Bioactivity-directed fractionation and isolation

Numerous important bioactive compounds have been, and continue to be, isolated worldwide from natural sources. These compounds include both primary and secondary metabolites isolated mainly from plants, as well as from the animal and mineral kingdoms. The recent development of new bioassay methods has facilitated progress in the BDFI (bioactivity-directed fractionation and isolation) of many useful bioactive compounds from natural sources (1). These active principles could be developed or additionally modified to enhance the biologic profiles as clinical trials candidates. Many natural pure compounds have become medicines, dietary supplements, and other useful commercial products. This article summarizes research on many different useful compounds isolated or developed from plants with an emphasis on those discovered recently by the laboratories of the authors as antitumor and anti-HIV clinical trial candidates. 

An herb or herbal prescription is chosen as the source of a potential new drug based on folk or clinical experiences. The initial research (new lead discovery) focuses on isolation of a bioactive natural lead compound(s), as illustrated graphically below. After extraction of the herbal medicine, activity is verified by pharmacological testing. Bioactivity-directed fractionation and isolation (BDFl)... 

A new method of bioactivity-directed fractionation, based on multidrug resistant pump (MDR) inhibition in Staphylococcus aureus, was reported for medicinal plants. This work resulted in the isolation, from berberine-containing Berberis species, two compounds that are themselves devoid of antibacterial activity, but that form potent synergistic couples with a sub-inhibitory concentration of berberine. The bacterial MDR pump inhibitors were identified as the flavonolignan 2 and the porphyrin 3 [98]. The isoflavones not only enhanced the antibacterial activity of the natural product, berberine. Fig. 4, but also the activity of synthetic... 

The structure of didehydrotuberostemonine (10) was identified by direct comparison of its physical and chemical data with those obtained from the oxidation products of tuberostemonine (4), but NMR data for didehydrotuberostemonine (10) are not available (72). Comparison of the H-NMR chemical shifts of bisdehydro-neotuberostemonine (11), isolated from S. tuberosa 14) and from S. collinsae (20), and epi-bisdehydroneotuberostemonine J (12), isolated from bioactivity-directed fractionation of the crude extract of S. tuberosa and whose structure was claimed to be established by X-ray crystallographic analysis (2), reveals a significant deshielding effect at H5 in both alkaloids when compared to the other members of this group as a result of the ring current of the pyrrole moiety. Rather unusual is the deshielding of one of the H6 (3.06 ppm) in epi-bisdehydroneotuberostemonine J (12) which also displays H8 and... 

Bioassay-directed fractionation of this plant led to the isolation of the known alkaloid verazine (16) and eight related compounds (six of them newly reported) as the bioactive constituents alkaloids 17 and 18 are examples of the new compounds isolated. The relative stereochemistry of the new alkaloids was established by careful ID- and 2D-NMR experiments. 

Isolation of potential anticancer compounds from bioactive extracts involves bioactivity-guided fractionation. The DNA-damaging natural products encountered in our studies were extracted by MEK and/or methanol, and the general methodology which we have employed in our bioassay-directed fractionation of these extracts is schematically presented in Fig. 7. These fractionations involved solvent-solvent partition, Sephadex LH-20 gel filtration, normal phase and reversed-phase (RP) column, preparative thin-layer and high pressure liquid chromatography (HPLC). Silica gel chromatography was employed only if bioactive compounds were found to be stable under these mildly acidic conditions. 

The most direct approach for testing the effects of a metabolite on a consumer is to isolate the compound, or a bioactive chemical fraction, incorporate it into a palatable food, and feed it (versus an equivalent control food without the metabolite) to consumers. Harpacticoid copepods have been fed on fish food with and without lipophilic extracts of diatoms (Shaw et al. 1994). This direct... 

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