Article pubs.acs.org/JAFC
Secondary Metabolites from the Endophytic Botryosphaeria dothidea of Melia azedarach and Their Antifungal, Antibacterial, Antioxidant, and Cytotoxic Activities Jian Xiao,∥ Qiang Zhang,∥ Yu-Qi Gao, Jiang-Jiang Tang, An-Ling Zhang, and Jin-Ming Gao* Shaanxi Engineering Center of Bioresource Chemistry and Sustainable Utilization, College of Science, Northwest A&F University, Yangling 712100, Shaanxi China S Supporting Information *
ABSTRACT: Two new metabolites, an α-pyridone derivative, 3-hydroxy-2-methoxy-5-methylpyridin-2(1H)-one (1), and a ceramide derivative, 3-hydroxy-N-(1-hydroxy-3-methylpentan-2-yl)-5-oxohexanamide (2), and a new natural product, 3-hydroxyN-(1-hydroxy-4-methylpentan-2-yl)-5-oxohexanamide (3), along with 15 known compounds including chaetoglobosin C (7) and chaetoglobosin F (8) were isolated from the solid culture of the endophytic fungus Botryosphaeria dothidea KJ-1, collected from the stems of white cedar (Melia azedarach L). The structures were elucidated on the basis of spectroscopic analysis (1D and 2D NMR experiments and by mass spectrometric measurements), and the structure of 1 was confirmed by X-ray single-crystal diffraction. These metabolites were evaluated in vitro for antimicrobial, antioxidant, and cytotoxicity activities. Pycnophorin (4) significantly inhibited the growth of Bacillus subtilis and Staphyloccocus aureus with equal minimum inhibitory concentration (MIC) values of 25 μM. Stemphyperylenol (5) displayed a potent antifungal activity against the plant pathogen Alternaria solani with MIC of 1.57 μM comparable to the commonly used fungicide carbendazim. Both altenusin (9) and djalonensone (10) showed markedly DPPH radical scavenging activities. In addition, stemphyperylenol (5) and altenuene (6) exhibited strong cytotoxicity against HCT116 cancer cell line with a median inhibitory concentration (IC50) value of 3.13 μM in comparison with the positive control etoposide (IC50 = 2.13 μM). This is the first report of the isolation of these compounds from the endophytic B. dothidea. KEYWORDS: endophyte, Botryosphaeria dothidea, cytotoxicity, antimicrobial activity
■
INTRODUCTION Plant-derived secondary metabolites have played an important role in medicinal and agricultural applications for centuries. However, due to the limitations of plant diversity, productivity, and sustainability, microorganisms, especially endophytic and symbiotic fungi, were considered as a readily renewable and inexhaustible source of novel bioactive secondary metabolites.1−3 Although endophytic fungi asymptomatically invade plant tissues, their secondary metabolites can stimulate plant growth or provide defense against plant pathogen attacks.3 These microorganisms have been thus recognized as potential sources of new bioactive compounds of possible agricultural, pharmaceutical, and industrial importance.4−6 Recent chemical studies of the endophytes associated with the medicinal plant white cedar (Melia azedarach L.) have led to the discovery of structurally unique and bioactive secondary metabolites, such as fusarimine,7 toxic fusariumin,8 insecticidal meroterpenes,9,10 allelopathic indole diketopiperazines,11 and antifungal alkaloids.12 As part of our program to investigate the chemical and biological diversity of the endophytic fungi isolated from M. azedarach, an endophytic fungus, Botryosphaeria dothidea KJ-1, was obtained from the stem bark of M. azedarach. The chemical investigation on the solid culture of B. dothidea led to the isolation and identification of three new natural products, one pyridone derivative, 3-hydroxy-2methoxy-5-methylpyridin-2(1H)-one (1), and two ceramide congeners, 3-hydroxy-N-(1-hydroxy-3-methylpentan-2-yl)-5© 2014 American Chemical Society
oxohexanamide (2) and 3-hydroxy-N-(1-hydroxy-4-methylpentan-2-yl)-5-oxohexanamide (3), together with 15 known compounds (Figure 1). Here, we report their structure elucidation and antimicrobial, antioxidant, and cytotoxic activities.
■
MATERIALS AND METHODS
General Experimental Procedures. Optical rotations were measured on a Rudolph Autopol III automatic polarimeter. UV measurements were obtained on a UV−vis Evolution 300 spectrometer. IR spectra were measured with a Bruker Tensor 27 spectrophotometer in KBr pellets. NMR spectra were performed on a Bruker Avance III spectrometer (Unity Plus 500 or 400 MHz). Chemical shifts were calculated using solvent residual as the internal standard. ESI-MS spectrometry data were obtained on a Thermo Fisher LTQ Fleet instrument spectrometer. HR-ESI-MS data were recorded on an Agilent 6520 Accurate-Mass Q-TOF LC-MS spectrometer. TLC and PTLC were performed on silica gel 60 GF254. Sephadex LH-20, MCI Gel CHP20P (75−150 μm), and silica gel (200−300 or 300−400 mesh and RP-C18) were used for column chromatography. Fractions were monitored by TLC, and spots were visualized by spraying with 5% H2SO4 in ethanol, followed by heating. All other chemicals used in this study are of analytical grade. Received: Revised: Accepted: Published: 3584
January 6, 2014 March 13, 2014 April 1, 2014 April 1, 2014 dx.doi.org/10.1021/jf500054f | J. Agric. Food Chem. 2014, 62, 3584−3590
Journal of Agricultural and Food Chemistry
Article
Figure 1. Structures of metabolites 1−18 isolated from B. dothidea.
Table 1. 1H NMR (500 MHz) and 13C NMR (125 MHz) Data for Compounds 1,a 2,b and 3b 1 δH 1 2 3 4 5 6 7 1′
162.7 s 132.0 s
6.05, s
159.2 s 100.5 d
2.21, s 4.02, s
141.0 s 19.0 q 59.7 q
2′ 3′ 4′ 5′ 6′ 1′-OH 3-OH NH a
2 δC
12.84, br s
δH, multi (J in Hz) 2.45, 2.36, 4.39, 2.73, 2.67,
dd dd m dd dd
(14.9, 3.7) (14.9, 7.7) (17.5, 8.1) (17.5, 4.3)
δH, multi (J in Hz)
172.1 s 42.3 t 65.0 d 49.1 t 209.3 s 30.8 q
2.19, s 3.71, 3.59, 3.81, 1.60, 1.48, 1.13, 0.88, 0.91, 3.20, 4.25, 6.52,
3 δC
dd (11.1, 3.3) dd (11.2, 6.8) m m m m d (7.4) d (6.9) s s d (8.5)
63.5 t 56.0 d 35.7 d 25.5 t 11.3 q 15.5 q
2.43, 2.35, 4.39, 2.73, 2.67,
dd dd m dd dd
(14.9, 3.6) (14.9, 7.7) (17.4, 8.1) (17.5, 4.0)
2.19, s 3.67, 3.49, 4.05, 1.34, 1.63,
d (10.4) m m m m
0.93, 0.89, 3.11, 4.18, 6.33,
d (6.6) d (6.6) s s d (7.3)
δC 172.0 s 42.2 t 64.9 d 49.1 t 209.2 s 30.8 q 66.0 t 50.0 d 40.0 t 24.9 d 23.0 q 22.1 q
In pyridine-d5. bIn CDCl3.
3585
dx.doi.org/10.1021/jf500054f | J. Agric. Food Chem. 2014, 62, 3584−3590
Journal of Agricultural and Food Chemistry
Article
to approximately 1 × 106 colony-forming units/mL (CFU/mL) with PD medium. In flat microtiter plates, tested compounds, fungal suspension, and sterile water were added to make up final concentrations of the compounds in the range of 1.57−200 μM. Each measurement consisted of three replicates. Cultures then grew 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. Antibacterial Bioassay. Metabolites were tested in vitro for the antibacterial activity against four pathogenetic bacteria: Escherichia coli, Bacillus subtilis, Staphyloccocus aureus, and Bacillus cereus. All of these tested pathogenetic bacteria were deposited at the College of Science, Northwest A&F University, China. Antibacterial activity was assessed by the microbroth dilution method in 96-well flat microtiter plates using a beef protein liquid (BP) medium.12 Antibacterial activity was assessed according to the same procedure as antifungal bioassay. Streptomycin sulfate, ampicillin, and toosendanin were used as positive controls, and the solution of equal concentration of DMSO was used as a negative control. Cultures were grown for 24 h at 37 ± 0.5 °C in the dark without shaking, in a moist chamber. 1,1-Diphenyl-2-picrylhydrazyl (DPPH) Radical Scavenging Assay. A previously described method13 was used to evaluate the radical scavenging capability of metabolites using DPPH. The reaction mixture (total volume = 4 mL), consisting of 1 mL of 0.2 mM DPPH in methanol and 3 mL of sample solution in methanol, was shaken vigorously and left to stand for 30 min in the dark; each measurement consisted of three replicates, and the absorbance was then measured at 517 nm. Tertiary butylhydroquinone (TBHQ) and vitamin C (VC) were used as positive controls. Lower absorbance of the reaction mixture indicated higher free radical scavenging activity. The DPPH radical concentration was calculated using the equation
Fungal Material. The endophytic fungal strain KJ-1 was isolated from the symptomless tissue of stem bark of M. azedarach L., which was collected at Yangling, Shaanxi province, China, in August 2011. The species was identified on the basis of the phylogenetic taxonomy with sequence alignment of ITS and had a genetic closeness of 100% to the B. dothidea strain. This strain was defined as B. dothidea KJ-1 and was deposited at the College of Science, Northwest A&F University, China. Fermentation and Cultivation. The producing strain was cultured on a plate of potato dextrose agar (PDA) medium at 28 ± 0.5 °C for 5 days. Then one piece (approximately 7 mm2) of mycelium was inoculated aseptically to 50 mL Erlenmeyer flasks each containing 20 mL of PD liquid medium, and the seed liquids were incubated at 28 ± 0.5 °C for 3 days on a rotary shaker at 120 rpm. A suspension (100 μL) of the seed liquid was inoculated aseptically to 500 mL Erlenmeyer flasks each containing 60 g of rice and 90 mL of distilled water. There were 300 flasks in total, and the flask cultures were incubated at 28 ± 0.5 °C for 4 weeks. Extraction and Isolation. Strain cultures were ultrasonically extracted three times with methanol (MeOH). The solvent was removed and dried under vacuum to yield a crude extract. The extract was dissolved in 90% MeOH/H2O (4 L) and further extracted three times with petroleum ether, and the remaining layer was adjusted to 50% aqueous methanol and extracted by chloroform. The chloroform extract (60 g) was separated by silica gel CC with CHCl3/MeOH (100:0 to 0:100) to provide four fractions. Fraction 1 (CHCl3) was separated by PTLC and repeated CC on silica gel, Sephadex LH-20, to yield compounds 4 (25 mg), 14 (6.5 mg), and 18 (85 mg). Fraction 2 (CHCl3/MeOH = 50:1) was separated by repeated CC on silica gel, Sephadex LH-20, followed by purification with PTLC to yield compounds 5 (13 mg), 6 (12 mg), 10 (8.5 mg), 11 (9.5 mg), and 16 (2.5 mg). Fraction 3 (CHCl3/MeOH = 20:1) was chromatographed over repeated CC on MCI gel, silica gel, Sephadex LH-20, and RP-C18 to yield compounds 1 (8 mg), 2 (9 mg), 3 (9 mg), 12 (90 mg), 13 (2.5 mg), and 17 (5 mg). Fraction 4 (CHCl3/MeOH = 5:1) was separated by repeated CC on silica gel, Sephadex LH-20, and RP-C18 to yield compounds 7 (13 mg), 8 (13 mg), 9 (38 mg), and 15 (40 mg). 3-Hydroxy-2-methoxy-5-methylpyridin-2(1H)-one (1). Light orange crystal (MeOH); mp 272.5−273.1 °C; UV (MeOH) λmax (log ε) = 207 (2.30), 285 nm (0.60); IR (KBr) νmax = 3442, 3017, 2927, 1630, 1571, 1461, 1221 cm−1; 1H and 13C NMR data are shown in Table 1; ESI-MS m/z 156.0 [M + H]+; HR-ESI-MS m/z 156.0647 ([M + H]+, C7H10NO3+; calcd 156.0655). 3-Hydroxy-N-(1-hydroxy-3-methylpentan-2-yl)-5-oxohexanamide (2). Colorless solid; [α]D23 −6.33 (c 0.14, CHCl3); IR (KBr) νmax = 3373, 3315, 1695, 1651, 1558, 1360, 1281 cm−1; 1H and 13C NMR data, see Table 1; ESI-MS m/z 245.81 [M + H]+; HR-ESI-MS m/z 268.1507 ([M + Na]+, C12H23NaNO4+; calcd 268.1519). 3-Hydroxy-N-(1-hydroxy-4-methylpentan-2-yl)-5-oxohexanamide (3). Colorless solid; [α]D23 −4.51 (c 0.12, CHCl3); IR (KBr) νmax = 3325, 3278, 1707, 1657, 1575 cm−1; 1H and 13C NMR data, see Table 1; ESI-MS m/z 267.98 [M + Na]+; HR-ESI-MS m/z 268.1506 ([M + Na]+, C12H23NaNO4+; calcd 268.1519). Antifungal Bioassay. Metabolites were tested in vitro for the antifungal activity against four different phytopathogenic fungi: Colletotrichum gloeosporiodes, Gibberella saubinetti, Botrytis cinerea, and Alternaria solani. All of these phytopathogenic fungi tested were provided by the College of Plant Protection, Northwest A&F University, and were deposited at the College of Science, Northwest A&F University, China. Antifungal activity was assessed by the microbroth dilution method in 96-well flat-microtiter plates using a potato dextrose (PD) medium.12 Compounds were made up to 4 mM in DMSO. Two commercial fungicides, carbendazim and hymexazol (Aladdin Chemistry Co. Ltd.), together with toosendanin, the efficient pesticide from M. azedarach L., were used as positive controls. The solution of equal concentration of DMSO 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 spore concentrations of different microorganism were diluted
DPPH scavenging effect (%) = (1 − A1/ A 0) × 100% where A0 and A1 are the 517 nm absorption of the control and the absorption of the sample reaction mixture, respectively. Brine Shrimp Bioassay. Metabolites were tested in vitro for the brine shrimp (Artemia salina) bioassay, processed according to the previously identified method.12 The final concentrations of the tested compounds were 10, 50, and 100 μM. Toosendanin was used as the positive control, and DMSO as the negative control. The lethality of each concentration was recorded. Cytotoxic Assay. In vitro cytotoxicity was assessed by using the SRB colorimetric assay.14 Briefly, 100 μL of HCT116 cells containing 2.5 × 104 cells/mL was added to each well of the 96-well flat microtiter plates and allowed to attach for 24 h. Then the medium was replaced by fresh medium, and cells were incubated with various amounts of the test compounds. After incubation for an additional 72 h at 37 °C, sulforhodamine B solution 0.4% (w/v) in 1% acetic solution was added to each well, and then bound sulforhodamine B was subsequently solubilized with 10 mM Tris base (pH 10.0), and the absorbance was read at 560 nm using an Epoch microplate reader. Each measurement consisted of three replicates. The percentage of cell viability was calculated relative to control wells designated as 100% viable cells. The IC50 values were measured by sigmoidal fit using Origin 7.5 software. X-ray Single-Crystal Diffraction. Crystals of compound 1 were grown in methanol at room temperature. Crystallographic data for 1 were collected on a SuperNova Eos diffractometer with graphite monochromatic Cu Kα radiation at 298(2) K. All calculations were performed using the SHELXL-97 program.15 Crystallographic data and experimental details for structure are listed in Table S1 in the Supporting Information. Parameters in CIF format are available as Electronic Supplementary Publication from Cambridge Crystallographic Data Centre (CCDC978829). These data can be obtained from http://www.ccdc.cam.ac.uk/conts/retrieving.html. 3586
dx.doi.org/10.1021/jf500054f | J. Agric. Food Chem. 2014, 62, 3584−3590
Journal of Agricultural and Food Chemistry
■
Article
[δH 2.19 (3H, s); δC 30.8 (q)], four aliphatic methylenes [δH 1.13 (1H, m, H-4′b), 1.48 (1H, m, H-4′a), 2.36 (1H, dd, J = 14.9, 7.7 Hz, H-2b), 2.45 (1H, dd, J = 14.9, 3.7, H-2a), 2.67 (1H, dd, J = 17.5, 4.3 Hz, H-4b), 2.73 (1H, dd, J = 17.5, 8.1 Hz, H-4a), 3.59 (1H, dd, J = 11.2, 6.8 Hz, H-1′b), 3.71 (1H, dd, J = 11.1, 3.3 Hz, H-1′a); δC 25.5 (t), 42.3 (t), 49.1 (t), 63.5 (t)], three aliphatic methines [δH 1.60 (1H, m), 3.81 (1H, m), 4.39 (1H, m); δC 35.7 (d), 56.0 (d), 65.0 (d)], two hydroxyls [δH 3.20, 4.25 (1H each, s)], one amide proton [δH 6.52 (1H, d, J = 8.5 Hz)], and two carbonyl carbons including an amide carbonyl one (δC 172.1) and a ketone carbonyl carbon (δC 209.3). The above evidence suggests that this compound is a short-chain fatty amide derivative. The correlations observed in the 1H−1H COSY and HSQC spectra of 2 indicate the presence of the following distinct spin systems: HOCH2− CH(NH)−CH(CH 3 )−CH 2 CH 3 (C-1′ to C-5′), and −CH2CH(OH)CH2− (C-4 to C-2). These data and HMBC correlations of NH/C-1, -2′, -3′, H-1′a/C-2′, -3′, H-2′/C-1, -1′, -3′, -4′, -6′, H-3′/C-4′, H-4′b/C-2′, -3′, -5′, -6′, H-6′/C-2′, -3′, -4′, H-2/C-1, -3, -4, H-4/C-2, -3, -5, and H-6/C-4 revealed a fatty amide of an isoleucinol and locations of a hydroxyl at C-3 and one ketone carbonyl group at C-5, as shown in Figure 3.
RESULTS AND DISCUSSION Structure Elucidation. Compound 1 was obtained as a light orange crystal, and its molecular formula was assigned as C7H9NO3 on the basis of the pseudomolecular ion at m/z 156.0647 [M + H]+ in HR-ESI-MS, requiring four degrees of unsaturation. The IR spectrum showed absorption for hydroxyl group and/or amide proton (3442 cm−1) and a conjugated amide carbonyl group (1630 cm−1). A strong UV absorption appeared at 285 nm, indicating that 1 contained a chromophoric moiety. The 1H NMR spectra of 1 (Table 1) displayed the presence of one methyl (δ 2.21, s), one methoxyl (δ 4.02, s), and one double-bond proton (δ 6.05, s). The 13C NMR and DEPT spectra of 1 (Table 1) revealed seven carbon signals, including one methyl (δ 19.0 q), one methoxyl (δ 59.7 q), one olefinic methine (δ 100.5 d), and four sp2 quaternary carbons (including one oxygenated olefin at δ 159.2 and 132.0 (two oxygenated olefinic carbons), a trisubstituted olefin carbon (δ 141.0 s), and an amide carbonyl (δ 162.7 s)). The assignment of all 1H and 13C NMR signals for 1 was achieved by analysis of HSQC and HMBC spectral data. In the HMBC spectrum of 1, correlations of H-4 (δ 6.05) with C-2, C-3, C-5, and C-6, H3-6 with C-4, C-5, and H3-7 to C-2 indicated locations of the methyl at C-5 (δ 141.0) and the methoxy group at C-2 (δ 132.0) in the molecule. This evidence suggests the existence of one 2-methoxy-3-hydroxypenta-2,4-diene moiety. Considering the molecular formula of 1, the remaining carbonyl and secondary amino NH groups should form an amide −CONH− unit that was attached to C-2 and C-5, respectively. However, no correlations from NH to C-1, C-2, C-5, and C-6 in the HMBC spectrum were observed. From the abovementioned data, the planar structure of 1 was deduced as shown in Figure 2a. X-ray single-crystal diffraction of 1 with Cu
Figure 3. Key HMBC and 1H−1H COSY correlations of 2 and 3.
These findings also confirmed the presence of an acyl residue, namely, 3-hydroxy-5-oxohexanoyl group (CH3COCH2CH(OH)CH2CO−). Thus, the structure of 2 was determined to be 3-hydroxy-N-(1-hydroxy-3-methylpentan-2-yl)-5-oxohexanamide. Compound 3 was obtained as a colorless solid. Its molecular formula was assigned as C12H23NO4 on the basis of the molecular ion at m/z 268.1506 [M + Na]+ in the HR-ESI-MS. Comparison of its 1H and 13C NMR spectra (Table 1) with those of 2 suggests that compound 3 had the same framework as 2. A significant difference was that the aliphatic C-6′ methyl should be placed at C-4′ (δC 24.9 d) in 3 rather than at C-3′ (δC 35.7 d) as in 2. The structure of 3 was further confirmed by analysis of 1H−1H COSY correlations of NH/H-2′, H-2′/H-1′, -3′, H-1′/1′-OH, H-4′/H-3′, -5′, -6′, H-3/H-2, 3-OH, -4, and the HMBC correlations of NH/C-1, -2′, H-3′/C-2′, -4′, -5′, -6′, H-4′/C-3′, -5′, -6′, H-2/C-1, -3, -4, H-4/C-2, -3, -5, H-6/C-4, -5 (Figure 3). Thus, the structure of 3 was elucidated as 3hydroxy-N-(1-hydroxy-4-methylpentan-2-yl)-5-oxohexanamide, which possesses a leucinol unit. Although it can be searched in SciFinder, it is a new natural product, and this is the first report of its spectral data. Compounds 2 and 3 belong to a group of ceramides. Absolute stereochemistries of 2 and 3 remain undetermined due to limited amounts. The structures of 15 known compounds were identified as pycnophorin (4),16 stemphyperylenol (5),17 altenuene (6),18 chaetoglobosin C (7),19,20 chaetoglobosin F (8),20 altenusin (9),21 djalonensone (10),22 alternariol (11),23,24 5′-methoxy-6methylbiphenyl-3,4,3′-triol (12),14 7-hydroxy-1-isochromanone
Figure 2. Key HMBC (2a) and ORTEP diagram (2b) of 1.
Kα radiation confirmed the proposed structure (Figure 2b; for further details see Table S1 in the Supporting Information). Thus, the structure of 1 was elucidated as 3-hydroxy-2methoxy-5-methylpyridin-2(1H)-one. Compound 2 was obtained as a colorless solid and analyzed for the molecular formula C12H23NO4 by HR-ESI-MS (m/z 268.1507 [M + Na]+), requiring two degrees of unsaturation. The 1H NMR and 13C NMR spectra of 2 (Table 1) showed two aliphatic methyl doublets [δH 0.88 (3H, d, J = 7.4 Hz), 0.91 (3H, d, J = 6.9 Hz); δC 11.3 (q), 15.5 (q)], one methyl singlet 3587
dx.doi.org/10.1021/jf500054f | J. Agric. Food Chem. 2014, 62, 3584−3590
Journal of Agricultural and Food Chemistry
Article
(13),25 ergosterol peroxide (14),26 β-sitosterol glucoside (15),27 5-(hydroxymethyl)-1H-pyrrole-2-carbaldehyde (16),28 5-hydroxymethylfurfural (17),29 and cerebroside C (18)30 by comparison of their spectroscopic data with those reported in the literature, including UV, IR, NMR, and MS data. To our knowledge, this is the first report of the isolation of these compounds from B. dothidea as endophytic fungus. Interestingly, perylenequinone derivatives, for example, stemphyperylenol (5), have been previously reported from the genera Alternaria, Stemphylium, and Talaromyces.31−34 Altenuene derivatives, for example, altenuene (6), have been reported from the genera Alternaria,35−37 Ulocladium,14 Nigrospora, and Phialophora.38 In addition, alternariol analogues (10 and 11) have been previously reported from several genera of microorganisms,36−43 especially from the common fungal genus Alternaria.35 Chaetoglobosins (7 and 8) are a family of cytochalasans with a wide range of biological activities targeting cytoskeletal processes, which have been produced by several genera of microorganisms, especially by the common fungal genus Chaetomium.44,45 According to previous investigations, alternariol and alternariol monomethyl ether were reported to have antimicrobial, cytotoxic, and antitumor activities,35,36 and alternariol (11) showed antiviral effects against herpes simplex virus in vitro.38 Some perylenequinone derivatives are phytotoxins and mutagenic.46,47 Antimicrobial Assays. Metabolites 1−18 were tested in vitro for antifungal activity against four phytopathogenic fungi: Colletotrichum gloeosporiodes, Gibberella saubinetti, Botrytis cinerea, and Alternaria solani. None of the tested compounds displayed any obvious antifungal activities against C. gloeosporiodes and G. saubinetti (Supporting Information Table S2). Compounds 4, 5, 7, 10, 11, 15, and 17 showed strong to moderate antifungal activities against A. solani (MICs of 1.57− 25 μM) and did not have effects against B. cinerea (Table 2).
fungicides to A. solani. Compound 10 also displayed moderate antifungal activities against B. cinerea (MIC of 25 μM). Compounds 1−18 were tested in vitro for their antibacterial activity against pathogenetic bacteria Escherichia coli, Bacillus subtilis, Staphyloccocus aureus, and Bacillus cereus, and the results are listed in Table S3 in the Supporting Information. The results showed that compound 4 mildly inhibited the growth of B. subtilis and S. aureus with both having MICs of 25 μM; the effects are much weaker than those of two positive controls, ampicillin and streptomycin sulfate, whereas the other compounds had weak activity (MICs of >50 μM). DPPH Radical Scavenging Assay. Both compounds 9 and 12 showed marked DPPH radical scavenging activities with IC50 values of 17.6 ± 0.23 and 18.7 ± 0.18 μM, respectively, which are weaker than those of the positive controls TBHQ and Vc (IC50 of 10 μM). These results are in agreement with those reported by Wang et al.14 Compounds 1 and 4 were found to be weak DPPH scavengers at concentrations of 50 μM, with scavenging rates of 22.5 and 30.9%, respectively, and the remaining metabolites showed no activity. From a comparison of the structures of compounds 6, 10, and 11 to compounds 9 and 12, it can be found that opening of the middle ring can result in enhanced DPPH scavenging capacity. Brine Shrimp Bioassay. Brine shrimp can be used as a convenient bioassay model for insecticidal activity.48 Compounds 1−18 were evaluated for insecticidal activity, and brine shrimp lethality is shown in Table S4 in the Supporting Information. Among the compounds tested, compound 4 was found to show the best activity with lethality of 43.8% at a concentration of 100 μM, whereas the other compounds were inactive. Cytotoxicity Bioassay. Compounds 1−18 were also evaluated in vitro against cancer cell line HCT116 by the sulforhodamine B (SRB) colorimetric assay, and the results are summarized in Table 3. Among them, compounds 5 and 6
Table 2. Inhibitory Effects of Compounds 1−18 on Phytopathogenic Fungia
Table 3. Cytotoxicity of Compounds 1−18 against Human Colorectal HCT 116 Cell Linea
phytopathogenic fungi (MIC, μM) a
compd
B. cinerea
A. solani
compd
IC50a (μM)
4 5 7 10 11 12 15 16 17 carbendazim hymexazol toosendanin
100 100 200 25 NA NA 100 100 50 12.5 200 200
6.25 1.57 12.5 25 12.5 50 6.25 50 6.25 1.57 6.25 6.25
1 2 3 4 5 6 7 8 9 10
>100 >100 >100 21.4 3.13 3.13 >100 26.5 28.9 NA
a
a
NA, inactive. Compounds with no antifungal activity are not shown in the table.
compd 11 12 13 14 15 16 17 18 etoposide
IC50a (μM) 33.9 73.4 NA 72.3 NA >100 NA NA 2.13
NA, inactive.
exhibited the strongest cytotoxicity against HCT116 with an IC50 value of 3.13 μM, which is comparable to that of the positive control etoposide (IC50 of 2.13 μM), whereas compounds 4, 8, 9, and 11 exhibited moderate activity (IC50 of 10−40 μM), and the remaining compounds were inactive (IC50 of >40 μM). These results indicate that compounds 5 and 6 could be promising lead compounds for the development of new antitumor drugs and need to be studied for their mechanisms of action. In recent years, many metabolites having plant protection have been identified from plant endophytes, such as ambuic
Methylation of 5-OH in alternariol (11) resulted in diminished activity (MIC of 25 μM for djalonensone (10) vs MIC of 12.5 μM for 11) to A. solani. Of these compounds, stemphyperylenol (5), with the lowest MIC value of 1.57 μM, had the best inhibition, which is comparable to that of the positive control, carbendazim (MIC of 1.57 μM), or better than those of hymexazol and toosendanin (MICs of 6.25 μM), indicating that it could be used as a fungicide or as a lead compound of new 3588
dx.doi.org/10.1021/jf500054f | J. Agric. Food Chem. 2014, 62, 3584−3590
Journal of Agricultural and Food Chemistry
Article
acid and phenazine-1-carboxylic acid.49,50 In the present study, the secondary metabolites produced by the endophyte B. dothidea were found to have some antimicrobial and antioxidant activities. Stemphyperylenol (5) was found to display significant antifungal activity against A. solani. Altenusin (9) and 5′-methoxy-6-methylbiphenyl-3,4,3′-triol (12) showed marked DPPH radical scavenging activities. These bioactive metabolites in B. dothidea could protect the host plant against pathogenic attacks and oxidation stress in nature, which may play a defensive role.
■
(11) 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. (12) 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. (13) Wang, Q. X.; Bao, L.; Yang, X. L.; Guo, H.; Yang, R. N.; Ren, B.; Zhang, L. X.; Dai, H. Q.; Guo, L. D.; Liu, H. W. Polyketides with antimicrobial activity from the solid culture of an endolichenic fungus Ulocladium sp. Fitoterapia 2012, 83, 209−214. (14) Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J.; Vistica, D.; Warren, J. T.; Bokesch, H.; Kenney, S.; Boyd, M. R. New colorimetric cytotoxicity assay for anticancer-drug screening. J. Natl. Cancer Inst. 1990, 82, 1107−1112. (15) Sheldrick, G. M. SHELXTL-97 − A Program for Refining Crystal Structure; University of Goettingen: Goettingen, Germany, 1997. (16) Sassa, T.; Kato, H.; Kajiura, H. Isolation and structure of pycnophorin, a novel diterpene α-pyrone with antimicrobial activity, produced by phytopathogenic Macrophoma kuwatsukai. Tetrahedron Lett. 1986, 27, 2121−2124. (17) Arnone, A.; Nasini, G.; Merlini, L.; Assante, G. Secondary mould metabolites. Part 16. Stemphyltoxins, new reduced perylenequinone metabolites from Stemphylium botryosum var. Lactucum. J. Chem. Soc., Perkin Trans. 1 1986, 525−530. (18) Bradburn, N.; Coker, R. D.; Blunden, G.; Turner, C. H.; Crabb, T. A. 5′-Epialtenuene and neoaltenuene, dibenzo-α-pyrones from Alternaria alternata cultured on rice. Phytochemistry 1994, 35, 665− 669. (19) Sekita, S.; Yoshihira, K.; Natori, S.; Kuwano, H. Chaetoglobosins, cytotoxic 10-(indol-3-yl)-[13] cytochalasans from Chaetomium spp. III. Structures of chaetoglobosins C, E, F, G, and J. Chem. Pharm. Bull. 1982, 30, 1629−1638. (20) Sekita, S.; Yoshihira, K.; Natori, S. Chaetoglobosins, cytotoxic 10-(indol-3-yl)-[13] cytochalasans from Chaetomium spp. IV. 13Cnuclear magnetic resonance spectra and their application to a biosynthetic study. Chem. Pharm. Bull. 1983, 31, 490−498. (21) Singh, S. B.; Jayasuriya, H.; Dewey, R.; Polishook, J. D.; Dombrowski, A. W.; Zink, D. L.; Guan, Z.; Collado, J.; Platas, G.; Pelaez, F.; Felock, P. J.; Hazuda, D. J. Isolation, structure, and HIV-1 integrase inhibitory activity of structurally diverse fungal metabolites. J. Ind. Microbiol. Biotechnol. 2003, 30, 721−731. (22) Onocha, P. A.; Okorie, D. A.; Connolly, J. D.; Roycroft, D. S. Monoterpene diol, iridoid glucoside and dibenzo-α-pyrone from Anthocleista djalonensis. Phytochemistry 1995, 40, 1183−1189. (23) Qin, J. C.; Zhang, Y. M.; Hu, L.; Ma, Y. T.; Gao, J. M. Cytotoxic metabolites produced by Alternaria no. 28, an endophytic fungus isolated from Ginkgo biloba. Nat. Prod. Commun. 2009, 4, 1473−1476. (24) Koch, K.; Podlech, J.; Pfeiffer, E.; Metzler, M. Total synthesis of alternariol. J. Org. Chem. 2005, 70, 3275−3276. (25) Odasso, G.; Winters, G. Cyclic hydrazides. IV. Synthesis of 2amino-3,4-dihydroisoquinolin-1(2H)-one. Farmaco 1978, 33, 148− 155. (26) Chen, Y. K.; Kuo, Y. H.; Chiang, B. H.; Lo, J. M.; Sheen, L. Y. Cytotoxic activities of 9,11-dehydroergosterol peroxide and ergosterol peroxide from the fermentation mycelia of Ganoderma lucidum cultivated in the medium containing leguminous plants on Hep 3B cells. J. Agric. Food Chem. 2009, 57, 5713−5719. (27) Kojima, H.; Sato, N.; Hatano, A.; Ogura, H. Sterol glucosides from Prunella vulgaris. Phytochemistry 1990, 29, 2351−2355. (28) Sudhakar, G.; Kadam, V. D.; Bayya, S.; Pranitha, G.; Jagadeesh, B. Total synthesis and stereochemical revision of acortatarins A and B. Org. Lett. 2011, 13, 5452−5455. (29) Jogia, M. K.; Vakamoce, V.; Weavers, R. T. Synthesis of some furfural and syringic acid derivatives. Aust. J. Chem. 1985, 38, 1009− 1016.
ASSOCIATED CONTENT
S Supporting Information *
1D and 2D NMR, HR-ESI-MS spectra of compounds 1−3, Xray crystallographic data of 1, and some bioassays results. This material is available free of charge via the Internet at http:// pubs.acs.org.
■
AUTHOR INFORMATION
Corresponding Author
*(J.-M.G.) Phone: +86-29-87092515. E-mail: jinminggao@ nwsuaf.edu.cn. Author Contributions ∥
J.X. and Q.Z. contributed equally to this work.
Funding
This work is supported by the Program for New Century Excellent Talents in University (NCET-05-0852). Notes
The authors declare no competing financial interest.
■
REFERENCES
(1) Chandra, S. Endophytic fungi: novel sources of anticancer lead molecules. Appl. Microbiol. Biotechnol. 2012, 95, 47−59. (2) Ebrahim, W.; Aly, A. H.; Mandi, A.; Totzke, F.; Kubbutat, M. H. G.; Wray, V.; Lin, W.-H.; Dai, H.; Proksch, P.; Kurtan, T.; Debbab, A. Decalactone derivatives from Corynespora cassiicola, an endophytic fungus of the mangrove plant Laguncularia racemosa. Eur. J. Org. Chem. 2012, 18, 3476−3484. (3) Gao, J. M.; Yang, S. X.; Qin, J. C. Azaphilones: chemistry and biology. Chem. Rev. 2013, 113, 4755−4811. (4) Schulz, B.; Boyle, C.; Draeger, S.; Römmert, A.-K.; Krohn, K. Endophytic fungi: a source of novel biologically active secondary metabolites. Mycol. Res. 2002, 106, 996−1004. (5) Qin, J. C.; Zhang, Y. M.; Gao, J. M.; Bai, M. S.; Yang, S. X.; Laatsch, H.; Zhang, A. L. Bioactive metabolites produced by Chaetomium globosum, an endophytic fungus isolated from Ginkgo biloba. Bioorg. Med. Chem. Lett. 2009, 19, 1572−1574. (6) 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. (7) Yang, S. X.; Xiao, J.; Laatsch, H.; Holstein, J. J.; Dittrich, B.; Zhang, Q.; Gao, J. M. Fusarimine, a novel polyketide isoquinoline alkaloid, from the endophytic fungus Fusarium sp. LN12, isolated from Melia azedarach. Tetrahedron Lett. 2012, 53, 6372−6375. (8) Yang, S. X.; Gao, J. M.; Zhang, Q.; Laatsch, H. Toxic polyketides produced by Fusarium sp., an endophytic fungus isolated from Melia azedarach. Bioorg. Med. Chem. Lett. 2011, 21, 1887−1889. (9) Fill, T. P.; Pereira, G. K.; dos Santos, R. G.; Rodrigues-Fo, E. Four additional meroterpenes produced by Penicillium sp. found in association with Melia azedarach. Possible biosynthetic intermediates to austin. Z. Naturforsch. 2007, 62B, 1035−1044. (10) Geris dos Santos, R. M.; Rodrigues-Fo, E. Meroterpenes from Penicillium sp. found in association with Melia azedarach. Phytochemistry 2002, 61, 907−912. 3589
dx.doi.org/10.1021/jf500054f | J. Agric. Food Chem. 2014, 62, 3584−3590
Journal of Agricultural and Food Chemistry
Article
cyclohexenone with antifungal activity from Pestalotiopsis spp. and Monochaetia sp. Phytochemistry 2001, 56, 463−468. (50) Delaney, S. M.; Mavrodi, D. V.; Bonsall, R. F.; Thomashow, L. S. phzO, a gene for biosynthesis of 2-hydroxylated phenazine compounds in Pseudomonas aureofaciens 30-84. J. Bacteriol. 2001, 183, 318−327.
(30) Koga, J.; Yamauchi, T.; Shimura, M.; Ogawa, N.; Oshima, K.; Umemura, K.; Kikuchi, M.; Ogasawara, N. Cerebrosides A and C, sphingolipid elicitors of hypersensitive cell death and phytoalexin accumulation in rice plants. J. Biol. Chem. 1998, 273, 31985−31991. (31) Gao, S. S.; Li, X. M.; Wang, B. G. Perylene derivatives produced by Alternaria alternata, an endophytic fungus isolated from Laurencia species. Nat. Prod. Commun. 2009, 4, 1477−1480. (32) Davis, V. M.; Stack, M. E. Mutagenicity of stemphyltoxin III, a metabolite of Alternaria alternata. Appl. Environ. Microbiol. 1991, 57, 180−182. (33) Arone, A.; Nasini, G. Secondary mould metabolites. Part 16. Stemphyltoxins, new reduced perylenequione metabolites from Stermphylium botryosum var. Lactucum. J. Chem. Soc., Perkin Trans. 1 1986, 525−530. (34) Liu, F.; Cai, X. L.; Yang, H.; Xia, X. K.; Guo, Z. Y.; Yuan, J.; Li, M. F.; She, Z. G.; Lin, Y. C. The bioactive metabolites of the mangrove endophytic fungus Talaromyces sp. ZH-154 isolated from Kandelia candel (L.) Druce. Planta Med. 2010, 76, 185−189. (35) Aly, A. H.; Edrada-Ebel, R.; Indriani, I. D.; Wray, V.; Müller, W. E. G.; Totzke, F.; et al. Cytotoxic metabolites from the fungal endophyte Alternaria sp. and their subsequent detection in its host plant Polygonum senegalense. J. Nat. Prod. 2008, 71, 972−980. (36) Kjer, J.; Wray, V.; Edrada-Ebel, R.; Ebel, R.; Pretsch, A.; Lin, W.; Proksch, P. Xanalteric acids I and II and related phenolic compounds from an endophytic Alternaria sp. isolated from the mangrove plant Sonneratia alba. J. Nat. Prod. 2009, 72, 2053−2057. (37) Stack, M. E.; Mazzola, E. P.; Page, S. W.; Pohland, A. E.; Highet, R. J.; Tempesta, M. S.; Corley, D. G. Mutagenic perylenequinone metabolites of Alternaria alternata: altertoxins I, II and III. J. Nat. Prod. 1989, 49, 866−871. (38) He, J. W.; Chen, G. D.; Gao, H.; Yang, F.; Li, X. X.; Peng, T.; Guo, L. D.; Yao, X. S. Heptaketides with antiviral activity from three endolichenic fungal strains Nigrospora sp., Alternaria sp. and Phialophora sp. Fitoterapia 2012, 83, 1087−1091. (39) Matumoto, T.; Hosoya, T.; Shigemori, H. Palmariols A and B, two new chlorinated dibenzo-α-pyrones from Discomycete Lachnum palmae. Heterocycles 2010, 81, 1231−1237. (40) Hussain, H.; Krohn, K.; Ullah, Z.; Draeger, S.; Schulz, B. Bioactive chemical constituents of two endophytic fungi. Biochem. Syst. Ecol. 2007, 35, 898−900. (41) Zhang, H. W.; Huang, W. Y.; Song, Y. C.; Chen, J. R.; Tan, R. X. Four 6H-dibenzo[b,d]pyran-6-one derivatives produced by the endophyte Cephalosporium acremonium IFB-E007. Helv. Chim. Acta 2005, 88, 2861−2864. (42) Dai, J. Q.; Krohn, K.; Flörke, U.; Gehle, D.; Aust, H. J.; Draeger, S.; Schulz, B.; Rheinheimer, J. Novel highly substituted biraryl ethers, phomosines D−G, isolated from the endophytic fungus Phomopsis sp. from Adenocarpus foliolosus. Eur. J. Org. Chem. 2005, 23, 5100−5105. (43) Oyama, M.; Xu, Z. H.; Lee, K. H.; Spitzer, T. D.; Kitrinos, P.; Mcdonald, O. B.; Jones, R. R. J.; Garvey, E. P. Fungal metabolites as potent protein kinase inhibitors: identification of a novel metabolite and novel activities of known metabolites. Lett. Drug. Des. Discovery 2004, 1, 24−29. (44) 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. (45) 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. (46) Stack, M. E.; Prival, M. J. Mutagenicity of the Alternaria metabolites altertoxins I, II, and III. Appl. Environ. Microbiol. 1986, 52, 718−722. (47) Hradil, C. M.; Hallock, Y. F.; Clardy, J.; Kenfield, D. S.; Strobel, G. Phytoxins from Alternaria cassiae. Phytochemistry 1989, 28, 73−75. (48) Blizzard, T. A.; Ruby, C. L.; Mrozik, H.; Preiser, F. A.; Fisher, M. H. Brine shrimp (Artemia salina) as a convenient bioassay for avermectin analogs. J. Antibiot. (Tokyo) 1989, 42, 1304. (49) Li, J.; Harper, J. K.; Grant, D. M.; Tombe, B. O.; Bashyal, B.; Hess, W.; Strobel, G. A. Ambuic acid, a highly functionalized 3590
dx.doi.org/10.1021/jf500054f | J. Agric. Food Chem. 2014, 62, 3584−3590
