Plant materials
Brachiaria
brizantha (Hochst. ex A. Rich.) Stapf. (common name; palisade grass, bread grass, syn; Urochloa brizantha Hochst. ex A. Rich.) was grown in the research field of National Institute for Agro-Environmental Sciences, Tsukuba, Japan. Shoots of the plants were harvested in June and October 2011 and January 2012, and stored at −20 °C until extraction. Cress (Lepidum sativum L.) and lettuce (Lactuca sativa L.) were used due to their uniform establishment and sensitivity as a seedling indicator (Kato-Noguchi et al. 2010, 2014). Weed species, Phleum pratense L. and Lolium multiflorum Lam were also used for bioassay.
Extraction and bioassay
B.
brizantha (100 g dry weight) shoots were extracted with 1 L of 70 % (v/v) aqueous methanol for 2 days. After filtration with filter paper (No. 2; Toyo, Tokyo, Japan), the residue was extracted again with 1 L methanol for 2 days and filtered, and the two filtrates were combined. An aliquot of the extract (final assay concentration of tested samples corresponded to the extracts obtained from 0.3, 1, 3, 10, 30, 100 and 300 mg dry weight of B.
brizantha shoots per mL) was evaporated to dryness, dissolved in a 0.2 mL of methanol and added to a sheet of filter paper (No. 2) in a 3-cm Petri dish. The methanol was evaporated in a fume hood. Then, the filter paper in the Petri dishes was moistened with 0.8 mL of 0.05 % (v/v) polyoxyethylene sorbitan monolaurate (Tween 20). Ten seeds of cress or lettuce, or 10 seedlings of P. pratense or L. multiflorum after germination in the darkness at 25 °C for 36–48 h were placed in the Petri dishes. The length of roots and shoots of these seedlings were measured after 48 h of incubation in the darkness at 25 °C. Controls were treated exactly as described above, with the exception that 0.2 mL methanol was used instead of B.
brizantha extracts. Inhibitory activity (%) was determined by the formula: [(control plant length − plant length incubated with extract)/control plant length] × 100. The bioassay was repeated five times using a randomized design with 10 plants for each determination. Significant differences were examined by Duncan’s multiple comparison tests.
Quantification of allelopathic active substances
Brachiaria
decumbens shoots was extracted as described above and the extracts were concentrated at 40 °C in vacuo to produce an aqueous residue. The aqueous residue was adjusted to pH 7.0 with 1 M phosphate buffer and partitioned three times against an equal volume of ethyl acetate as described by Kato-Noguchi et al. (2014). The ethyl acetate fraction was evaporated to dryness and separated on a column of silica gel (100 g, silica gel 60, 70–230 mesh; Merck), eluted with 20, 30, 40, 50, 60, 70 and 80 % ethyl acetate in n-hexane (100 mL per step) and ethyl acetate (100 mL). Allelopathic active substances, (6R,9R)-3-oxo-α-ionol and (6R,9S)-3-oxo-α-ionol, were obtained by the elution with 70 % ethyl acetate in n-hexane on the silica gel column, and 4-ketopinoresinol was obtained by the elution with 80 % ethyl acetate in n-hexane on the silica gel column (Kato-Noguchi et al. 2014).
For quantification of (6R,9R)-3-oxo-α-ionol and (6R,9S)-3-oxo-α-ionol, the fraction obtained with 70 % ethyl acetate in n-hexane on the silica gel column was evaporated. The residue was then dissolved in 20 % (v/v) aqueous methanol (2 mL) and loaded onto reverse-phase C18 cartridges (YMC Ltd., Kyoto, Japan). The cartridge was eluted with 20, 40, 60 and 80 % (v/v) aqueous methanol (15 mL per step). The active fraction was eluted by 40 % aqueous methanol and evaporated to dryness. The residue was injected into reverse-phase HPLC (4.6 mm i.d. × 250 mm, Inertsil ODS-3, GL Sciences, Osaka, Japan) eluted at a flow rate of 0.8 mL min−1 with 55 % aqueous methanol and detected at 220 nm. Retention time of (6R,9R)-3-oxo-α-ionol and (6R,9S)-3-oxo-α-ionol was 65 and 70 mm, respectively. Quantification of those compounds was performed by measuring their peak areas on the chromatogram of HPLC. The quantification was repeated three times independently with three assays for each determination. Significant differences were examined by Duncan’s multiple comparison tests.
The 1H-NMR spectrum of (6R,9R)-3-oxo-α-ionol, δH: 1.00 (3H, s), 1.03 (3H, s), 1.24 (3H, d, J = 6.8 Hz), 1.94 (3H, d, J = 1.0 Hz), 2.05 (1H, d, J = 16.6 Hz), 2.40 (1H, d, J = 16.6 Hz), 2.67 (1H, d, J = 9.3 Hz), 4.27 (1H, m), 5.58 (1H, dd, J = 15.1, 8.8 Hz), 5.70 (1H, dd, J = 15.2, 5.9 Hz), 5.88 (1H, s). The specific rotation of the compound \(\left( {\left[ \alpha \right]^{ 2 5}_{\text{D}} } \right)\) was +210° (c 0.03, CH2Cl2). The 1H-NMR spectrum of (6R,9S)-3-oxo-α-ionol, δH: 0.98 (3H, s), 1.03 (3H, s), 1.24 (3H, d, J = 6.4 Hz), 1.96 (3H, d, J = 1.5 Hz), 2.05 (1H, d, J = 16.6 Hz), 2.42 (1H, d, J = 17.1 Hz), 2.66 (1H, d, J = 8.8 Hz), 4.28 (1H, m), 5.55 (1H, dd, J = 15.1, 8.8 Hz), 5.69 (1H, dd, J = 15.1, 5.9 Hz), 5.89 (1H, s). The specific rotation of the compound \(\left( {\left[ \alpha \right]^{ 2 5}_{\text{D}} } \right)\) was +214° (c 0.05, CH2Cl2).
For quantification of 4-ketopinoresinol obtained with 80 % ethyl acetate in n-hexane on the silica gel column was evaporated. The residue was then dissolved in 20 % (v/v) aqueous methanol (2 mL) and loaded onto reverse-phase C18 cartridges. The cartridge was eluted with 20, 40, 60 and 80 % (v/v) aqueous methanol (15 mL per step). The active fraction was eluted by 40 % aqueous methanol and evaporated to dryness. The residue was injected into reverse-phase HPLC (10 mm i.d. × 50 cm, ODS AQ-325; YMC Ltd.) eluted at a flow rate of 1.5 mL min−1 with 40 % aqueous methanol and detected at 220 nm. Retention time of 4-ketopinoresinol was 145 mm and quantification of the compound was performed by measuring their peak areas as described above.
The 1H-NMR spectrum of 4-ketopinoresinol, δH: 3.25 (m, 1 H, H1), 3.47 (dd, J = 9.2, 3.8 Hz, 1 H, H5), 3.90 (s, 3 H, OCH3), 3.92 (s, 3 H, OCH3), 4.04 (dd, J = 9.4, 4.5 Hz, 1 H, H8a), 4.34 (dd, J = 9.3, 6.8 Hz, 1 H, H8b), 5.34 (d, J = 4.0 Hz, 1 H, H2), 5.35 (d, J = 3.6 Hz, 1 H, H6), 6.8 (m, 2H, aromatic H), 6.9 (m, 4H, aromatic H). The 13C NMR spectrum of the compound, δC: 50.1 (C1), 53.5 (C5), 56.2 (OCH3), 56.2 (OCH3), 72.8 (C8), 83.5 (C2), 84.8 (C6), 107.9 (C2′), 108.2 (C2″), 114.5 (C5″), 114.8 (C5′), 118.1 (C6″), 118.5 (C6′), 131.2(C1′), 132.4 (C1″), 145.4 (C4″), 146.2 (C4′), 146.9 (C3″), 147.1 (C3′), 177.1 (C4). The specific rotation of the compound \(\left( {\left[ \alpha \right]^{ 2 5}_{\text{D}} } \right)\) was +5.7° (c 0.1, CH3OH).