Bioassay-directed isolation of the allelopathic constituents of the aquatic plant Nymphaea odorata
An aqueous extract of the aquatic plant Nymphaea odorata exhibited high allelopathic potential in Lemna minor frond growth and lettuce seedling radicle growth bioassays. Hexane, diethyl ether, acetone, and 95% ethanol were used in succession to extract the dried and ground leaves and stems of the plant. After removal of solvent under reduced pressure, all of the concentrated extracts were bioassayed. The 95% ethanol extract exhibited the highest activity, limiting lettuce seedling radicle growth to 12% of control at 1000 ppm. The purpose of this project was to completely investigate the 95% ethanol extract to determine the compounds responsible for plant growth inhibition. Solvent fractionation of the 95% ethanol extract yielded an ethyl acetate soluble fraction which suppressed radicle growth to 10% of control at 1000 mg/L or parts per million (ppm). Acid/base fractionation of the ethyl acetate fraction yielded an acidic fraction that suppressed radicle growth to 38% of control and frond growth to 24% of control, at 100 ppm. Silica gel column chromatography of the acidic fraction yielded active fractions which were separated further by preparative TLC on silica gel. Gallic acid was isolated and at 100 ppm, limited radicle growth to 10% of control and frond growth to 50% of control. Polyamide column chromatography of the ethyl acetate fraction yielded myricitrin (myricetin 3-O-rhamnoside). Myricitrin exhibited no activity in the lettuce seedling bioassay and only minimal activity in the L. minor bioassay. Repeated Sephadex column chromatography of the ethyl acetate fraction yielded myricetin. Myricetin did not exhibit lettuce radicle growth inhibition and displayed only minimal L. minor frond growth inhibition. Sephadex column fractions also yielded the gallotannin 1,2,3,4,6-penta-O-galloyl-$\beta$-D-glucose (1,2,3,4,6-PGG), which held radicle growth to 52% of control and frond growth to 63% of control at 100 mg/L. Reversed-phase high performance liquid chromatography (RP-HPLC) of remaining active fractions produced 2,3,4,6-tetra-O-galloyl glucose (2,3,4,6-TGG), which reduced radicle growth to 51% of control and frond growth to 52% of control at 100 ppm.