Identification of UV-Induced Diterpenes Including a New Diterpene

Article pubs.acs.org/JAFC

Identification of UV-Induced Diterpenes Including a New Diterpene Phytoalexin, Phytocassane F, from Rice Leaves by Complementary GC/MS and LC/MS Approaches Kiyotaka Horie,† Yasuno Inoue,‡ Miki Sakai,‡ Qun Yao,‡,# Yosuke Tanimoto,‡ Jinichiro Koga,§ Hiroaki Toshima,†,‡ and Morifumi Hasegawa*,†,‡ †

United Graduate School of Agricultural Science, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo 183-8509, Japan ‡ College of Agriculture, Ibaraki University, 3-21-1 Chuo, Ami, Inashiki, Ibaraki 300-0393, Japan § Department of Biosciences, Teikyo University, 1-1 Toyosatodai, Utsunomiya, Tochigi 320-8551, Japan S Supporting Information *

ABSTRACT: Rice phytoalexins are regarded as one of the most important weapons against pathogenic microorganisms. We attempted to identify novel phytoalexins and their derivatives using GC/MS and LC/MS analyses. Diterpene derivatives, 9βpimara-7,15-diene-3β,6β,19-triol, 1, stemar-13-en-2α-ol, 2, and 1α,2α-dihydroxy-ent-12,15-cassadiene-3,11-dione, 3, were isolated from UV-irradiated rice leaves by chromatographic methods. These structures were confirmed by 1D- and 2D-NMR and MS analyses. Interestingly, all three compounds were accumulated following an infection by the rice blast pathogen Magnaporthe oryzae. Compounds 1 and 2 exhibited weak antifungal activity and may be the biosynthetic intermediates of rice phytoalexins momilactones and oryzalexin S, respectively. Compound 3 exhibited relatively high inhibitory activity against the fungal mycelial growth of M. oryzae to the same extent as the known phytoalexin phytocassane A. We conclude that 3 is a member of the cassane-type phytoalexin family and propose the name phytocassane F. KEYWORDS: ultraviolet irradiation, mass spectrometry, phytoalexin, rice, phytocassane, Magnaporthe oryzae

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INTRODUCTION Plants respond to pathogen infection through a variety of defense responses that include hypersensitive response, cell wall reinforcement, production of pathogenesis-related proteins, and the induced accumulation of antimicrobial metabolites (i.e., phytoalexins).1 Phytoalexins are low-molecular-weight compounds that accumulate in plants invaded mostly by pathogenic microorganisms.2−10 Rice (Oryza sativa) is known to produce 16 phytoalexins, including 15 diterpenes and a flavanone, sakuranetin, 4 (Figure 1).2−11 The diterpene phytoalexins are classified into five groups according to their structure: momilactones A, 5, and B,11 oryzalexins A−E and F, 6,2−5 oryzalexin S, 7,6 phytocassanes A−E, 8−12,7,8 and ent-10oxodepressin.9 These phytoalexins have been reported to exert an in vitro inhibitory activity on the spore germination and germ tube elongation of the rice blast fungus Magnaporthe oryzae.2−11 The importance of these phytoalexins in imparting resistance to the rice plant against the rice blast fungus has been suggested in several studies. Hasegawa et al.12,13 showed that exogenously applied 4 and 5 decreased M. oryzae growth in detached rice leaves. Toyomasu et al.14 demonstrated that a functional deficient mutant for an enzyme involved in the biosynthesis of momilactones and 7 was impaired in the resistance against M. oryzae. In view of the agricultural significance of improving the resistance of rice against pathogens, the biosynthesis of rice phytoalexins has been extensively studied and has been summarized in several reviews.15−17 The hydrocarbon skeletons © XXXX American Chemical Society

of diterpenoid phytoalexins are biosynthesized from a common precursor, geranylgeranyl diphosphate, by dual cyclizations via ent- or syn-copalyl diphosphate. These aliphatic and inactive intermediates, ent-cassa-12,15-diene, ent-sandaracopimaradiene, 9β-pimara-7,15-diene and stemar-13-ene, have been considered to be oxidized by cytochrome P450 or dehydrogenase to produce the bioactive phytoalexins phytocassanes, oryzalexins A−F, momilactones, and 7, respectively.18−21 The production of phytoalexins is induced not only by biotic elicitors, including exogenously applied plant hormones, but also by abiotic elicitors such as heavy-metal ions or UV irradiation.22−26 All rice phytoalexins isolated from UVirradiated rice leaves were also detected from M. oryzaeinfected rice leaves.4,5,9,10 It is considered that the compound repertoire induced by UV irradiation is similar to that induced by M. oryzae infection. The treatment of rice leaves by UV irradiation is a useful method to isolate phytoalexins because it is easy to perform and results in the accumulation of a large amount of these compounds. Recently, Inoue et al.9 found a novel phytoalexin, ent-10-oxodepressin, by comparing the GC/ MS chromatogram of an extract from UV-irradiated rice leaves with that of an extract from healthy leaves. Although GC/MS is a powerful technique for identifying organic compounds from a Received: February 10, 2015 Revised: April 10, 2015 Accepted: April 11, 2015

A

DOI: 10.1021/acs.jafc.5b00785 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

Figure 1. Structures of compounds.

operated in positive ESI mode. Preparative HPLC was carried out with a PU-980 HPLC pump and an MD-910 photodiode array detector (Jasco) under the following conditions; flow rate, 2.0 mL/min; detection, UV 210−350 nm. Chemicals. Compound 4 was chemically synthesized from naringenin (Sigma-Aldrich, St. Louis, MO) according to the method reported by Aida et al.27 Compound 5 was purified from rice husks according to the method reported by Kato et al.28 with some modifications. Compounds 6 and 7 were purified from UV-irradiated rice leaves according to previously described methods with some modifications.5,6 The standard solution for the quantitation of 8−12 was kindly gifted by Dr. Kazunori Okada at the University of Tokyo. Plant Materials. Rice plants (Oryza sativa cv. Koshihikari) were grown in a phytotron at 27 °C under natural light conditions. The leaf blades of the seventh and eighth leaf stages were detached, and the middle regions of those leaf blades were used in the subsequent experiments. Fungal Materials and Spore Preparation. The rice blast fungus (Magnaporthe oryzae strain P-2) had been maintained on a potato dextrose agar (PDA) (Nissui Pharmaceutical, Tokyo, Japan) medium as a stock culture in our laboratory. Spores of the fungus were prepared by the previously described method with some modifications.29 The mycelia cultured on the oatmeal medium were transplanted to a vegetable-juice medium in a Petri dish and cultured for 2 weeks at 26 °C in the dark. For synchronously produced spores, aerial hyphae were removed by soft scratching with a paint brush in distilled water. The Petri dish was incubated for 48 h at 26 °C under a BLB 20 W near-UV light (Toshiba, Tokyo, Japan) in dry conditions. The produced spores were harvested with distilled water for a time course infection experiment or with a spore germination medium (4 g of glucose, 2 g of yeast extract, 7.4 g of Na2HPO4· 12H2O and 2 g of citric acid monohydrate dissolved in 1 L of distilled water) for an inhibition assay using a paint brush. The spore suspension was filtered through cheesecloth and stored on ice until use.

complex mixture of biological origin, it is not suitable for analyzing heat-labile, highly polar, or nonvolatile compounds. In this context, LC/MS can be complementarily used for the analysis of such compounds. In this study, we used both the GC/MS and LC/MS techniques to find novel phytoalexins from UV-irradiated rice leaves, followed by complementary spectroscopic techniques to identify them.

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MATERIALS AND METHODS

General Analytical Methods. 1H NMR (400 MHz), 13C NMR (100 MHz), and 2D-NMR spectra were acquired on an AVANCE III FT-NMR spectrometer (Bruker BioSpin, Rheinstetten, Germany) equipped with a 5 mm BBFO probe. The chemical shifts were referenced to TMS (δH 0.00; δC 0.00) or C6D6 (δH 7.16; δC 128.39) as an internal standard. The IR spectra were acquired using an FT/IR-4100 spectrometer (Jasco, Tokyo, Japan); the samples for IR were prepared as KBr disks. The UV spectra were acquired using a V-550 spectrometer (Jasco); the samples for UV spectroscopy were dissolved in MeOH. The specific rotations were measured using a P-2100 polarimeter equipped with a halogen lamp and a 589 nm filter (Jasco); the samples for polarimetry were dissolved in CHCl3. The electron ionization (EI)-mass spectra (70 eV) were recorded with a JMS-BU25 mass spectrometer (Jeol, Tokyo, Japan) operated in direct inlet mode. GC/MS was performed with a JMS-BU25 mass spectrometer coupled with an HP6890 gas chromatograph (Agilent Technologies, Santa Clara, CA) under the following conditions: column, 30 m × 0.32 mm i.d., 0.25-μm film thickness, J&W Scientific HP-5 (Agilent Technologies); injection port temperature, 280 °C; carrier gas, He; flow rate, 1.0 mL/min; temperature program of column oven, 70 °C for 1 min before heating to 300 °C at 10 °C/min and then maintained at 300 °C for 3 min; ionization mode, EI (70 eV); ion chamber temperature, 200 °C; scan range, m/z 61−627; scan rate, 1 scan/s. LC/MS was performed with a 3200 QTRAP LC/MS/MS system (AB Sciex, Framingham, MA) coupled with a Prominence UFLC system (Shimadzu Co., Kyoto, Japan). The Turbo V ion source was B


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Journal of Agricultural and Food Chemistry UV Irradiation of the Rice Leaves. UV irradiation was conducted by the previously described method.9 UV-irradiated rice leaves were incubated at 26 °C under continuous light condition for 3 days for the screening and isolating of UVinducible substances, and for 0, 1, 2, 3, 4, and 5 days for the quantitation of 1−12 in the UV irradiation time course experiment. The UV-irradiated leaves were stored at −20 °C before use. Purification of 9β-Pimara-7,15-diene-3β,6β,19-triol, 1, and Stemar-13-en-2α-ol, 2, from UV-Irradiated Rice Leaves. The 400 g of UV-irradiated rice leaves were cut and shaken in 6 L of 80% (v/v) MeOH with a reciprocal shaker for 24 h. The homogenate was filtered to obtain an extract, and the residual leaves were then re-extracted with 4 L of 80% MeOH in the same manner. The combined extract was concentrated in vacuo to remove the MeOH. The resulting aqueous concentrate was extracted with EtOAc, and the organic layer was evaporated to dryness in vacuo. The EtOAc extract was subjected to column chromatography on 50 g of 63-210-μm particle size, silica gel 60N (Kanto Chemical Co., Tokyo, Japan) and eluted with a stepwise gradient of n-hexane/EtOAc (95:5, 80:20, 60:40, 20:80, 0:100; 300 mL of each). Each fraction was analyzed by GC/MS to detect the UV-induced substances. Compounds 1 and 2, exhibiting plausible molecular ions at m/z 302 and 288, were detected in the fraction eluted with 80:20 and 0:100 n-hexane/EtOAc, respectively. Their MS and tR did not correspond to those of known phytoalexins. The fraction eluted with 0:100 n-hexane/EtOAc was applied to a 20 cm × 20 cm, 1.0 mm thick, silica gel 60 F254 preparative TLC plate (Merck Millipore, Billerica, MA) developed with 1:1 benzene/EtOAc. The Rf 0.10−0.40 TLC region, which contained 1, was scraped off and extracted with EtOAc. The EtOAc extract was evaporated in vacuo to dryness to give a 127 mg of TLC-purified fraction. The fraction was dissolved in MeOH at a concentration of 4% (w/v) and separated by ODSHPLC under the following conditions: column, 25 cm × 1 cm i.d., 5 μm particle size, COSMOSIL 5C18AR (Nacalai Tesque, Kyoto, Japan); solvent, 70% (v/v) MeOH; injection volume in a run, 200 μL. The peak containing 1 (tR 30−35 min) was collected and evaporated to dryness in vacuo to afford 2.1 mg of purified 1. Compound 1 was identified as 9β-pimara-7,15diene-3β,6β,19-triol by spectroscopic methods. High-resolution electron ionization mass spectrum (HREIMS) (m/z): (M+) calcd for C20H32O3, 320.2352; found, 320.2377; 1H NMR (400 MHz, CDCl3, δ): 0.87 (3H, s, H-17), 1.26 (3H, s, H-20), 1.29 (3H, s, H-18), 1.91 (dd, J = 12.1, 2.3 Hz, 1H, H-14), 2.08 (d, J = 12.1 Hz, 1H, H-14), 3.32 (m, 1H, H-3), 4.03 (d, J = 11.6 Hz, 1H, H-19), 4.32 (d, J = 11.6 Hz, 1H, H-19), 4.46 (dd, J = 5.5, 3.8 Hz, 1H, H-6), 4.90 (dd, J = 10.7, 1.2, 1H, H-16), 4.94 (dd, J = 17.4, 1.2, 1H, H-16), 5.56 (br d, J = 5.5 Hz, 1H, H-7), 5.82 (dd, J = 17.4, 10.7 Hz, 1H, H-15); 13C NMR (100 MHz, CDCl3, δ): 21.77 (q, C-17), 21.85 (q, C-18), 24.81 (t, C-11), 26.39 (q, C-20), 27.25 (t, C-2), 35.00 (s, C-10), 35.61 (t, C-1), 37.54 (t, C-12), 39.35 (s, C-13), 44.15 (s, C-4), 47.88 (t, C-14), 48.42 (d, C-5), 53.49 (d, C-9), 62.77 (t, C-19), 65.01 (d, C-6), 80.59 (d, C-3), 109.68 (t, C-16), 121.66 (d, C-7), 141.14 (s, C8), 149.61 (d, C-15); EIMS (70 eV) m/z: M+ 320 (2), 302 (21), 287 (50), 269 (43), 149 (59), 105 (54), 91 (60), 81 (66), 61 (83), 55 (100); IR (KBr) ṽmax (cm−1): 3370, 1459, 1377, 1342, 1185, 959, 907; [α]20D −150 (c 0.16, CHCl3). The fraction eluted with 80:20 n-hexane/EtOAc was subjected to preparative TLC and developed with 3:1 benzene/EtOAc. The Rf 0.55−0.82 TLC region, which

contained 2, was scraped off and extracted with EtOAc. The EtOAc extract was evaporated in vacuo to dryness to give a 59 mg of TLC-purified fraction. The fraction was dissolved in MeOH at a concentration of 1% (w/v) and separated by ODSHPLC under the following conditions: column, COSMOSIL 5C18AR; solvent, 70% (v/v) MeOH; injection volume in a run, 100 μL. A peak containing 2 (tR 60−65 min) was collected and evaporated to dryness in vacuo to afford 1.3 mg of purified 2. Compound 2 was identified as stemar-13-en-2α-ol by spectroscopic methods. HREIMS (m/z): (M+) calcd for C20H32O, 288.2452; found, 288.2446; 1H- and 13C NMR (C6D6): see Table 1; EIMS (70 eV) m/z: M+ 288 (33), 270 Table 1. 13C and 1H NMR Data for Stemar-13-en-2α-ol, 2, in C 6D 6 position

δC (multiplicity)

1

42.95 (t)

2 3

65.23 (d) 52.09 (t)

4 5 6

35.29 (s) 49.11 (d) 22.56 (t)

7

32.33 (t)

8 9 10 11

44.63 51.53 41.07 30.52

(d) (s) (s) (t)

12 13 14 15

43.79 139.06 124.64 33.92

(d) (s) (d) (t)

16

32.37 (t)

17 18 19 20

22.59 34.35 23.71 18.12

(q) (q) (q) (q)

δH (multiplicity, J in Hz) 1.20 1.68 3.74 1.03 1.65 1.00 1.45 1.20 1.64 1.28 1.95 1.63 1.36 2.13 5.05 1.66 1.47 1.71 1.22 1.63 0.83 0.79 0.89

(m, overlapped, 1H) (m, overlapped, 1H) (tt, 11.6, 3.9, 1H) (t, 11.6, 1H) (m, overlapped, 1H) (dd, 12.2, 2.6, 1H) (m, overlapped, 1H) (m, overlapped, 1H) (m, overlapped, 1H) (m, overlapped, 1H) (ddd, 11.7, 4.5, 3.9, 1H)

(m, overlapped, 1H) (dd, 10.6, 4.3) (br t, 4.3, 1H) (dqd, 4.5, 1.5, 0.6, 1H) (m, overlapped, 1H) (m, overlapped, 1H) (m, overlapped, 1H) (m, overlapped, 1H) (t, 1.5, 3H) (s, 3H) (s, 3H) (br s, 3H)

(57), 255 (100), 131 (43), 119 (48), 105 (92), 91 (83), 79 (55), 69 (53), 58 (86); IR (KBr) ṽmax (cm−1): 3348, 2954, 1459, 1389, 1317, 1255, 1162, 1039; [α]20D +60 (c 0.1, CHCl3). Screening of UV-Inducible Substances with LC/MS. The control and UV-irradiated rice leaves were cut, weighed, and shaken in 80% (v/v) MeOH [5 mL/0.1 g fresh weight (FW)] using a reciprocal shaker. The homogenate was filtered through a cotton-plugged Pasteur pipet, and the extract was refiltered through a 13 mm diameter, 0.22-μm pore size, cellulose acetate membrane filter (Lab Lab Company, Tokyo, Japan). A 10 μL volume of the solution was subjected to LC/ MS analysis. The liquid chromatographic separation of the analytes was achieved in a 150 mm × 2.1 mm i.d., 3.0-μm particle size, Atlantis T3 column (Waters, Milford, MA) with a binary gradient of 0.1% (v/v) aqueous formic acid (solvent A) and MeOH containing 0.1% (v/v) formic acid (solvent B) at flow rate of 0.2 mL/min and at a temperature of 40 °C. The solvent gradient elution was programmed as follows: initial, C


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Table 2. 13C and 1H NMR Data for 1α,2α-Dihydroxy-ent12,15-cassadiene-3,11-dione, 3, in CDCl3

20% B; 0−5 min, isocratic elution by 20% B; 5−35 min, a linear gradient from 20% B to 100% B; 35−40 min, isocratic elution by 100% B. The scan range of MS was set as m/z 100−711 (enhanced MS scan). Every extracted ion chromatogram obtained from the extracts of the control and UV-irradiated leaves were compared using LightSight 2.2 software (AB Sciex) in an attempt to discover novel UV-induced substances. An unknown compound exhibiting an m/z of 333, which was estimated as [M + H]+, was detected at a tR of 29.7 min in the chromatogram of the UV-treated leaves. Therefore, suspecting a novel phytoalexin candidate, we attempted to purify compound 3. Purification of 1α,2α-Dihydroxy-ent-12,15-cassadiene-3,11-dione, 3, from UV-Irradiated Rice Leaves. The LC/MS/MS analysis conditions were defined for a rapid monitoring of 3 during the purification procedure. The liquid chromatographic separation of the analytes was conducted using a 50 mm × 2.0 mm i.d., 3.0 μm particle size, TSKgel ODS-100V column (Tosoh, Tokyo, Japan) with a binary gradient of 0.1% (v/v) aqueous formic acid (solvent A) and MeOH containing 0.1% (v/v) formic acid (solvent B) at a flow rate of 0.2 mL/min and at a temperature of 40 °C. The solvent gradient elution was programmed as follows: initial, 20% B; 0− 10 min, a linear gradient from 20% B to 100% B; 10−12 min, isocratic elution by 100% B. The scan range of MS was set as m/z 80−338 (enhanced MS scan). Compound 3 was detected at a tR of 9.4 min under these conditions. EtOAc extract was prepared from 400 g of UV-irradiated rice leaves in the same manner as the purification of 1 and 2. The EtOAc extract was subjected to column chromatography on 60 g of silica gel and eluted with a stepwise gradient of n-hexane/ EtOAc (95:5, 80:20, 60:40, 40:60, 20:80, 0:100; 300 mL of each). Each fraction was analyzed by LC/MS/MS under the previously described conditions to detect compound 3. Compound 3 was detected in the fraction eluted with 20:80 n-hexane/EtOAc. The fraction was subjected to preparative TLC and developed with 10:1 chloroform/MeOH. The Rf 0.41−0.76 TLC region, which contained 3, was scraped off and extracted with EtOAc. The EtOAc extract was evaporated in vacuo to dryness to give a 65 mg of TLC-purified fraction. The fraction was subjected to reversed-phase column chromatography using 6 g of 200−300 mesh, Chromatorex ODS (Fuji Silysia Chemical Ltd., Kasugai, Japan) and eluted with a stepwise gradient of MeOH/H2O (20, 50, 80, 100% MeOH; 96 mL of each). Each fraction was also analyzed by LC/MS/MS in the same manner as the previous purification step. The fraction containing 3, which was eluted with 80% MeOH, was then evaporated in vacuo to dryness to give a 26 mg of reversedphase-chromatography-purified fraction. The fraction was dissolved in MeOH at a concentration of 2% (w/v) and separated by ODS-HPLC under the following conditions: column, 25 cm × 1 cm i.d., 5 μm particle size, Luna 5 μm C18(2) (Phenomenex, Torrance, CA); solvent, 60% (v/v) MeOH; injection volume in a run, 100 μL. A peak containing 3 (tR 70−74 min) was collected and evaporated to dryness in vacuo to afford 1.9 mg of purified 3. Compound 3 was identified as 1α,2α-dihydroxy-ent-12,15-cassadiene-3,11-dione by spectroscopic methods, as described in the Results and Discussion section. 3. HREIMS (m/z): (M+) calcd for C20H28O4, 332.1988; found, 332.1955; 1H- and 13C NMR (CDCl3): see Table 2; EIMS (70 eV) m/z: M+ 332 (2), 315 (10), 279 (4), 229 (5), 203 (6), 167 (11), 149 (85), 83 (42), 57 (100); UV (MeOH) λmax (log ε): 269 (4.0); IR (KBr) ṽmax

position 1 2 3 4 5 6 7

δC (multiplicity) 78.47 70.57 214.58 47.02 48.27 21.97

(d) (d) (s) (s) (d) (t)

30.32 (t)

8 9 10 11 12 13 14 15 16

38.42 56.78 42.49 202.67 127.91 163.37 33.38 135.97 122.09

(d) (d) (s) (s) (d) (s) (d) (d) (t)

17 18 19 20 1-OH 2-OH

13.42 18.79 29.25 11.49 -

(q) (q) (q) (q)

δH (multiplicity, J in Hz) 4.36 4.74 1.91 1.53 1.75 1.54 1.75 2.16 2.31 5.86 2.72 6.39 5.57 5.76 1.14 1.10 1.17 0.74 5.24 3.52

(dd, 8.0, 2.8, 1H) (br d, 8.0, 1H)

(dd, 11.6, 3.3, 1H) (m, overlapped, 1H) (m, overlapped, 1H) (m, overlapped, 1H) (m, overlapped, 1H) (m, 1H) (d, 13.1, 1H)

(s, 1H) (qd, 7.2, 4.2, 1H) (dd, 17.6, 10.8, 1H) (d, 10.8, 1H) (d, 17.6, 1H) (d, 7.2, 3H) (s, 3H) (s, 3H) (s, 3H) (d, 2.8, 1H) (br s, 1H)

(cm−1): 3453, 2963, 2857, 1726, 1634; [α]20D −147 (c 0.17, CHCl3). During the purification of 3, we also purified 2.6 mg of 8 from the 60:40 n-hexane/EtOAc-eluted silica gel column chromatography fraction, followed by reversed-phase chromatography and ODS-HPLC. The 1H- and 13C NMR data of 8 coincided with those previously reported.7 Assay of the Antifungal Activity against the Rice Blast Fungus. For the fungal colony growth inhibition assay, each test compound was dissolved in an unsolidified PDA medium containing 1% (v/v) dimethyl sulfoxide at a concentration of 75 or 300 μM. The PDA medium was poured into a 9 cm Petri dish and solidified at room temperature. A PDA medium containing 1% (v/v) dimethyl sulfoxide was used as a control. Four 25 mm2 pieces of the blast fungus, which had been cultured on a PDA medium, were transplanted to the PDA medium. The inoculated PDA medium was incubated at 26 °C in the dark. After 0, 1, 2, 3, 4, and 5 days of incubation, the distance between the front of the planted pieces and the front of the mycelium colony was measured using a ruler. For the spore germination and germ tube growth inhibition assay, the concentration of spore suspension was adjusted to 6 × 104 spores/mL with a prechilled spore germination medium. The samples were dissolved in distilled water containing 0.2% (v/v) dimethyl sulfoxide and 0.1% (v/v) Tween 80. The 12.5 μL of spore suspension and the 12.5 μL of sample solution were mixed on a concavity slide and then incubated for 16 h at 26 °C in a highly humidified box in the dark. The germination rate and germ tube length of the M. oryzae spores were measured under a microscope. The inhibitory activity against spore germination and germ tube elongation was determined according to the previously reported method.9 Quantitative LC/MS/MS Analysis of 9β-Pimara-7,15diene-3β,6β,19-triol, 1, Stemar-13-en-2α-ol, 2, 1α,2αD


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C20H32O3 and five degrees of unsaturation. The NMR spectra of 1 suggested that it contains two oxygenated methines and one oxygenated methylene. The acetylation of 1 using acetic anhydride gave a triacetylated compound, indicating that 1 has three hydroxyl groups (data not shown). The 1D- and 2DNMR spectra indicated that the structure of 1 was 9β-pimara7,15-diene-3β,6β,19-triol, which had been reported as a LiAlH4reduction product of 5.30 We also synthesized the compound from 5 using the method reported in the literature, and the 1Hand 13C NMR data of the synthetic 1 coincided with those of 1 obtained and purified from the UV-irradiated rice leaves (data not shown). Therefore, UV-induced compound 1 from rice leaves was identified as 9β-pimara-7,15-diene-3β,6β,19-triol. The HREIMS analysis of 2 suggested a molecular formula C20H32O and five degrees of unsaturation. The number of carbon atoms agreed with the 13C NMR data (Table 1). IR data indicated the presence of a hydroxyl group, whose proton was not observed in the 1H NMR spectrum. 13C NMR and distortionless enhancement by polarization transfer (DEPT) spectra indicated the presence of one olefinic methine (C-14; δC 124.64 ppm) and one olefinic quaternary carbon (C-13; δC 139.06 ppm), suggesting that 2 has one C−C double bond. Five degrees of unsaturation and the presence of one C−C double bond indicated that 2 has four rings. 13C NMR and DEPT data showed that 2 consists of four methyl, seven methylene, five methine, and four quaternary carbons. These results suggested that 2 should be a monohydroxylated tetracyclic diterpene. The 13C chemical shifts of 2 were very similar to those of 7, a rice stemarane-type phytoalexin, except that 2 lacks an oxymethylene signal corresponding to the C-19 of 7.31 Moreover, four methyl signals were observed in the 1Hand 13C NMR spectra of 2, whereas three methyl signals were observed in those of 7. Typical signals of a stemar-13-ene skeleton, such as δH 5.05 (H-14), 2.13 (H-12), 1.63 (H-17), 0.89 (H-20), 0.83 (H-18), and 0.79 (H-19), were observed in the 1H NMR spectrum.31−36 2D-NMR correlations confirmed the stemar-13-ene skeleton (Figure 2). A methine carbon at δC 64.5 ppm (C-2) should be connected to a hydroxyl group. The coupling constants between H-2 and H-1/H-3 as well as nuclear Overhauser enhancement and exchange spectroscopy (NOESY) correlations between H-2 and H-19/H-20 indicated that the 2-hydroxyl group should be equatorial (α-orientation). These results suggested that the structure of 2 should be 19deoxyoryzalexin S. Although the absolute stereochemistry of 2 was not determined experimentally, given its origin, it likely has the same stemarane skeleton as 7. We thus concluded that 2 must be stemar-13-en-2α-ol. UV-induced compounds 1 and 2 were successfully found using GC/MS analysis. We subsequently attempted to search for novel phytoalexins using LC/MS analysis. As a result, we discovered compound 3, which showed an m/z 333 estimated as [M + H]+ and a tR of 29.7 min in the chromatogram of the UV-treated leaves. Its tR and mass spectrum did not correspond to those of known phytoalexins or their biosynthetic precursors, as determined by a comparison with authentic samples and theoretical m/z values. Moreover, we estimated that the accumulated amount of 3 was as high as that of 5 based on the peak area, whose induction level was reported to be relatively high among known phytoalexins in UV-irradiated and M. oryzae-infected rice leaves.9 Compound 3 was not detected in healthy rice leaves that had not been UV-irradiated. Therefore, we performed a large-scale isolation of 3 to determine its structure. Compound 3 (1.9 mg, a pale-yellow

Dihydroxy-ent-12,15-cassadiene-3,11-dione, 3, and Other Phytoalexins, 4−12. The liquid chromatographic separation of the analytes was achieved in an Atlantis T3 column with a binary gradient of 0.1% (v/v) aqueous formic acid (solvent A) and MeCN containing 0.1% (v/v) formic acid (solvent B) at a flow rate of 0.2 mL/min and at a temperature of 40 °C. The solvent gradient elution was programmed as follows: initial, 40% B; 0−3 min, isocratic elution by 40% B; 3− 23 min, a linear gradient from 40% B to 100% B; 23−28 min, isocratic elution by 100% B. The selective reaction monitoring (SRM) transitions and the parameters of the mass spectrometer were optimized for detecting 1−12 using Analyst 1.5.1 software (AB Sciex). Calibration curves were obtained from the SRM peak areas of the standards for 1−12 in a concentration range of 1−1000 ng/mL. The concentrations of the analytes were calculated from their individual SRM peak area based on the calibration curves. Quantitation of 9β-Pimara-7,15-diene-3β,6β,19-triol, 1, Stemar-13-en-2α-ol, 2, 1α,2α-Dihydroxy-ent-12,15cassadiene-3,11-dione, 3, and Other Phytoalexins, 4− 12, from UV-Irradiated and M. oryzae-Inoculated Rice Leaves. UV-irradiated rice leaves (ca. 3 leaves) were cut, weighed and shaken in 80% (v/v) MeOH (5 mL/0.1 g FW) using a reciprocal shaker. The extract was filtered through a cotton-plugged Pasteur pipet and was refiltered through a membrane filter. The spore suspension of M. oryzae was diluted with water to a concentration of 2 × 105 spores/mL and 25 μL of the spore suspension was then applied to press-injured spots with a diameter of 1 mm on the rice leaves. The leaves were maintained in a highly humidified box and incubated for 1, 2, 3, 4, and 5 days at 26 °C under light irradiation. The leaf tissues around the injured spots were excised with a 4 mm i.d., cork borer. Approximately 45 pieces of the excised leaf tissues (ca. 100 mg) were collected and shaken in 5 mL of 80% MeOH using a reciprocal shaker. The homogenate was filtered through a cotton-plugged Pasteur pipet and a membrane filter. A 5 μL volume of the sample solution was subjected to the aforementioned LC/MS/MS analysis to quantitate 1−12.

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RESULTS AND DISCUSSION Purification and Structural Elucidation of 9β-Pimara7,15-diene-3β,6β,19-triol, 1, Stemar-13-en-2α-ol, 2, and 1α,2α-Dihydroxy-ent-12,15-cassadiene-3,11-dione, 3, from UV-Irradiated Rice Leaves. We detected UV-induced compounds 1 and 2 in the silica gel column chromatographypurified fractions of UV-irradiated rice leaves by GC/MS analysis. Compounds 1 and 2 were detected at tR 19.2 and 18.2 min and their mass spectra showed plausible molecular ions at m/z 302 and 288, respectively. These tR and mass spectra did not correspond to those of known phytoalexins or to their biosynthetic precursors when compared to the tR and mass spectra of authentic samples. Compounds 1 and 2 were not detected in healthy rice leaves that had not been UV-irradiated. Therefore, we isolated 1 and 2 to determine their structures. Compounds 1 (2.1 mg, a white solid) and 2 (1.3 mg, a colorless oil) were successfully purified from the 400 g of UV-irradiated rice leaves by chromatographic methods. Although we estimated m/z 302 as the molecular ion of 1 in the GC/MS analysis, m/z 320 was detected as a more reasonable molecular ion in the MS analysis using the direct inlet mode, suggesting that m/z 302 should be [M−H2O]+. The HREIMS analysis of 1 suggested a molecular formula of E


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bonds. Seven degrees of unsaturation and the presence of two C−C double bonds and two carbonyl groups indicated that 3 had three rings. Correlated spectroscopy (COSY) and heteronuclear single quantum coherence (HSQC) correlations suggested OH-1/C-1/C-2/OH-2, C-15/C-16 and C-5/C-6/C7/C-8/C-9/C-14/C-17 connectivities (Figure 2). Heteronuclear multiple bond correlation (HMBC) data (Figure 2), as well as UV (269 nm) and IR (1634 cm−1) absorptions indicated the presence of an α,β,γ,δ-unsaturated ketone (C-11/ C-12/C-13/C-15/C-16). The HMBC data suggested a quaternary carbon (C-4) bearing geminal methyls (C-18 and C-19) at the α-position of a ketone (C-3). The HMBC data were used to connect these fragment structures and the remaining fractions, including a quaternary carbon (C-10) and a methyl group (C-20). The skeleton of the deduced structure coincided with phytocassanes, the rice ent-cassadiene-type diterpene phytoalexins.7,8,37 The 13C chemical shifts of 3 were very similar to those of 12 except for the C-2, which bore a hydroxyl group in 3 but not in 12. Moreover, NOESY correlations indicated that H-1/H-2/H-5/H-9/H-17/H-19 were located on the same side of the ring and that H-8/H14/H-20/1-OH were located on the other side. These results suggested that 3 had the same relative configuration as phytocassanes. Although the absolute stereochemistry of 3 was not determined experimentally, given its origin, 3 likely has the same ent-cassadiene skeleton as phytocassanes. Therefore, the structure of 3 was determined as 1α,2α-dihydroxy-ent12,15-cassadiene-3,11-dione. Accumulation of 9β-Pimara-7,15-diene-3β,6β,19-triol, 1, Stemar-13-en-2α-ol, 2, and 1α,2α-Dihydroxy-ent12,15-cassadiene-3,11-dione, 3, in UV-Irradiated and M. oryzae-Inoculated Rice Leaves. We subsequently developed a quantitation method for 1, 2, and 3. The leaf extract was subjected to LC/MS/MS analysis, which resulted in the detection of these compounds in the SRM mode by positive ESI. The change in the accumulated levels of 1−12 after UV irradiation was analyzed using this method. We observed that brown spots dramatically increased in number and area 24−48 h after UV irradiation, and this change was not observed in the control leaves. Corresponding to this apparent change, the amounts of 1, 2, and 3 significantly increased after UV irradiation, reaching a maximum after 48 h and then slightly decreasing after 72−120 h (Figure 3). The leaves that had not been UV-irradiated did not show the presence of these compounds. In the case of M. oryzae infection, brown spots

Figure 2. 2D-NMR correlations for stemar-13-en-2α-ol, 2, and 1α,2αdihydroxy-ent-12,15-cassadiene-3,11-dione, 3.

oil) was successfully purified from the 400 g of UV-irradiated rice leaves by chromatographic methods. Compound 3 could not be detected by GC/MS, likely because of its high polarity or heat-labile property. The HREIMS analysis of 3 suggested a molecular formula C20H28O4 and seven degrees of unsaturation. The numbers of carbons and hydrogens agreed with the 1H and 13C NMR data (Table 2). 13C NMR and IR data indicated that two of four oxygens belonged to two carbonyl groups and the two remaining oxygens belonged to two hydroxyl groups. 13C NMR and DEPT data indicated the presence of two olefinic methines, one terminal olefinic methylene and one olefinic quaternary carbon, suggesting that 3 had two C−C double

Figure 3. Time-dependent accumulation of 9β-pimara-7,15-diene-3β,6β,19-triol, 1, stemar-13-en-2α-ol, 2, and 1α,2α-dihydroxy-ent-12,15-cassadiene3,11-dione, 3, in UV-irradiated and rice blast fungus-inoculated rice leaves. Values are presented as the mean ± SD (n = 3). F


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inoculated leaves were not substantially different from those of known phytoalexins, 6 and 8−12, but were lower than those of 5 and 7. Antifungal Activities of 9β-Pimara-7,15-diene3β,6β,19-triol, 1, Stemar-13-en-2α-ol, 2, and 1α,2αDihydroxy-ent-12,15-cassadiene-3,11-dione, 3. The antifungal activities of 1, 2, 3 and phytoalexins against M. oryzae were determined by a spore germination and germ tube growth assay. Initially, we tested the solubility of these compounds in an aqueous liquid medium and observed that 3 and 5 were insoluble at high concentrations. To ensure the effective concentration of insoluble compounds in the test, we attempted to add dimethyl sulfoxide and Tween 80 to the test solution in final concentrations of 0.1% and 0.05%, respectively. The additives did not affect the spore germination rate (95%) or germ tube growth of the control experiment. The spore germination was completely suppressed by the addition of 3 or 4 at a concentration of 300 μM. In the germ tube growth assay, 4, 5, 7, and 3 exerted a dose-dependent inhibitory activity (Figure 4). The IC50 values of these compounds were calculated as 37, 74, 122, and 16 μM, respectively. Compounds 1 and 2 inhibited germ tube growth only in high doses, and their IC50 values were calculated to be approximately equal to or greater than 300 μM. Furthermore, we investigated whether 3 inhibited the fungal mycelial colony growth of M. oryzae on a solid medium. Compound 3 showed dose-dependent inhibition of fungal colony growth, and the inhibition rate was calculated as 26% at 300 μM after 5 days of incubation (Figure 5). We also examined the inhibitory activity of known phytoalexins, 4, 5, and 8. The inhibition rates of 4 and 8 were calculated as 51% and 22% at 300 μM after 5 days of incubation, respectively. However, 5 only slightly inhibited fungal growth. The inhibitory activities of 4 and 5 were approximately the same as that observed in previous reports.12,13 Thus, the antifungal

emerged at the inoculated site 24 h after inoculation, and their areas gradually spread up to 120 h after inoculation. The brown spots were not observed in the mock-treated leaves. The amounts of 1, 2, and 3 also gradually increased up to 96 h after inoculation and were almost stable until 120 h after inoculation (Figure 3). The mock-treated leaves did not accumulate 1, 2, and 3. The contents of 1−12 in the UV-irradiated and M. oryzaeinoculated leaves are summarized in Table 3. A flavonoid-type Table 3. Accumulation of Phytoalexins and 9β-Pimara-7,15diene-3β,6β,19-triol, 1, Stemar-13-en-2α-ol, 2, and 1α,2αDihydroxy-ent-12,15-cassadiene-3,11-dione, 3, in UVIrradiated Rice Leaves after 48 h of Incubation and in M. oryzae-Inoculated Rice Leaves after 120 h of Incubationa accumulation (μg/g FW) compd 1 2 3 4 5 6 7 8 9 10 11 12 a

UV irradiation 73.0 52.9 59.2 352.9 96.1 60.9 53.4 75.9 23.0 12.8 10.8 15.9

± ± ± ± ± ± ± ± ± ± ± ±

19.9 8.4 15.6 79.3 25.4 9.0 7.5 20.2 4.9 4.6 1.7 4.5

M. oryzae inoculation 8.5 9.6 12.0 75.2 29.3 10.6 26.4 7.7 8.1 2.5 10.3 10.2

± ± ± ± ± ± ± ± ± ± ± ±

3.2 1.8 2.7 30.7 6.8 5.5 3.5 2.4 1.9 0.4 1.9 2.3

Values are presented as the mean ± SD (n = 3).

phytoalexin, 4, accumulated at the highest level in the UVirradiated and M. oryzae-inoculated leaves. The levels of diterpenoid-type compounds in the UV-irradiated leaves were similar. The accumulation levels of 1, 2, and 3 in the M. oryzae-

Figure 4. Antifungal activity of 9β-pimara-7,15-diene-3β,6β,19-triol, 1, stemar-13-en-2α-ol, 2, and 1α,2α-dihydroxy-ent-12,15-cassadiene-3,11-dione, 3, against the germ tube growth of rice blast fungus. Values are presented as the mean ± SD (n = 100). G


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antibacterial and antifungal components, such as oxidized fatty acids,39 tryptophan-derived metabolites or phenylamides,40,41 have been discovered in rice leaves. We found that 4 and 5 could be metabolized and detoxified by M. oryzae.12,13,42 On the basis of these findings, we speculate that rice does not depend on only one or a few defensive compounds but rather divides its own resistant capacity among diverse compounds.

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ASSOCIATED CONTENT

S Supporting Information *

Figure S1, Time-dependent accumulation of known phytoalexins in UV-irradiated and rice blast fungus-inoculated rice leaves; Figure S2, Representative images used for measuring the length of elongated germ tubes of rice blast fungus M. oryzae; Table S1, The ion source parameters for LC/MS/MS analyses; Table S2, LC/MS/MS conditions for quantitating 1−12 in the SRM mode. These materials are available free of charge via the Internet at http://pubs.acs.org.

Figure 5. Antifungal activity of 1α,2α-dihydroxy-ent-12,15-cassadiene3,11-dione, 3, and phytoalexins against the mycelium growth of the rice blast fungus. Data are presented as means ± SD (n = 4) from four independent samples.

activity of 3 on mycelial growth was estimated to be approximately the same as that of 8 and lower than that of 4. The formation of compound 1 and 2 were induced by both UV irradiation and M. oryzae infection (Figure 3). However, 1 and 2 exhibited only slight antifungal activity on M. oryzae (Figure 4). Thus, these are not recognized as rice phytoalexins. Its structure clearly indicated that 1 should be biosynthesized by the oxidation of 9β-pimara-7,15-diene, which is a biosynthetic precursor of 5. We speculated that 1 may be a biosynthetic intermediate of 5. The structure of compound 2 suggested that 7 can be produced from 2 by the hydroxylation of the C-19. Kato et al.20 identified a potential precursor of oryzalexins E and F, ent-isopimara-8(14),15-dien-3β-ol, from UV-irradiated rice leaves. They also confirmed that the enzymatic hydroxylation of each precursor was an important activation step for the antifungal activity of oryzalexins E and F.19 In the case of 7, C-19 oxidation is likely important for its antifungal activity. The structure of 3 is regarded as 1-hydroxyphytocassane A, 3-dehydrophytocassane B, or 2-hydroxyphytocassane E. The accumulated level of 3 was approximately the same as that of 8 in the UV-irradiated and M. oryzae-inoculated leaves (Figure 3 and Table 3). Compound 8 has been reported to be the most abundant phytocassane in M. oryzae-inoculated rice leaves.8 Thus, 3 is considered a major compound among rice cassanetype diterpenes. Phytocassanes A−D were isolated from Rhizoctonia solani-infected rice leaf stems,7 and phytocassane E was isolated from elicitor-treated rice suspension cells.8 We speculated that the difference between the materials may be critical to the isolation of 3 in this study. Moreover, compound 3 has a strong inhibitory activity on the spore germination and germ tube elongation of M. oryzae, to the same extent as 4 (Figure 4). The antifungal activity of 3 on fungal colony growth was greater than that of 5, lower than that of 4 but approximately the same as that of 8 (Figure 5). Therefore, 3 is recognized as a phytoalexin with a ent-cassane-type diterpene skeleton and should be named phytocassane F. Our study should contribute to greater understanding of the resistance of rice against M. oryzae and strengthen the foundation of phytoalexin biosynthetic pathways. In total, a flavanone 4 and 16 diterpene phytoalexins, including 3, were identified from rice leaves.2−11 Obara et al.29 reported that many monoterpenes and sesquiterpenes were emitted from rice leaves in response to M. oryzae infection. Taniguchi et al.38 also reported a volatile sesquiterpene, β-elemene, with an antifungal effect on M. oryzae. In addition to terpenoids, diverse

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel.: +81-29-888-8660. Fax: +81-29-888-8525. Present Address #

(Q.Y.) Aikoku Gakuen University, 1532 Yotsukaido, Yotsukaido, Chiba 284-0005, Japan Funding

This work was supported by JSPS KAKENHI Grant no. 22580113. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Compounds 1 and 2 were discovered in our laboratory by Hideki Kato for the first time. Unfortunately, he passed away on May 13, 2010. We would like to express our respect for his pioneering work. We thank Shigeru Tamogami and Randeep Rakwal for critical reading of the manuscript. We thank Kazunori Okada for providing the standard solution of phytocassanes. We thank Hiroshi Kawaide for his helpful advice.

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ABBREVIATIONS USED COSY, correlated spectroscopy; DEPT, distortionless enhancement by polarization transfer; EI, electron ionization; FW, fresh weight; HMBC, heteronuclear multiple bond correlation; HSQC, heteronuclear single quantum coherence; HREIMS, high-resolution electron ionization mass spectrometry; NOESY, nuclear Overhauser enhancement and exchange spectroscopy; PDA, potato dextrose agar; SRM, selected reaction monitoring

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REFERENCES

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