thioglycerol matrix. Gas chromatographic sugar analysis was performed on a Hewlett Packard 5890A gas chromatograph with a 7673A autosampler, 3393A integrator, and ChromPerfect software, using a Restek Rtx-1 100% dimethyl polysiloxane column (0.25 µm, 3.0 m, 0.32 mm ID). The samples were silylated and compared with silylated known standards. All NMR spectra were obtained using a Varian VXR-500S spectrometer with standard Varian pulse sequences and parameters, and standard 5 mm tubes and 20 mg samples at ambient temperature in pyridine-d5.
Materials. Plant material (Barringtonia asiatica) used for this study was collected on the island of Upolu near Soluafata, Western Samoa. The seeds were removed from the fibrous mesocarp, placed in 70% ethanol, stored under refrigeration, and shipped to Brigham Young University. Upon arrival at BYU, the seeds were stored at 10oC in 70% ethanol to inhibit fungal growth. A voucher specimen is maintained by the L.D.S. Church College of Western Samoa, Sauniatu, Western Samoa.
Bioassays. The freshwater fish bioassays were conducted using small native fish (mosquito fish) collected from the Sauniatu river (Western Samoa). Each well of a standard 12-well sample plate was filled with river water (5 mL), and three fish were added to each well. Initial biological evaluation of the crude fraction at a concentration of 250 µL mL-1 showed that the non-water-soluble, 1-butanol fraction was responsible for most of the activity (Figure 2).
Brine shrimp bioassays were conducted in triplicate.15 Following the hatching of brine shrimp eggs, a solution (5 mL) containing the brine shrimp was pipetted into individual test tubes and the number of shrimp in each tube was determined. Following removal of the solvent a 100 µg sample of each isolated HPLC peak (1, 2, 3, 4, 5, 6, and 7) was added to each tube. A control set (also in triplicate) was used, in which only water (5 mL) was added to the brine shrimp. The total number of living shrimp in each tube was again measured after 24 hr and compared to the initial number to determine toxicity. Although activity was found in each of the HPLC peaks, peak 7 was the most active (Figure 3).
