Quantitation of annonaceous acetogenins in Asimina triloba and bioactivity directed fractionation of Monarda fistulosa and Citrus paradisi
Abstract
The Annonaceous acetogenins are a growing class of compounds endogenous to the plant family Annonaceae that have been shown to have significant cytotoxic, in vivo antitumor, and pesticidal properties. Monthly twig samples of Asimina triloba extracted with CH2Cl2 were monitored for biological activity using the brine shrimp lethality test (BST). The LC50 values determined by the BST correlated with the concentration of acetogenins in the twigs and hence showed the relative bioactivity of the monthly extracts. The results revealed that the months of May, June, and July were the top three months for acetogenin production. There was a one to two order of magnitude decrease in activity in late fall to winter. A single extract sample from each month was then evaluated using liquid chromatography/(+)electrospray ionization/tandem mass spectrometry (LC/(+)ESI-MS/MS). Concentrations of the three major and most active Annonaceous acetogenins, bullatacin, asimicin, and trilobacin, were directly evaluated without any further sample preparation in the crude CH2Cl2 extracts of the twigs. All three compounds (MW 622), possess a 4-OH and showed a characteristic neutral loss of 112 amu from the sodiated parent molecule (m/z 645). The m/z 533 daughter ions were monitored and quantified using 'spiked' amounts of pure analytes to construct calibration curves. These results showed that the contents of bullatacin and the mixture of asimicin and trilobacin increased significantly in May and June, with bullatacin at 13 and 15 ppm, respectively, and the mixture of asimicin and trilobacin at 47 and 51 ppm, respectively in the dry weight of the twigs. Monarda fistulosa L. (Lamiaceae), also known as wild bergamot, was subjected to bioactivity-directed fractionation using the BST to identify the in vitro bioactive constituents. Purification of the F005 (partitioned fraction) sample yielded thymoquinone (C10H 12O2, MW164) and thymol (C10H14O, MW 150) as the most bioactive compounds in the BST. Thymoquinone showed selective cytotoxicity towards the PC-3 (prostate) human tumor cancer cell line with an ED50 comparable to Adriamycine® a standard chemotherapeutic agent. In the National Cancer Institute (NCI) 60 human tumor cell panel, thymoquinone displayed interesting selectivities towards SF-539 (CNS), M-14 (melanoma), PC-3, and OVCAR-5 (ovarian). Thymol was not found to be cytotoxic, but did show pesticidal activity comparable to rotenone.
Degree
Ph.D.
Advisors
McLaughlin, Purdue University.
Subject Area
Analytical chemistry|Pharmacology
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