Article pubs.acs.org/jnp
Cite This: J. Nat. Prod. 2018, 81, 1143−1147
Identification of Lycopodium Alkaloids Produced by an UltravioletIrradiated Strain of Paraboeremia, an Endophytic Fungus from Lycopodium serratum var. longipetiolatum Kan’ichiro Ishiuchi,*,† Dai Hirose,‡ Takuma Suzuki,† Waka Nakayama,† Wen-Ping Jiang,§ Orawan Monthakantirat,⊥ Jin-Bin Wu,§ Susumu Kitanaka,‡ and Toshiaki Makino† †
Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1, Tanabe-Dori, Mizuho-ku, Nagoya 467-8603, Aichi, Japan School of Pharmacy, Nihon University, 7-7-1, Narashinodai, Funabashi, 274-8555, Chiba, Japan § School of Pharmacy, China Medical University, No. 91, Hsueh-Shih R., Taichung 40402, Taiwan ⊥ Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand ‡
S Supporting Information *
ABSTRACT: 12-epi-Lycopodine (1), a Lycopodium alkaloid, along with lycopodine (2) and huperzine A (3), were discovered in the mycelium of Paraboeremia sp. Lsl3KI076, a UV-irradiated strain of Paraboeremia sp. Lsl3, an endophytic fungus from Lycopodium serratum Thunb. var. longipetiolatum Spring. Additionally, a trace of 1 was isolated from Phlegmariurus nummulariifolius (Blume) Ching, and the structure was elucidated on the basis of spectroscopic data. This is the first report proving that a new naturally occurring Lycopodium alkaloid can be obtained from an endophytic fungus. Lycopodium alkaloids1 are known to be a structural diversity of constituents that are produced by clubmosses, Lycopodiaceae plants. Lycopodium alkaloids possess unique heterocyclic ring systems such as C16N1, C16N2, and C27N3 and have attracted great interest from biological,2 biogenetic,3 and chemical4 viewpoints. Although over 400 Lycopodium alkaloids have been characterized, their biosynthetic pathways have not been fully revealed yet.3 Huperzine A (3),2 a Lycopodium alkaloid isolated from Huperzia serrata, is a potent, reversible, and selective acetylcholinesterase inhibitor and, thus, has promise in treating some of the symptoms of Alzheimer’s disease. There is a need for a more productive source to generate a larger supply of huperzine A.5 Recently, endophytic fungi producing huperzine A (3) have been isolated from H. serrata,6 although there are no other reports of Lycopodium alkaloids being produced by fungi. Since a paclitaxel-producing fungus, Taxomyces andreanae, from Taxus brevifolia was discovered by Stierle et al.,7 some endophytic fungi producing plant-derived secondary metabolites, including camptothecin, vincristine, podophyllotoxin, hypericin, and emodin, have been reported,8 however, these are produced in extremely small amounts. During our investigations into new Lycopodium alkaloids,9 we have successfully identified 12-epi-lycopodine (1), a new naturally © 2018 American Chemical Society and American Society of Pharmacognosy
occurring Lycopodium alkaloid produced by Paraboeremia sp. Lsl3KI076, obtained as a UV-radiation-mutated isolate of Paraboeremia sp. Lsl3, which had been isolated from Lycopodium serratum var. longipetiolatum,9c along with lycopodine (2)10 and huperzine A (3).2
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RESULTS AND DISCUSSION Colonies of the fungus Lsl3 grown on oatmeal agar, potato dextrose agar, and malt extract agar are shown in Figure S1. The molecular phylogenetic trees, traced from both the rRNA internal transcribes spacer (ITS) region and the β-tubulin gene, strongly supported the view that this fungus belongs to the Received: July 20, 2017 Published: April 20, 2018 1143
DOI: 10.1021/acs.jnatprod.7b00627 J. Nat. Prod. 2018, 81, 1143−1147
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genus Paraboeremia, which could be clearly distinguished from other related genera by molecular phylogenetic analysis (Figure S2). As Lsl3 does not form pycnidia on agar plates, the micromorphological comparison of this genus with known species could not be made. Protoplasts were prepared from mycelia of Paraboeremia sp. Lsl3. Approximately 106 protoplasts were spread on potato dextrose agar (PDA) plates, irradiated by ultraviolet (UV) light, and then incubated in the dark at 28 °C. Under these conditions, only approximately 10% of the protoplasts survived. From this procedure, 211 UV-irradiated strains (Paraboeremia sp. Lsl3KI001−077 and TS001−134) were obtained, which were cultured and analyzed using electrospray ionization (ESI)MS. ESIMS analysis of a basic extract of Paraboeremia sp. Lsl3KI076 indicated the presence of 1 showing a protonated molecule at m/z 248 [M + H]+ not observed from the wildtype fungus (Figure S3). HRESIMS established the molecular formula, C16H25NO, which is the same as that of lycopodine (2),10 a representative member of the C16N1-type Lycopodium alkaloid family. However, LC-MS analysis revealed that the retention time of 1 was not identical with that of 2 (Figure 1).
subjected to an amino-silica gel column and C18 HPLC to afford 12-epi-lycopodine (1). 12-epi-Lycopodine (1) [[α]21D −34.7 (c 1.0, MeOH)] showed a protonated molecule at m/z 248 (M + H)+ in the ESIMS, and the molecular formula, C16H25NO, was established by HRESIMS. 1H and 13C NMR data (Table 1) and the HSQC Table 1. NMR Spectroscopic Data (C5D5N) for 12-epiLycopodine position
δC,a type
1a 1b 2 3 4 5 6a 6b 7 8 9a 9b 10 11 12 13 14 15 16
47.5, CH2 22.1, CH2 17.7, CH2 55.1, CH 207.0, C 47.4, CH2 35.5, CH 34.2, CH2 47.9, CH2 22.8, CH2 22.8, CH2 41.5, CH 64.0, C 26.6, CH2 24.6, CH 22.3, CH3
δH,b multi (J in Hz) 3.12, 2.91, 2.09, 2.13, 3.28,
dd (12.5, 4.5) ddd (13.0, 13.0, 4.0) 1.65, ndc 1.55, ndc d (12.0)
2.65, 2.38, 1.94, 1.25, 3.19, 2.87, 2.18, 1.65, 2.55,
dd (16.0, 6.5) dd (16.0, 1.5) ndc ndc dd (12.5, 4.0) ndc 1.69, ndc 1.28, ndc brd (12.5)
HMBC 3 1a 1a, 2 2, 6, 14 4, 6b
6a, 6b, 8 6a, 6b, 14, 16 11
9a, 10 6, 8, 11, 14 1a, 3, 4, 11, 14 4, 16 8, 14 14
1.91, 1.52, ndc 1.44, m 0.75, d (6.0)
a 125 MHz. b500 MHz. cnd: J-values were not determined because of overlapping with other signals.
spectrum revealed 16 carbon signals due to one carbonyl group, one nonprotonated sp3 carbon, four sp3 methine groups, nine sp3 methylene groups, and one sp3 methyl group. Among them, one nonprotonated sp 3 carbon (δC 64.0) and two sp3 methylenes (δC 47.9; δH 3.19 and 2.87, and δC 47.5; δH 3.12 and 2.91) were attributed to those attached to a nitrogen atom. The planar structure of 1 was elucidated by the analysis of 2D NMR data including the 1H−1H COSY, TOCSY, HSQC, and HMBC data. Three structural units, namely, a (C-1−C-4), b (C-6−C-8, C-8/C-15, and C-14−C-16), and c (C-9−C-12), were disclosed by 1H−1H COSY and TOCSY data (Figure 2). Connectivities of C-1, C-9, and C-13 through a nitrogen atom were revealed by HMBC correlations for H-1a (δH 3.12) and H-9a (δH 3.19) to C-13 (δC 64.0). HMBC cross-peaks of H11b (δH 1.28) to C-13, and H-8 (δH 1.25) to C-12 (δC 41.5),
Figure 1. Single-ion chromatograms of (a) the basic extract of L. serratum var. longipetiolatum, (b) the basic extract of Paraboeremia sp. Lsl3KI076, and (c) lycopodine (2) in condition 1. All LC traces were monitored at m/z = 248.
Interestingly, 1 was almost undetectable in L. serratum var. longipetiolatum,9 the host plant of Paraboeremia sp. Lsl3 (Figure 1). Lsl3KI076 subsequently lost the ability to produce 1. This strain was cultivated on a large scale but did not produce 1, although the production of lycopodine (2) and huperzine A (3) was observed by LC-MS analyses (Figures S4 and S5). As a result, insufficient alkaloid 1 was obtained from Paraboeremia sp. Lsl3KI076 to elucidate the structure by spectroscopic techniques. The presence of alkaloid 1 in other unidentified Lycopodiaceae plants (Th1−Th9) was investigated. The results of LC-MS analyses showed 1 was present in the basic extract of Th1 identified as Phlegmariurus nummulariifolius (Blume) Ching (Figure S6). The MeOH extract of P. nummulariifolius was partitioned between EtOAc and 3% tartaric acid. The water-soluble fraction after the pH was adjusted to pH 10 with saturated Na2CO3(aq) was partitioned with CHCl3. CHCl3-soluble materials were
Figure 2. Selected 2D NMR correlations for 1. 1144
DOI: 10.1021/acs.jnatprod.7b00627 J. Nat. Prod. 2018, 81, 1143−1147
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revealed that C-7 (δC 35.5) is connected to C-13 through C-12. An HMBC correlation for H-3a (δH 2.13) to C-13 suggested that C-4 (δC 55.1) is connected to C-13. Connectivities of C-4 and C-6 through C-5 were indicated by HMBC cross-peaks of H-6b (δH 2.38) to C-4 and C-5 (δC 207.0). Finally, an HMBC correlation for H-14b (δH 1.52) to C-13 revealed that C-14 (δC 26.6) connected to C-13. The planar structure of 1, shown in Figure 2, is the same as that of lycopodine (2). The relative stereochemistry of 1 was deduced from NOESY data (Figure 3). The chairlike shape of the piperidine ring (C-
Figure 4. Single-ion chromatograms of (a) the basic extract of Paraboeremia sp. Lsl3KI076, (b) 12-epi-lycopodine (1), and (c) lycopodine (2) in condition 1. All LC traces were monitored at m/z = 248.
Thasana et al. reported that 12-epi-lycodoline N-oxide exhibited strong acetylcholine esterase (AChE) inhibitory activity (IC50 0.59 μM).15 We prepared 12-epi-lycopodine Noxide (4) from 1 treated with m-chloroperbenzoic acid (mCPBA) and evaluated the AChE inhibitory activities16 of 1 and 4. However, these two compounds did not show similar inhibitory activities (IC50 > 100 μM). In this study, it was discovered that Paraboeremia sp. Lsl3KI076, a UV-irradiated strain of an endophytic fungus from L. serratum var. longipetiolatum, produces three Lycopodium alkaloids, namely, 12-epi-lycopodine (1), lycopodine (2), and huperzine A (3). These results infer that Paraboeremia sp. Lsl3 possesses not only the genes involved in the biosynthesis of Lycopodium alkaloids but also those similar to the Lycopodium alkaloid-producing host plant. The genomic analysis of the Lsl3 strain is currently in progress now.
Figure 3. Selected NOESY correlations and relative stereochemistry for 1.
1−C-4, C-13, and N) was revealed from NOESY correlations for H-1b/H-14a. NOESY cross-peaks of H-14b/H-9b and H11a suggested that the cyclohexane ring (C-7−C-8, C-12−C15) and the piperidine ring (C-9−C-13 and N) have a chairlike structure and that these two rings are cis-fused with each other. The equatorial position of the methyl group at C-15 was revealed by a NOESY correlation for H-14b/H3-16. NOESY cross-peaks of H-4/H-6a and H-12 indicated that the cyclohexanone ring (C-4−C-7, C-12−C-13) has a pseudochair form. The relative stereochemistry of 1 is shown in Figure 3. The absolute configuration of 1 was elucidated using electronic circular dichroism (ECD) analysis. The ECD spectrum of 1 displayed a negative Cotton effect at 293 nm by the n → π* transition of the keto group (Figure S17), the same as 12-epi-lycodoline.11 Thus, the absolute configuration of 1 was established as 4S, 7S, 12S, 13R, and 15R. Subsequently, the basic extract of Paraboeremia sp. Lsl3KI076 containing 1 was analyzed by LC-MS, using 12epi-lycopodine isolated from P. nummulariifolius as a reference. All the peaks detected at m/z 248 under three different elution conditions were observed at the same retention time with those of 12-epi-lycopodine (Figure 4 and Figure S18). Thus, the Lycopodium alkaloid produced by Paraboeremia sp. Lsl3KI076 was identified as 12-epi-lycopodine. 1 was reported as a synthetic derivative in the course of structure elucidation of lycodoline,12 but it has never been isolated from nature. There are over a hundred lycopodine-type species in Lycopodium alkaloids, and almost all possess the same stereochemistry as lycopodine. The 12-epi-lycopodine-type alkaloid is very rare, and only three examples of the latter, 12-epi-lycodoline,11 12epi-lycodoline N-oxide,13 and 11β-hydroxy-12-epi-lycodoline,14 have been reported.
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EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotation was recorded on a JASCO P-2100 polarimeter. The ECD spectrum was recorded on a JASCO J-725 spectropolarimeter. IR data were recorded on a Shimadzu IR Affinity-1 spectrometer. NMR spectra were recorded on an Agilent Varian VNS500 spectrometer. Chemical shifts (ppm) were referenced to the residual solvent peaks (δH 7.21 and δC 135.5 for pyridine-d5). Positive-mode ESITOFMS was obtained on a JEOL JMS-T100LP AccuTOF LC-plus 4G spectrometer using a sample dissolved in MeOH. LC-MS analyses were performed using a tandem mass spectrometer (Quattro Premier XE, Waters) connected to an ACQUITY UPLC system (Waters) on a 2.1 × 100 mm i.d., 1.8 μm, ACQUITY UPLC HSS T3 column (Waters Corp., Milford, MA, USA) at room temperature. The mobile phase consisted of H2O containing 0.1% HCO2H (A) and CH3CN containing 0.1% HCO2H (B). A linear gradient elution was applied under four conditions as follows: 0−1.0 min hold at 5% B, then a 1.0−8.0 min linear gradient to 100% B, followed by 8.0−9.0 min hold at 100% B, then a 9.0−10.0 min linear gradient to 5% B, followed by 10.0−11.0 min hold at 5% B (condition 1). Plant Material. Lycopodium serratum Thunb. var. longipetiolatum Spring was collected in Miaoli County, Taiwan.9c Phlegmariurus nummulariifolius (Blume) Ching was purchased at a flower market in Bangkok, Thailand. The botanical identification of P. nummulariifolius was performed by Dr. Santi Watthana at the School of Biology, 1145
DOI: 10.1021/acs.jnatprod.7b00627 J. Nat. Prod. 2018, 81, 1143−1147
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Compound 1 in basic extract of Paraboeremia sp. Lsl3KI076: ESIMS m/z 248 [M + H]+; HRESIMS m/z 248.2016 [M + H]+ (calcd for C16H26NO, 248.2014). Large-Scale Culture, Extraction, and Metabolite Separation of Paraboeremia sp. Lsl3KI076. Paraboeremia sp. Lsl3KI076 was cultivated in PDA (20 mL × 100) at 28 °C for 14 days, and the mycelia were extracted with MeOH. The extract was partitioned between EtOAc and 3% tartaric acid. The water-soluble fraction after the pH was adjusted to pH 10 with saturated Na2CO3(aq) was partitioned with CHCl3. The CHCl3 layer was separated by an aminosilica gel column (n-hexane/EtOAc, 1:0 → 1:1, then CHCl3/MeOH, 1:0 → 1:1) to obtain 24 fractions. Two fractions (fr. 5 and fr. 14) eluted with n-hexane/EtOAc (4:1) and CHCl3/MeOH (30:1), respectively, were analyzed by LC-MS analyses. Isolation of 12-epi-Lycopodine (1). The whole plant of Phlegmariurus nummulariifolius (740 g, wet weight) was extracted with MeOH. The extract was partitioned between EtOAc and 3% tartaric acid. Water-soluble materials adjusted to pH 10 with saturated Na2CO3(aq) were extracted with CHCl3. CHCl3-soluble materials were subjected to an amino-silica gel column (n-hexane/EtOAc, 1:0 → 1:1, and then CHCl3/MeOH, 1:0 → 1:1). A fraction eluted with nhexane/EtOAc (4:1) was purified by C18 HPLC (CAPCELL PAK C18 AQ (SHISEIDO), 5 μm, 10 mm i.d. × 250 mm, solvent MeCN/ MeOH/H2O/TFA, 7:0:93:0.01 → 0:100:0:0.01, flow rate 2.5 mL/ min) to afford 12-epi-lycopodine (1, 0.003%). 12-epi-Lycopodine (1): colorless, amorphous solid; [α]21D −34 (c 1.0, MeOH); ECD (MeOH) λ (Δε) 229 (−2.82), 293 (−4.55) nm; IR (ATR) νmax 2951, 2924, 1701 cm−1; 1H NMR (pyridine-d5, 500 MHz) and 13C NMR (pyridine-d5, 125 MHz), see Table 1; ESIMS m/ z 248 [M + H]+; HRESIMS m/z 248.2008 [M + H]+ (calcd for C16H26NO, 248.2014).
Institute of Science, Suranaree University of Technology, Nakhon Ratchasima, Thailand, and by Dr. Kazumi Fujikawa at the Kochi Prefectural Makino Botanical Garden, Kochi, Japan. A voucher specimen (No. 20130801) is stored at Nagoya City University. Isolation of Paraboeremia sp. Lsl3. Fresh stems of L. serratum var. longipetiolatum were first sterilized in 70% ethanol for 1 min, subsequently immersed for 20 min in a 180 mL solution of 1% sodium hypochlorite and two drops of Tween 20, and finally rinsed three times with sterile distilled water. The stems were then placed on Murashige and Skoog (MS) basal medium (Duchefa Biochemie) containing 3% sucrose and 0.3% Gelrite. Two weeks later, mycelia were observed growing from the cut edge. The hyphal tips with agar were transferred to the PDA media (Difco) and incubated at 28 °C in total darkness. The fungal isolate is stored at Nagoya City University. Identification of Paraboeremia sp. Lsl3. Fungal identification was based on morphological observations and molecular phylogenetic analyses. Genomic DNA was extracted from mycelia that had been cultured on 2% malt extract agar overlaid with a cellophane membrane using an ISOPLANT kit (Nippon Gene, Tokyo, Japan). Polymerase chain reactions (PCR) were performed using a Quick Taq HS DyeMix (Toyobo, Osaka, Japan). The rRNA ITS region (ITS1−5.8S−ITS2) was amplified with primer pairs ITS4/ITS5.17 The β-tubulin gene was amplified with primer pairs T118/Bt2b.19 PCR products were purified with a FastGene Gel/PCR extraction kit (Nippon Genetics, Tokyo, Japan) according to the manufacturer’s instructions. Purified PCR products were sequenced by Fasmac Co., Ltd. (Kanagawa, Japan). The sequences determined in this study were deposited in the DNA Data Bank of Japan (DDBJ) (accession nos. LC310980 and LC310981). For phylogenetic analysis, related sequences were obtained from the DDBJ after BLASTN searches.20 The multiple alignments of nucleotide sequences were generated with MAFFT ver. 7 software.21 A phylogenetic tree was constructed using maximum likelihood methods22 with a best fit nucleotide substitution model based on the lowest Bayesian Information Criterion score. In order to estimate clade support, the bootstrap procedure23 was employed with 500 replicates. These analyses were carried out using MEGA7.24 Morphological observations were carried out using the procedure provided in refs 25 and 26. Preparation of UV-Irradiated Strains of Paraboeremia sp. Lsl3. Paraboeremia sp. Lsl3 was initially cultured on a PDA plate at 28 °C for 7 days. The mycelia were blended in 100 mL of sterilized water using a Waring blender, and 5 mL of the suspension was added into 100 mL of potato dextrose broth medium, which was then shaken at 28 °C for 10 h. The grown cells were collected by filtration and washed with 0.8 M sodium chloride. The cells were incubated with 1 mL of 10 mM sodium phosphate buffer (pH 8.0), containing 0.8 M sodium chloride, 50 mg/mL lysing enzymes from Trichoderma harzianum (Sigma-Aldrich), and 850 units of β-glucuronidase from Helix pomatia (Sigma-Aldrich) at 30 °C for 3 h. The resulting protoplasts were filtered and subsequently centrifuged at 1500g for 5 min at room temperature. The collected protoplasts were washed with 0.8 M sodium chloride and centrifuged to remove the washing solution. Approximately 106 protoplasts were suspended in 200 μL of STC buffer at pH 8.0 (0.8 M sorbitol, 10 mM calcium chloride, and 10 mM Tris-HCl) and spread on PDA plates containing 1 M sorbitol. The plates with protoplasts were placed on an UV-illuminator (NIPPON Genetics), irradiated for 30 or 60 s, and then cultured in total darkness at 28 °C for 3−7 days. In these conditions, the lethal ratio of the protoplasts reached over 90%. The surviving colonies were isolated, and 211 UV-irradiated strains (Paraboeremia sp. Lsl3KI001− 077 and TS001−134) were obtained. Extraction and Partition of a Wild-Type and UV-Irradiated Strains of Paraboeremia sp. Lsl3. The mycelia of a wild-type and UV-irradiated strains of Paraboeremia sp. Lsl3 cultured on PDA media at 28 °C for 14 days were extracted with MeOH. The extracts were partitioned between EtOAc (1 mL × 2) and 3% tartaric acid (1 mL). Water-soluble materials were adjusted to pH 10 with 28% ammonia and extracted with EtOAc (1 mL × 2) to obtain the basic EtOAc layers.
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ASSOCIATED CONTENT
* Supporting Information S
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.7b00627. Morphology and molecular phylogenetic trees of the Lsl3 strain; NMR, MS, and ECD spectra of 1; and LC-MS (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail (K. Ishiuchi):
[email protected]. ORCID
Kan’ichiro Ishiuchi: 0000-0003-2048-7592 Toshiaki Makino: 0000-0002-2524-8745 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors thank Dr. K. Fujikawa, Kochi Prefectural Makino Botanical Garden, Dr. S. Watthana, School of Biology, Suranaree University of Technology, for identification of Phlegmariurus nummulariifolius (Blume) Ching, and Dr. T. Nakazawa, Graduate School of Agriculture, Kyoto University, for advice about production of UV-irradiated strains of Paraboeremia sp. Lsl3. This work was partly supported by a Grant-in Aid for Young Scientists B (No. 25860080 and No. 17K15467 to K.I.) and The Research Foundation for Pharmaceutical Sciences (to K.I.).
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REFERENCES
(1) For reviews and recent literature on the Lycopodium alkaloids, see: (a) Ma, X.; Gang, D. R. Nat. Prod. Rep. 2004, 21, 752−772.
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DOI: 10.1021/acs.jnatprod.7b00627 J. Nat. Prod. 2018, 81, 1143−1147
Journal of Natural Products
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DOI: 10.1021/acs.jnatprod.7b00627 J. Nat. Prod. 2018, 81, 1143−1147
