Plant materials Fresh and mature leaves of L. chinensis were collected from Bangladesh during January and February 2011. After collection, leaves were washed with tap water for 2–3 times followed by sun drying, and then kept in a refrigerator at 2°C until extraction. Three dicotyledonous, cress (Lepidium sativum L.), lettuce (Lactuca sativa L.), alfalfa (Medicago sativa L.), and three monocotyledonous, timothy (Phleum pratense L.), Italian ryegrass (Lolium multiflorum Lam.) and barnyard grass (Echinochloa crusgalli (L.) Beauv.), test plant species were selected for this experiment. Cress, lettuce, alfalfa and timothy were chosen due to their known seedling growth, and Italian ryegrass and barnyard grass were selected due to their worldwide acceptance as deleterious weeds.
Extraction Dried leaves (100 g) of L. chinensis were ground into powder by a grinding machine and extracted with 500 mL of 70% (v/v) aqueous methanol for 48 h. The extract was then filtered through one layer of filter paper (No. 2; Advantec® Toyo Roshi Kaisha, Ltd., Tokyo, Japan), using a vacuum pump. The residue was re-extracted with equal amount of methanol for 24 h and filtered. The two filtrates were then combined and evaporated to dryness using a rotary evaporator at 40°C.
Germination bioassay Lettuce and barnyard grasses were selected for germination bioassay because lettuce is highly sensitive to allelopathic substances (Xuan et al. 2005), while barnyard grass is one of the worst weeds throughout the world and has developed high resistance against many herbicides (Dilipkumar et al. 2012; Heap 2013; Islam & Kato-Noguchi 2013a). The germination bioassay was performed according to Islam and Kato-Noguchi (2013b) with some modifications. An aliquot of the extract was evaporated to dryness, dissolved in methanol and added to a sheet of filter paper in a 2.8 cm Petri dish. The final assay concentration was 0.003, 0.01, 0.03 and 0.1 g dry wt. eq. extract mL−1. The filter paper was moistened with 0.6 mL of 0.05% (v/v) aqueous solution of polyoxyethylene sorbitan monolaurate (Tween 20, Nacalai Tesque, Inc., Kyoto, Japan; a surfactant that is not toxic) after evaporating the methanol in a draft chamber. Ten seeds of lettuce or ten pre-soaked barnyard grass (soaked in distilled water for 24 h at 25°C to imbibe the seeds) were arranged on the filter paper in Petri dishes. A control was also maintained by sowing the seeds on the filter paper moistened with Tween 20 without plant extracts. Then the Petri dishes were incubated in the dark chamber at 25°C. Seeds that showed the emergence of the radical by rupturing the seed coat were considered to be germinated as described by Faria et al. (2005). Germination of seeds was recorded at every 12-h interval for four days until the number becomes constant. The percentage of germination in each treatment over control was calculated using the following equation as prescribed by Islam and Kato-Noguchi:
Germination ð% of control = GT/ GO x 100
GT = average number of germinated seed in the treatment in each time of measurements G0 = average number of germinated seed in the control at the same time of measurements
Growth bioassay Test samples of plant extracts and the Petri dishes were prepared by the aforesaid procedure. Ten seeds
of cress, lettuce, alfalfa or ten pre-germinated seeds of timothy (germinated at the darkness at 25°C for 48 h), Italian ryegrass (germinated in darkness at 25°C for 36 h) or barnyard grass (germinated in darkness at 25°C for 24 h) were arranged on the filter paper in the Petri dishes containing 0.6 mL of 0.05% (v/v) Tween 20. All of the Petri dishes were kept in a growth chamber in dark condition at 25°C. The hypocotyl/coleoptile and root lengths of the seedlings were measured after 48 h incubation. The growth bioassay was performed as per Islam and Kato-Noguchi (2012) with slight modifications. The inhibition percentage (IP) was calculated using the following formula: IP = (1- LE/ LC) x 100
IP = inhibition% LE = length of seedlings in aqueous methanol plant extract LC = length of seedlings in control (without extract)
Statistical analysis All the bioassay experiments were conducted in a completely randomized design with three replications and repeated twice. Experimental data were analysed using predictive analytics software (PASW) statistics 17.0 (SPSS Inc., USA). All measured variables were subjected to two-way analysis of variance (ANOVA). The concentration required for 50% inhibition (defined as I50) of the test species in the assay was calculated from the regression equation of the concentration-response curves, using GraphPad Prism 6.0 (GraphPad Software, Inc., USA).