Lycosides, Unusual Carotenoid-Derived Terpenoid Glycosides from a

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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

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‡

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:


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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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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,

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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).

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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

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200 mL with 50% MeOH. A vegetable juice (100 mL) was mixed with MeOH (100 mL). In

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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

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thoroughly suspended in MeOH (0.5 mL) and centrifuged at 30,000 rpm for 3 min. A portion

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(1 µL) of the supernatant was analyzed by LC/MS. The analytical conditions were as follows:

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LC: Unison UK-C8 column (2 × 75 mm、Imtakt, Kyoto Japan), 50% MeOH (0.3 mM

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HCOOH/0.3 mM HCOONa), flow rate 0.1 mL/min. MS: ionization and detection ESI-TOF,

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positive mode, nebulizer gas 0.2 L/min, nozzle potential 200 V, flow rate 5 µL/min (split ratio

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of LC/MS, 20:1). The lycosides were detected on an extracted ion chromatograph (XIC) by

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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

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and 4 were used to obtain the standard curves for quantitative analysis.

212 213

RESULTS AND DISCUSSION

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Isolation. The inhibitory activity against the zoosporangia formation was evaluated

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with P. capsici NBRC 30696. The strain was inoculated on a potato-sucrose-agar medium that

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contained a sample to be tested and incubated for 6−10 d. The number of zoosporangia

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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

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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

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formation.

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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.

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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



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


Page 12 of 31

Page 13 of 31


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



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

Page 14 of 31

Page 15 of 31


323

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.



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


Page 16 of 31

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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



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


Page 18 of 31

Page 19 of 31


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)



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

Page 20 of 31

Page 21 of 31


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.



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)


Page 22 of 31

Page 23 of 31


Figure graphics

Figure 1



A

B *

* * * *

*

Figure 2


Page 24 of 31

Page 25 of 31


Figure 3



Figure 4


Page 26 of 31

Page 27 of 31


Figure 5



A

B

Figure 6


Page 28 of 31

Page 29 of 31


A

m/z 639.4

% Intensity

XIC

B

XIC

% Intensity

m/z 477.3

tR (min)

Mass (m/z)

Figure 7



A

Page 30 of 31

B

tomato cultivars mixed juices

Figure 8


tomato juices

Page 31 of 31


Table of Contents Graphic


Lycosides, Unusual Carotenoid-Derived Terpenoid Glycosides from a

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