A method, utilizing brine shrimp (Artemia salina Leach), is proposed as a simple bioassay for natural product research. The procedure determines LC (50) values in microg/ml of active compounds and extracts in the brine medium. Activities of a broad range of known active compounds are manifested as toxicity to the shrimp. Screening results with seed extracts of 41 species of Euphorbiaceae were compared with 9KB and 9PS cytotoxicities. The method is rapid, reliable, inexpensive, and convenient as an in-house general bioassay tool.

EtOH extracts of the seeds of Euphorbia lagascae inhibited crown gall tumors on potato discs, were active against 3PS (in vivo) and 9PS (in vitro) mouse leukemia, and were toxic to brine shrimp. Using the potato disc and brine shrimp bioassays to monitor fractionation, piceatannol (3,4,3',5'- tetrahydroxystilbene ) was isolated as the 3PS active principle. Aesculetin (6,7-dihydroxycoumarin) was isolated in large quantity, was weakly cytotoxic in 9KB , but was inactive in the other bioassay systems. Sucrose was also isolated. A combination of the simple crown gall and brine shrimp bioassays can thus be used both to detect and to isolate plant antitumor substances, minimizing the need for extensive antitumor testing in vivo.

Galsky et al. (10, 11) have reported that the inhibition and growth of crown gall tumors, initiated on potato discs by Agrobacterium tumefaciens, apparently has good agreement with 3PS in vivo antileukemic activity in mice. We have now modified and evaluated this assay for its potential use as a prescreen and fractionation monitor of plant extracts for 3PS activity. The modified assay was performed on a series of natural compounds and plant extracts (known to have 3PS activity) and on extracts of seeds of 41 Euphorbiaceae species. The results suggest that the potato disc assay is a safe, simple, rapid, in-house, low cost, prescreen for 3PS antitumor activity; it detects some false positives, but few false negatives; it is statistically much more predictive (p = 0.002) of 3PS activity than either the 9KB (p = 0.140) or the 9PS (p = 0.114) cytotoxicity assays. Its extended use could easily obviate the expense and extensive need for animals in the initial stages of antitumor screening and plant fractionation.

Bioactive Annonaceous acetogenins have been isolated from the EtOH extract of the bark of Xylopia aromatica by bioactivity-directed fractionation using lethality to brine shrimp. These acetogenins include xylopianin [1 , xylopiacin [2], and xylomaticin [3], which are three new mono-tetrahydrofuran ring type acetogenins, in addition to the known compounds, annomontacin, gigantetronenin, gigantetrocin A, and annonacin. Compounds 1 and 2 are unusual in having hydroxylation at C-8; 3 has the same functionalities as annonacin but with 37 carbons instead of 35 carbons. The structures were elucidated by spectral analysis of the parent compounds and/or simple chemical derivatives. These acetogenins showed cytotoxicities, comparable to adriamycin, against three human solid tumor cell lines.

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