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Lycosides, Unusual Carotenoid-derived Terpenoid Glycosides from a Vegetable Juice, Inhibit Asexual Reproduction of the Plant Pathogen Phytophthora Rika Iwai, Chunguang Han, Sudhakar V. S. Govindam, and Makoto Ojika J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b04766 • Publication Date (Web): 09 Dec 2017 Downloaded from http://pubs.acs.org on December 12, 2017
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Journal of Agricultural and Food Chemistry
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Lycosides, Unusual Carotenoid-derived Terpenoid Glycosides from a Vegetable Juice,
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Inhibit Asexual Reproduction of the Plant Pathogen Phytophthora
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Rika Iwai,† Chunguang Han,‡ Sudhakar V. S. Govindam,† and Makoto Ojika*,†
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†
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Nagoya 464-8601, Japan
8
‡
9
Japan
Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku,
Research Center for Materials Science, Nagoya University, Chikusa-ku, Nagoya 464-8602,
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*Corresponding
author
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[email protected])
(Tel:
81-527894116;
Fax:
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1 ACS Paragon Plus Environment
81-527894118;
E-mail:
Journal of Agricultural and Food Chemistry
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ABSTRACT: Vegetable juices, typical culture media for the plant pathogen Phytophthora,
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effectively induce its asexual reproduction (zoosporangia formation). However, some
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chromatographic fractions from a vegetable juice were found to inhibit the asexual
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reproduction. Bioassay-guided chromatographic steps led to the isolation of four novel
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compounds named lycosides A–D, 1–4, that could be metabolic products from a carotenoid.
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They showed 50% inhibitory activity against the asexual reproduction of P. capsici at 2.1 –
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7.6 µM. The structure-activity relationship and the universality of the inhibitory activity
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within the Phytophthora genus were also investigated. In addition, the quantitative analysis of
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lycosides in fresh vegetables and vegetable juices revealed that tomato is the source of these
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active substances. These food-derived chemicals could help provide safe agents to control the
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outbreak of the agricultural pest Phytophthora in fields.
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KEYWORDS: Plant pathogen, Phytophthora, inhibitor, asexual reproduction, natural
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products, vegetable juice
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INTRODUCTION
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The genus Phytophthora represents a group of filamentous fungus-like oomycetes
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and includes more than 100 species, most of which are deleterious to a broad range of
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economically and ecologically important plant species.1-3 P. infestans is notorious as the
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causal agent of the Great Famine during the mid-1840s that wiped out the potato crop in
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Ireland and eventually led to mass starvation.3 The potato late blight caused by this
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microorganism is one of the biggest world-wide problems in the agricultural sector, resulting
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in economic losses of several billion dollars annually.4 Farmers are forced to spray synthetic
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fungicides for about ten or more times to control the plant pest.5,6 The acute damages caused
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by this pathogen are primarily due to its asexual spores, zoosporangia, that produce thousands
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of motile zoospores as weapons.7 Instead of using synthetic fungicides, other approaches such
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as developing genetically resistant plant strains, chemically inducing plant resistance with
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elicitors and other compounds, and biocontrol could have fewer harmful effects on the
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environment. An additional possibility could involve using natural products that selectively
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suppress the reproduction of Phytophthora, especially zoosporangia formation, although such
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examples are rare.8
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Phytophthora is generally cultured on vegetable juices (typically V8 juice) that
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promote hyphal growth and reproduction (both asexual and sexual ones, as indicated by
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zoosporangia and oospores formation, respectively). During our search for the vegetable juice
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components that promote Phytophthora reproduction, we found an interesting phenomenon.
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Some chromatographic fractions from an extract of V8 vegetable juice inhibited the formation
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of zoosporangia. This suggests that the vegetable juice includes not only promoters but also
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inhibitors of the Phytophthora reproduction. In this study, we report the isolation of the
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Phytophthora asexual reproduction inhibitors named lycosides A–D, 1–4 (Figure 1) from V8
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vegetable juice, as well as their structure-activity relationship (SAR), universality of their 3 ACS Paragon Plus Environment
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inhibitory activity within the Phytophthora genus, and distribution in vegetables, especially
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tomatoes.
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MATERIALS AND METHODS
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General Experimental Procedures. Specific rotations were recorded on a DIP-370
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spectrometer (JASCO, Tokyo, Japan). Infrared (IR) spectra were measured using an
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FT-IR-7000S spectrometer (JASCO). Ultraviolet (UV) spectra were obtained using a V-530
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spectrometer (JASCO). Circular dichroism (CD) spectra were obtained on a J-720WI
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spectrometer (JASCO). Mass spectra (MS) were recorded on a Mariner Biospectrometry
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Workstation (Applied Biosystems, Foster City, CA) in the positive electrospray ionization
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(ESI) mode. High-resolution MS was performed by infusion method with 50% MeCN/0.1%
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HCOOH as the solvent. For LC/MS measurements, the equipment was connected to an
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Agilent 1100 HPLC system (Hewlett Packard, Palo Alto, CA) under the conditions described
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below. Nuclear magnetic resonance (NMR) spectra were recorded on an AMX2 600 (600
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MHz for 1H) or ARX 400 (400 MHz for 1H) spectrometer (Bruker BioSpin, Yokohama,
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Japan). The chemical shifts (ppm) were referenced to the tetramethylsilane (TMS) peak.
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Preparative high-performance liquid chromatography (HPLC) was performed using a
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high-pressure gradient system (JASCO) composed of a PU-1586 pump, DG-1580-53 degasser,
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and UV 1570 detector.
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Strains and Culture. Phytophthora capsici NBRC 30696, P. cinnamomi NBRC
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33180, and P. nicotianae NBRC 9049 were purchased from the Biological Resource Centre,
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National Institute of Technology and Evaluation (NBRC, Chiba, Japan). P. infestans PI
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1234-1 (race 1.2.3.4) was provided by Prof. K. Kawakita at the Graduate School of
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Bioagricultural Sciences, Nagoya University. The strains were kept on PSA medium (soup
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stock from 200 g fresh sliced potato in 1 L water, 10 g sucrose, 20 g agar) at 15 °C and 4 ACS Paragon Plus Environment
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subcultured every 6 months. Prior to the zoosporangia formation test, the strains were
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precultured on the PSA medium in a 9-cm dish in an Eyela KCL-2000A incubator (Tokyo
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Rikakikai Co., Ltd., Tokyo, Japan) at 25 °C and 60% humidity except for P. infestans, which
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was precultured at 20 °C and 70% humidity. The preculture was continued until the surface
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was completely covered by the colony, which took 6-14 d depending on the strains.
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Zoosporangia Formation Test. A piece of precultured Phytophthora mycelium with
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agar (5 x 5 mm) was inoculated on one of the following media (5 mL in 6-cm dish): (a) 0.4%
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(v/v) potato/1% sucrose (w/v)/1.5% agar (w/v) ("0.4P medium"), (b) 4% or 3% V8 vegetable
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juice (v/v)/1% sucrose/2% agar ("4V or 3V medium", respectively), or (c) 5% V8 juice/0.02%
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(w/v) CaCO3/1% sucrose/2% agar ("5V-Ca medium"). All the media contained 1% (v/v)
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DMSO or a sample solution in DMSO. The strains were incubated at 25 °C, 60% humidity
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(20 °C, 70% humidity for P. infestans) for 6−10 d depending on the strains and media. The
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optimized culture conditions are as follows: 0.4P (8 d culture) or 4V (6 d culture) media for
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the tests of chromatographic fractions; 3V medium (7 d culture) for the activity of the
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compounds against P. capsici; 3V medium (6 and 8 d culture) for P. cinnamomi and P.
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nicotianae, respectively; and 5V-Ca medium (10 d culture) for P. infestans. Three or four
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circular areas (12 mm diam.) at a distance of 1.5 cm from the colony center were cut out, and
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the number of zoosporangia was counted under a microscope.
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Preliminary Fractionation of Vegetable Juice and Zoosporangia Formation
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Activity. A can of V8 vegetable juice (340 mL) (Campbell Soup Company, Camden, NJ) was
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diluted with MeOH (220 mL) and stirred at room temperature for 1 h. The mixture was
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separated into supernatant and precipitate by centrifugation (2,000 rpm, 5 min). The latter was
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washed with 40% MeOH (100 mL) by mixing and centrifugation under the same conditions.
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The supernatant and washing were combined and concentrated. The aqueous residue was
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dissolved in 40% MeOH (200 mL in a final volume) and applied to an ODS column (100 g of 5 ACS Paragon Plus Environment
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Cosmosil 75C18-OPN) (Nacalai Tesque, Kyoto, Japan) that was eluted with 40, 60, and 80%
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MeOH (4 fr. × 100 mL each) and then MeOH (7 fr. × 100 mL) to produce 19 fractions. A
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portion (1.25 mL V8 juice equivalent) of each fraction was dissolved in 0.4P medium (5 mL)
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and tested for the zoosporangia formation of P. capsici, which was incubated for 8 d (Figure
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2A).
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The fractions 6−8, which were eluted with 60% MeOH, were combined and
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concentrated to produce a solid (68.5 mg). The material was subjected to flash
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chromatography on a Hi-Flash size S column (silica gel 6 g) (Yamazen, Kyoto, Japan), which
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was eluted with MeOH/H2O/CHCl3 (9:1:90 to 45:5:50, 40 min linear gradient) at a flow rate
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of 3 mL/min to generate 5 fractions. A portion (2.5 mL V8 juice equivalent) of each fraction
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was dissolved in 4V medium (5 mL) and tested for the zoosporangia formation of P. capsici,
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which was incubated for 6 d (Figure 2B).
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Isolation of 1–4. Twenty cans of V8 vegetable juice (6.8 L) were diluted with MeOH
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(4.4 L) and stirred at room temperature for 1 h. The mixture was filtered by suction, and the
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residue was washed with 40% MeOH (0.7 L). The supernatant and washing were combined
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and concentrated to a MeOH-free solution. The aqueous solution was dissolved in 40%
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MeOH (1 L in total) and chromatographed on ODS (1 kg of Cosmosil 75C18-OPN), which
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was eluted with 40, 60, 80% MeOH (4 L each). The 60% MeOH fraction (3.24 g) was
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chromatographed on silica gel (50 g of Wakogel C300) (Wako Pure Chemicals Industries,
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Osaka, Japan) eluted with MeOH/H2O/CHCl3 (27:3:70, 325 mL and then 45:5:50, 200 mL). A
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fraction (673 mg) eluted with MeOH/H2O/CHCl3 (27:3:70) was subjected to flash
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chromatography on a Hi-Flash size M column (silica gel 14 g) (Yamazen), which was eluted
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with MeOH/H2O/CHCl3 (4.5:0.5:95 to 40.5:4.5:55, 40 min linear gradient) at a flow rate of 6
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mL/min. The active fraction (111 mg) eluted at 14-24 min was purified by HPLC. The column
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used was a 250 mm × 20 mm i.d., 5 µm, YMC-Pack D-ODS-5 (YMC, Kyoto, Japan), which 6 ACS Paragon Plus Environment
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was eluted with MeOH in water (50 to 75%, 75 min linear gradient) at a flow rate of 5
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mL/min with detection at 210 nm. The active fraction (8.4 mg) eluted at 48-52 min was
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further purified by HPLC. The column used was a 250 mm × 10 mm i.d., 5 µm, Develosil
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ODS-UG-5 (Nomura Chemical, Seto, Aichi, Japan), which was eluted with 23% MeCN at a
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flow rate of 3 mL/min with detection at 234 nm to produce 1 (0.5 mg, tR = 48.6 min), 2 (1.2
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mg, tR = 53.1 min), 3 (0.6 mg, tR = 54.9 min), and 4 (0.7 mg, tR = 57.5 min).
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Lycoside A, 1: Colorless oil, [α]30D +109 (c 0.021, MeOH); UV (MeOH) λmax(ε) 236 (24,000)
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nm; CD λext (∆ε) 251 (+36), 227 (-39) nm; IR (KBr) νmax 3398, 1653, 1596, 1159, 1103, 1074
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cm−1; HR ESIMS m/z 617.3178 [M+H]+ (calcd for C30H49O13 617.3168). NMR data are listed
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in Tables 1 and 2.
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Lycoside B, 2: Colorless oil; [α]30D +126 (c 0.034, MeOH); UV (MeOH) λmax(ε) 236 (22,000)
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nm; CD (MeOH) λext (∆ε) 250 (+31), 226 (-33) nm; IR (film) νmax 3397, 1655, 1594, 1159,
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1075, 1026 cm−1; HR ESIMS m/z 617.3203 [M+H]+ (calcd for C30H49O13 617.3168). NMR
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data are listed in Tables 1 and 2.
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Lycoside C, 3: Colorless oil, [α]30D + 133 (c 0.010, MeOH); UV (MeOH) λmax(ε) 236 nm
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(25,000) nm; CD λext (∆ε) 249 (+35), 227 (-30) nm; IR (film) νmax 3419, 1650, 1593, 1077,
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1024 cm−1; HR ESIMS m/z 455.2651 [M+H]+ (calcd for C24H39O8 455.2639). NMR data are
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listed in Tables 1 and 2.
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Lycoside D, 4: Colorless oil, [α]30D +144 (c 0.023, MeOH); UV (MeOH): λmax(ε) 236 nm
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(29,000) nm; CD λext (∆ε) 250 (+30), 227 (-30) nm; IR (film) νmax 3396, 1655, 1596, 1076,
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1024 cm−1; HR ESIMS m/z 455.2646 [M+H]+ (calcd for C24H39O8 455.2639). NMR data are
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listed in Tables 1 and 2.
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Preparation of Aglycons 5 and 6. β-D-Glucosidase (from almond, 11 units/mg)
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(Oriental Yeast, Tokyo, Japan) was dissolved in ammonium acetate buffer (0.1 M, pH 5) to 7 ACS Paragon Plus Environment
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prepare a stock solution of 20 units/mL. A portion (50 µL) of the enzyme stock solution was
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added to a solution of lycoside D, 4 (0.6 mg) in the same buffer (0.2 mL). The mixture was
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incubated at 37 °C for 13.5 h. The mixture was extracted twice with EtOAc (0.5 mL). The
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extracted material was purified by HPLC. The column used was a 250 mm × 10 mm i.d., 5
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µm, Develosil ODS-UG-5, which was eluted with 55% MeOH (55 min) and then 60% MeOH
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at a flow rate of 1.5 mL/min with detection at 235 nm to produce (13S)-aglycon, 6 (tR = 80.5
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min, 0.083 mg). The same procedure was conducted for a mixture of lycoside A, 1 (0.4 mg)
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and C, 3 (0.5 mg) to produce (13R)-aglycon, 5 (tR = 77.3 min, 0.105 mg). The yields were
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calculated by UV measurement with a molar absorption coefficient of 25,000.
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(13R)-Aglycon, 5: Colorless powder, [α]28D +254 (c 0.0079, MeOH); UV (MeOH) λmax (ε)
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236 nm (24,000) nm; CD (MeOH); λext (∆ε) 249 (+28), 228 (-37) nm; IR (film) νmax 3421,
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1655, 1126 cm-1; HR ESIMS m/z 293.2104 [M+H]+ (calculated for C18H29O3 293.2111).
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NMR data are listed in Table 3.
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(13S)-Aglycon, 6: Colorless powder, [α]30D +246 (c 0.0052, MeOH); UV (MeOH) λmax 236
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nm (ε 24000); CD (MeOH) λext 247 (∆ε +27), 225 (∆ε -38) nm; IR (film) νmax 3421, 1652,
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1594, 1125 cm-1; HR ESIMS m/z 293.2096 [M+H]+ (calculated for C18H29O3 293.2111).
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NMR data are listed in Table 3.
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Preparation of Mosher's Esters of Aglycons. (13R)-Aglycon, 5 (0.1 mg, 0.35
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µmol) was treated with (S)-methoxy(trifluoromethyl)phenylacetyl (MTPA) chloride (15 µL,
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80 µmol) in dry pyridine (50 µL) at a room temperature for 5 h. The reaction was quenched
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by adding water (10 µL) and dried. The resulting residue was chromatographed on alumina (1
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g, aluminum oxide 90 activity II-III) (Merck, Kenilworth, NJ) using CHCl3 to produce
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(R)-MTPA ester 7r (ca. 0.1 mg). E,Z-(13S)-Aglycon, 6 (0.1 mg, 0.35 µmol) was converted to
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(R)-MTPA ester 8r (ca. 0.1 mg) and (S)-MTPA ester 8s (ca. 0.1 mg) by the same procedure
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with (S)-MTPA chloride and (R)-MTPA chloride, respectively. 8 ACS Paragon Plus Environment
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7r: 1H NMR (CDCl3, 600 MHz) δ 7.53 (m, 2H, Ph), 7.41 (m. 3H, Ph), 6.70 (d, J = 15.6 Hz,
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1H, H-8), 5.89 (s, 1H, H-4), 5.75 (d, J = 15.6 Hz, 1H, H-7), 5.38 (t, J = 7.5 Hz, 1H, H-10),
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5.16 (m, 1H, H-13), 3.56 (s, 3H, OMe), 2.48 (d, J = 17.1 Hz, 1H, H-2), 2.27 (d, J = 17.1 Hz,
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1H, H-2), 2.26 (m, 1H, H-11), 2.20 (m, 1H, H-11), 1.88 (s, 3H, H-16), 1.81 (s, 3H, H-15),
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1.79 (m, 1H, H-12), 1.63 (m, 1H, H-12), 1.27 (d, J = 6.6 Hz, 3H, H-14), 1.10 (s, 3H, H-18),
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1.00 (s, 3H, H-17).
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8r: 1H NMR (CDCl3, 400 MHz) δ 7.52 (m, 2H, Ph), 7.41 (m. 3H, Ph), 6.66 (d, J = 15.9 Hz,
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1H, H-8), 5.91 (s, 1H, H-4), 5.73 (d, J = 15.9 Hz, 1H, H-7), 5.35 (t, J = 7.2 Hz, 1H, H-10),
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5.17 (m, 1H, H-13), 3.56 (s, 3H, OMe), 2.45 (d, J = 16.5 Hz, 1H, H-2), 2.26 (d, J = 16.5 Hz,
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1H, H-2), 2.18 (m, 1H, H-11), 2.10 (m, 1H, H-11), 1.90 (s, 3H, H-16), 1.79 (s, 3H, H-15),
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1.72 (m, 1H, H-12), 1.51 (m, 1H, H-12), 1.36 (d, J = 6.6 Hz, 3H, H-14), 1.10 (s, 3H, H-18),
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1.00 (s, 3H, H-17).
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8s: 1H NMR (CDCl3, 600 MHz) δ 7.52 (m, 2H, Ph), 7.41 (m. 3H, Ph), 6.69 (d, J = 15.3 Hz,
189
1H, H-8), 5.90 (s, 1H, H-4), 5.74 (d, J = 15.3 Hz, 1H, H-7), 5.39 (t, J = 7.2 Hz, 1H, H-10),
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5.15 (m, 1H, H-13), 3.56 (s, 3H, OMe), 2.46 (d, J = 17.1 Hz, 1H, H-2), 2.28 (m, 1H, H-11),
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2.26 (d, J = 17.1 Hz, 1H, H-2), 2.19 (m, 1H, H-11), 1.89 (d, J = 1.2 Hz, 3H, H-16), 1.81 (s,
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3H, H-15), 1.79 (m, 1H, H-12), 1.64 (m, 1H, H-12), 1.28 (d, J = 6.6 Hz, 3H, H-14), 1.09 (s,
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3H, H-18), 0.99 (s, 3H, H-17).
194
Quantitative Analysis of Lycosides in Vegetables and Juices. A fresh vegetable
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(100 g) was homogenized in MeOH (100 mL) in a blender, and the volume was adjusted to
196
200 mL with 50% MeOH. A vegetable juice (100 mL) was mixed with MeOH (100 mL). In
197
both cases, the mixture was stirred at room temperature for 1 h. The solid part was separated
198
by centrifugation and washed with 10% MeOH (50 mL). The supernatant and washing
199
solutions were combined and concentrated to a MeOH-free aqueous solution that was
200
dissolved in 30% MeOH (50 mL in total) and chromatographed on ODS (Cosmosil 9 ACS Paragon Plus Environment
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75C18-OPN, 10 g). The fraction eluted with 60% MeOH was concentrated to dryness and was
202
thoroughly suspended in MeOH (0.5 mL) and centrifuged at 30,000 rpm for 3 min. A portion
203
(1 µL) of the supernatant was analyzed by LC/MS. The analytical conditions were as follows:
204
LC: Unison UK-C8 column (2 × 75 mm、Imtakt, Kyoto Japan), 50% MeOH (0.3 mM
205
HCOOH/0.3 mM HCOONa), flow rate 0.1 mL/min. MS: ionization and detection ESI-TOF,
206
positive mode, nebulizer gas 0.2 L/min, nozzle potential 200 V, flow rate 5 µL/min (split ratio
207
of LC/MS, 20:1). The lycosides were detected on an extracted ion chromatograph (XIC) by
208
using m/z 639.4 [M+Na]+ for 1 and 2 or 477.3 [M+Na]+ ion for 3 and 4 at approximately
209
8.8 and 9.7 min, respectively. The ionization efficiency of 1 and 2 (and 3 and 4) was assumed
210
to be same in this analysis. The peak areas of XICs obtained by 15 and 47 pmol/injection of 1
211
and 4 were used to obtain the standard curves for quantitative analysis.
212 213
RESULTS AND DISCUSSION
214
Isolation. The inhibitory activity against the zoosporangia formation was evaluated
215
with P. capsici NBRC 30696. The strain was inoculated on a potato-sucrose-agar medium that
216
contained a sample to be tested and incubated for 6−10 d. The number of zoosporangia
217
formed on the mycelia was compared to a control. In a small-scale fractionation, one can of
218
V8 vegetable juice was extracted with 40% MeOH, and the extract was chromatographed on
219
ODS to produce 19 fractions, a few of which showed significant inhibitory activity (fractions
220
7−9) (Figure 2A). The active fractions were then subjected to silica gel column
221
chromatography to generate one inhibitory fraction (fraction 1) (Figure 2B). This preliminary
222
result prompted us to identify the natural inhibitors of the Phytophthora zoosporangia
223
formation.
224
For the separation of the target compounds, V8 vegetable juice (6.8 L) was extracted
225
with MeOH, and the soluble part was chromatographed on ODS. The inhibitory fraction was 10 ACS Paragon Plus Environment
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then subjected to further chromatographic steps by using the bioassay described above. The
227
final HPLC produced four components named lycosides A–D, 1–4, in the yields of 0.5, 1.2,
228
0.6, and 0.7 mg, respectively.
229
Structural Elucidation. The most abundant compound, lycoside B, 2 was first
230
utilized to analyze its structure. The molecular formula C30H48O13 was determined by the
231
[M+H]+ ion peak
232
and intense infrared (IR) absorption at 3397 cm-1 suggested the presence of a number of
233
hydroxy groups. Two IR bands at 1655 and 1594 cm-1 and the ultraviolet (UV) maximum at
234
236 nm could indicate conjugated diene and/or enone functionalities. 1H
235
1) and a 1H-1H correlation spectroscopy (DQF-COSY) experiment revealed the following
236
substructures: CH3-CH(O)-CH2-CH2-CH=, -CH=CH-, and some sugar-related connectivities
237
(Figure 3). 13C NMR data (Table 2) indicated the presence of five quaternary carbons due to a
238
ketone (δ 201.2, C3), two sp3 carbons (δ 42.7, C1; 80.7, C6), and two olefinic carbons (δ
239
154.6, C5; 132.5, C9). The connectivities of these substructures, as well as two sugar moieties,
240
were finally revealed by Heteronuclear Multiple Bond Coherence (HMBC) experiments
241
(Figure
242
4-O-β-D-glucopyranosyl-β-D-glucopyranose (β-cellobiose) based on the J values of 7.6
243
(H1'-H2') and 8.0 Hz (H1"-H2"), nuclear Overhauser effects correlation spectroscopy
244
(NOESY) correlations of H1'-H5', H1"-H3", and H1"-H5" (Figure 3), and the comparison of
245
13
246
bond at C9 was concluded by the NOESY correlations of H8-H11 and H10-H15 (Figure 3).
247
Based on these analyses, the planar structure of 2 was determined as shown in Figure 3.
248
Lycoside A, 1, C30H48O13, was found to be an isomer of 2 based on the
249
3).
in high-resolution electrospray ionization mass spectra (ESI MS). A broad
The
disaccharide
structure
was
NMR data (Table
determined
to
be
C chemical shifts (Table 2) with a database.9 The Z geometry of the trisubstituted double
high-resolution ESI MS data. The NMR data (Tables 1 and 2) were very similar to those of 2
11 ACS Paragon Plus Environment
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250
except for the small 1H chemical shift difference through H11 to H14, suggesting that 1 is the
251
13-epimer of 2. The 2D NMR data also confirmed the planar structure of 1.
252
Lycoside C, 3, has the molecular formula of C24H38O8 as determined by
253
high-resolution ESI MS. The NMR data indicated that 3 possessed the same aglycon as that of
254
1 and only one β-D-glucose (Tables 1 and 2). This was strongly supported by the distinct high
255
field shifts at the position 4' of the glucose moiety: δ 3.57 to 3.30 (H4') and δ 80.7 to 71.8
256
(C4'). The 2D NMR analysis confirmed the structure of 3.
257
Lycoside D, 4, C24H38O8, was found to be an isomer of 3 based on the
258
high-resolution ESI MS data. The NMR data (Tables 1 and 2) were very similar to those of 3
259
except for the small chemical shift difference through H11 to H14, indicating that 4 is the
260
13-epimer of 3 as the relationship between 1 and 2. The 2D NMR analyses including the
261
NOESY experiment confirmed the structure of 4.
262
To confirm the D-glucoside structures, lycosides A, 1, and C, 3, were treated with a
263
β-D-glucosidase that produced the same aglycon 5. The structure of 5 was then confirmed by
264
1D and 2D NMR analysis. Lycoside D, 4, was also treated with the same enzyme to produce
265
aglycon 6. The NMR data for these aglycons are indistinguishable as summarized in Table 3.
266
The reactivity of the lycosides against β-D-glucosidase supports that the sugar moieties
267
consist of D-glucose.
268
The aglycons, 5 and 6, share the major part of their structure with abscisic acid
269
(Figure 4). To determine the absolute configuration at the position C-6, the circular dichroism
270
(CD) spectra of all the lycosides, 1− −4, and their aglycons, 5 and 6, were first obtained and
271
compared with a reference CD data of abscisic acid. All the compounds showed highly
272
similar positive (~250 nm) and negative (~227 nm) Cotton effects that were similar to that of
273
(+)-abscisic acid10 (Table 4). This Cotton effect could reflect the spacial arrangement of the
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274
two conjugated systems and not be affected by the sugar moiety and the C9 geometry. This
275
result strongly indicates the 6S configuration of the lycosides.
276
The absolute configuration at C-13 was next examined by the modified Mosher's
277
method.11 The aglycon 6 was converted to (S)- and (R)-MTPA esters, 8s and 8r, respectively,
278
and the chemical shift difference, δ(8s) − δ(8r), was calculated (Figure 5). The result clearly
279
demonstrated the 13S configuration of 6. To confirm the 13R configuration of the aglycon 5, 5
280
was also converted to (R)-MTPA ester 7r. The 1H NMR data of 7r was almost identical to 8s
281
(Figure 5), indicating that the C7−C14 portions of 7r and 8s are mirror images each other. To
282
summarize the above analyses, the absolute stereochemistry of the lycosides is shown in
283
Figure 1.
284
The aglycons possess a rare carbon skeleton, and only two examples are known.12
285
Glycosides of this type of aglycon have not been reported previously. The skeleton is possibly
286
biosynthesized by the degradation of a carotenoid such as zeaxanthin in the manner in which
287
abscisic acid (ABA) biosynthesis occurs in higher plants, although the oxidative cleavage
288
occurs at C13-C14 in this case but not at C11-C12 for the ABA biosynthesis.
289
Inhibition of Zoosporangia Formation. The inhibitory activity of the lycosides 1−4,
290
and aglycons 5 and 6 against the zoosporangia formation of P. capsici was evaluated by the
291
biological test described above. The inhibitory curves resulted in IC50 values of 2.1, 3.5, 4.1,
292
7.6, 1.2, and 1.8 µM for 1−6, respectively (Figure 6A). Among the lycosides, 1 was most
293
active, and the two β-cellobiosides 1 and 2, were more active than the β-glucosides 3 and 4.
294
Interestingly, the aglycons, 5 and 6, were more active than the lycosides, suggesting that the
295
sugar moiety of the lycosides is not necessarily important for the inhibitory activity. The
296
compounds with the 13R configuration always showed slightly higher activity than the
297
corresponding 13S isomers. However, the SAR is not very clear because the difference was
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298
not significant, and another bioassay test produced somewhat different activity (data not
299
shown).
300
The universality of the activity within the Phytophthora genus was also examined by
301
using other species. Lycoside D, 4, was administered to three additional strains, P. cinnamomi
302
NBRC 30697, P. nicotianae NBRC 9049, and P. infestans PI 1234-1. Their asexual
303
reproduction was inhibited by 4 at the IC50 values of 34, 5.5, and 12 µM, respectively (Figure
304
6B). Although P. cinnamomi was slightly susceptible, the lycosides could be universal
305
inhibitors against Phytophthora.
306
Distribution of Lycosides in Vegetables. The distribution of the lycosides was then
307
examined by using several vegetable juices and fresh vegetables. To quantitate the lycosides,
308
lycosides A, 1, and D, 4, were analyzed by LC/MS. These compounds were clearly detected at
309
15 pmol/injection (Figure 7). Since lycoside B, 2 and C, 3 showed the same retention times as
310
those of 1 and 4, respectively, the lycoside contents were obtained as the sum of 1 + 2 and the
311
sum of 3 + 4.
312
Several commercial vegetable juices and fresh vegetables were extracted with MeOH,
313
and the extracts were roughly separated by the ODS column. The 60% MeOH fraction was
314
used for the quantitative analysis of the lycosides. Among the six fresh vegetables (tomato,
315
lettuce, egg plants, carrot, parsley, celery, and paprika) that are used to prepare V8 vegetable
316
juice, only tomato contained the lycosides (Figure 8A). Several commercial tomato cultivars
317
were also analyzed, revealing that the lycoside contents largely depended on the cultivars
318
(Figure 8A). In addition, several commercial vegetable juices were found to universally
319
contain the lycosides at approximately 0.2−0.8 µM in total (Figure 8B).
320
These results suggest that the lycosides are widely distributed at least in tomatoes
321
and stable enough to be preserved in a can for foods. Since the lycosides are food chemicals,
322
they or their derivatives could be a promising candidate to be ecologically friendly pesticides. 14 ACS Paragon Plus Environment
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In addition, the quantitative analysis of the lycosides in pathogen-resistant tomatoes will be
324
valuable to know the correlation between the resistance and the lycoside productivity in
325
tomatoes.
326 327
ASSOCIATED CONTENT
328
Supporting Information
329
This material is available free of charge via the Internet at http://pubs.acs.org.
330
Key 2D NMR correlations, spectral data for new compounds, and inhibition data of
331
zoosporangia formation by compounds (PDF)
332 333
AUTHOR INFORMATION
334
Corresponding Author
335
*E-mail:
[email protected]. Phone: +(81) 527894116. Fax: +(81) 527894118.
336
ORCID
337
Makoto Ojika: 0000-0002-6671-8598
338
Funding
339
This work was supported by Japan Society for the Promotion of Science (JSPS) for
340
KAKENHI (No. 26252015).
341
Notes
342
The authors declare no competing financial interest.
343 344
REFERENCES
345
(1) Fry W. Phytophthora infestans: the plant (and R gene) destroyer. Mol. Plant Pathol. 2008,
346
9, 385-402.
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347
(2) Hansen, E. M.; Reeser, P. W.; Sutton, W. Phytophthora beyond agriculture. Annu. Rev.
348
Phytopathol. 2012, 50, 359-378.
349
(3) Erwin D. C.; Ribeiro O.
350
Phytopathological Society, MN, USA, 1996.
351
(4) Haverkort, A. J., Struik, P. C., Visser, R. G. F., Jacobsen, E. Applied biotechnology to
352
combat late blight in potato caused by Phytophthora infestans. Potato Res. 2009, 52, 249-264.
353
(5) Forbes, G. Global overview of late blight. Proceedings regional workshop on potato late
354
blight for east and southeast Asia and the Pacific, Yezin Agricultural University, 2004, 3-10.
355
(6) Liljeroth, E.; Lankinen, A.; Wiik, L.; Burra, D. D.; Alexandersson, E.; Andreasson, E.,
356
Potassium phosphite combined with reduced doses of fungicides provides efficient protection
357
against potato late blight in large-scale field trials. Crop Prot. 2016, 86, 42-55.
358
(7) Judelson, H. S.; Blanco, F. A. The spores of Phytophthora: weapons of the plant destroyer.
359
Nat. Rev. Microbiol. 2005, 3, 47-58.
360
(8) Vedenyapina, E. G.; Safir, G. R.; Niemira, B. A.; Chase, T. E., Low concentrations of the
361
isoflavone genistein influence in vitro asexual reproduction and growth of Phytophthora sojae.
362
Phytopathology, 1996, 86, 144-148.
363
(9) Bock, K.; Pedersen, C.; Pedersen, H. Carbon-13 nuclear magnetic resonance data for
364
oligosaccharides. Adv. Carbohydr. Chem. Biochem. 1984, 42, 193-225.
365
(10) Koreeda, M.; Weiss, G.; Nakanishi, K. Absolute configuration of natural (+)-abscisic acid.
366
J. Am. Chem. Soc. 1973, 95, 239-240.
367
(11) Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H. High-field FT NMR application of
368
Mosher's method. The absolute configurations of marine terpenoids. J. Am. Chem. Soc. 1991,
369
113, 4092-4096.
370
(12) Seger, C.; Hofer, O.; Vajrodaya, S.; Greger, H. Two new nor-diterpenes from Glycosmis
371
cf. cyanocarpa. Nat. Prod. Lett. 1998, 12, 117-124.
K. Phytophthora diseases worldwide. American
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372 373
Figure captions
374
Figure 1. Structures of lycosides A–D, 1–4
375
Figure 2. Zoosporangia formation rate of P. capsici in the presence of chromatographic
376
fractions from V8 vegetable juice
377
(A) Activity of the first step (ODS) column fractions. The strain was incubated on 0.4%
378
potato medium for 8 d. The sample dose was a 25% juice equivalent. The values are the
379
average of triplicate or duplicate data with ± s.d. The asterisks indicate the activity higher than
380
120%; (B) Activity of the second step (silica gel) column fractions from the ODS fractions
381
6−8. The strain was incubated on 4% V8 juice medium for 6 d. The sample dose was a 50%
382
juice equivalent. The values are average of triplicate data with ±s.d.
383
Figure 3. Key 2-dimensional NMR correlations of 2
384
Bold lines: DQF-COSY; arrows: HMBC; dotted arrows: NOESY
385
Figure 4. Structures of aglycons, 5 and 6, and (+)-abscisic acid
386
Figure 5. Determination of C-13 configuration by modified Mosher's method
387
(A) Chemical shift difference of (S)-MTPA ester 8s and (R)-MTPA ester 8r derived from
388
aglycon 6. (B) Chemical shifts of (R)-MTPA ester 7r derived from 5 were almost identical to
389
those of 8s.
390
Figure 6. Inhibition of Phytophthora zoosporangia formation by lycosides and aglycons
391
(A) Inhibitory activity of lycosides A−D, 1−4, and their aglycons, 5 and 6, against P. capsici
392
NBRC 30697. (B) Inhibitory activity of lycoside D, 4, against four Phytophthora strains. The
393
curves are drawn by the sigmoid curve fitting. Error bars are omitted here.
394
Figure 7. LC/MS analysis of lycosides A, 1, and D, 4
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395
(A) ESI-TOF mass spectrum of 1 and extracted ion chromatogram (XIC, inset) using the m/z
396
639.4 [M+Na]+ ion at a 15 pmol injection; (B) Mass spectrum of 4 and XIC (inset) using the
397
m/z 477.3 [M+Na]+ ion at a 15 pmol injection.
398
Figure 8. Lycoside contents in fresh vegetables and vegetable juices
399
(A) Lycosides in fresh vegetables; (B) Lycosides in Japanese vegetable juices. Each data point
400
is obtained from a single experiment.
401
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Table 1. 1H NMR Data for Lycosides A–D, 1–4, in CD3OD (400 MHz) position
1
2
3
4
2.17 (d, 16.2),
2.19 (d, 16.8),
2.17 (d, 16.8),
2.19 (d, 16.5),
2.46 (d, 16.2)
2.47 (d, 16.8)
2.47 (d, 16.8)
2.48 (d, 16.5)
4
5.91 (s)
5.90 (s)
5.91 (s)
5.90 (s)
7
5.77 (d, 15.6)
5.77 (d, 15.8)
5.77 (d, 16.2)
5.77 (d, 15.6)
8
6.76 (d, 15.6)
6.80 (d, 15.8)
6.76 (d, 16.2)
6.79 (d, 15.6)
10
5.47 (t, 7.4)
5.45 (t, 7.6)
5.48 (t, 7.2)
5.45 (t, 7.5)
11
2.18, 2.34 (m)
2.30 (m)
2.17, 2.35 (m)
2.28 (m)
12
1.51, 1.60 (m)
1.50, 1.65 (m)
1.51, 1.60 (m)
1.49, 1.65 (m)
13
3.86 (m)
3.80 (sext, 6.0)
3.88 (m)
3.81 (sext, 6.0)
14
1.17 (d, 6.0)
1.24 (d, 6.0)
1.16 (d, 6.0)
1.23 (d, 6.0)
15
1.81 (s)
1.82 (s)
1.81 (s)
1.82 (s)
16
1.92 (d, 1.2)
1.92 (d, 1.2)
1.92 (s)
1.92 (d, 0.6)
17
1.01 (s)
1.00 (s)
1.01 (s)
1.00 (s)
18
1.06 (s)
1.05 (s)
1.06 (s)
1.06 (s)
1'
4.35 (d, 7.8)
4.35 (d, 7.6)
4.32 (d, 7.8)
4.32 (d, 7.8)
2'
3.23 (dd, 9.0, 7.8)
3.22 (dd, 9.0, 7.6)
3.16 (dd, 9.0, 7.8)
3.16 (dd, 9.0, 7.8)
3'
3.52 (t, 9.0)
3.51 (t, 9.0)
3.35 (t, 9.0)
3.34 (t, 9.0)
4'
3.57 (t, 9.0)
3.56 (t, 9.0)
3.30 (m)
3.28 (t, 9.0)
5'
3.38 (m)
3.38 (m)
3.24 (m)
3.24 (m)
3.67 (dd, 12.0,
3.66 (dd, 12.0,
5.4), 3.85 (dd,
5.4), 3.84 (12.0,
12.0, 2.0)
1.8)
2
6'
3.86 (m)
3.86 (m)
1''
4.42 (d, 7.8)
4.41 (d, 8.0)
2''
3.22 (9.0, 7.8)
3.21 (dd, 8.8, 8.0)
3''
3.37 (m)
3.36 (m)
4''
3.33 (m)
3.33 (m)
5''
3.30 (m)
3.31 (m)
6''
3.66 (dd, 11.7, 5.7), 3.87 (m)
3.65 (dd, 11.6, 5.2), 3.87 (dd, 11.6, 2.0)
19 ACS Paragon Plus Environment
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Table 2. 13C NMR Data for Lycosides A–D, 1–4, in CD3OD (100 MHz) position
1
2
3
4
1
42.7
42.6
42.7
42.6
2
50.8
50.8
50.8
50.8
3
201.2
201.2
201.2
201.3
4
127.1
127.1
127.1
127.1
5
167.6
167.3
167.6
167.5
6
80.0
80.7
80.7
80.7
7
130.1
130.3
130.1
130.3
8
128.8
128.8
128.8
128.8
9
132.4
132.6
132.4
132.6
10
132.5
132.4
132.6
132.4
11
24.5
24.3
24.6
24.3
12
38.7
37.9
38.7
37.9
13
75.4
77.3
75.1
77.2
14
19.9
22.0
19.8
22.0
15
20.8
20.8
20.8
20.8
16
19.7
19.7
19.7
19.7
17
24.6
24.7
24.6
24.7
18
23.6
23.6
23.6
23.6
1'
102.1
103.9
102.2
104.0
2'
75.0
75.1
75.2
75.4
3'
76.5a
76.4
77.8
77.8
4'
80.7
80.7
71.8
71.7
5'
a
76.4
76.4
78.2
78.1
6'
62.1
62.0
62.9
62.8
1''
104.7
104.6
2''
74.8
74.9
3''
77.9
77.9
4''
71.4
71.4
5''
78.2
78.1
6''
62.4
62.4
a
Interchangeable signals 20 ACS Paragon Plus Environment
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Table 3. 1H and 13C NMR Data for Aglycons, 5 and 6, in CDCl3 (13R)-aglycon, 5 (13S)-aglycon, 6 position
δC
δH (mult., J in Hz)
δC
δH (mult., J in Hz)
1
41.4
-
41.0
-
2
49.8
2.26 (d, 17.2), 2.47 (d, 17.2)
49.8
2.25 (d, 17.0), 2.46 (d, 17.0)
3
198.1
-
198.0
-
4
126.7
5.92 (s)
126.7
5.93 (s)
5
163.1
-
163.0
-
6
79.8
-
79.7
-
7
128.6
5.73 (d, 15.8)
128.7
5.71 (d, 15.6)
8
127.7
6.78 (d, 15.8)
127.8
6.77 (d, 15.6)
9
131.5
-
131.6
-
10
131.5
5.45 (t, 7.4)
131.6
5.44 (t, 7.8)
11
23.8
2.29 (m)
23.7
2.30 (m)
12
39.2
1.52 (m)
39.0
1.50 (m)
13
67.4
3.77 (m)
67.3
3.77 (m)
14
23.7
1.20 (d, 6.0)
23.7
1.20 (d, 6.0)
15
20.6
1.82 (s)
20.6
1.82 (s)
16
19.0
1.91 (d, 1.2)
19.0
1.91 (d, 1.2)
17
24.1
1.01 (s)
24.2
1.02 (s)
18
23.0
23.0
1.10 (s)
1.10 (s) 1
13
Observed at 400 MHz for H and 100 MHz for C.
21 ACS Paragon Plus Environment
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Table 4. CD Data for Lycosides and Aglycons in MeOH λext, nm (∆ε)
compounds lycoside A (1)
251 (+36), 227 (-39)
lycoside B (2)
250 (+31), 226 (-33)
lycoside C (3)
249 (+35), 227 (-30)
lycoside D (4)
250 (+30), 227 (-30)
(13R)-aglycon (5)
249 (+28), 228 (-37)
(13S)-aglycon (6)
247 (+27), 228 (-38)
(+)-abscisic acid10
261 (+34.5), 229 (-28.0)
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Figure graphics
Figure 1
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A
B *
* * * *
*
Figure 2
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Figure 3
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Figure 4
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Figure 5
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A
B
Figure 6
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A
m/z 639.4
% Intensity
XIC
B
XIC
% Intensity
m/z 477.3
tR (min)
Mass (m/z)
Figure 7
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A
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B
tomato cultivars mixed juices
Figure 8
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tomato juices
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Table of Contents Graphic
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