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New sesquiterpenes from the endophyte Microdiplodia sp. TT-12 and their antimicrobial activity
Phytochemistry Letters 14 (2015) 143–147
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New sesquiterpenes from the endophyte Microdiplodia sp. TT-12 and their antimicrobial activity Yoshihito Shiono* , Hiromasa Koyama, Tetsuya Murayama, Takuya Koseki Department of Food, Life, and Environmental Science, Faculty of Agriculture, Yamagata University, Tsuruoka, Yamagata 997-8555, Japan
A R T I C L E I N F O
A B S T R A C T
Article history: Received 23 May 2015 Received in revised form 6 October 2015 Accepted 8 October 2015 Available online xxx
Four new compounds, phomadecalin F (1), 8a-monoacetoxyphomadecalin D (2), 3-epi-phomadecalin D (3), and 13-hydroxylmacrophorin A (4), were isolated from the endophyte, Microdiplodia sp. TT12 together with two known compounds, phomadecalins C (5) and D (6). The structures of the compounds were elucidated by NMR spectroscopic and mass spectrometric analyses in combination with chemical means. The antibacterial activities of the isolated compounds were evaluated. Compound 4 was weakly active against Raffaelea quercivora. ã 2015 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
Keywords: Endophyte Raffaelea quercivora Microdiplodia sp. TT-12 Phomadecalin Macrophorin A
1. Introduction Japanese oak wilt (JOW) is a fungal disease that affects Japanese oak, Quercus serrata, on the Sea of Japan side of Japan. JOW is caused by a fungal pathogen, Raffaelea quercivora, which is vectored by female ambrosia beetles, namely Platypus quercivorus (Kubono and Ito, 2002). Detailed observation of the distribution of R. quercivora hyphae in JOW-infested Quercus saplings revealed that the hyphal distribution overlapped with areas where vessel function was disturbed. Accordingly, it is thought that the mortality caused by JOW might be induced by the severe dysfunction of vessel lumens (Takahashi et al., 2010). Since the late 1980s, JOW has steadily been spreading across mainland Japan (Kuroda, 2001). Due to the rapid expansion of JOW, Japanese oaks have died at a rate of approximately 1000 ha/year (Takahashi et al., 2010). Although the number of affected trees has not increased in the last several years, the invasiveness of JOW has caused a significant loss of biodiversity in Japan. Several endophytes have been reported to support the growth and improve the health of plants, and therefore may serve as important biocontrol agents (Haggag, 2010; O’Hanlon et al., 2012; Zhanga et al., 2014). Interestingly, it has been reported that endophytic bacteria isolated from surviving live oaks, Quercus fusiformis, in Texas could be used as effective biocontrol agents
* Corresponding author. Fax: +81 235 28 2873. E-mail address: [email protected] (Y. Shiono).
against the oak wilt pathogen Ceratocystis fagacearum (Brooks et al., 1994). In this study, we investigated the endophytes associated with surviving live Japanese oaks in Yamagata Prefecture, where JOW is epidemic, and we evaluated the antifungal activity of metabolites from the endophytes against the fungal pathogen of JOW, R. quercivora. Of the 50 fungal isolates tested, a methanol extract of a culture of the fungal strain Microdiplodia sp. TT-12 inhibited R. quercivora in vitro. Herein, we describe the fermentation, isolation, structure determination, and biological activities of the secondary metabolites produced by Microdiplodia sp. TT-12. 2. Results and discussion Steamed unpolished rice was used as the cultivation substrate for the endophyte Microdiplodia sp. TT-12. Cultivated media were extracted with methanol (1.5 L) for 24 h. The organic extract was concentrated, and the aqueous residue was extracted with ethyl acetate. The organic layer was subjected to silica gel column chromatography using a gradient of n-hexane–EtOAc as the eluent. Further chromatographic studies revealed the presence of six compounds: phomadecalin F (1), 8a-monoacetoxyphomadecalin D (2), 3-epi-phomadecalin D (3), 13-hydroxylmacrophorin A (4), and phomadecalins C (5) and D (6). Known compounds 5 and 6 were identified as phomadecalins C and D on the basis of their 1H and 13C NMR, 1H–1H COSY, HMQC, and HMBC data (Che et al., 2002). The molecular formula of 1, C15H20O4, was determined by HRESITOFMS data (m/z 289.1289 [M + Na]+). The IR spectrum of 1
http://dx.doi.org/10.1016/j.phytol.2015.10.004 1874-3900/ ã 2015 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
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Y. Shiono et al. / Phytochemistry Letters 14 (2015) 143–147
indicated the presence of a carbonyl group. The UV spectrum of 1 in MeOH exhibited the presence of a triene moiety. The gross structure of 1 was deduced from 1H and 13C NMR data, as well as 2D NMR experiments. Close inspection of the 13C NMR (Table 1) and DEPT spectra of 1 revealed carbon signals that were attributed to two methyl groups, three sp3 methylene moieties (two of them bearing an oxygen atom), two sp3 methine moieties (one of them bearing an oxygen atom), one sp3 quaternary carbon, three sp2 quaternary carbons, three sp2 methine moieties, and one a,b-unsaturated ketone. The 1H NMR and HMQC spectra of 1 exhibited proton signals which were attributed to one secondary methyl group [dH 1.05 (d, J = 6.3 Hz, Me-15)], one singlet methyl [dH 0.92 (s, Me-14)], coupled olefinic protons [dH 6.83 (d, J = 10.3 Hz, H8), 6.29 (d, J = 10.3 Hz, H-9)], one singlet olefinic methine moiety [dH 5.96 (s, H-1)], two methane moieties [dH 1.54 (m, H-4), 4.57 (s, H-6)], and three methylene groups [dH 2.39 (m, H-3), 4.36 (d, J = 12.7 Hz, H-12), 4.50 (d, J = 12.7 Hz, H-12), 4.30 (d, J = 12.7 Hz, H13), 4.50 (d, J = 12.7 Hz, H-13)]. Overall, the NMR data suggested that the structure of 1 was closely related to a eremophilane sesquiterpene analogue. The 1H–1H COSY spectrum revealed the partial structures that comprised 1 (Fig. S19). The connections between the partial structures and functional groups were determined on the basis of key HMBC correlations, as summarized in Fig. S19. HMBC correlations from H-1 to C-3, from Me-14 to C-4, C-5, and C-10, and from Me-15 to C-3, C-4, and C-5 revealed the presence of an a,b-unsaturated carbonyl functionality (4,5dimethyl-2-cyclohexen-1-one). Since six out of eight unsaturation equivalents were accounted for in the aforementioned 13C NMR data, 1 was inferred to contain two rings. Three olefinic protons in the 1H NMR spectrum and HMBC correlations from H-9 to C-1 and C-7, from H-8 to C-6, and from Me-14 to C-6, indicated that the 2cyclohexen-1-one moiety was fused to the hydroxycyclohexene unit (C-5 to C-10). The oxymethylene protons (H2-12, 13) showed HMBC correlations with the carbons at C-7 and C-11, which indicated that the 1,3-propanediol moiety was attached at the C-7position. The relative configuration of 1 was verified by NOE and comparison to similar compounds. NOE correlations from Me-15 to H-6 and from Me-14 to H-6 revealed that Me-14, Me-15, and H6 were on the same side of the molecular plane as the b-configuration. Thus, the structure of 1 was unambiguously determined to be 6a-hydroxy-1(10), 9-trien-2-oxoeremophilane,
which represented a novel phomadecalin-like compound and was named phomadecalin F. NMR spectroscopy was used to determine the absolute stereochemistry of the chiral center at C-6, by employing a modified version of Mosher's method (Ohtani et al., 1991). The chiral derivatizing agents used in the esterification reaction were (S)- and (R)-a-methoxy-a-(trifluoromethyl) phenylacetic acid [(S)- and (R)-MTPA]. Unfortunately, when compound 1 was treated with (S)- and (R)-MTPA, the desired esterified products at C-6 could not be obtained due to the low reactivity of MTPA with OH-6. 8a-Monoacetoxyphomadecalin D (2) was determined to have the molecular formula C17H22O5 using HRESITOFMS data (m/z 329.1395 [M + Na]+). The structure of 2 was determined using 1H and 13 C NMR spectral data (Tables 1 and 2). Furthermore, a mass unit difference of 42 was revealed between 2 and 6. The IR spectrum of 2, which exhibited an ester carbonyl absorption, as well as the appearance of a three-proton singlet at dH 2.09, a methyl carbon at dH 20.7, and an ester carbonyl at dC 170.9 indicated the presence of an additional acetyl group. HMBC correlations from H-8 to the ester carbonyl carbon confirmed the substitution of the acetoxy group at C-8 (Fig. S19). The relative configuration of 2 was determined by NOE correlations (Fig. S20). In addition, when 2 was treated with (S)and (R)-MTPA in the presence of DCC-DMAP at room temperature, 3,13-bis-MTPA esters (2a/2b) were produced. The differences in the 1 H chemical shifts between the (R)- and (S)-MTPA esters (2a/2b) of 2 are shown in Fig. S21. These results suggested that the asymmetric center at C-3 had the R-configuration. The molecular formula of 3 was established to be C15H20O4 by HRESITOFMS data (m/z 264.1332 [M + Na]+), indicating that 3 had the same molecular formula as 6. The IR, 1H, and 13C NMR spectra (Tables 1 and 2) of 3 resembled those of 6. Accordingly, we hypothesized that 3 was a diastereomer of 6. A detailed comparison of the chemical shifts and coupling constants in the 1 H NMR spectra of 3 and 6 revealed that the only significant difference was in the signal of the methine proton at C-3 [dH 3.95 (d, J = 6.1 Hz) in 3, 3.82 (d, J = 10.0 Hz) in 6], while the other signals remained unchanged. This difference suggested that 3 was the C3 epimer of 6. In addition, NOEs between H-3 and H3-14 were not observed in 3, although an NOE between H-3 and H3-14 was visible in 6. Furthermore, the small coupling constant of the methine proton indicated that the 3-hydroxy group assumed the
Table 1 13 C NMR data for 1,2 and 3.
Table 2 1 H NMR data for 1, 2 and 3.
dC (mult.)
dH (mult., J in Hz)
Position
1a
2b
3c
Position
1a
2b
3c
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 CH3C(O) CH3C(O)
126.4 d 200.7 s 41.5 t 31.1 d 41.7 s 67.4 d 134.7 s 129.2 d 127.6 d 161.6 s 141.4 s 59.1 td 59.2 td 15.2 q 13.7 q
132.2 d 127.8 d 70.0 d 41.2 d 38.6 s 65.2 d 62.5 s 68.6 d 118.3 d 139.9 s 144.2 s 114.1 t 63.1 t 16.4 q 10.4 q 20.7 q 170.9 s
130.7 d 125.3 d 68.9 d 38.6 d 38.4 s 67.8 d 64.0 s 66.9 d 125.3 d 140.2 s 148.3 s 113.1 t 64.8 t 19.0 q 11.4 q
1 2 3 4 5 6 7 8 9 10 11 12
5.96 (1H, s) 2.39 (2H, m) 1.54 (1H, m)
5.96 (1H, d, 10.1) 5.75 (1H, d, 10.1) 3.95 (1H, d, 10.0) 1.81 (1H, m)
6.03 (1H, d, 10.3) 5.77 (1H, dd, 10.3, 4.9) 3.99 (1H, t, 4.9) 1.90 (1H, m)
4.57 (1H, s)
3.15 (1H, s)
3.04 (1H, s)
6.83 (1H, d, 10.3) 6.29 (1H, d, 10.3)
5.19 (1H, br. s) 6.06 (1H, d, 1.4)
4.72 (1H, d, 2.4) 5.29 (1H, d, 2.4)
4.36 (1H, d, 12.7) 4.50 (1H, d, 12.7) 4.30 (1H, d, 12.7) 4.50 (1H, d, 12.7) 0.92 (3H, s) 1.05 (3H, d, 6.3)
5.25 (1H, s) 5.28 (1H, s) 4.18 (1H, d, 14.0) 4.25 (1H, d, 14.0) 1.00 (3H, s) 1.20 (3H, d, 6.3) 2.09 (3H, s)
5.19 (1H, s) 5.29 (1H, s) 4.14 (1H, d, 13.7) 4.27 (1H, d, 13.7) 1.17 (3H, s) 1.21 (3H, d, 6.3)
a b c d
Measured in CDCl3/CD3OD (2:1). Measured in CDCl3. Measured in CD3OD. Assignment of carbons can be interchangeable.
13 14 15 CH3C(O) a b c
Measured in CDCl3/CD3OD (2:1). Measured in CDCl3. Measured in CD3OD.
Y. Shiono et al. / Phytochemistry Letters 14 (2015) 143–147
b-configuration. The signals in the 1H and 13C NMR spectra could be unambiguously assigned using HMBC experiments. Compound 4 was isolated as an amorphous powder. The molecular formula of 4 was determined to be C22H32O5 by HRESITOFMS data (m/z 399.2189 [M + Na]+), which revealed seven degrees of unsaturation. The IR spectrum of 4 revealed the presence of hydroxyl and carbonyl groups. The UV spectrum of 4 indicated the presence of a conjugated enone. The 13C NMR (Table 3) and DEPT spectra of 4 revealed carbon signals due to two methyl carbons, nine methylene carbons, five methine moieties, and six quaternary carbons, one of which was a carbonyl group. The aforementioned data suggested that 4 had four rings. The 1H NMR of 4 (Table 3) showed signals due to the exo-methylene group [dH 1.86 (dd, J = 14.5, 11.0 Hz, H-12) and 2.35 (d, J = 14.5 Hz, H-12)], two tertiary methyl groups [dH 0.72 (s, Me-14), 0.76 (s, Me-15)], two oxygenated methine protons [dH 4.59 (br. s, H-50 ), 3.07 (d, J = 2.7 Hz, H-60 )], two primary alcohols [dH 2.97 (d, J = 11.5 Hz, H-13), 3.31 (d, J = 11.5 Hz, H-13), 4.25 (d, J = 15.5 Hz, H-70 ), 4.32 (d, J = 15.5 Hz, H70 )], and one olefinic proton [dH 5.93 (d, J = 2.0 Hz, H-30 )]. The 1H–1H COSY spectrum of 4 demonstrated that 4 contained the partial structures represented by thick lines in Fig. S19. The connectivity of these fragments was determined by HMBC (Fig. S19). In the HMBC spectrum of 4, the singlet methyl protons (Me-15) correlated with C-1, C-5, C-9, and C-10, while another singlet methyl proton and the oxygenated methylene protons correlated with C-3, C-4, and C5, and the exo-methylene protons correlated with C-7, C-8, and C-9. These data suggested the presence of a 2-methylene-5,8adimethyl-5-hydroxymethyl-decahydro-1-naphtylmethyl (a sesquiterpene drimane skeleton) structure. Further analysis of the HMBC of 4 revealed correlations from H-30 to C-10, C-50 and C-70, and H-50 to C-10. These data suggested the presence of an oxygenated cyclohexenone nucleus as well as hydroxymethyl and hydroxyl moieties. The presence of an epoxy group at C-10 and C-60 was deduced from the chemical shifts of the 13C NMR signals
145
at these positions. The attachment of 2-methylene-5,8a-dimethyl5-hydroxymethyl-decahydro-1-naphtylmethyl to C-10 was established by the HMBC correlation from H-11 to C-20 and C-60 . The planar structure of 4 was deduced from these results and is shown in Fig. 1. The relative configuration of the drimane skeleton of 2 was determined by NOE correlations (Fig. S20). This planar structure was in accord with those of myrothecols B (7) and C (8) isolated from Myrothecium sp. (Fu et al., 2014). Comparison of the chemical shifts and coupling constants between 4, 7, and 8 revealed that the only substantial difference was in the coupling constant between H-50 and H-60 (J50 ,60 = 2.7 Hz, 1.6 Hz and 1.3 Hz in 4, 7 and 8, respectively). Therefore, we hypothesized that 4 was a diastereomer of 7 (10 S, 50 R, 60 S) and 8 (10 R, 50 S, 60 R). The absolute stereochemistry of the epoxycyclohexene was deduced by circular dichroism (CD) measurements. The CD spectrum of 4 showed a negative Cotton effect at 240 nm and a positive Cotton effect at 334 nm, suggesting that with respect to 7 [CD (MeOH) De: 249 (5.5), 334 (3.8)], the orientation of the hydroxy group at C-50 was the same, while that of the oxiran at C-10, 60 had the opposite configuration (Fig. 1). This was supported by the close relationship between the coupling constants and CD spectra of macrophorin A [CD (MeOH) De: 243 (4.4), 334 (+2.76)], where the epoxy and hydroxy groups (10 R, 50 R, 60 R) are oriented syn to each other (Fang et al., 2012). Thus, 4 was determined to be 13-hydroxylmacrophorin A. The antifungal activities of compounds 1–4 were tested against the pathogenic fungus R. quercivora JCM 11526 (Table 4). Compound 4 exhibited inhibitory activity against R. quercivora,
O 2 3
1
9
10
8
5
4
7
OH
OH
14
12
15
Table 3 1 H and 13C NMR data of 4 in CD3OD.
13
11
6
OH
1
Position
dC (mult.)
dH (mult., J in Hz)
1
39.6 t
2
19.8 t
3
36.5 t
1.21 1.77 1.56 1.65 1.23 1.56
4 5 6
38.9 s 48.5 d 25.3 t
7
39.1 t
8 9 10 11
150.6 s 52.9 d 40.6 s 22.0 t
12
107.3 t
13
71.9 t
14 15 10 20 30 40 50 60 70
18.1 q 15.5 q 61.3 s 195.5 s 120.3 d 161.2 s 66.2 d 62.5 d 62.2 t
(1H, td, 13.5, 3.4) (1H,br. d, 13.5) (m) (m) (m) (m)
R3
R2 OH
O R1
2 : R1 = OH, R2 = H, R3 = OAc 3 : R1 = H, R2 = OH, R3 = OH 5 : R1 = O, R2 = O, R3 = OH 6 : R1 = OH, R2 = H, R3 = OH
1.51 (1H, dd, 12.5, 2.4) 1.31 (1H, qd, 13.0, 4.0) 1.65 (m) 1.99 (1H, td, 13.4, 3.5) 2.33 (1H,br. d, 13.4) 1.80 (1H, d, 11.0)
2'
1
4
OH
12
9 5
OH
4' 5'
6'
8
10
3 5.93 (1H, d, 2.0)
O
1'
11 15
2
7'
3'
O
1.86 (1H,dd, 14.5, 11.0) 2.35 (1H,d, 14.5) 4.59 (1H, s) 4.79 (1H, s) 2.97 (1H, d, 11.5) 3.31 (1H, d, 11.5) 0.72 (3H, s) 0.76 (3H, s)
7
6
H 4.59 3.07 4.25 4.32
(1H, (1H, (1H, (1H,
br. s) d, 2.7) d, 15.5) d, 15.5)
14
13
OH
4 Fig. 1. Structures of metabolites isolated from Microdiplodia sp. TT-12.
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Y. Shiono et al. / Phytochemistry Letters 14 (2015) 143–147
Table 4 Antimicrobial activity of 1, 2, 3 and 4.
3.3. Fermentation, extraction and isolation
Microorganisms
1
2
3
4
Raffaelea quercivora JCM 11526 Pseudomonas aeruginosa ATCC 15442 Staphylococcus aureus NBRC 13276 Aspergillus clavatus F318a Candida albicans ATCC 2019
ND 8 ND ND ND
ND 10 13 ND ND
ND 10 11 ND ND
12 10 15 ND ND
Diameter of the inhibition areas (mm) using the plate diffusion assay (50 mg of each tested compound soaked in a 8 mm filter disk). ND: not detectable.
whereas compounds 2–4 showed moderate antimicrobial activity against both Pseudomonas aeruginosa ATCC 15442 and Staphylococcus aureus NBRC 13276. The results of this study conclude that 4 is ingredient as a antimicrobial against R. quercivora in the culture of Microdiplodia sp. TT-12 isolated from the plant hosts. This fungal strain TT-12 may play an important role in the inhibition growth of Japanese oak pathogen R. quercivora. However, the kind of interaction between this fungal strain TT-12 and R. quercivora at the wounded site needs to be investigated in order to understand whether or not TT-12 is able to produce 4 when testing this fungus as a biological control agent toward JOW. 3. Experimental procedures 3.1. Instrumentation Optical rotation values were measured with a Horiba SEPA300 polarimeter, and IR, and UV spectra were respectively recorded with Jasco J-20A, Shimadzu UV mini-1240, and Jasco J-20A spectrophotometers. CD data was collected by J-820CD spectrometer. Mass spectra were obtained with a Jeol JMS-700 and a Synapt G2 mass spectrometer instruments. NMR data were recorded on a Jeol ECZ-600 at 600 MHz for 1H and 150 MHz for 13C and an a Jeol EX-400 spectrometer at 400 MHz for 1H and 100 MHz for 13C. Chemical shifts are given on a d (ppm) scale with TMS as an internal standard. 1H, 13C, COSY, HMQC and HMBC spectra were recorded using standard Jeol standard pulse sequences. Semipreparative HPLC was carried out with Shimadzu pump and UV LC-10A detector (set at 210 nm) on Mightysil ODS column (250 6.0 mm i. d.) at the flow rate of 1.5 mL/min1. Column chromatography was conducted on silica gel 60 (Kanto Chemical Co., Inc., Japan) and ODS (Fuji Silysia, Japan). TLC was carried out on Merck precoated silica gel plates (silica gel 60 F254), and spots were detected by spraying with 10% vanillin in H2SO4 followed by heating, or by UV irradiation. 3.2. The producing strain The fungal strain Microdiplodia sp. TT-12 was isolated from Japanese oak wilt collected from a forest in Tsuruoka, Yamagata in November 2012. The branch samples were surface-sterilized successively with 70% EtOH for 1 min, 5% sodium hypochlorite for 5 min and 70% EtOH for 1 min, then rinsed in sterile water for two times. The sterilized samples were dried on sterilized paper and cut into 1 cm pieces. The pieces were placed on plates of Potato–Dextrose–Agar (PDA) containing chloramphenicol (100 mg/L). After incubation at 25 C for 7 days, the hyphal tips of the fungi on the plates were removed from the agar plates and transferred to PDA plates (slant). The strain TT-12 was isolated and grew on slants of PDA as white colored culture. This strain was identified to be Microdiplodia sp. by BEX Co., Ltd., Japan, using a DNA analysis of the 18S rDNA regions. This fungus has been deposited at our laboratory in the Faculty of Agriculture of Yamagata University.
The fungal strain TT-12 was cultivated on sterile steamed unpolished rice (total 600 g, 20 g/petri dish 30) at 25 C for four weeks. The moldy unpolished rice was extracted with MeOH (1.5 L), and MeOH extract was concentrated. The resulting aqueous concentrated was partitioned into n-hexane layer (0.5 L), EtOAc layer (1.0 L) and aqueous layer (0.3 L). Purifications of eluates were monitored by the characteristic intense blue coloration with 10% vanillin in H2SO4 on TLC plates. The EtOAc layer (250 mg) was chromatographed on a silica gel column with stepwise elution of nhexane–EtOAc (100:0–0:100) and EtOAc–MeOH (50:50, 0:100), respectively, to afford fractions 1-1 to 1-13. Fractions 1-5 and 16 were combined and further rechromatographed on a silica gel column using CHCl3–EtOAc (80:20) to yield phomadecalin F (1, 10.5 mg; 0.0018% yield) and 8a-monoacetoxyphomadecalin D (2, 13.5 mg; 0.0023% yield). Fraction 1-11 (EtOAc–MeOH, 50:50, 400 mg) was subjected to silica gel column chromatography by eluting with CHCl3 and an increasing ratio of EtOAc (100:0–0:100) to afford fractions 2-1 to 2-13. Fractions 2-8 and 2-9 (CHCl3–EtOAc, 30:70, 20:80, 130 mg) was further separated by ODS chromatography eluted with MeCN–H2O (60:40) to give 3-epi-phomadecalin D (3, 8.5 mg; 0.0014% yield) and phomadecalin C (5, 5.4 mg; 0.0009% yield). Fraction 1-12 (EtOAc–MeOH, 0:100, 225 mg) was subjected to silica gel column chromatography by eluting with CHCl3 and an increasing ratio of EtOAc (100:0–0:100) to afford fractions 3-1 to 313. Fractions 3-6 and 3-7 (CHCl3–EtOAc, 50:50, 40:60, 80 mg) was further separated by semipreparative ODS HPLC (MeOH–H2O, 80:20) to give 13-hydroxylmacrophorin A (4, 25.0 mg, tR = 11.9 min; 0.0042% yield) and phomadecalin D (6, 13.2 mg; 0.0022% yield). 3.3.1. Phomadecalin F (1) White amorphous powder; [a]D20 87.5 (c 0.32, CH3OH); UV (MeOH) lmax (log e): 208 (4.9), 268 (4.7), 317 (3.7); IR (KBr) nmax cm1; 3444, 1720, 1658, 1438, 1315, 1268, 1174; HRESITOFMS (positive ion mode) m/z 289.1289 [M + Na]+ (calculated for C15H20O4Na 289.1259). 3.3.2. 8a-Monoacetoxyphomadecalin D (2) White amorphous powder; [a]D20 +89.1 (c 0.73, CH3OH); UV (MeOH) lmax (log e): 224 (3.0), 275 (3.2); IR (KBr) nmax cm1; 3320, 2968, 2950, 1730, 1680, 1470, 1242, 1031; HRESITOFMS (positive ion mode) m/z 329.1395 [M + Na]+ (calculated for C17H22O5Na 329.1359). 3.3.3. 3-epi-Phomadecalin D (3) White amorphous powder; [a]D20 +309.8 (c 0.13, CH3OH); UV (MeOH) lmax (log e): 235 (3.9), 283 (3.3); IR (KBr) nmax cm1; 3310, 29950, 2943, 1630, 1457, 1400, 1031; HRESITOFMS (positive ion mode) m/z 264.1332 [M + Na]+ (calculated for C15H20O4Na 264.1362). 3.3.4. 13-Hydroxylmacrophorin A (4) White amorphous powder; [a]D20 +31.4 (c 0.48, CHCl3); UV (MeOH) lmax (log e): 239 (3.7); CD (MeOH) De: 214 (+0.21), 240 (4.62), 334 (+2.86); IR (KBr) nmax cm1; 3410, 2939, 2886, 2866, 2845, 1670, 1450, 1437, 1366, 1270, 1200, 1030; HRESITOFMS (positive ion mode) m/z 399.2189 [M + Na]+ (calculated for C22H32O5Na 399.2147). 3.4. Preparation of MTPA ester derivatives (2a and 2b) from 2 To 2 (1.3 mg) in CH2Cl2 (1.0 ml) were added (S)-()-a-methoxy (trifluoromethyl)phenylacetic acid (MTPA, 1.5 mg), dicyclohexylcarbodiimide (2.0 mg) and 4-(dimethylamino)pyridine (5.0 mg), and the mixture was stirred at room temperature for 24 h. EtOAc
Y. Shiono et al. / Phytochemistry Letters 14 (2015) 143–147
was added to the reaction mixture, before the resulting solution was washed with a saturated solution of aqueous NaHCO3 and brine, and concentrated in vacuo. Purification by column chromatography on silica gel (n-hexane-EtOAc) gave the (S)-()-MTPA ester (2a, 1.0 mg) of 2. Compound 2 (1.5 mg) was treated with (R)(+)-MTPA (1.5 mg) in the same manner to afford the (R)-(+)-MTPA ester (2b, 1.0 mg). (S)-()-MTPA ester 2a: oil; HRESITOFMS (positive ion mode) m/ z 739.2359 [M + H]+ (calculated for C37H37F6O9 739.2342); 1H NMR dH (600 MHz, CDCl3): d 6.09 (1H, d, J = 10.0 Hz, H-2), 5.70 (1H, d, J = 10.0 Hz, H-1), 5.33 (1H, d, J = 10.0 Hz, H-3), 2.12 (1H, q, J = 6.9 Hz, H-4), 2.89 (1H, s, H-6), 5.26 (1H, d, J = 2.4 Hz, H-8), 6.05 (1H, s, H-9), 5.41 (1H, s, H-12), 5.47 (1H, s, H-12), 4.82 (1H, d, J = 11.0 Hz, H-13), 4.99 (1H, d, J = 11.0 Hz, H-13), 0.82 (3H, s, H3-14), 0.84 (3H, d, J = 6.9 Hz, H3-15), 2.08 (3H, s, CH3C(O) ), 7.39 (6H, m, MTPA–ArH), 7.49 (2H, m, MTPA–ArH), 7.55 (2H, m, MTPA–ArH), 3.54 (3H, s, MTPA–OCH3), 3.56 (3H, s, MTPA–OCH3). (R)-(+)-MTPA ester 2b: oil; HRESITOFMS (positive ion mode) m/ z 739.2363 [M + H]+ (calculated for C37H37F6O9 739.2342); 1H NMR dH (600 Hz, CDCl3): 6.01 (1H, d, J = 10.1 Hz, H-2), 5.58 (1H, d, J = 10.0 Hz, H-1), 5.37 (1H, d, J = 10.0 Hz, H-3), 2.14 (1H, q, J = 6.9 Hz, H-4), 3.07 (1H, s, H-6), 5.18 (1H, d, J = 2.3 Hz H-8), 5.93 (1H, s, H-9), 5.37 (1H, s, H-12), 5.47 (1H, s, H-12), 4.87 (1H, d, J = 11.0 Hz, H-13), 4.91 (1H, d, J = 11.0 Hz, H-13), 0.85 (3H, s, H3-14), 1.11 (3H, d, J = 6.9 Hz, H3-15), 2.08 (3H, s, CH3C(O) ), 7.39 (3H, m, MTPA–ArH), 7.42 (3H, m, MTPA–ArH), 7.50 (2H, m, MTPA–ArH), 7.55 (2H, m, MTPA–ArH), 3.54 (3H, s, MTPA–OCH3), 3.55 (3H, s, MTPA–OCH3). 3.5. Antimicrobial activity Test microorganisms were Staphylococcus aureus NBRC 13276, Pseudomonas aeruginosa ATCC 15442, Aspergillus clavatus F 318a, Raffaelea quercivora JCM 11526 and Candida albicans ATCC 2019. Antimicrobial assays were carried out by the paper disk diffusion method using a published protocol (Shiono et al., 2005). Acknowledgements We would like to thank Professor Dr. Masaru Hashimoto and Mr. Atsushi Ito, Hirosaki University, for CD measurement. Financial
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assistance from ‘JSPS KAKENHI Grant Numbers (24580154)’ has gratefully been acknowledged. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.phytol.2015 .10.004. References Brooks, D.S., Gonzalez, C.F., Appel, D.N., Filer, T.H., 1994. Evaluation of endophytic bacteria as potential biological-control agents for oak Wilt. Biol. Control 4, 373–381. Che, Y., Gloer, J.B., Wicklow, D.T., 2002. Phomadecalins A-D and phomapentenone A: new bioactive metabolites from Phoma sp. NRRL 25697, a fungal colonist of Hypoxylon stromata. J. Nat. Prod. 65, 399–402. Fang, S.M., Cui, C.B., Li, C.W., Wu, C.J., Zhang, Z.J., Li, L., Huang, X.J., Ye, W.C., 2012. Purpurogemutantin and purpurogemutantidin, new drimenyl cyclohexenone derivatives produced by a mutant obtained by diethyl sulfate mutagenesis of a marine-derived Penicillium purpurogenum G59. Mar. Drugs 10, 1266–1287. Fu, Y., Wu, P., Xue, J., Wei, X., 2014. Cytotoxic and antibacterial quinone sesquiterpenes from a myrothecium fungus. J. Nat. Prod. 77, 1791–1799. Haggag, W.M., 2010. Role of entophytic microorganisms in biocontrol of plant diseases. Life Sci. J. 7, 69–78. Kubono, T., Ito, I., 2002. Raffaelea quercivora sp. nov. associated with mass mortality of Japanese oak, and the ambrosia beetle (Platypus quercivorus). Mycoscience 43, 255–260. Kuroda, K., 2001. Responses of Quercus sapwood to infection with the pathogenic fungus of a new wilt disease vectored by the ambrosia beetle Platypus quercivorus. J. Wood Sci. 47, 425–429. Ohtani, I., Kusumi, T., Kashman, H., Kakisawa, H., 1991. High-field FT NMR application of Mosher’s method. The absolute configurations of marine terpenoids. J. Am. Chem. Soc. 113, 4092–4096. O’Hanlon, K.A., Knorr, K., Jørgensen, L.N., Nicolaisen, M., Boelt, B., 2012. Exploring the potential of symbiotic fungal endophytes in cereal disease suppression. Biol. Control 63, 69–78. Shiono, Y., Murayama, T., Takahashi, K., Okada, K., Katohda, S., Ikeda, M., 2005. Three oxygenated cyclohexenone derivatives, produced by an endophytic fungus. Biosci. Biotechnol. Biochem. 69, 287–292. Takahashi, Y., Matsushita, N., Hogetsu, T., 2010. Spatial distribution of Raffaelea quercivora in xylem of naturally infested and inoculated oak trees. Phytopathology 100, 747–755. Zhanga, Q., Zhanga, J., Yanga, L., Zhangb, L., Jianga, D., Chenc, W., Lia, G., 2014. Diversity and biocontrol potential of endophytic fungi in Brassica napus. Biol. Control 72, 98–108.
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Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol
New sesquiterpenes from the endophyte Microdiplodia sp. TT-12 and their antimicrobial activity Yoshihito Shiono* , Hiromasa Koyama, Tetsuya Murayama, Takuya Koseki Department of Food, Life, and Environmental Science, Faculty of Agriculture, Yamagata University, Tsuruoka, Yamagata 997-8555, Japan
A R T I C L E I N F O
A B S T R A C T
Article history: Received 23 May 2015 Received in revised form 6 October 2015 Accepted 8 October 2015 Available online xxx
Four new compounds, phomadecalin F (1), 8a-monoacetoxyphomadecalin D (2), 3-epi-phomadecalin D (3), and 13-hydroxylmacrophorin A (4), were isolated from the endophyte, Microdiplodia sp. TT12 together with two known compounds, phomadecalins C (5) and D (6). The structures of the compounds were elucidated by NMR spectroscopic and mass spectrometric analyses in combination with chemical means. The antibacterial activities of the isolated compounds were evaluated. Compound 4 was weakly active against Raffaelea quercivora. ã 2015 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
Keywords: Endophyte Raffaelea quercivora Microdiplodia sp. TT-12 Phomadecalin Macrophorin A
1. Introduction Japanese oak wilt (JOW) is a fungal disease that affects Japanese oak, Quercus serrata, on the Sea of Japan side of Japan. JOW is caused by a fungal pathogen, Raffaelea quercivora, which is vectored by female ambrosia beetles, namely Platypus quercivorus (Kubono and Ito, 2002). Detailed observation of the distribution of R. quercivora hyphae in JOW-infested Quercus saplings revealed that the hyphal distribution overlapped with areas where vessel function was disturbed. Accordingly, it is thought that the mortality caused by JOW might be induced by the severe dysfunction of vessel lumens (Takahashi et al., 2010). Since the late 1980s, JOW has steadily been spreading across mainland Japan (Kuroda, 2001). Due to the rapid expansion of JOW, Japanese oaks have died at a rate of approximately 1000 ha/year (Takahashi et al., 2010). Although the number of affected trees has not increased in the last several years, the invasiveness of JOW has caused a significant loss of biodiversity in Japan. Several endophytes have been reported to support the growth and improve the health of plants, and therefore may serve as important biocontrol agents (Haggag, 2010; O’Hanlon et al., 2012; Zhanga et al., 2014). Interestingly, it has been reported that endophytic bacteria isolated from surviving live oaks, Quercus fusiformis, in Texas could be used as effective biocontrol agents
* Corresponding author. Fax: +81 235 28 2873. E-mail address: [email protected] (Y. Shiono).
against the oak wilt pathogen Ceratocystis fagacearum (Brooks et al., 1994). In this study, we investigated the endophytes associated with surviving live Japanese oaks in Yamagata Prefecture, where JOW is epidemic, and we evaluated the antifungal activity of metabolites from the endophytes against the fungal pathogen of JOW, R. quercivora. Of the 50 fungal isolates tested, a methanol extract of a culture of the fungal strain Microdiplodia sp. TT-12 inhibited R. quercivora in vitro. Herein, we describe the fermentation, isolation, structure determination, and biological activities of the secondary metabolites produced by Microdiplodia sp. TT-12. 2. Results and discussion Steamed unpolished rice was used as the cultivation substrate for the endophyte Microdiplodia sp. TT-12. Cultivated media were extracted with methanol (1.5 L) for 24 h. The organic extract was concentrated, and the aqueous residue was extracted with ethyl acetate. The organic layer was subjected to silica gel column chromatography using a gradient of n-hexane–EtOAc as the eluent. Further chromatographic studies revealed the presence of six compounds: phomadecalin F (1), 8a-monoacetoxyphomadecalin D (2), 3-epi-phomadecalin D (3), 13-hydroxylmacrophorin A (4), and phomadecalins C (5) and D (6). Known compounds 5 and 6 were identified as phomadecalins C and D on the basis of their 1H and 13C NMR, 1H–1H COSY, HMQC, and HMBC data (Che et al., 2002). The molecular formula of 1, C15H20O4, was determined by HRESITOFMS data (m/z 289.1289 [M + Na]+). The IR spectrum of 1
http://dx.doi.org/10.1016/j.phytol.2015.10.004 1874-3900/ ã 2015 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
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Y. Shiono et al. / Phytochemistry Letters 14 (2015) 143–147
indicated the presence of a carbonyl group. The UV spectrum of 1 in MeOH exhibited the presence of a triene moiety. The gross structure of 1 was deduced from 1H and 13C NMR data, as well as 2D NMR experiments. Close inspection of the 13C NMR (Table 1) and DEPT spectra of 1 revealed carbon signals that were attributed to two methyl groups, three sp3 methylene moieties (two of them bearing an oxygen atom), two sp3 methine moieties (one of them bearing an oxygen atom), one sp3 quaternary carbon, three sp2 quaternary carbons, three sp2 methine moieties, and one a,b-unsaturated ketone. The 1H NMR and HMQC spectra of 1 exhibited proton signals which were attributed to one secondary methyl group [dH 1.05 (d, J = 6.3 Hz, Me-15)], one singlet methyl [dH 0.92 (s, Me-14)], coupled olefinic protons [dH 6.83 (d, J = 10.3 Hz, H8), 6.29 (d, J = 10.3 Hz, H-9)], one singlet olefinic methine moiety [dH 5.96 (s, H-1)], two methane moieties [dH 1.54 (m, H-4), 4.57 (s, H-6)], and three methylene groups [dH 2.39 (m, H-3), 4.36 (d, J = 12.7 Hz, H-12), 4.50 (d, J = 12.7 Hz, H-12), 4.30 (d, J = 12.7 Hz, H13), 4.50 (d, J = 12.7 Hz, H-13)]. Overall, the NMR data suggested that the structure of 1 was closely related to a eremophilane sesquiterpene analogue. The 1H–1H COSY spectrum revealed the partial structures that comprised 1 (Fig. S19). The connections between the partial structures and functional groups were determined on the basis of key HMBC correlations, as summarized in Fig. S19. HMBC correlations from H-1 to C-3, from Me-14 to C-4, C-5, and C-10, and from Me-15 to C-3, C-4, and C-5 revealed the presence of an a,b-unsaturated carbonyl functionality (4,5dimethyl-2-cyclohexen-1-one). Since six out of eight unsaturation equivalents were accounted for in the aforementioned 13C NMR data, 1 was inferred to contain two rings. Three olefinic protons in the 1H NMR spectrum and HMBC correlations from H-9 to C-1 and C-7, from H-8 to C-6, and from Me-14 to C-6, indicated that the 2cyclohexen-1-one moiety was fused to the hydroxycyclohexene unit (C-5 to C-10). The oxymethylene protons (H2-12, 13) showed HMBC correlations with the carbons at C-7 and C-11, which indicated that the 1,3-propanediol moiety was attached at the C-7position. The relative configuration of 1 was verified by NOE and comparison to similar compounds. NOE correlations from Me-15 to H-6 and from Me-14 to H-6 revealed that Me-14, Me-15, and H6 were on the same side of the molecular plane as the b-configuration. Thus, the structure of 1 was unambiguously determined to be 6a-hydroxy-1(10), 9-trien-2-oxoeremophilane,
which represented a novel phomadecalin-like compound and was named phomadecalin F. NMR spectroscopy was used to determine the absolute stereochemistry of the chiral center at C-6, by employing a modified version of Mosher's method (Ohtani et al., 1991). The chiral derivatizing agents used in the esterification reaction were (S)- and (R)-a-methoxy-a-(trifluoromethyl) phenylacetic acid [(S)- and (R)-MTPA]. Unfortunately, when compound 1 was treated with (S)- and (R)-MTPA, the desired esterified products at C-6 could not be obtained due to the low reactivity of MTPA with OH-6. 8a-Monoacetoxyphomadecalin D (2) was determined to have the molecular formula C17H22O5 using HRESITOFMS data (m/z 329.1395 [M + Na]+). The structure of 2 was determined using 1H and 13 C NMR spectral data (Tables 1 and 2). Furthermore, a mass unit difference of 42 was revealed between 2 and 6. The IR spectrum of 2, which exhibited an ester carbonyl absorption, as well as the appearance of a three-proton singlet at dH 2.09, a methyl carbon at dH 20.7, and an ester carbonyl at dC 170.9 indicated the presence of an additional acetyl group. HMBC correlations from H-8 to the ester carbonyl carbon confirmed the substitution of the acetoxy group at C-8 (Fig. S19). The relative configuration of 2 was determined by NOE correlations (Fig. S20). In addition, when 2 was treated with (S)and (R)-MTPA in the presence of DCC-DMAP at room temperature, 3,13-bis-MTPA esters (2a/2b) were produced. The differences in the 1 H chemical shifts between the (R)- and (S)-MTPA esters (2a/2b) of 2 are shown in Fig. S21. These results suggested that the asymmetric center at C-3 had the R-configuration. The molecular formula of 3 was established to be C15H20O4 by HRESITOFMS data (m/z 264.1332 [M + Na]+), indicating that 3 had the same molecular formula as 6. The IR, 1H, and 13C NMR spectra (Tables 1 and 2) of 3 resembled those of 6. Accordingly, we hypothesized that 3 was a diastereomer of 6. A detailed comparison of the chemical shifts and coupling constants in the 1 H NMR spectra of 3 and 6 revealed that the only significant difference was in the signal of the methine proton at C-3 [dH 3.95 (d, J = 6.1 Hz) in 3, 3.82 (d, J = 10.0 Hz) in 6], while the other signals remained unchanged. This difference suggested that 3 was the C3 epimer of 6. In addition, NOEs between H-3 and H3-14 were not observed in 3, although an NOE between H-3 and H3-14 was visible in 6. Furthermore, the small coupling constant of the methine proton indicated that the 3-hydroxy group assumed the
Table 1 13 C NMR data for 1,2 and 3.
Table 2 1 H NMR data for 1, 2 and 3.
dC (mult.)
dH (mult., J in Hz)
Position
1a
2b
3c
Position
1a
2b
3c
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 CH3C(O) CH3C(O)
126.4 d 200.7 s 41.5 t 31.1 d 41.7 s 67.4 d 134.7 s 129.2 d 127.6 d 161.6 s 141.4 s 59.1 td 59.2 td 15.2 q 13.7 q
132.2 d 127.8 d 70.0 d 41.2 d 38.6 s 65.2 d 62.5 s 68.6 d 118.3 d 139.9 s 144.2 s 114.1 t 63.1 t 16.4 q 10.4 q 20.7 q 170.9 s
130.7 d 125.3 d 68.9 d 38.6 d 38.4 s 67.8 d 64.0 s 66.9 d 125.3 d 140.2 s 148.3 s 113.1 t 64.8 t 19.0 q 11.4 q
1 2 3 4 5 6 7 8 9 10 11 12
5.96 (1H, s) 2.39 (2H, m) 1.54 (1H, m)
5.96 (1H, d, 10.1) 5.75 (1H, d, 10.1) 3.95 (1H, d, 10.0) 1.81 (1H, m)
6.03 (1H, d, 10.3) 5.77 (1H, dd, 10.3, 4.9) 3.99 (1H, t, 4.9) 1.90 (1H, m)
4.57 (1H, s)
3.15 (1H, s)
3.04 (1H, s)
6.83 (1H, d, 10.3) 6.29 (1H, d, 10.3)
5.19 (1H, br. s) 6.06 (1H, d, 1.4)
4.72 (1H, d, 2.4) 5.29 (1H, d, 2.4)
4.36 (1H, d, 12.7) 4.50 (1H, d, 12.7) 4.30 (1H, d, 12.7) 4.50 (1H, d, 12.7) 0.92 (3H, s) 1.05 (3H, d, 6.3)
5.25 (1H, s) 5.28 (1H, s) 4.18 (1H, d, 14.0) 4.25 (1H, d, 14.0) 1.00 (3H, s) 1.20 (3H, d, 6.3) 2.09 (3H, s)
5.19 (1H, s) 5.29 (1H, s) 4.14 (1H, d, 13.7) 4.27 (1H, d, 13.7) 1.17 (3H, s) 1.21 (3H, d, 6.3)
a b c d
Measured in CDCl3/CD3OD (2:1). Measured in CDCl3. Measured in CD3OD. Assignment of carbons can be interchangeable.
13 14 15 CH3C(O) a b c
Measured in CDCl3/CD3OD (2:1). Measured in CDCl3. Measured in CD3OD.
Y. Shiono et al. / Phytochemistry Letters 14 (2015) 143–147
b-configuration. The signals in the 1H and 13C NMR spectra could be unambiguously assigned using HMBC experiments. Compound 4 was isolated as an amorphous powder. The molecular formula of 4 was determined to be C22H32O5 by HRESITOFMS data (m/z 399.2189 [M + Na]+), which revealed seven degrees of unsaturation. The IR spectrum of 4 revealed the presence of hydroxyl and carbonyl groups. The UV spectrum of 4 indicated the presence of a conjugated enone. The 13C NMR (Table 3) and DEPT spectra of 4 revealed carbon signals due to two methyl carbons, nine methylene carbons, five methine moieties, and six quaternary carbons, one of which was a carbonyl group. The aforementioned data suggested that 4 had four rings. The 1H NMR of 4 (Table 3) showed signals due to the exo-methylene group [dH 1.86 (dd, J = 14.5, 11.0 Hz, H-12) and 2.35 (d, J = 14.5 Hz, H-12)], two tertiary methyl groups [dH 0.72 (s, Me-14), 0.76 (s, Me-15)], two oxygenated methine protons [dH 4.59 (br. s, H-50 ), 3.07 (d, J = 2.7 Hz, H-60 )], two primary alcohols [dH 2.97 (d, J = 11.5 Hz, H-13), 3.31 (d, J = 11.5 Hz, H-13), 4.25 (d, J = 15.5 Hz, H-70 ), 4.32 (d, J = 15.5 Hz, H70 )], and one olefinic proton [dH 5.93 (d, J = 2.0 Hz, H-30 )]. The 1H–1H COSY spectrum of 4 demonstrated that 4 contained the partial structures represented by thick lines in Fig. S19. The connectivity of these fragments was determined by HMBC (Fig. S19). In the HMBC spectrum of 4, the singlet methyl protons (Me-15) correlated with C-1, C-5, C-9, and C-10, while another singlet methyl proton and the oxygenated methylene protons correlated with C-3, C-4, and C5, and the exo-methylene protons correlated with C-7, C-8, and C-9. These data suggested the presence of a 2-methylene-5,8adimethyl-5-hydroxymethyl-decahydro-1-naphtylmethyl (a sesquiterpene drimane skeleton) structure. Further analysis of the HMBC of 4 revealed correlations from H-30 to C-10, C-50 and C-70, and H-50 to C-10. These data suggested the presence of an oxygenated cyclohexenone nucleus as well as hydroxymethyl and hydroxyl moieties. The presence of an epoxy group at C-10 and C-60 was deduced from the chemical shifts of the 13C NMR signals
145
at these positions. The attachment of 2-methylene-5,8a-dimethyl5-hydroxymethyl-decahydro-1-naphtylmethyl to C-10 was established by the HMBC correlation from H-11 to C-20 and C-60 . The planar structure of 4 was deduced from these results and is shown in Fig. 1. The relative configuration of the drimane skeleton of 2 was determined by NOE correlations (Fig. S20). This planar structure was in accord with those of myrothecols B (7) and C (8) isolated from Myrothecium sp. (Fu et al., 2014). Comparison of the chemical shifts and coupling constants between 4, 7, and 8 revealed that the only substantial difference was in the coupling constant between H-50 and H-60 (J50 ,60 = 2.7 Hz, 1.6 Hz and 1.3 Hz in 4, 7 and 8, respectively). Therefore, we hypothesized that 4 was a diastereomer of 7 (10 S, 50 R, 60 S) and 8 (10 R, 50 S, 60 R). The absolute stereochemistry of the epoxycyclohexene was deduced by circular dichroism (CD) measurements. The CD spectrum of 4 showed a negative Cotton effect at 240 nm and a positive Cotton effect at 334 nm, suggesting that with respect to 7 [CD (MeOH) De: 249 (5.5), 334 (3.8)], the orientation of the hydroxy group at C-50 was the same, while that of the oxiran at C-10, 60 had the opposite configuration (Fig. 1). This was supported by the close relationship between the coupling constants and CD spectra of macrophorin A [CD (MeOH) De: 243 (4.4), 334 (+2.76)], where the epoxy and hydroxy groups (10 R, 50 R, 60 R) are oriented syn to each other (Fang et al., 2012). Thus, 4 was determined to be 13-hydroxylmacrophorin A. The antifungal activities of compounds 1–4 were tested against the pathogenic fungus R. quercivora JCM 11526 (Table 4). Compound 4 exhibited inhibitory activity against R. quercivora,
O 2 3
1
9
10
8
5
4
7
OH
OH
14
12
15
Table 3 1 H and 13C NMR data of 4 in CD3OD.
13
11
6
OH
1
Position
dC (mult.)
dH (mult., J in Hz)
1
39.6 t
2
19.8 t
3
36.5 t
1.21 1.77 1.56 1.65 1.23 1.56
4 5 6
38.9 s 48.5 d 25.3 t
7
39.1 t
8 9 10 11
150.6 s 52.9 d 40.6 s 22.0 t
12
107.3 t
13
71.9 t
14 15 10 20 30 40 50 60 70
18.1 q 15.5 q 61.3 s 195.5 s 120.3 d 161.2 s 66.2 d 62.5 d 62.2 t
(1H, td, 13.5, 3.4) (1H,br. d, 13.5) (m) (m) (m) (m)
R3
R2 OH
O R1
2 : R1 = OH, R2 = H, R3 = OAc 3 : R1 = H, R2 = OH, R3 = OH 5 : R1 = O, R2 = O, R3 = OH 6 : R1 = OH, R2 = H, R3 = OH
1.51 (1H, dd, 12.5, 2.4) 1.31 (1H, qd, 13.0, 4.0) 1.65 (m) 1.99 (1H, td, 13.4, 3.5) 2.33 (1H,br. d, 13.4) 1.80 (1H, d, 11.0)
2'
1
4
OH
12
9 5
OH
4' 5'
6'
8
10
3 5.93 (1H, d, 2.0)
O
1'
11 15
2
7'
3'
O
1.86 (1H,dd, 14.5, 11.0) 2.35 (1H,d, 14.5) 4.59 (1H, s) 4.79 (1H, s) 2.97 (1H, d, 11.5) 3.31 (1H, d, 11.5) 0.72 (3H, s) 0.76 (3H, s)
7
6
H 4.59 3.07 4.25 4.32
(1H, (1H, (1H, (1H,
br. s) d, 2.7) d, 15.5) d, 15.5)
14
13
OH
4 Fig. 1. Structures of metabolites isolated from Microdiplodia sp. TT-12.
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Y. Shiono et al. / Phytochemistry Letters 14 (2015) 143–147
Table 4 Antimicrobial activity of 1, 2, 3 and 4.
3.3. Fermentation, extraction and isolation
Microorganisms
1
2
3
4
Raffaelea quercivora JCM 11526 Pseudomonas aeruginosa ATCC 15442 Staphylococcus aureus NBRC 13276 Aspergillus clavatus F318a Candida albicans ATCC 2019
ND 8 ND ND ND
ND 10 13 ND ND
ND 10 11 ND ND
12 10 15 ND ND
Diameter of the inhibition areas (mm) using the plate diffusion assay (50 mg of each tested compound soaked in a 8 mm filter disk). ND: not detectable.
whereas compounds 2–4 showed moderate antimicrobial activity against both Pseudomonas aeruginosa ATCC 15442 and Staphylococcus aureus NBRC 13276. The results of this study conclude that 4 is ingredient as a antimicrobial against R. quercivora in the culture of Microdiplodia sp. TT-12 isolated from the plant hosts. This fungal strain TT-12 may play an important role in the inhibition growth of Japanese oak pathogen R. quercivora. However, the kind of interaction between this fungal strain TT-12 and R. quercivora at the wounded site needs to be investigated in order to understand whether or not TT-12 is able to produce 4 when testing this fungus as a biological control agent toward JOW. 3. Experimental procedures 3.1. Instrumentation Optical rotation values were measured with a Horiba SEPA300 polarimeter, and IR, and UV spectra were respectively recorded with Jasco J-20A, Shimadzu UV mini-1240, and Jasco J-20A spectrophotometers. CD data was collected by J-820CD spectrometer. Mass spectra were obtained with a Jeol JMS-700 and a Synapt G2 mass spectrometer instruments. NMR data were recorded on a Jeol ECZ-600 at 600 MHz for 1H and 150 MHz for 13C and an a Jeol EX-400 spectrometer at 400 MHz for 1H and 100 MHz for 13C. Chemical shifts are given on a d (ppm) scale with TMS as an internal standard. 1H, 13C, COSY, HMQC and HMBC spectra were recorded using standard Jeol standard pulse sequences. Semipreparative HPLC was carried out with Shimadzu pump and UV LC-10A detector (set at 210 nm) on Mightysil ODS column (250 6.0 mm i. d.) at the flow rate of 1.5 mL/min1. Column chromatography was conducted on silica gel 60 (Kanto Chemical Co., Inc., Japan) and ODS (Fuji Silysia, Japan). TLC was carried out on Merck precoated silica gel plates (silica gel 60 F254), and spots were detected by spraying with 10% vanillin in H2SO4 followed by heating, or by UV irradiation. 3.2. The producing strain The fungal strain Microdiplodia sp. TT-12 was isolated from Japanese oak wilt collected from a forest in Tsuruoka, Yamagata in November 2012. The branch samples were surface-sterilized successively with 70% EtOH for 1 min, 5% sodium hypochlorite for 5 min and 70% EtOH for 1 min, then rinsed in sterile water for two times. The sterilized samples were dried on sterilized paper and cut into 1 cm pieces. The pieces were placed on plates of Potato–Dextrose–Agar (PDA) containing chloramphenicol (100 mg/L). After incubation at 25 C for 7 days, the hyphal tips of the fungi on the plates were removed from the agar plates and transferred to PDA plates (slant). The strain TT-12 was isolated and grew on slants of PDA as white colored culture. This strain was identified to be Microdiplodia sp. by BEX Co., Ltd., Japan, using a DNA analysis of the 18S rDNA regions. This fungus has been deposited at our laboratory in the Faculty of Agriculture of Yamagata University.
The fungal strain TT-12 was cultivated on sterile steamed unpolished rice (total 600 g, 20 g/petri dish 30) at 25 C for four weeks. The moldy unpolished rice was extracted with MeOH (1.5 L), and MeOH extract was concentrated. The resulting aqueous concentrated was partitioned into n-hexane layer (0.5 L), EtOAc layer (1.0 L) and aqueous layer (0.3 L). Purifications of eluates were monitored by the characteristic intense blue coloration with 10% vanillin in H2SO4 on TLC plates. The EtOAc layer (250 mg) was chromatographed on a silica gel column with stepwise elution of nhexane–EtOAc (100:0–0:100) and EtOAc–MeOH (50:50, 0:100), respectively, to afford fractions 1-1 to 1-13. Fractions 1-5 and 16 were combined and further rechromatographed on a silica gel column using CHCl3–EtOAc (80:20) to yield phomadecalin F (1, 10.5 mg; 0.0018% yield) and 8a-monoacetoxyphomadecalin D (2, 13.5 mg; 0.0023% yield). Fraction 1-11 (EtOAc–MeOH, 50:50, 400 mg) was subjected to silica gel column chromatography by eluting with CHCl3 and an increasing ratio of EtOAc (100:0–0:100) to afford fractions 2-1 to 2-13. Fractions 2-8 and 2-9 (CHCl3–EtOAc, 30:70, 20:80, 130 mg) was further separated by ODS chromatography eluted with MeCN–H2O (60:40) to give 3-epi-phomadecalin D (3, 8.5 mg; 0.0014% yield) and phomadecalin C (5, 5.4 mg; 0.0009% yield). Fraction 1-12 (EtOAc–MeOH, 0:100, 225 mg) was subjected to silica gel column chromatography by eluting with CHCl3 and an increasing ratio of EtOAc (100:0–0:100) to afford fractions 3-1 to 313. Fractions 3-6 and 3-7 (CHCl3–EtOAc, 50:50, 40:60, 80 mg) was further separated by semipreparative ODS HPLC (MeOH–H2O, 80:20) to give 13-hydroxylmacrophorin A (4, 25.0 mg, tR = 11.9 min; 0.0042% yield) and phomadecalin D (6, 13.2 mg; 0.0022% yield). 3.3.1. Phomadecalin F (1) White amorphous powder; [a]D20 87.5 (c 0.32, CH3OH); UV (MeOH) lmax (log e): 208 (4.9), 268 (4.7), 317 (3.7); IR (KBr) nmax cm1; 3444, 1720, 1658, 1438, 1315, 1268, 1174; HRESITOFMS (positive ion mode) m/z 289.1289 [M + Na]+ (calculated for C15H20O4Na 289.1259). 3.3.2. 8a-Monoacetoxyphomadecalin D (2) White amorphous powder; [a]D20 +89.1 (c 0.73, CH3OH); UV (MeOH) lmax (log e): 224 (3.0), 275 (3.2); IR (KBr) nmax cm1; 3320, 2968, 2950, 1730, 1680, 1470, 1242, 1031; HRESITOFMS (positive ion mode) m/z 329.1395 [M + Na]+ (calculated for C17H22O5Na 329.1359). 3.3.3. 3-epi-Phomadecalin D (3) White amorphous powder; [a]D20 +309.8 (c 0.13, CH3OH); UV (MeOH) lmax (log e): 235 (3.9), 283 (3.3); IR (KBr) nmax cm1; 3310, 29950, 2943, 1630, 1457, 1400, 1031; HRESITOFMS (positive ion mode) m/z 264.1332 [M + Na]+ (calculated for C15H20O4Na 264.1362). 3.3.4. 13-Hydroxylmacrophorin A (4) White amorphous powder; [a]D20 +31.4 (c 0.48, CHCl3); UV (MeOH) lmax (log e): 239 (3.7); CD (MeOH) De: 214 (+0.21), 240 (4.62), 334 (+2.86); IR (KBr) nmax cm1; 3410, 2939, 2886, 2866, 2845, 1670, 1450, 1437, 1366, 1270, 1200, 1030; HRESITOFMS (positive ion mode) m/z 399.2189 [M + Na]+ (calculated for C22H32O5Na 399.2147). 3.4. Preparation of MTPA ester derivatives (2a and 2b) from 2 To 2 (1.3 mg) in CH2Cl2 (1.0 ml) were added (S)-()-a-methoxy (trifluoromethyl)phenylacetic acid (MTPA, 1.5 mg), dicyclohexylcarbodiimide (2.0 mg) and 4-(dimethylamino)pyridine (5.0 mg), and the mixture was stirred at room temperature for 24 h. EtOAc
Y. Shiono et al. / Phytochemistry Letters 14 (2015) 143–147
was added to the reaction mixture, before the resulting solution was washed with a saturated solution of aqueous NaHCO3 and brine, and concentrated in vacuo. Purification by column chromatography on silica gel (n-hexane-EtOAc) gave the (S)-()-MTPA ester (2a, 1.0 mg) of 2. Compound 2 (1.5 mg) was treated with (R)(+)-MTPA (1.5 mg) in the same manner to afford the (R)-(+)-MTPA ester (2b, 1.0 mg). (S)-()-MTPA ester 2a: oil; HRESITOFMS (positive ion mode) m/ z 739.2359 [M + H]+ (calculated for C37H37F6O9 739.2342); 1H NMR dH (600 MHz, CDCl3): d 6.09 (1H, d, J = 10.0 Hz, H-2), 5.70 (1H, d, J = 10.0 Hz, H-1), 5.33 (1H, d, J = 10.0 Hz, H-3), 2.12 (1H, q, J = 6.9 Hz, H-4), 2.89 (1H, s, H-6), 5.26 (1H, d, J = 2.4 Hz, H-8), 6.05 (1H, s, H-9), 5.41 (1H, s, H-12), 5.47 (1H, s, H-12), 4.82 (1H, d, J = 11.0 Hz, H-13), 4.99 (1H, d, J = 11.0 Hz, H-13), 0.82 (3H, s, H3-14), 0.84 (3H, d, J = 6.9 Hz, H3-15), 2.08 (3H, s, CH3C(O) ), 7.39 (6H, m, MTPA–ArH), 7.49 (2H, m, MTPA–ArH), 7.55 (2H, m, MTPA–ArH), 3.54 (3H, s, MTPA–OCH3), 3.56 (3H, s, MTPA–OCH3). (R)-(+)-MTPA ester 2b: oil; HRESITOFMS (positive ion mode) m/ z 739.2363 [M + H]+ (calculated for C37H37F6O9 739.2342); 1H NMR dH (600 Hz, CDCl3): 6.01 (1H, d, J = 10.1 Hz, H-2), 5.58 (1H, d, J = 10.0 Hz, H-1), 5.37 (1H, d, J = 10.0 Hz, H-3), 2.14 (1H, q, J = 6.9 Hz, H-4), 3.07 (1H, s, H-6), 5.18 (1H, d, J = 2.3 Hz H-8), 5.93 (1H, s, H-9), 5.37 (1H, s, H-12), 5.47 (1H, s, H-12), 4.87 (1H, d, J = 11.0 Hz, H-13), 4.91 (1H, d, J = 11.0 Hz, H-13), 0.85 (3H, s, H3-14), 1.11 (3H, d, J = 6.9 Hz, H3-15), 2.08 (3H, s, CH3C(O) ), 7.39 (3H, m, MTPA–ArH), 7.42 (3H, m, MTPA–ArH), 7.50 (2H, m, MTPA–ArH), 7.55 (2H, m, MTPA–ArH), 3.54 (3H, s, MTPA–OCH3), 3.55 (3H, s, MTPA–OCH3). 3.5. Antimicrobial activity Test microorganisms were Staphylococcus aureus NBRC 13276, Pseudomonas aeruginosa ATCC 15442, Aspergillus clavatus F 318a, Raffaelea quercivora JCM 11526 and Candida albicans ATCC 2019. Antimicrobial assays were carried out by the paper disk diffusion method using a published protocol (Shiono et al., 2005). Acknowledgements We would like to thank Professor Dr. Masaru Hashimoto and Mr. Atsushi Ito, Hirosaki University, for CD measurement. Financial
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assistance from ‘JSPS KAKENHI Grant Numbers (24580154)’ has gratefully been acknowledged. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.phytol.2015 .10.004. References Brooks, D.S., Gonzalez, C.F., Appel, D.N., Filer, T.H., 1994. Evaluation of endophytic bacteria as potential biological-control agents for oak Wilt. Biol. Control 4, 373–381. Che, Y., Gloer, J.B., Wicklow, D.T., 2002. Phomadecalins A-D and phomapentenone A: new bioactive metabolites from Phoma sp. NRRL 25697, a fungal colonist of Hypoxylon stromata. J. Nat. Prod. 65, 399–402. Fang, S.M., Cui, C.B., Li, C.W., Wu, C.J., Zhang, Z.J., Li, L., Huang, X.J., Ye, W.C., 2012. Purpurogemutantin and purpurogemutantidin, new drimenyl cyclohexenone derivatives produced by a mutant obtained by diethyl sulfate mutagenesis of a marine-derived Penicillium purpurogenum G59. Mar. Drugs 10, 1266–1287. Fu, Y., Wu, P., Xue, J., Wei, X., 2014. Cytotoxic and antibacterial quinone sesquiterpenes from a myrothecium fungus. J. Nat. Prod. 77, 1791–1799. Haggag, W.M., 2010. Role of entophytic microorganisms in biocontrol of plant diseases. Life Sci. J. 7, 69–78. Kubono, T., Ito, I., 2002. Raffaelea quercivora sp. nov. associated with mass mortality of Japanese oak, and the ambrosia beetle (Platypus quercivorus). Mycoscience 43, 255–260. Kuroda, K., 2001. Responses of Quercus sapwood to infection with the pathogenic fungus of a new wilt disease vectored by the ambrosia beetle Platypus quercivorus. J. Wood Sci. 47, 425–429. Ohtani, I., Kusumi, T., Kashman, H., Kakisawa, H., 1991. High-field FT NMR application of Mosher’s method. The absolute configurations of marine terpenoids. J. Am. Chem. Soc. 113, 4092–4096. O’Hanlon, K.A., Knorr, K., Jørgensen, L.N., Nicolaisen, M., Boelt, B., 2012. Exploring the potential of symbiotic fungal endophytes in cereal disease suppression. Biol. Control 63, 69–78. Shiono, Y., Murayama, T., Takahashi, K., Okada, K., Katohda, S., Ikeda, M., 2005. Three oxygenated cyclohexenone derivatives, produced by an endophytic fungus. Biosci. Biotechnol. Biochem. 69, 287–292. Takahashi, Y., Matsushita, N., Hogetsu, T., 2010. Spatial distribution of Raffaelea quercivora in xylem of naturally infested and inoculated oak trees. Phytopathology 100, 747–755. Zhanga, Q., Zhanga, J., Yanga, L., Zhangb, L., Jianga, D., Chenc, W., Lia, G., 2014. Diversity and biocontrol potential of endophytic fungi in Brassica napus. Biol. Control 72, 98–108.