New Phytotoxic Cassane-Like Diterpenoids from Eragrostis plana

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Bioactive Constituents, Metabolites, and Functions

New Phytotoxic Cassane-Like Diterpenoids from Eragrostis plana Adriana Favaretto, Charles Lowell Cantrell, Frank R Fronczek, Stephen O. Duke, David E. Wedge, Abbas Ali, and Simone Meredith Scheffer-Basso J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b06832 • Publication Date (Web): 28 Jan 2019 Downloaded from http://pubs.acs.org on January 29, 2019

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Journal of Agricultural and Food Chemistry

New Phytotoxic Cassane-Like Diterpenoids from Eragrostis plana

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Adriana Favaretto†*, Charles L. Cantrell‡*, Frank R. Fronczek§, Stephen O.

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Duke‡, David E. Wedge‡, Abbas Ali# and Simone M. Scheffer-Basso†

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†University

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Brazil.

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‡United

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Unit, Mississippi, USA.

of Passo Fundo, Agronomy Graduate Program, Passo Fundo, Rio Grande do Sul,

States Department of Agriculture, USDA-ARS, Natural Products Utilization Research

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§Department

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‡United

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Unit, Mississippi, USA.

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#National

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38677, USA.

of Chemistry, Louisiana State University, Baton Rouge, Louisiana 70803, USA.

States Department of Agriculture, USDA-ARS, Natural Products Utilization Research

Center for Natural Products Research, The University of Mississippi, University, MS

15 16 17 18 19 20 1 ACS Paragon Plus Environment


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ABSTRACT

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Eragrostis plana (Nees) is an allelopathic plant with invasive potential in South American

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pastures. To isolate and identify phytotoxic compounds from leaves and roots of E. plana, a

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bioassay-directed isolation of the bioactive constituents was performed. This is the first report on

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a new diterpene carbon skeleton, the neocassanes, and of three new neocassane diterpenes,

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neocassa-1,12(13),15-triene-3,14-dione, 1, 19-norneocassa-1,12(13),15-triene-3,14-dione, 2,

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and 14-hydroxyneocassa-1,12(17),15-triene-3-one, 3, identified from the roots. Compounds 1, 2,

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and 3 inhibited the growth of duckweed 50% at concentrations of 109 ± 28, 200 ± 37, and 59 ±

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15 µM, respectively. Compound 2 was fungicidal to Colletotrichum fragariae, C. acutatum and

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C. gloeosporioides. The compounds identified here could explain the allelopathy of E. plana.

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The description of the newly discovered compounds, besides contributing to the chemical

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characterization of the species, may be the first step in the study of the potential of these

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compounds as bioherbicides.

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KEYWORDS: allelopathy, diterpenes, Eragrostis plana, phytotoxicity

2 ACS Paragon Plus Environment

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INTRODUCTION

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Eragrostis plana (Nees), known as tough lovegrass and South African lovegrass (or capim-annoni

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in Brazil), is an African grass initially introduced in Argentina, later in Brazil (1950-1960) and

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dispersed throughout Uruguay. In 1971, it was considered as a good forage species and for that

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purpose was propagated in several Brazilian states. Unfortunately, the grass has low forage quality

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compared with native species and has several undesirable traits. It is considered the main invasive

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plant of pastures of the Pampa Biome, with competitive characteristics that stand out over local

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species. Beyond South America, E. plana is currently present in several regions of Asia, India and

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the USA.1,2

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The allelopathic effect of this species é is responsible for the ability to colonize extensive

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areas.3-5 The use of allelopathy in weed management has been considered promising6-8 and one of

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the major expected applications is discovery and development of bioherbicides.9 The first step in

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the search for bioherbicides from plants is the knowledge about the chemical composition of plants

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and the phytotoxic activity of isolated compounds. Bioassay-guided fractionation is the preferred

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method for determining which compounds are related to phytotoxicity .10

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Most reported allelochemicals are phenolic or terpenoid compounds.11 Terpenoids are the

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largest and most diverse class of the plant biochemicals, with over 25,000 known structures.12 In

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E. plana, some phenolic compounds were identified previously: caffeic, ferulic, vanillic and p-

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coumaric acids, catechin, epicatechin, and coumarin,13 as well as rutin, quercetin, and chlorogenic,

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ellagic and gallic acids. These compounds are present in many plant species (both allelopathic and

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non allelopathic), and the role of most of them in allelopathy has been questioned.15 Even though

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the allelopathic activity of E. plana and the phytotoxicity of some of its chemical constituents have

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been described previously, the compounds responsible for the activity of the crude extracts were



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not isolated and identified. Therefore, the objective of this study was to isolate and identify the

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phytotoxic compounds from E. plana by bioassay-directed isolation of the most potent bioactive

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constituents.

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MATERIALS AND METHODS Chemicals. All the solvents used in this work were HPLC grade and purchased from SigmaAldrich (St. Louis, MO).

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Plant Material and Extraction Procedures. Leaves and roots were collected from E. plana

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plants in vegetative stage in the city of Passo Fundo, Rio Grande do Sul state, Brazil (28°13'28.8"S

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52°23'00.3"W), in December 2016. Identification of E. plana was according to the voucher

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specimen deposited at the herbarium of Zoobotanical Museum Augusto Ruschi of the University

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of Passo Fundo, under the code RSPF 11832. The material was dried in an oven at 40 °C with air

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circulation; and then crushed in a Wiley mill. Ground roots (100.07 g) and leaves (100.31 g) were

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consecutively extracted by soaking with solvents of increasing polarity: hexane (HEX),

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dichloromethane/methanol (dichloromethane/MeOH) (1:1) and water (H2O), for 24 h each using

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800 mL for each solvent. After the extraction, the extracts were filtered through 0.22 µm paper in

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a Büchner funnel coupled to a vacuum pump and the solvents were evaporated in a rotary

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evaporator, except for the aqueous extract, which was freeze-dried. This process yielded the

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following extracts: LeavesHEX (2.91 g), Leavesdichloromethane/MeOH (2.89 g), LeavesH2O (5.39 g),

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RootsHEX (1.85 g), Roots dichloromethane/MeOH (3.12 g), RootsH2O (1.57 g), which were stored at -20 oC.

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Phytotoxicity Bioassays with Lactuca sativa and Agrostis stolonifera. The phytotoxic

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activity of the extracts from the initial fractionation, column chromatography fractions and pure

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compounds were evaluated using lettuce (Lactuca sativa) and creeping bentgrass (Agrostis


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stolonifera) as test plants, according described Dayan et al.16 In each well of a 24-well plate were

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placed a filter paper (Whatman No. 1) and five lettuce seeds (L. sativa L. cv. iceberg A from

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Burpee Seeds, Warminster, PA) or 10 mg of creeping bentgrass (A. stolonifera var. penncross from

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Turf-Seed Inc., Gervais, OR). The test extracts or fractions were dissolved in acetone for

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preparation of the stock solutions (10 mg/mL). Plus the 20 µL of the stock solution or acetone as

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solvent control, 180 µL of distilled water was added to each well. The final concentration per well

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was 1 mg/mL for extracts or fractions and 10% v/v for acetone. Plates were sealed with Parafilm,

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and incubated at 26 °C in a growth chamber set at 173 µmol/m2/s continuous photosynthetically

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active radiation. The visual comparison in each well with solvent control at 7 d determined the

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phytotoxic activity. The qualitative estimation of phytotoxicity was evaluated by using a rating

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scale of 0 to 5, where 0 = no effect and 5 = no growth or germination of the seeds. Each experiment

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was repeated in duplicate.

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Phytotoxicity-Guided Fractionation. Guided by the lettuce and creeping bentgrass bioassays,

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RootsHEX and Leavesdichloromethane/MeOH extracts of E. plana were subjected to column

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chromatography using an Isolera One system (Biotage) (Uppsala, Sweden), equipped with a UV

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detector (254 and 280 nm) and an automatic fraction collector. Separation was performed by

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normal-phase chromatography. The column used was a SNAP Cartridge KP-Sil, 37 mm x 157

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mm, 50 μm irregular silica, 100 g (Biotage) and a pre-packaged SNAP Samplet Cartridge KP-Sil,

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37 mm x 17 mm (Biotage). The separation for both extracts was performed using a gradient of

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hexane (solvent A) and ethyl acetate (solvent B), beginning with 0-10% B, over 2310 mL, followed

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by 20-50% B over 800 mL, then 50-100% B over 400 mL, and finishing with a MeOH wash (300

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mL). Flow rate was 40 mL min. Portions of 22 mL each were collected in 16x150 mm test tubes.

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According their TLC and chromatogram profiles fractions were recombined, giving seventeen



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fractions from RootsHex, named A to Q, and thirteen fraction from Leavesdichloromethane/MeOH, named

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A to M. These fractions were evaluated for their phytotoxicity activity.

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Fraction L from RootsHex was submitted to normal-phase chromatography as above, using a

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linear gradient from 100% CHCl3 to 80/20 (CHCl3/Et2O) over 2400 mL. Flow rate was 40 mL/min.

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By TLC similarity test tubes were grouped into three new subfractions: La, Lb and Lc. Subfraction

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Lb was purified by HPLC on a model 1200 system (Agilent Technologies, Santa Clara, CA)

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equipped with a quaternary pump, autosampler, diode-array detector, and vacuum degasser, using

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a Zorbax RX-SIL (Agilent) 5 µm 9.4 x 250 nm HPLC column. Running at 4.5 mL/min. A linear

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gradient of 100% hexane to 25% hexane/75% Et2O over 40 min at a flow rate of 4.5 mL/min and

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with the diode array detector set at 254 and 280 nm was used to afford compound 1 (14.1 mg). 254

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and 280 nm were used by the diode array detector for collection.

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Fraction M from the RootsHex was also directly purified by HPLC using the same equipment

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and HPLC column as described above for purification of fraction Lb. The linear gradient consisted

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of 100% hexane (solvent A) and hexane/isopropyl alcohol (97:3, v/v) (solvent B) changed from

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20-80%B over 35 min at a flow rate of 4.5 mL/min, with diode array detection at 254 and 280 nm,

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providing compounds 2 (11.4 mg) and 3 (13.2 mg). 254 and 280 nm were used by the diode array

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detector for collection.

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Fraction J from Leavesdichloromethane/MeOH was identified as compound 4 (20.3 mg).

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Chemical Analysis and Compound Identification. The isolated compounds were analyzed

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by GC/MSD on a 7890A GC system coupled to a 5975C Inert XL MSD (Agilent). The GC was

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equipped with a DB-5 fused silica capillary column (30 m × 0.25 mm, film thickness of 0.25 μm)

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operated using the following conditions: injector temperature, 240 °C; column temperature, 60–

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240 °C at 3 °C/min then held at 240 °C for 5 min; carrier gas, He; injection volume, 1 μL (splitless).


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The MS range was m/z 40-650, with a filament delay of 3 min, target TIC of 20,000, a prescan

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ionization of 100 μs, an ion-trap temperature of 150 °C, a manifold temperature of 60 °C, and a

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transfer line temperature of 170 °C. 1H- and 13C-NMR spectra were recorded in CDCl3 on a Bruker

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400 MHz spectrometer (Bruker, Billerica, MA). High-resolution mass (ESI-MS) spectra of

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isolated compounds in MeOH were acquired by direct injection of 20 μl of sample (approximately

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0.1 mg/mL) on an JMS-T100LC AccuTOF liquid chromatograph (JEOL, Peabody, MA).

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Neocassa-1,12(13),15-triene-3,14-dione, 1: High-resolution ESI-MS m/z 299.19964 [M+H]+

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calculated for C20H27O2 299.20110, mass difference (mmu) -1.46; 1H NMR (400 MHz, CDCl3)

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and 13C NMR (101 MHz, CDCl3) (Table 1).

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Neocassa-1,12(13),15-triene-3,14-dione X-ray structure. The crystal structure of 1 was

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determined using X-ray data collected at 180K with CuKα radiation (1.54184 Å), on a Kappa

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Apex-II DUO diffractometer (Bruker, Billerica, MA) equipped with a microfocus Cu source.

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C20H26O2, orthorhombic space group P212121, a=7.2346(2), b=9.1010(3), c=25.1969(8) Å, Z=4,

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Dcalcd=1.195 g/cm3. A total of 20,857 data was collected to =68.3˚, R=0.032 for 2961 data with

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I>2(I) of 3018 unique data (Rint=0.033) and 203 refined parameters. H atoms were visible in

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difference maps, but were placed in idealized positions for refinement. Maximum and minimum

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residual densities were 0.22 and -0.17 e/Å3. The absolute configuration was confirmed from the

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Flack parameter, x=0.00(7), based on 1203 Friedel pair quotients. The CIF has been deposited at

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the Cambridge Crystallographic Data Centre, CCDC 1821493.

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19-norneocassa-1,12(13),15-triene-3,14-dione, 2: High-resolution ESI-MS m/z 285.18744

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[M+H]+ calculated for C19H25O2 285.18545, mass difference (mmu) 1.99; 1H NMR (400 MHz,

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CDCl3) and 13C NMR (101 MHz, CDCl3) (Table 1).



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14-hydroxyneocassa-1,12(17),15-triene-3-one, 3: High-resolution ESI-MS m/z 301.21826

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[M+H]+ calculated for C20H29O2 301.21675, mass difference (mmu) 1.51; 1H NMR (400 MHz,

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CDCl3) and 13C NMR (101 MHz, CDCl3) (Table 1).

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9,12,15 Octadecatrienoic acid, 4: High-resolution ESI-MS m/z 277.22184 [M+H]+ calculated

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for C18H29O2 277.21675, mass difference (mmu) 5.08; 1H-NMR and 13C-NMR data in agreement

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with that previously reported.17-18

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Phytotoxicity Bioassay with Duckweed. Phytotoxicity of isolated compounds was evaluated

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on duckweed (Lemna paucicostata (L.) Hegelm.).19 Briefly, modified Hoagland media was used

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to grown duckweed stocks from a single colony constituted by a mother and two daughter fronds.

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The medium was adjusted to pH 5.5 with 1 M NaOH and filtered through a 0.2 µm filter. Each

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well of CoStar 3506 non-pyrogenic polystyrene sterile 6-well plates (Corning Inc., Corning, NY)

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was filled with 4,850 µL of Hoagland media and, 150 µL of acetone in solvent control or 150 µL

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of acetone containing the appropriate concentration of test compound. The final concentration of

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acetone was 1 % (v/v). The pure compounds were tested at the concentrations 0.1; 0.3; 1; 3; 10;

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33; 100 and 333 (µM), with three replicates for each concentration. A positive control of atrazine

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(Sigma-Aldrich, St. Louis, MO) at the same concentrations was used. Two, three-frond colonies

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from 4 to 5 days-old stock cultures were placed in each well. Plates were placed in an incubator

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with white light (94.2 µE/m2/s). Total frond area per well was recorded by Scanalyzer image

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analysis system (LemnaTec, Würselen, Germany) from days 0-7. Percentage of increase between

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days 1 and 7 was determined relative to baseline area at day zero.

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Electrolyte Leakage Assay. The pure compounds isolated from E. plana were tested on

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membrane stability using cucumber cotyledon disks. Experimental units were Petri plates (60mm

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x 15 mm, sterile, polystyrene) containing 4,850 µL of 1µM MES buffer, pH 6.5, with 2% sucrose


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plus 150 µL acetone as control, or 150 µL acetone containing the appropriate concentration of test

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compound. The pure compounds were tested at the concentrations 10, 33, 100, 333, and 1000 µM,

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with three replicates for each concentration and compared with the solvent control (acetone) and

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the herbicide acifluorfen (50 μM) (Chem Service. West Chester, PA). Acifluorfen causes rapid

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plasma membrane leakage in the light. Four-mm leaf disks were cut from cucumber cotyledons

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with a cork borer under dim green light and 50 disks placed in each plate. Fifty disks were also

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deposited in 5 mL of buffer in test tubes. The tubes were placed in boiling water for 15 min, and

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allowed to cool to room temperature. A model 3082 electrical conductivity meter with an 865

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multi-cell (Pt) probe (Amber Science, Eugene, OR) was used to take electrical conductivity

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measurements. Conductivity measurements were carried out at the beginning of the dark

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incubation period, after 18 h, when the samples were placed under high light intensity, and

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subsequently, evaluated at 19, 20, 22 and 24 h from the beginning of the experiment. The data

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were subtracted from the initial reading values, and a graph with conductivity versus time was

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plotted. The maximal possible conductivity was determined by measuring the conductivity of the

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solution in which the cotyledon disks were boiled.

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Fungicide Bioassay with Colletotrichum spp. Isolates of Colletotrichum acutatum Simmonds,

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C. fragarie Brooks, and C. gloeosporioides (Penz.) Penz & Sacc. were used by bioautography TLC

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technique to detect antifungal activity of the compounds isolated from E. plana according to

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published methods.20 Isolated substances were tested at 10 and 100 µg/spot, in duplicate. Each

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plate was subsequently sprayed with a spore suspension (106 spores/mL) of the fungus and

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incubated in a moisture chamber for 4 d at 26 oC with a 12 h photoperiod. Clear zones of fungal

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growth inhibition on the TLC plate indicated the presence of antifungal constituents21 and their

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diameter measured.



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Statistical Analysis. Data from dose–response experiments were analyzed using the dose–

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response curve module22 of R version 2.2.1.23 This software calculates IC50 values. Data from

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phytotoxicity bioassays were analyzed by ANOVA using the Assistat software version 7.7,24 with

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comparison of means by Tukey test at a 5% probability.

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RESULTS AND DISCUSSION

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Phytotoxicity Bioassay-guided Isolation. Initially, the leaves and roots of E. plana were

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extracted with hexane, dichloromethane/MeOH and water, sequentially, giving three extracts for

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leaves and three for roots, which were which were evaluated for their phytotoxicity. Evaluation

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against lettuce and creeping bentgrass at 1 mg/mL indicated that RootsHEX and Leaves

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dichloromethane/MeOH were

the most phytotoxic (Table 2).

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Based on these results, the RootsHEX was subjected to normal-phase flash column

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chromatography, giving 17 fractions (A-Q), which were, in the same way, evaluated for their

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phytotoxicity on lettuce and creeping bentgrass at 1 mg/mL. Fractions L and M were the most

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active. Fraction L was submitted again to normal-phase flash column chromatography followed

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by normal phase HPLC purification providing compound 1 (Figures 1 and 2). Fraction M was

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purified by normal phase HPLC providing compounds 2 and 3 (Figure 1).

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Identification of phytotoxic compounds. Compound 1 was identified as neocassa-

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1,12(13),15-triene-3,14-dione based on its spectroscopic data, both one- and two-dimensional

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NMR spectroscopy and X-ray diffraction analysis. Its molecular formula was C20H26O2, as

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revealed by the pseudomolecular ion [M+H]+ acquired in positive ion mode. The 1H NMR

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spectrum of 1 showed the presence of four methyl singlets (δ 2.08, 1.15, 1.14, and 1.09), one of

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which was an olefinic methyl, and olefinic protons (δ 6.40, 5.46, and 5.41) of a monosubstituted


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double bond, in addition to olefinic protons of an ,-unsaturated double bond ( 7.00 and 5.94).

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The COSY, 13C NMR and DEPT spectra confirmed the presence of the ,unsaturated double

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bond as well as the presence of a 1,3-diene (δ 154.5, 132.6, 130.1 and 120.3) containing the

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monosubstituted double bond. The above together with the presence of three methyl singlets (δ

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27.1, 22.1, and 16.2) and one olefinic singlet at δ 22.5 suggested a cassane-type skeleton. Upon

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inspection of the X-ray crystallographic data it was realized that the structure was not a cassane

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skeleton but rather a new carbon skeleton containing the C-17 olefinic methyl at C-12 rather than

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at C-14 (Figures 2 and 3). This was further supported by the HMBC correlations between H-5 and

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C-4, C-6, C-7, and C-10 as well as those between H-1 and C-3 and C-5; and H-20 and C-1, and C-

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10 (Figure 4). Critical to the establishment of the diene moiety at C-11 were the HMBC

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correlations between H-17 and C-11, C-12, and C-13 as well as those between H-16 and C-13.

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COSY and HSQC correlations helped establish all assignment data in Table 1. X-ray

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crystallographic data established both the relative and absolution configuration of 1.

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Literature analysis revealed the presence mainly of the isopimarane, labdane, and cassane

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diterpene skeletal types from the genus Eragrostis.25 They also isolated two new diterpenes from

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the roots of E. ferruginea (Thunb.): isopimara-9(11),15-dien-19-ol-3-one and cassa-13(14),15-

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diene-3,12-dione. They have also isolated the known diterpene diol, isopimara-9(11),15-diene-3β,

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19-diol. Four labdanes with a 8a,15-epoxy ring (8a,15-epoxylabdan-16b-oic acid; 8a,15-epoxy-

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16-norlabdan-13-one; 8a,15-epoxy-16-norlabdane; and 16-acetoxy-8a,15-epoxylabdane) and the

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known compound ambreinolide were isolated from the hexane extract of the aerial parts of the E.

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viscosa (Retz.).26 It appears that our diterpene carbon skeletons for the compound 1 does not fit

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any of these isopimaranes, labdanes, and cassanes structural types. However, they are very similar

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to that of the cassanes (Figure 3) aside from the position of the C-17 methyl group. In the cassanes,



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the C-17-methyl group is positioned at the C-14 carbon; however, the C-17 methyl group in

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compound 1 is attached to the C-12 carbon giving rise to an entirely new diterpene carbon skeleton.

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Due to the similarity to the cassanes, we have chosen to name this new carbon skeleton

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neocassanes. Figure 3 shows an example of the typical dienes found with a cassane carbon

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skeleton, compared with the typical corresponding diene found in the neocassanes which

248

corresponds to those we isolated.

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Compound 2 from fraction M was identified as 19-norneocassa-1,12(13),15-triene-3,14-dione

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according of its one- and two-dimensional NMR spectroscopic data. Its molecular formula was

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C19H24O2 as revealed by the pseudomolecular ion at m/z 285.18744 [M+H]+ acquired in positive

252

ion mode, which suggested one fewer carbons than compound 1. The 1H NMR spectrum of 2

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looked nearly identical to that observed for 1 apart from the absence of a C-19 methyl singlet and

254

presence of a methyl doublet at δ 1.17 (C-18). The 13C NMR and 90° and 135° DEPT specta of 2

255

looked nearly identical to that observed for 1 except for the presence of only 19 carbons with the

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clear absence of a C-19 methyl and a singlet carbon signal. Instead of the singlet carbon, we

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observed a doublet carbon signal corresponding to C-4 at δ 42.1. The above suggested a 19-nor

258

neocassane and led us to assign the structure of 2 as that drawn in Figure 1. COSY and HMBC

259

correlations were in agreement with those observed for compound 1 in the C ring and the diene

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with the addition of a correlation between H-16 and C-14. HMBC correlations between H-4 and

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C-3; H-18 and C-3 and C-5 confirmed the absence of the dimethyl group at C-4 as was found in

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compound 1. NOESY correlations between H-5 and H-9 and those between H-5 and H-18

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established the H-18 methyl at C-4 with an α-orientation. The remaining relative configurations

264

were all in agreement with that observed for compound 1 as shown by NOESY correlations

265

between H-4 and H-20 and those between H-20 and H-8.


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Compound 3 from fraction M was identified as 14-hydroxyneocassa-1,12(17),15-triene-3-one

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on the basis of its one- and two-dimensional NMR spectroscopic data. The pseudomolecular ion

268

at m/z 301.21826 [M+H]+acquired in positive ion mode, indicated the molecular formula as

269

C20H28O2, suggesting the same number of carbons as compound 1 but with one less site of

270

unsaturation. The

271

ketone (δ 204.9), three olefins (δ 155.6, 145.5, 136.8, 126.2, 116.8, 114.3), and one hydroxylated

272

carbon (δ 75.5). Two of the olefinic carbon signals are triplets (δ 116.8, 114.3) suggesting the

273

presence of two exocyclic methylenes. Based on reasoning described above for compound 1, it

274

was clear that compound 3 was a neocassane-type with both A and B rings similar to 1. HMBC

275

data correlations observed are listed in Figure 4 and clearly agree with the A and B ring correlations

276

observed for compound 1. The C ring of 3 was missing the C-14 ketone found in 1 which was

277

replaced by a hydroxylated carbon. 1H-NMR, HSQC, and HMBC spectral data confirmed this and

278

the presence of two exocyclic methylenes with protons at C-16 (δ 5.18, 5.15) and C-17 (δ 5.04,

279

4.94). COSY correlations clearly established connections between H-16 to H-15 and H-15 to H-

280

13. H-14 HMBC correlations were instrumental in the establishment of the location of the hydroxyl

281

group. H-14 correlated to C-7, C-13, and C-9. The above suggested a neocassane reduced isomer

282

of 1 and led us to assign the structure of 3 as that show in Figure 1.

13C

NMR and 90° and 135° DEPT spectra of 3 revealed the presence of one

283

By normal-phase flash column chromatography were obtained 13 fractions (A-M) from

284

Leavesdichloromethane/MeOH, which were evaluated for their phytotoxicity on lettuce and creeping

285

bentgrass at 1 mg/mL. The fractions I, K and L had good activity, but contained a mixture of

286

different compounds. Fraction J was analyzed by GC-MS, LC-MS and 1H and 13C NMR data, and

287

by comparison with the literature it was identified as 9,12,15 octadecatrienoic acid, 4.17-18,27



Page 14 of 38

288

Phytotoxic Effect of the Pure Compounds on Lettuce, Creeping Bentgrass and Duckweed.

289

All the compounds isolated from E. plana leaves and roots were tested in a dose-response

290

bioassays on lettuce and creeping bentgrass. This bioassay provides a determination of the

291

phytotoxicity of a compound on both a monocotyledonous (creeping bentgrass) and a

292

dicotyledonous (lettuce) plant species using small amounts of compounds. The concentration of

293

333 µM was inhibitory for 3 and 4 (Table 3). At 1000 µM, all the compounds were inhibitory,

294

especially on monocot creeping bentgrass. Compound 4 (-linolenic acid) is a ubiquitous

295

compound in plants, and therefore was not tested further. After 7 d the fresh weight of the plants

296

was measured. We found a statistical difference only for compound 3, where at 333 µM the lowest

297

fresh weight within creeping bentgrass plants was measured.

298

During dose-response bioassays on duckweed, the pure compounds 1, 2 and 3 inhibited growth.

299

Compound 3 was the most phytotoxic, but all of them significantly reduced the growth at higher

300

concentrations as in Figure 5. Duckweed (Lemna spp.) has several advantages as test organism,

301

among other things its simple structure and small size, allowing small volumes of sample toxicants

302

to be used.28 Effects on growth can be monitored over time without harvesting or making

303

destructive measurements.19, 29 Furthermore, duckweed has a rapid rate of growth, ease of culture

304

and handling, and a good homogeneity with in a clone.30 It is sensitive to most phytotoxins and is

305

uniquely suitable for testing herbicides.19, 29

306

Based on growth inhibition of duckweed, IC50 values were 109 ± 28, 200 ± 37, and 59 ± 15 µM

307

for 1, 2 and 3, respectively. In a repeated study, the IC50 value for 3 was 68 ± 23 µM and that for

308

atrazine, a commercial herbicide was 1.95 ± 46 µM. Statistically, the IC50 value for 3 was the same

309

as in Figure 5, and that for atrazine was close to that reported before (0.93 µM) by Michel et

310

al.19 using the same bioassay. Studying the phytotoxicity of 26 herbicides with as many as 19


Page 15 of 38


311

different modes of action, Michel et al.19 found the IC50 values ranged from 0.003 μM for

312

sulcotrione and 0.005 μM for chlorsulfuron to 388 μM for glyphosate isopropylamine salt and 407

313

μM for asulam. Such growth differences are due physicochemical properties, uptake, metabolic

314

degradation, and molecular target sites of the herbicides. Therefore, the IC50 values of the isolated

315

compounds in this work are within the range of values found for commercial herbicides.

316

Duckweed can exhibit many symptoms when it is under stress. These include chlorosis,

317

necrosis, colony breakup, root destruction, loss of buoyancy, and gibbosity (humpback or

318

swelling).29 In this work, the image analysis by the LemnaTec software allowed the determination

319

of the percentage of the healthy, chlorotic, and necrotic tissues at duckweed plants. There was a

320

reduction on the percentage of healthy tissues at the 333 µM, and there was an increase on the

321

percentage of necrotic tissues of 12, 47 and 26% for compounds 1, 2, and 3, respectively (Figure

322

6).

323

Compounds 1, 2 and 3 are diterpenes. Many phytotoxic compounds are derived from the

324

mevalonic acid pathway,31 but few of these phytotoxins have a mode of action completely

325

understood. 32 Modes of action of various plant-derived terpenoids have been related to inhibition

326

of ATP formation, alkylation of nucleophiles, disruption of hormonal activity, complexation with

327

protein, binding with free sterols, inhibition of respiration, and increasing relative electron

328

partitioning to the alternative oxidase pathway.33 Despite many studies with terpenoids, most of

329

them are related with diterpenes from fungi while few phytotoxins are known from plants.32

330

Because of their phytotoxicity when compared to commercial herbicides, these compounds could

331

be used as herbicide models34. An example of active natural diterpenes is the quassinoids

332

chaparrinune and glaucaxubulone, which inhibit growth and germination of dicots and monocots

333

at concentrations below 10 μM.31 Moreover, several diterpenoids act as phytoalexins



Page 16 of 38

334

(antimicrobial compounds) in monocots35 such as momilactones A and B,36 oryzalexins A–F37,

335

and oryzalexin S38 from rice (Oryza sativa L.). Momilactone B is a well established allelochemical

336

found in the most allelopathic rice varieties, however its mode of action is unknown.39

337

Electrolyte Leakage Assay. None of the compounds isolated in this work caused the kind of

338

changes in the electrolyte leakage (Figure 7) expected with compounds that directly modify the

339

integrity of the plasma membrane, which is a good biomarker to help identify modes of action of

340

herbicides and their dependence on light.40 The fact that the isolated compounds from E. plana did

341

not influence the membrane integrity indicates that the mechanism of action of these compounds

342

is not directly related to cell membrane damage but to other, unknown molecular targets.

343

Bioassay against Colletotrichum spp. A bioautographic technique can be used to identify

344

fungitoxic substances. Visible inhibition zones observed after incubation indicate the presence of

345

fungitoxic compounds.41

346

In this work, a direct antifungal bioautography was performed against the strawberry pathogens

347

C. acutatum, C. fragariae and C. gloeosporioides. These pathogens work can occur singly or in

348

combination in strawberry crops, causing a disease loosely referred to as anthracnose.42 Compound

349

2 was the most active, especially at 100 µg/spot, although at 10 µg/spot it was possible to observe

350

activity (Table 4). Compounds 1 and 3 it produced a diffuse inhibition zone (Table 4). Previous

351

work reported antifungal activity of crude extracts from E. plana leaves on the plant pathogen

352

Drechslera tritici-repentis.43 Another species from the same genus, E. cynosuroides (Retz.), has

353

antifungal activity against Aspergillus niger.44

354

In the present study, three new diterpenes were extracted, isolated and identified for the first

355

time as potential allelochemicals produced by E. plana. Previously, only phenolic compounds were

356

identified as potential allelochemicals in this species.13 Many phenolics and terpenoids have been


Page 17 of 38


357

reported as allelopathic compounds.45-47 However, simply finding a phytotoxic compound in a

358

plant extract does not prove that it is an allelochemical.48 Bioassay-guided isolation of active

359

compounds is a first step in proof of an allelochemical. A potential complicating factor is the

360

interaction of potential allelochemicals. For example, little attention has been paid to interactions

361

of phenolics and terpenoids from the same plant.49 Compounds identified in this work should be

362

studied in combination. The description of the newly discovered compounds 1, 2 and 3, besides

363

contributing to the chemical characterization of the species, may be the first step in the study of

364

potential of these compounds as bioherbicides. Allelochemicals such as diterpenes or

365

sesquiterpene lactones could be used as models in the development of herbicides of natural origin.

366 367 368 369

ACKNOWLEDGMENTS

370

We are grateful to Amber Reichley, Solomon Green, Robert Johnson, Jesse Linda Robertson and

371

Ramona Pace for their excellent technical assistance.

372 373

FUNDING SOURCES

374

We acknowledge the CAPES – Coordination for the Improvement of Higher Education Personnel

375

(Brazil) (Project PDSE-No 19/2016).

376 377

SUPPORTING INFORMATION

378

The Supporting Information is available free of charge on the ACS Publications website at DOI:



Page 18 of 38

379

X-ray crystallography information for compound 1 including crystal data, data collection,

380

refinement, fractional atomic coordinates and isotropic or equivalent isotropic displacement

381

parameters (Å2), atomic displacement parameters (Å2), and geometric parameters (Å, º). 1H, 13C,

382

1H-1H

383

material is available free of charge via the Internet at http://pubs.acs.org.

COSY, 1H-13C HSQC, and 1H-13C HMBC NMR spectra for compounds 1, 2, and 3. This

384 385

CORRESPONDING AUTHOR

386

*Corresponding author. Tel.: 55 54 991702687. Email: [email protected].

387

*Co-corresponding author. Tel.: +1-662-915-5898. Email: [email protected].

388 389


Page 19 of 38

390


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391 392 393 394 395 396 397

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H. G., Eds.; CRC Press: Boca Raton, Florida, 2004, pp. 201-216.

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493 43. Favaretto, A.; Tonial, F.; Bertol, C. D.; Scheffer-Basso, S. M. Antimicrobial activity of leaf and 494

root extracts of tough lovegrass. Comun. Sci., 2016, 7, 420-427.

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501 47. Waller, G. R. Allelochemicals: role in agriculture and forestry. Amer. Chem. Soc. Symp. Ser. 502

#330. Washington, D. C., 1987. 606 pp. 48. Duke, S. O. Proving allelopathy in crop-weed

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interactions. Weed Sci., 2015, 63, 121-132.

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phenolics, and their mixtures, and their recovery from soil. Can. J. Bot., 1997, 75, 888-891.


Page 25 of 38

506


FIGURE CAPTIONS

507 508

Figure 1. Structures of compounds isolated from E. plana roots: neocassa-1,12(13),15-triene-

509

3,14-dione, 1, 19-norneocassa-1,12(13),15-triene-3,14-dione, 2, and 14-hydroxyneocassa-

510

1,12(17),15-triene-3-one, 3.

511 512

Figure 2. Molecular structure of neocassa-1,12(13),15-triene-3,14-dione, 1, from X-ray analysis,

513

with 50% ellipsoids.

514 515

Figure 3. Carbon skeleton examples for cassa-13(14),15-diene and neocassa-12(13),15-diene

516

analogs.

517 518

Figure 4. HMBC correlation data for neocassa-1,12(13),15-triene-3,14-dione, 1, 19-norneocassa-

519

1,12(13),15-triene-3,14-dione, 2, and 14-hydroxyneocassa-1,12(17),15-triene-3-one, 3.

520 521

Figure 5. Growth-response curve of the effects of neocassa-1,12(13),15-triene-3,14-dione, 1, 19-

522

norneocassa-1,12(13),15-triene-3,14-dione, 2, 14-hydroxyneocassa-1,12(17),15-triene-3-one, 3,

523

on L. pauciscostata growth 7 d after treatment. The solvent control value is 0 µM.

524 525

Figure 6. Proportion of healthy, chlorotic and necrotic tissues in L. pauciscostata plants, when

526

submitted to different concentrations of the compounds 1: neocassa-1,12(13),15-triene-3,14-

527

dione; 2: 19-norneocassa-1,12(13),15-triene-3,14-dione; 3: 14-hydroxyneocassa-1,12(17),15-

528

triene-3-one.



Page 26 of 38

529

Figure 7. Electrolyte leakage induced by the compounds neocassa-1,12(13),15-triene-3,14-

530

dione,

531

1,12(17),15-triene-3-one, 3, submitted to 18 h dark + 6 h light (arrows indicate the start of the

532

light exposure). Data represent means of three replications ± SD.

1,

19-norneocassa-1,12(13),15-triene-3,14-dione,

2,

and

14-hydroxyneocassa-

533 534


Page 27 of 38


Table 1. 13C and 1H NMR Assignmenta Data for Compounds 1, 2, and 3 in CDCl3. position

δC

1 2 3 4 5 6

154.2 126.9 204.7 44.6 51.6 21.0

7

26.9

8

45.2b

9 10 11

46.2b 38.9 32.8

12 13 14 15

154.5 132.6 199.5 130.1

16

120.3

17 18 19 20

1 δH (multiplicity, J in Hz)

2c δH (multiplicity, J in Hz)

δC

7.00 (d, 10.2) 5.94 (d, 10.2) 1.66 (dd, 12.0, 2.0) 1.53 (ddd, 12.0, 12.8, 3.6) 2.41 (m) 1.31 (m) 2.29 (ddd, 13.4, 11.5, 4.8) 1.77 (m) 2.52 (dd, 18.1, 4.6) 2.43 (m) 6.40 (dd, 17.9, 11.6)

153.6 127.5 201.3 42.1 49.2 23.4

120.2

22.5

5.46 (dd, 17.9, 2.1) 5.41 (dd, 11.6, 2.1) 2.08 (s)

22.1 27.1 16.2

1.09 (s) 1.15 (s) 1.14 (s)

δC

7.03 (d, 10.2) 5.95 (d, 10.2) 2.35 (m) 1.58 (dd, 12.2, 2.8) 1.88 (m) 1.33 (m) 2.38 (m) 1.31 (m) 2.33 (m)b

155.6 126.2 204.9 44.5 51.7 21.4

44.7 38.9 29.5

22.3

1.81 (m)b 2.60 (dd, 18.1, 4.4) 2.46 (m) 6.43 (ddd, 17.9, 11.6, 11.6) 5.50 (dd, 17.9, 2.2) 5.44 (dd, 11.6, 2.2) 2.11 (s)

12.1 13.6 -

1.17 (d, 6.7) 1.11 (s) -

22.0 27.1 15.7

26.0 44.8 44.6 38.9 32.9 154.3 132.5 199.4 129.9

29.9 36.1

145.5 55.1 75.5 136.8 116.8 114.3

a

Assignments of NMR data are based on 1H, 13C, DEPT, 1H−1H COSY, HSQC, and HMBC NMR experiments.

b

Signals in same columns interchangeable.

3c δH (multiplicity, J in Hz) 7.07 (d, 10.3) 5.93 (d, 10.3) 1.62 (m) 1.56 (m) 1.72 (m) 1.65 (m) 1.30 (m) 1.76 (m) 1.49 (m) 2.35 (dd, 12.9, 3.8) 2.14 (t, 12.9) 3.13 (m) 3.73 (dd, 2.4) 5.87 (ddd, 16.5, 10.5, 5.4) 5.18 (dd, 10.5, 1.9) 5.15 (dd, 16.5, 1.9) 5.04 (t, 2.0) 4.94 (t, 2.0) 1.10 (s) 1.16 (s) 1.08 (s)



Page 28 of 38

Table 2. Phytotoxic Activity of Extracts Prepared with Leaves and Roots of Eragrostis plana Using Different Solvents phytotoxicity at 7 da

extracts (1mg/mL)

lettuce

creeping bentgrass

rootsHEX

2

4

rootsdichloromethane/MeOH

0

2

rootsH2O

0

0

leavesHEX

0

1

leavesdichloromethane/MeOH

1

3

leavesH2O

0

0

Bioassay rating based on scale of 0 to 5: 0 = no eﬀect and 5 = no growth or germination. a


Page 29 of 38


Table 3. Phytotoxic Activity of Novel Pure Compounds Isolated From Roots of Eragrostis plana compounds

1

2

3

tested concentration (µM) 0* 0** 10 33 100 333 1000 0* 0** 10 33 100 333 1000 0* 0** 10 33 100 333 1000

phytotoxicity at 7 da lettuce creeping bentgrass 0 0 0 0 0 0 0 0 0 0 0 0 1 3 0 0 0 0 0 0 0 0 0 0 0 0 1 4 0 0 0 0 0 0 0 0 0 0 2 3 1 4

a Bioassay * Water

rating based on scale of 0 to 5: 0 = no eﬀect and 5 = no growth or germination. control. ** Solvent control.

Table 4. Antifungal Activity of Pure Compounds Isolated from Leaves and Roots of Eragrostis plana 29 ACS Paragon Plus Environment


compounds

1 2 3 Benomyl Captan Cyprodinil Azoxystrobin

tested concentration (µg) 10 100 10 100 10 100

Page 30 of 38

inhibitory zone diameter (mm) Colletotrichum Colletotrichum Colletotrichum acutatum fragariae gloeosporioides *5.0±0.0 0.0±0.0 4.0±0.0 *7.5±0.8 *11.5±0.7 *10.0±0.0 *4.5±0.6 3.5±0.7 5.5±0.7 9.0±0.0 12.7±1.0 7.6±0.5 0.0±0.0 0.0±0.0 0.0±0.0 *8.5±0.7 0.0±0.0 0.0±0.0 0±0 0±0 0±0 15 14 17 *20 *21 0 *33 *26 *23

* diffuse.


Page 31 of 38


FIGURES

17 15 20 9 1 3

5

19

18

O

H

H

13

H

16

O

H

7

O

H

H

1

O

H 2

H H H O

H

OH

H 3

Figure 1.



Page 32 of 38

Figure 2.


Page 33 of 38

Journal of Agricultural and Food Chemistry 15 13

20 9

H

1 3

5

19

18

H

12 16

15

H

17

H

7

H

H

cassa-13(14),15-diene

neocassa-12(13),15-diene

Figure 3.



Page 34 of 38

H O

H O

1

H

H O

H O

H H CH3

2

H OH

H O

H

3

Figure 4.


Page 35 of 38

Lemna growth (%)

600


1

500 400 300 200 100 0 0 0.1 0.3 1

3

10 33 100 333

Concentration (µM)

Lemna growth (%)

2 400 300 200 100 0 0 0.1 0.3 1

3

10 33 100 333

Concentration (µM)

3

Lemna growth (%)

500 400 300 200 100 0 0 0.1 0.3 1

3

10 33 100 333

Concentration (µM) Figure 5.



healthy

chlorotic

Page 36 of 38

1

necrotic

100 tissue (%)

80 60 40 20 0 C

CS 0.1 0.3

1

3

10

33 100 333

Concentration (µM) 2

tissue (%)

100 80 60 Figure 6.

40 20

3 33

0 10

33

10

3

1

0. 3

0. 1

S C

C

0 3

Concentration (µM)

80 60 40

33

10

Figure 6. 3

1

0. 3

0. 1

C

S

0

10 0 33 3

20 C

tissue (%)

100

Concentration (µM)


Page 37 of 38


Figure 7. 37 ACS Paragon Plus Environment


Page 38 of 38

Table of Contents Graphic

Eragrostis plana

New neocassanes


New Phytotoxic Cassane-Like Diterpenoids from Eragrostis plana

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