Characterization of Cytochalasins from the Endophytic Xylaria sp. and

Oct 28, 2014 - ... that of the positive control glyphosate. Additionally, the fungicidal effect of 2 against the phytopathogen Gibberella saubinetti w...
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Characterization of Cytochalasins from the Endophytic Xylaria sp. and Their Biological Functions Qiang Zhang,† Jian Xiao,†,‡ Qing-Qing Sun,† Jian-Chun Qin,§ Gennaro Pescitelli,∇ and Jin-Ming Gao*,† †

College of Science, Northwest A&F University, Yangling 712100, Shaanxi P. R. China Shaanxi Key Laboratory of Phytochemistry, College of Chemistry and Chemical Engineering, Baoji University of Arts and Sciences, Baoji 721013, P. R. China § College of Plant Sciences, Jilin University, Changchun 130062, P. R. China ∇ Dipartimento di Chimica e Chimica Industriale, Università di Pisa, Via Risorgimento 35, 56126, Pisa, Italy ‡

S Supporting Information *

ABSTRACT: Bioassay-guided fractionation of the fermentation extract of Xylaria sp. XC-16, an endophyte from Toona sinensis led to the isolation of two new cytochalasans cytochalasin Z27, 1, and cytochalasin Z28, 2, along with three known compounds seco-cytochalasin E, 3, and cytochalasin Z18, 4, and cytochalasin E, 5. The structures of 1 and 2 were elucidated by spectroscopic and electronic circular dichroism methods. Compound 5 was shown to be potently cytotoxic against brine shrimp (LC50 = 2.79 μM), comparable to that of the positive agent toosendanin (LC50 = 4.03 μM), and also exhibited potential phytotoxic effects on Lactuca sativa and Raphanus sativus L. seedlings, which are higher than that of the positive control glyphosate. Additionally, the fungicidal effect of 2 against the phytopathogen Gibberella saubinetti was better than that of hymexazol. This is the first report of the three types of cytochalasins present in genus Xylaria. A structure−phytotoxicity activity relationship is also discussed. KEYWORDS: natural products, plant growth regulator, cytochalasins, Xylaria sp., absolute configuration, toxicity, phytotoxins

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INTRODUCTION Plant endophytic fungi have attracted considerable attention as promising sources of new bioactive compounds, which are often structurally unique and display various biological activities.1 The endophytic fungi in the genus Xylaria have proved to be a rich source of secondary metabolites with diverse chemical structures and biological,2,3 for example antifungal sordaricins,4 and antifungal lactones multiplolides A and B.5 The cytochalasins are a family of fungal natural products first discovered in 1966.6,7 To date, there are more than 80 cytochalasins, isolated from a variety of fungal species, including Aspergillus, Phomopsis, Penicillium, Zygosporium, Chaetomium, Phoma spp., Xylaria spp., Hypoxylon spp., and Rhinocladiella spp.8−10 Structurally, cytochalasins are composed of a highly substituted isoindolone ring with a benzyl group at the C-3 position and fused to an 11- to 14-membered macrocyclic ring. Cytochalasins exhibit a broad spectrum of bioactivities, such as phytotoxic activity.11,12 Particularly, cytochalasins have been used extensively to probe the role of F-actin in different aspects of cellular function.10 Chinese toon (Toona sinensis (A. Juss.) Roem, synonym Cedrela sinensis) is a rapidly growing deciduous tree, belonging to family Meliaceae, that has a long history of consumption as a delicious vegetable and medicinal plant exhibiting anticancer and anti-inflammatory effects.13,14 In continuation of our search for agriculturally active cytochalasan alkaloids from medicinal plant endophytes,15−20 an extract of the endophytic fungus Xylaria sp. XC-16, isolated from the healthy leaves of Toona sinensis, was found to exhibit potent toxicity against brine shrimp (Artemia cysts). A brine shrimp toxicity-guided © XXXX American Chemical Society

fractionation of an ethyl acetate extract of the same fungus XC-16 resulted in the isolation of five cytochalasins 1−5 (Figure 1) including two new compounds 1 and 2 and a new natural product 3. Herein, we report the bioassay guidedisolation and structure elucidation of the new compounds, as well as their brine shrimp toxicity, phytotoxicity, and antifungal effects.

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MATERIALS AND METHODS

General Experimental Procedures. Optical rotations were measured on an Autopol III automatic polarimeter (Rudolph Research Analytical, NJ, USA). UV spectra were obtained using a Thermo Scientific Evolution 300 UV/vis spectrophotometer (Thermo Fisher Scientific Inc., MA, USA). CD spectra were measured with a Chirascan circular dichroism spectrometer (Applied Photophysics Ltd., Leatherhead, Surrey, U.K.), using spectroscopic grade solvent and a 0.5 mm quartz cell with 50 nm/min scan rate and 1 nm bandwidth. IR spectra were recorded on a Bruker Tensor 27 spectrophotometer (Bruker Optics, Rheinstetten, Germany) in KBr pellets. ESI-MS spectra were performed on a Thermo Fisher LTQ Fleet instrument spectrometer (Thermo Fisher Scientific Inc., MA, U.S.). HR-ESI-MS spectra were performed on a VG Autospec-3000 spectrometer (VG, Oakville, Ontario, Canada). Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker AVANCE III (500 MHz) instrument (Bruker BioSpin, Rheinstetten, Germany). Chemical shifts are reported using solvent residual peak as the internal standard. Column chromatography (CC) was performed on silica gel (90−150 μm) (Qingdao Marine Chemical Inc., Qingdao, China), MCI gel (75−150 μm)

Received: August 9, 2014 Revised: October 26, 2014 Accepted: October 28, 2014

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Figure 1. Structures of cytochalasins 1−5 isolated from Xylaria sp. XC16 and toosendanin. (8.7 mg), 4 (5.8 mg), and 5 (3.9 mg). Fraction B was purified with 82% MeOH/H2O to afford compounds 1 (12.3 mg) and 2 (8.2 mg). Cytochalasin Z27, 1: White solid; [α]D22 + 77.8 (c 0.16, CH3OH); UV (MeOH) λmax (log ε) 206 (4.30), 252 (3.96); CD (MeOH) λmax (Δε) 198 (+48.4), 238 (−12.8), 265 (+4.9), 315 (+4.2); IR (KBr) νmax 3395, 2922, 1707, 1668, 1516, 1451, 1265, 1225, 1135, 980 cm−1; 1 H and 13C NMR data, see Table 1; ESI MS (positive) m/z 502.30 [M + Na] + ; HR-ESI-MS m/z 502.2212 ([M + Na] + , calcd. C28H33NO6Na+, 502.2206). Cytochalasin Z28, 2: White solid; [α]D22 + 68.9 (c 0.14, CH3OH); UV (MeOH) λmax (log ε) 206 (4.21), 251 (3.82); CD (MeOH) λmax (Δε) 194 (+25.6), 229 (−5.4), 262 (+3.3), 317 (+2.1); IR (KBr) νmax 3425, 3258, 2866, 2931, 1701, 1559, 1516, 1440, 1269, 1228, 1019 cm−1; 1H and 13C NMR data, see Table 1; ESI MS (positive) m/z 502.29 [M + Na]+; HR-ESI-MS m/z 502.2205 ([M + Na]+, calcd. C28H33NO6Na+, 502.2206). seco-Cytochalasin E, 3: Colorless oily solid; [α]D25 +114.8 (c 0.08, MeOH); UV (MeOH) λmax (log ε) 222 (2.61), 258 (0.69); IR (KBr) νmax 3368, 2969, 1694, 1454, 1015, 697 cm−1; 1H and 13C NMR data see Table 1; ESI MS m/z 550 [M + Na]+. Computational Section. MMFF and DFT calculations were run with Spartan 10 (Wave function Inc., Irvine, CA) with standard parameters and convergence criteria. TDDFT calculations were run with Gaussian 09,22 with default grids and convergence criteria. Conformational searches were run with the Monte Carlo algorithm implemented in Spartan 10 using Merck molecular force field (MMFF). All structures thus obtained were optimized with DFT method using B3LYP functional, using first the 6-31G(d) basis set and then 6-311+G(d,p) basis set. TDDFT calculations were run using B3LYP and CAM-B3LYP functionals and TZVP basis set, including 64 and 42 excited states, respectively. UV and CD spectra were generated using the program SpecDis23 by applying a Gaussian band shape with 0.3−0.4 eV exponential half-width, from dipole-length rotational strengths. Toxicity Bioassay. All the isolated metabolites were tested for their toxicity activity against brine shrimp (Artemia cysts) as previously reported method.19 Phytotoxicity Bioassay. The seeds of two herbaceous plants, lettuce (Lactuca sativa) and radish (Raphanus sativus) were used for the bioassay. The procedure was conducted according to the

(Mitsubishi Chemical Corp., Tokyo, Japan), Sephadex LH-20 (40−70 μm) (Amersham Pharmacia Biotech AB, Uppsala, Sweden), and Lichroprep RP-18 gel (40−63 μm) (Merck, Darmstadt, Germany). GF254 plates (Qingdao Marine Chemical Inc., Qingdao, China) were used for thin-layer chromatography (TLC). High-performance liquid chromatography (HPLC) analysis was performed on an Agilent TCC18 column (250 mm × 4.6 mm, 5 μm, Agilent Technologies Ltd., CA, USA) on a Waters 1525 instrument (Waters Corp., MA, USA). Brine shrimp eggs (Artemia cysts) (Advanced Hatchery Technology Inc., Xiamen, China) were used for bioguided assay. Fungal Material. Plant material was collected in Yangling, Shaanxi province, China, in April 2011. Isolation, maintenance, and preservation of endophytic fungi were performed according to the reported protocols.21 All isolated fungal strains (28 in total, XC1− XC28) were prescreened for their toxicity toward brine shrimp.19 The most toxic strain XC16 belonged to the genus Xylaria, identified by internal transcribed spacer (ITS) rDNA region sequence comparison, and was designated as Xylaria sp. Fermentation, Extraction, and Isolation. All 28 isolated fungal strains (XC1−XC28) were fermented on solid rice medium. Toxicity of the fermented culture (20 μg/mL) against brine shrimp were assayed according to our previously reported protocol.19 The most toxic strain XC16 was selected to be cultivated on rice medium (40 g rice and 100 mL distilled water, sterilized under 121 °C for 30 min) in 200 Erlenmeyer flasks (500 mL each). The flask cultures were incubated under 28 °C for 3 weeks. The fungal cultures of Xylaria sp. XC16 grown on rice medium were extracted with petroleum ether and EtOAc three times, respectively, and tested by brine shrimp lethality assay. The toxic EtOAc crude extract was fractionated by silica gel CC using CHCl3− MeOH gradient elution to provide five fractions (Fr.1−Fr.5). Fr.2 (4.93 g) indicated the highest activity and was further chromatographed on Sephadex LH-20 eluted with MeOH as an eluent to give two fractions A and B. These two fractions were analyzed by a Thermo BDS Hypersil column (250 × 10 mm, 5 μm, Thermo Fisher Scientific Inc., MA, U.S.), eluted with gradient 60−90% MeOH/H2O at a flow rate of 2.0 mL/min over 30 min. Then fractions A and B were chromatographed on the same HPLC column, respectively. Fraction A was separated with 70% MeOH/H2O as eluent to yield compounds 3 B

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Table 1. 1H and 13C NMR Data of Compounds 1−3 1b δH, multi (J in Hz) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 1′ 2′ 3′ 4′ 5′ 6′ a

2b δC

δH, multi (J in Hz)

172.4 9.56, s 3.86, t (7.1) 4.02, br. s

4.56, d (9.8) 3.81, t (9.8) 3.21, 3.10, 1.52, 1.88,

dd (13.9, 7.1) dd (13.9, 7.1) s s

6.67, dd (15.1, 10.1) 5.66, ddd (15.1, 10.1, 3.8) 2.18,a m 2.01, ddd (25.2, 11.4, 0.63) 3.38, m

6.53, dd (10.6, 6.1) 3.74, t (10.9) 3.18, dd (11.4, 6.3) 1.131 d (6.6) 2.15, s

7.30, d (8.2) 7.17, d (8.2) 7.17, d (8.2) 7.30, d (8.2)

59.9 49.6 125.4 134.8 70.1 50.9 85.3 43.3 17.5 14.8 128.6 134.5 40.5 40.0 205.4 142.6 133.0 37.8 169.4 17.3 13.0 128.6 131.0 116.2 157.6 116.2 131.0

3c δC

δH, multi (J in Hz)

171.3 9.21, s 3.64,a m 3.02,a m 3.44,a m

53.9 48.6 32.4 151.9 69.8 50.3 84.6 43.1

4.45, d (11.3) 3.68, t (10.4) 3.00,a m 1.06, d (6.6) 5.62, br. s (H-12Z) 5.21, br. s (H-12E) 6.27, ddd (15.0, 9.5, 1.3) 5.74, ddd (15.0, 11.1, 3.8) 2.23, br. d (11.0) 2.13, br. d (12.0) 3.44,a m

6.71, t (8.0) 3.51, dd (12.6, 9.8) 3.29, dd (12.6, 6.9)

14.2 112.7 128.7 134.2 40.6 39.4 202.0 142.3 133.7 36.7 169.3 17.7 12.6

1.13, d (6.6) 2.17, s

128.4 131.2 116.0 157.5 116.0 131.2

7.22, d (8.5) 7.12, d (8.5) 7.12, d (8.5) 7.22, d (8.5)

δC 170.1

8.52, br.s 3.66,a m 2.44, dd (5.2, 2.1) 1.84, m 2.67, d (5.2) 2.76, m 2.88, dd (13.0, 5.2) 2.63, m 0.59,d (7.8) 1.13, s 6.01, dd (15.8, 8.8) 5.35, m 2.27, ddd (13.5, 6.7, 2.1) 1.97, ddd (14.0, 7.3, 2.6) 3.31,a m

2.70, m 2.54, dd (16.1, 3.1) 9.55, t (2.1)

0.97, d (7.3) 1.26, s 3.68, s 7.16, 7.30, 7.22, 7.30, 7.16,

t t t t t

(7.3) (7.3) (7.3) (7.3) (7.3)

52.8 47.0 35.6 57.1 60.0 45.6 85.2 43.2 12.4 19.2 127.9 130.6 35.5 38.7 217.0 77.0 52.5 201.1 153.1 16.4 25.3 54.6 137.0 129.6 128.3 126.5 128.3 129.6

Overlapped signal. bpyridine-d5; cDMSO-d6.

If T > C , then RI = 1 − C /T ; if T < C , then RI = T /C − 1

(CFU/mL) with PD medium. In flat-microtiter plates, tested compounds, fungal suspension, and sterile water were added to made up final concentrations of the compounds in the range of 1.57− 200 μM. Cultures were then grown in the dark at 28 ± 0.5 °C for 48 h. Minimum inhibitory concentrations (MICs) were inspected as the lowest concentrations in which no fungal growth could be observed.

where T is the length of the treatment, C is the length of the blank control, and RI is the response index. RIs are expressed as averages ± standard deviation (SD) for three replicates. Antifungal Bioassay. All the isolated metabolites were tested in vitro for the antifungal activity against agriculturally important four phytopathogenic fungi: Fusarium solani, Gibberella saubinetti, Botrytis cinerea, and Alternaria solani, according to our reported bioassay.19 All these pathogenic organisms tested were deposited at the College of Science, Northwest A&F University. Antifungal activity was assessed by the microbroth dilution method in 96-well flat-microtiter plates using a potato dextrose (PD) medium. Initial concentrations of compounds tested were made up to 4 mM in DMSO. Two commercial fungicides, carbendazim and hymexazol (Aladdin Chemistry Co. Ltd., Shanghai, China) were used as positive control. A DMSO solution of equal concentration was used as a negative control. The fungi were incubated in the PD medium for 18−36 h at 28 ± 0.5 °C at 150 rpm, and spores of different microorganism concentrations were diluted to approximately 1 × 106 colony-forming units/mL

RESULTS AND DISCUSSION Identification of the Bioactive Fungal Endophytic Strain XC16. Brine shrimp test has been proved to be a simple and reliable benchtop bioassay for screening toxicity for natural extracts and synthetic products.27−29 Twenty-eight strains (XC1−XC28) of endophytic fungi were isolated from the healthy leaves of Toona sinensis. All isolates were screened for their toxicity against brine shrimp at 20 μg/mL level. Among the isolates, the secondary metabolites of the two strains XC16 and XC20 exhibited remarkable toxicity to the organism. The culture filtrate from strain XC16 killed all brine shrimp larvae, showing the greatest activity, and was therefore selected for the isolation and characterization of bioactive metabolites. To identify this active strain XC16, a GenBank search with the nuclear ribosomal ITS1 sequence and 18S rDNA of XC16 was

previously reported protocol.20,24−26 Glyphosate was selected as the positive control. Triplicate experiments were conducted on 6-well plates, and the phytotoxic effects [response index (RI)] were calculated according to the equation:

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C

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from Aspergillus f lavipes, an endophytic fungus of the mangrove plant Acanthus ilicifolius.30 The relative configuration of 1 was assigned by 13C NMR data in comparison with reported ones, and by NOESY results with the help of molecular modeling. As shown in Figure 2, the observed correlations of H-10a/H-4 and H-4/H-8 indicated that C-10, H-4, and H-8 are on the same side of the molecule and were arbitrarily assigned as β-oriented. The NOE correlation of H-7/H-13 indicated H-7 to be α-oriented. Further confirmation of the stereochemistry at C-7 and C-8 was provided by the NOE correlation of H-8/H-14. Moreover, the NOE correlation H-14/H-16, when checked against molecular modeling results (Figure 2), offered the proof of the αorientation of Me-22. Finally, the 18E configuration of the C18/C-19 double bond was proved by H-20/Me-23 NOE correlation and the absence of H-19/Me-23 correlation. Previous studies suggested that the stereochemistry of the essential elements of cytochalasins skeleton is well retained along the series. These include (1) the stereochemistry of the macrocyclic ring, with a trans C-8/C-9 junction and a cis C-4/ C-9 junction;31,32 (2) the β orientation of the oxygen at C7;11,33,34 (3) the α-orientation of Me-22;31,32 and (4) the E configuration of the C-18/C-19 double bond.30,34,35 The 13C NMR spectrum of 1 (Table 1) showed similar chemical shifts to that of cytochalasin Z17,30 especially of the macrocyclic ring moiety. In particular, the similarities between the chemical shifts of C-7, C-9, and C-16 indicated that the relative configurations of these centers in 1 are consistent with those in cytochalasin Z17, as shown in Figure 2. Furthermore, compound 1 showed a CD spectrum (Figure 3A) consistent with that of cytochalasin Z17.35 This is not surprising because of the structural analogy between the two compounds; still, an independent proof of the absolute configuration of 1 was obtained by quantum mechanical calculations of its CD spectrum. It was concluded that 1 has the same configuration of cytochalasin Z17, and its structure is determined as (+)-(3S,4S,7S,8S,9S,13E,16S,18E)-7-hydroxy-5,6,16,18- tetramethyl-10-(4′-hydroxyphenyl)-22-oxa-[12]-cytochalasa6,13,18-trien-1,17,21-trione. Molecular modeling of compound 1 was run through a wellestablished protocol.36 The most stable energy minima thus obtained had a very similar conformation of the macrocycle (Figure 2), which was in keeping with the NOE correlations discussed above, and differed only for the orientation of the 4′hydroxyphenylmethyl and the hydroxyl groups. CD calculations were run of DFT geometries with TDDFT method,37 using B3LYP and CAM-B3LYP functionals and TZVP basis set, and averaged at 300 K using Boltzmann populations. The weightedaverage CD spectra (Figure 3B) are in good agreement with the experimental one. In particular, the B3LYP functional performed better in the long-wavelength region, where TDDFT method is intrinsically more accurate.38 Conversely, the CAM-B3LYP functional reproduced better the shortwavelength region. In conclusion, the absolute configuration of cytochalasin Z 2 7 was definitely established as (+)-(3S,4S,7S,8S,9S,16S)-1. Cytochalasin Z28, 2, was obtained as white solid. Its molecular formula C28H33NO6 is the same as 1, as deduced from the pseudomolecular ion in HR-ESI-MS, suggesting that 2 was an isomer of 1. Comparison of its 1H and 13C NMR data with those of 1 (Table 1) revealed that compound 2 also had a quite similar skeleton to that of 1. A significant difference was that the two carbon signals at δC 125.4 (C-5, s) and 134.8 (C-6,

carried out. The results revealed a species of Xylaria as the closest match, with sequence identities 99%. The most similar sequence was that of Xylaria sp. JW38−4 (accession number GQ906959). On the basis of its ITS and 18S rDNA sequences, the fungus XC16 clearly belongs to the member of the genus Xylaria, identified as Xylaria sp. XC16, a mycelial fungus under scanning electron microscope. Isolation, Purification, and Structure Elucidation. The active EtOAc extracts of the cultures of this strain Xylaria sp. XC-16 were submitted to column chromatography on silica gel to provide five fractions (Fr. 1-Fr. 5). Among these fractions, Fr.2 showed strong toxicity to brine shrimp at 20 μg/mL level. As a result, Fr. 2 was fractionated through silica gel column chromatography, gel filtration over Sephadex LH-20, ODS column chromatography, and semipreparative HPLC to afford two new cytochalasins 1 and 2, together with three known cytochalasin derivatives, 3−5 (Figure 1). Their structures were elucidated by a combination of spectroscopic analyses (UV, CD, IR, MS, 1D, and 2D NMR) and by comparison to literature data. Cytochalasin Z27, 1, was obtained as white powder. Its molecular formula was assigned as C28H33NO6 based on the pseudomolecular ion in HR-ESI-MS, requiring 13 degrees of unsaturation. The IR absorptions at 1707, 1667, 1614, and 1516 cm−1 indicated the presence of carbonyl and benzene ring groups in the structure. The 1H and 13C NMR data of 1 (Table 1) suggested four methyls, three methenes, 12 methines (including seven unsaturated ones), and nine quaternary carbons (including eight unsaturated ones). The unsaturated carbon signals in 13C NMR suggested six carbon−carbon double bonds and three CO groups (δC 205.4, 172.4, and 169.4). In particular, the AA′XX′ spin system [δH 7.30 (d, J = 8.2 Hz), 7.17 (d, J = 8.2 Hz)] indicated a 1,4-disubstituted benzene ring in 1. Apart from the ten degrees of unsaturation due to the unsaturated bonds and the benzene ring, the remaining ones indicated that 1 possesses a tricyclic framework, which was elucidated by means of extensive analysis of the 2D NMR spectra (HSQC, 1H−1H COSY, and HMBC). All protons directly bonded to the carbon atoms were first assigned on the basis of the HSQC data. Five fragments were deduced from the COSY spectrum (Figure 2). Further detailed HMBC correlations (Figure 2) revealed the whole planar structure of 1, a 4′-hydroxyl derivative of cytochalasin Z17, which was obtained

Figure 2. Selected HMBC, 1H−1H COSY, and NOESY correlations of 1. D

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Figure 4. Selected HMBC, 1H−1H COSY, and NOESY correlations of 2.

methyl-10-(4′-hydroxy)phenyl-22-oxa-[12]-cytochalasa-6(12),13,18-trien-1,17,21-trione. seco-Cytochalasin E, 3, was isolated as colorless oily solid. Its molecular formula was tentatively established as C29H37NO8 based on ESI-MS peak at m/z 550.11 ([M + Na]+) associated with 13C NMR data. The 1H NMR signals (Table 1) at δH 7.15 (2H, d, J = 7.3 Hz), 7.22 (1H, d, J = 7.3 Hz), and 7.30 (2H, d, J = 7.3 Hz) indicated a phenyl group in the structure. The large coupling constant (J = 15.6 Hz) at δH 7.15 indicated a trans double bond in the structure. The similar 13C NMR data of compound 3 and cytochalasin Z18 (4)30 revealed a typical octahydro-1H-isoindol-1-one moiety of cytochalasin skeleton. Further detailed analysis of HSQC and 1H−1H COSY suggested fragments as shown in Figure 5. Connections

Figure 3. (A) Experimental CD spectra of (+)-1 and (+)-2 in methanol (0.5 mM, 0.5 mm cell). (B) CD spectra calculated for (3S,4S,7S,8S,9S,16S)-1 at B3LYP/TZVP//B3LYP/6-311+G(d,p) and CAM-B3LYP/TZVP//B3LYP/6-311+G(d,p) levels as Boltzmanns average on 8 structures. Gaussian band-shape with 0.3 (B3LYP) and 0.4 eV (CAM-B3LYP) exponential width; CAM-B3LYP spectrum redshifted by 30 nm.

s), attributable to the tetrasubstituted endocyclic double bond in 1, were replaced by the two exocyclic double-bond carbon signals at δC 151.9 (C-6, s) and 112.7 (C-12, t) in 2. This portion of the spectrum was also similar to that observed in the 13 C spectrum of 10-phenyl[12]cytochalasin Z16.35 Furthermore, in the 1H NMR spectrum of 2, the C-11 methyl proton signal (δH 1.06, d, J = 6.6 Hz), appearing as a doublet instead of the singlet (δH 1.52, s) as in 1, was also consistent with a methylsubstituted six-membered ring as seen in cytochalasin Z16.35 This difference between 1 and 2 was further confirmed by 1 H−1H COSY and HMBC correlations as shown in Figure 4. The similar 13C NMR data of the macrocyclic rings in 1 and 2 (Table 1) and the consistency of NOESY results (Figures 2 and 4) suggested that the cyclic moieties in both 1 and 2 possess the same relative configurations. Additionally, the relative configuration of the chirality center at C-5 in 2 was assigned through the NOE correlations H-3/H3-11, proving that Me-11 is α-oriented. The absolute configuration of compound (+)-2 was established by spectroscopic comparison. Compound (+)-2 has in fact a CD spectrum very similar to that of (+)-1 (Figure 3A), and also consistent with that reported for cytochalasin Z16.35 It can therefore be assumed that cytochalasins 1 and 2 have the same absolute configuration of the corresponding chirality centers, as established above for (+)-1. Thus, the structure of 2 was determined as (+)-(3S,4S,5S,7S,8S,9S,13E,16S,18E)-7-hydroxy-5,16,18-tri-

Figure 5. Selected HMBC and COSY correlations of 3.

among the fragments showing COSY cross-peaks, quaternary carbons and methyl groups were further confirmed by HMBC correlations (Figure 5). The whole planar structure was elucidated as seco-cytochalasin E, a ring-opened derivative of cytochalasin E, which had been previously obtained as a semisynthetic derivative of 5,39 but without detailed spectroscopic data reported in the literature. Herein, all the 1H NMR and 13C NMR data were assigned unambiguously according to the 2D NMR experiments (Table 1). This is the first report of seco-cytochalasin E as a natural product. Compounds 4 and 5 were identified as cytochalasin Z1830 and cytochalasin E, 30,33 respectively, on the basis of their spectroscopic features (NMR, MS) and by comparison with the published data in the literature. Biological Activity. All the compounds 1−5 were examined for toxicity against brine shrimp, for phytotoxicity and antiphytopathogens in vitro. E

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findings demonstrated again that the 13-membered macrocyclic vinyl carbonate ester moiety in cytochalasins could be a determinant feature for activity. In fact, the macrocyclic ring opening of 5 to yield seco-cytochalasin E, 3, leads to a loss of activity; 22-oxa[12]cytochalasins (e.g., cytochalasins Z27, 1, and Z28, 2), possessing the secondary hydroxyl group on C-7, were inactive; and open-chain cytochalasins (e.g., seco-cytochalasin E, 3, and cytochalasin Z18, 4) without a perihydroisoindolyl scaffold were also inactive. Interestingly, the bioassay results of cytochalasin E, 5, show an agreement between phytotoxic and insecticidal activity. Cytochalasins have been considered as potential mycotoxins, and some of them have been found to be phytotoxic to different plants. Cytochalasin H, a toxic [11]cytochalasan of Phomopsis sp. isolated from weevil-damaged pecans, was markedly inhibitory to the growth of the floral development of tobacco plants.40 Two [11]cytochalasans, pyrichalasin H, produced by the fungus Pyricularia grisea, and zygosporin D, isolated from the culture of the insect pathogenic fungus Metarrhizium anisopliae, were found to strongly inhibit the growth of rice seedlings.41,42 Cytochalasin Z6, an 24-oxa[14]cytochalasan isolated from the culture of Phoma exigua var. heteromorpha, caused inhibition of rootlet elongation of tomato seedlings.43 In addition, three 24-oxa[14]cytochalasans, namely cytochalasins Z3, B, F, and an [13]cytochalasan, deoxaphomin, from the culture of Pyrenophora semeniperda, a promising mycoherbicide for the biological control of grass weeds, showed a remarkable ability to inhibit root elongation of wheat and tomato seedlings.44 Of the test cytochalasins B, F, Z2, and Z3, and deoxaphomin, produced by Phoma exigua var. exigua, a potential mycoherbicide against the perennial weeds Sonchus arvensis and Cirsium arvense, deoxaphomin demonstrated the highest level of toxicity on leaves of S. arvensis, whereas other cytochalasins showed lower activity.11 These observations promoted structure−activity relationship (SAR) studies leading to the conclusions that a [11] or a [13]carbocyclic or a [14]lactonic macrocyclic ring fused to an intact perihydroisoindolyl-1-one motif is an important feature for toxicity, and that the role played by the hydroxyl group on C-7 also appears crucial in imparting toxicity.44−48 However, these results are not in accordance with our results, which indicated instead that the presence of 7-OH group in 22-oxa[12]cytochalasin motif has no major role in conferring activity. This discrepancy may depend on different crop species, varying sensitivity, and cytochalasin type. This is the first report of phytotoxicity of the tested cytochalasins, especially, a small type of 21,23-dioxa[13]cytochalasin containing the unique vinyl carbonate moiety (e.g., 5), to Lactuca sativa and Raphanus sativus seedlings. Antifungal Activity. The antiphytopathogenic activity of the isolated cytochalsins was also evaluated on four different pathogens, namely, Fusarium solani, Gibberella saubinetti, Botrytis cinerea, and Alternaria solani, and the results are shown in Table 2. The antifungal activity of cytochalsin derivatives strongly depends on the fungal species. Among the compounds tested, only compound 2 showed strong fungicidal effect (MIC of 12.5 μM) against G. saubinetti, compared with that of the positive control hymexazol (MIC of 25 μM). In contrast, other compounds displayed quite weak properties with MIC values of greater than 50 μM against the pathogens tested. In conclusion, five cytochalasins 1−5 have been characterized in the endophytic Xylaria sp., including two new compounds cytochalasin Z27, 1, and cytochalasin Z28, 2, and one new

Toxicity to Brine Shrimp. In the screening assay on brine shrimp, cytochalasin E, 5, at 50 μM displayed 100% lethality. In contrast, the other compounds 1−4 displayed very weak toxicity toward brine shrimp larvae with lethality below 10%. Compound 5 produced strong toxic effects on brine shrimp in a concentration-dependent manner (0.625−20.0 μM (Figure 6),

Figure 6. Brine shrimp lethality of 5 and the positive control drug toosendanin in concentrations 0.625−20.0 μM.

with a LC50 value of 2.79 μM, which was comparable to that of the commercial insecticidal agent toosendanin, showing a LC50 value of 4.03 μM. By comparison of the isolated structures of 1−5, the toxicity against brine shrimp of 5 may be rationalized as due to the 13-membered macrocycle with a vinyl carbonate ester group. Phytotoxic Activity. Among the five compounds tested, only cytochalasin E, 5, bearing a vinyl carbonate ester and an epoxy group, demonstrated the highest seedling growth inhibition on Lactuca sativa and Raphanus sativus at concentrations in the range of 10−80 μM (Figure 7). The phytotoxicity of 5 was higher than that of the positive control, glyphosate, a broad-spectrum systemic herbicide in agriculture. In contrast, the remaining compounds 1−4 showed no effect on both seedlings even at a concentration of 100 μM. These

Figure 7. Phytotoxic activities of 5 and glyphosate on (A) Lactuca sativa root, (B) shoot, (C) Raphanus sativus L. Root, and (D) shoot. F

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agents sordarins across filamentous fungi. Mycol. Res. 2009, 113, 754− 770. (5) Boonphong, S.; Kittakoop, P.; Isaka, M.; Pittayakhajonwut, D.; Tanticharoen, M.; Thebtaranonth, Y. Multiplolides A and B, New antifungal 10-membered lactones from Xylaria multiplex. J. Nat. Prod. 2001, 64, 965−967. (6) Rothweiler, W.; Tamm, C. Isolation and structure of phomin. Experientia 1966, 22, 750−752. (7) Aldridge, D. C.; Armstrong, J. J.; Speake, R. N.; Turner, W. B. Cytochalasins, A new class of biologically active mold metabolites. Chem. Commun. 1967, 26−27. (8) Zhang, Q.; Li, H. Q.; Zong, S. C.; Gao, J. M.; Zhang, A. L. Chemical and bioactive diversities of the genus Chaetomium secondary metabolites. Mini-Rev. Med. Chem. 2012, 12, 127−148. (9) Dagne, E.; Gunatilaka, A. A. L.; Asmellash, S.; Abate, D.; Kingston, D. G. I.; Hofmann, G. A.; Johnson, R. K. Two new cytotoxic cytochalasins from Xylaria obovata. Tetrahedron 1994, 50, 5615−5620. (10) Wagenaar, M. M.; Corwin, J.; Strobel, G.; Clardy, J. Three new cytochalasins produced by an endophytic fungus in the genus Rhinocladiella. J. Nat. Prod. 2000, 63, 1692−1695. (11) Cimmino, A.; Andolfi, A.; Berestetskiy, A.; Evidente, A. Production of phytotoxins by Phoma exigua var. exigua, A potential mycoherbicide against perennial thistles. J. Agric. Food Chem. 2008, 56, 6304−6309. (12) Li, H.; Xiao, J.; Gao, Y.-Q.; Tang, J.-J.; Zhang, A.-L.; Gao, J.-M. Chaetoglobosins from Chaetomium globosum, An endophytic fungus in Ginkgo biloba, and their phytotoxic and cytotoxic activities. J. Agric. Food Chem. 2014, 62, 3734−3741. (13) Li, J.-X.; Eidman, K.; Gan, X.-W.; Haefliger, O. P.; Carroll, P. J.; Pika, J. Identification of (S,S)-γ-glutamyl-(cis-S-1-propenyl)thioglycine, a naturally occurring norcysteine derivative, from the Chinese vegetable Toona sinensis. J. Agric. Food Chem. 2013, 61, 7470−7476. (14) Hseu, Y.-C.; Chen, S.-C.; Lin, W.-H.; Hung, D.-Z.; Lin, M.-K.; Kuo, Y.-H.; Wang, M.-T.; Cho, H.-J.; Wang, L.; Yang, H.-L. Toona sinensis (leaf extracts) inhibit vascular endothelial growth factor (VEGF)-induced angiogenesis in vascular endothelial cells. J. Ethnopharmacol. 2011, 134, 111−121. (15) Gao, J.-M.; Yang, S.-X.; Qin, J.-C. Azaphilones: Chemistry and biology. Chem. Rev. 2013, 113, 4755−4811. (16) Xue, M.; Zhang, Q.; Gao, J.-M.; Li, H.; Tian, J.-M.; Pescitelli, G. Chaetoglobosin Vb from endophytic Chaetomium globosum: Absolute configuration of chaetoglobosins. Chirality 2012, 24, 668−674. (17) Yang, S.-X.; Gao, J.-M.; Laatsch, H.; Tian, J.-M.; Pescitelli, G. Absolute configuration of fusarone, A new azaphilone from the endophytic fungus Fusarium sp isolated from Melia azedarach, and of related azaphilones. Chirality 2012, 24, 621−627. (18) Li, X.-J.; Gao, J.-M.; Chen, H.; Zhang, A.-L.; Tang, M. Toxins from a symbiotic fungus, Leptographium qinlingensis associated with Dendroctonus armandi and their in vitro toxicities to Pinus armandi seedlings. Eur. J. Plant Pathol. 2012, 134, 239−247. (19) Li, X.-J.; Zhang, Q.; Zhang, A.-L.; Gao, J.-M. Metabolites from Aspergillus f umigatus, an endophytic fungus associated with Melia azedarach, and their antifungal, antifeedant, and toxic activities. J. Agric. Food Chem. 2012, 60, 3424−3431. (20) Zhang, Q.; Wang, S.-Q.; Tang, H.-Y.; Li, X.-J.; Zhang, L.; Xiao, J.; Gao, Y.-Q.; Zhang, A.-L.; Gao, J.-M. Potential allelopathic indole diketopiperazines produced by the plant endophytic Aspergillus f umigatus using the one strain−many compounds method. J. Agric. Food Chem. 2013, 61, 11447−11452. (21) Schulz, B.; Wanke, U.; Draeger, S.; Aust, H. J. Endophytes from herbaceous plants and shrubs: Effectiveness of surface sterilization methods. Mycol. Res. 1993, 97, 1447−1450. (22) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G. A.; Nakatsuji, H.; Caricato, M.; Li, X.; Hratchian, H. P.; Izmaylov, A. F.; Bloino, J.; Zheng, G.; Sonnenberg, J. L.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.; Montgomery, J. A., Jr.; Peralta, J. E.; Ogliaro, F.; Bearpark, M.; Heyd, J. J.; Brothers, E.; Kudin,

Table 2. Inhibitory Effects of Compounds 1−5 on Phytopathogenic Fungi phytopathogenic fungi (MIC, μM) compound

Alternaria solani

Botrytis cinerea

Fusarium solani

Gibberella saubinetti

1 2 3 4 5 carbendazim hymexazol

50 50 100 50 50 3.13 12.5

100 100 100 >100 100 6.25 50

100 50 >100 100 >100 6.25 50

50 12.5 100 >100 100 3.13 25

natural product seco-cytochalasin E. To the best of our knowledge, this is the first report of these three types of cytochalasan alkaloids in the genus Xylaria.49 Cytochalasin E was identified as the most active component in brine shrimp toxicity and phytotoxicity potency in the fungal endophyte. The preliminary SAR analysis suggested that cytochalasin E with a characteristic 13-membered macrocyclic vinyl carbonate group could most likely result in strong activity. Fungal endophytes are well-known for their ability to produce a wide range of antimicrobial substances and enhance plant resistance to pathogens and pests. Therefore, a chemical interaction between the endophyte Xylaria sp. and the plant T. sinensis produces the mycotoxins as chemical defense compounds that could protect the host.

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ASSOCIATED CONTENT

S Supporting Information *

Photographs of isolated strains, SEM photographs and rDNA ITS sequence of XC16, bioassay-guided results of the fungal exteacts of Xylaria sp., NMR spectra of compounds 1−3. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

* Tel: +86-29-87092515. Fax: +86-29-87092226. E-mail: [email protected]. Funding

This work was financially supported by the Natural Science Foundation of Shaanxi Province (2015YFJZ0115), as well as by the National Natural Science Foundation of China (31371886 and 21102114) and the Chinese Universities Scientific Fund (QN2011118). Notes

The authors declare no competing financial interest.

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H

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