.7 | 3.72 (dd, 3.5, 2.6, 1H) | 78.8 | 3.73 (d, 2.0, 1H) |
| 8 | 83.1 | 4.74 (brs, 1H) | 83.2 | 4.74 (brs, 1H) |
| 9 | 128.7 | 5.85 (dd, 15.3, 4.8, 1H) | 129.4 | 5.86 (dd, 15.0, 5.0, 1H) |
| 10 | 134.3 | 6.64 (dd, 15.3, 10.8, 1H) | 134.2 | 6.63 (dd, 15.0, 11.5, 1H) |
| 11 | 135.6 | 6.48 (dd, 14.8, 10.8, 1H) | 136.5 | 6.49 (dd, 15.0, 11.0, 1H) |
| 12 | 133.3 | 6.40 (dd, 14.8, 11.1, 1H) | 133.0 | 6.40 (dd, 15.0, 11.0, 1H) |
| 13 | 133.4 | 7.11 (dd, 15.0, 11.1, 1H) | 135.5 | 7.16 (dd, 15.0, 11.0, 1H) |
| 14 | 120.5 | 6.29 (d, 15.0, 1H) | 120.4 | 6.40 (d, 15.0, 1H) |
| 15 | 152.9 | 154.3 | ||
| 16 | 112.8 | 108.6 | ||
| 17 | 144.5 | 6.98 (s, 1H) | 170.7 | |
| 18 | 124.3 | 89.2 | 5.51 (s, 1H) | |
| 19 | 163.1 | 163.9 | 1.97 (s, 3H) | |
| 20 | 18.0 | 1.39 (s, 3H) | 18.1 | 1.38 (s, 3H) |
| 21 | 16.2 | 1.19 (s, 3H) | 16.2 | 1.18 (s, 3H) |
| 22 | 15.2 | 2.05 (s, 3H) | 9.1 | 1.97 (s, 3H) |
| 23 | 16.8 | 2.09 (s, 3H) | 56.4 | 3.83 (s, 3H) |
a) 1H and 13C NMR spectra for 1 were measured at 600 and 150 MHz, respectively. b) 1H and 13C NMR spectra for asteltoxin were measured at 500 and 125 MHz, respectively; data from Ref. 6). c) Chemical shifts were not reported.
Insecticidal assay was carried out by employing agar gels as a matrix for test samples. The agar gels were prepared as follows: 380 µL of the sample solution in MeOH and 2820 µL of a 2.27% (w/v) agar solution (60°C) were placed in five wells of a 6-well plate and mixed uniformly. This mixture was kept at room temperature to allow the agar gels (φ36 mm) containing the test sample to solidify. Five doses of the test sample (measured in µg per mg of prepupal body weight) were tested. An agar gel prepared with MeOH only, instead of the sample solution, was used as a negative control.
To perform the insecticidal assay, agar gels were placed in separate covered Petri dishes (φ90 mm). Ten prepupae were laid on each agar gel. A filter paper sprayed with 0.26 g of water was placed correctly between the Petri dish and its lid to maintain the humidity. Petri dishes were kept in a CN-25C incubator (Mitsubishi Electric Engineering Co. Ltd., Tokyo, Japan) in dark conditions at 22±1°C for 7 days. Prepupae deaths were counted every day. The death of a prepupa was ascertained by the loss of mobility. The dose of the test sample that killed half of the prepupae on the seventh day was determined as its LD50. Each dose, as well as the control, was tested twice.
We obtained 1 as a yellowish amorphous solid. A sodium adduct peak at m/z 425.1933 in the positive ion mode of HRESIMS allowed the assignment of the molecular formula C23H30O6 to 1. The MeOH solution of 1 showed UV absorption maxima at 378, 283, and 212 nm, which indicated the presence of conjugated double bonds.
1H NMR data (Table 1) revealed the presence of five methyl groups, one methylene group, four methine protons, seven olefinic protons, and two hydroxy protons. Comparing spectroscopic data of 1 to those of asteltoxin,6,7) 1 is considered to be an analog of the asteltoxin. Hence, the position numbering of 1 has been given according to that of asteltoxin. The six olefinic proton signals resonating at δH 5.85 (H-9), 6.64 (H-10), 6.48 (H-11), 6.40 (H-12), 7.11 (H-13), and 6.29 (H-14) indicate the presence of a moiety with conjugated double bonds (Fig. 2). Considering the COSY correlations and the proton coupling constants, double bonds in this moiety should be in an all-trans conformation.
Another moiety was detected by analyzing HMBC correlations between H-1/C-2, C-3; H-2/C-1, C-3; H-3/C-1, C-4; H-20/C-3, C-4, C-5; H-21/C-4, C-5, C-6, C-7; and H-6/ C-3, C-5, C-7, C-8, C-21. These correlations confirmed the presence of a 2,8-dioxabicyclo [3.3.0] octane ring, a moiety characteristic of asteltoxins.7) This was supported by COSY correlations between spin systems H-1/H-2, H-2/H-3, H-3/4-OH, H-7/7-OH, and H-7/H-8. Furthermore, the COSY correlation between peaks at δH 5.85 (H-9) and 4.74 (H-8) suggested a linkage between the 2,8-dioxabicyclo [3.3.0] octane ring and the moiety with conjugated double bonds.
Detailed analysis of the HMBC spectrum revealed that the methyl proton H-23 was correlated to carbons C-16, C-17, and C-18 and the ester carbonyl carbon C-19. The HMBC spectrum also showed correlations from H-22 to the carbons sp2 C-15, C-16, and C-17. These data, along with an HSQC correlation observed between the olefinic methine proton H-17 and C-17, establish the presence of an α-pyrone moiety. That the α-pyrone moiety and the conjugated double bond moiety are connected was deduced from the HMBC correlation between H-14/C-15 and C-16. Accordingly, the structure of 1 was established as a new asteltoxin that differs from asteltoxin in its α-pyrone moiety. The α-pyrone moiety in asteltoxin has a methyl and a methoxy substituent, whereas the α-pyrone moiety in 1 has two methyl and no methoxy substituents.
The relative configuration of 1 was determined based on NOESY experiments. The NOESY correlations of H-3/H-20, H-6/H-8 and H-21, and H-7/H8, suggested that H-3 and H-20 lie on the same side of the ring, and H-6, H-7, H-8 and H-21 are on the same side of the ring. While no significant correlation was observed between H-20 and H-21 as well as between H-20 and H-2, it is suggested that H-3 and H-20 are on the opposite side of H-6, H-7, H-8 and H-21. Also, the NOE correlations of H-9/H-11, H-10/H-12, H-11/H-13, and H-12/H-14 confirmed the all-trans conformation of the moiety with conjugated double bonds (Fig. 2). This assignment showed that the relative stereochemistry of 1 is identical to that of asteltoxin. Moreover, 1 shows positive optical rotation with an [α]D19 value of +15.4 (c 0.55, MeOH), similar to that of asteltoxin, which has an [α]D23 value of +20.0 (c 1.15, MeOH).7) On the basis of these data, we concluded that the absolute configuration of 1 is as shown in Fig. 1.
The planar structure of 1 is the same as that of AB5529 (CAS Registry Number: 220383-76-6), an insecticidal metabolite isolated from Paecilomyces sp. AB5529, as shown in the patent application by Maruyama and colleagues.8) However, the application does not mention the stereochemistry of AB5529. Moreover, the specific rotation value of AB5529 has been mentioned as being [α]D25+32 (c 0.5, MeOH),8) which is considerably different from that of 1. Therefore, we conclude that AB5529 and 1 are two different molecules, and propose 1 to be named asteltoxin H.
Asteltoxins are a class of compounds featuring an α-pyrone attached to a 2,8-dioxabicyclo [3.3.0] octane ring via a triene linker, except for asteltoxin G which contains a tetrahydrofuran ring instead of a 2,8-dioxabicyclo [3.3.0] octane ring (Fig. 3). Asteltoxin, the first member of asteltoxins, was isolated from Aspergillus stellatus Curzi.7) Asteltoxins B, C, and D were produced by Pochonia bulbillosa 8-H-28.9) Asteltoxins E and F were purified from Aspergillus sp. SCSIO XWS02F40,6) and asteltoxin G was obtained from Aspergillus ochraceopetaliformis.10) These compounds share the same α-pyrone moiety (4-methoxy-5-methyl-2-pyrone-based structure), but asteltoxin H has another type of α-pyrone moiety (3,5-dimethyl-2-pyrone-based structure), indicating a new structural variation of asteltoxins (Fig. 3). Therefore, asteltoxin H would be considered to be the first member of a new series of asteltoxins.
Asteltoxin H was examined for its insecticidal activity against prepupae of the blowfly, L. sericata. By employing agar gel as the matrix for test samples, the LD50 value of asteltoxin H was found to be 0.94 µg/mg prepupal body weight. This is the first report, to the best of our knowledge, of an insecticidal asteltoxin compound produced by genus Pochonia sp. Previously, asteltoxin C isolated from Pochonia bulbillosa 8-H-28 was reported to have potent antiproliferative activity against NIAS-SL64 cells, an insect cell line derived from the fat body of Spodoptera litura larvae.9) However, the mechanism of this activity has not been mentioned.
Much of the research on asteltoxins has focused on their antiviral activity11) and antiproliferative activity against cancer cells.12) The detailed mechanisms of these activities have not yet been reported. Kawai et al.13) reported that asteltoxin inhibited the energy transfer system of rat liver mitochondria, primarily by depressing the activity of Mg2+-ATPase. The insecticidal action of asteltoxin H against blowfly prepupae might be a result of ATPase inhibition. Several natural insecticidal products, such as decaleside I (O-α-D-gulofuranosyl-(1→4)-O-β-D-altropyranosyl-(1→1)-α-D-psicofuranose) and decaleside II (O-β-D-galactopyranosyl-(1→4)-O-α-D-altropyranosyl-(1→1)-α-D-psicofuranose), have been reported to inhibit Na+, K+–ATPase of the housefly.14)
The authors express their gratitude to the Division of Instrumental Analysis, Department of Instrumental Analysis and Cryogenics, Advanced Science Research Center, Okayama University, for NMR and MS measurements. In addition, the MEXT scholarship provided to S.S. is gratefully acknowledged.
The online version of this article contains supplementary materials (Supplemental Figs. S1–S8), which are available at https://www.jstage.jst.go.jp/browse/jpestics/.
