J 1 I 1 1 J 1 1 J I I

f1 f2 f3 f4 f5 f6 f7 f8 f9 f10

f1 f2 f3 f4 f5 f6 f7 f8 f9 f10

Crude extract

Screening

•••ooooooooo oooooooooooo 000ooooooooo oooooooooooo oooooooooooo oooooooooooo oooooooooooo oooooooooooo

Crude extract

Screening

-100X in DMSO

•••ooooooooo oooooooooooo 000ooooooooo oooooooooooo oooooooooooo oooooooooooo oooooooooooo oooooooooooo

FIGURE 6.8 Prefractionation of natural products to generate improved samples for screening.

respiration inhibitors that are highly toxic to eukaryotic cells. Therefore, if one were screening in a rodent model for antiparasitic activity, a crude extract containing both the milbemycin and oligomycin would likely only show the toxicity. On the contrary, if the components were resolved chromatographically prior to screening the cryptic antiparasitic effect of the milbemycin would also be observed.

Apart from unmasking activities, there are benefits that accrue from enhancing the concentration of minor components present in a complex mixture. This is particularly true if in the preparation of fractionated screening samples the effort is made to normalize the concentration of the samples. The benefits of obtaining the maximum positive responses from these samples, often representing precious material collected under unique conditions, argue for expending the extra effort required.

In the course of resolving "hits" from natural product screening through bioassay-guided frac-tionation, as shown in Figure 6.6, it was emphasized that this is an empirical and highly experimental process, each new extract requiring an individual strategy for the isolation of its biologically active principles. If one has prefractionated the extract prior to initial screening, and the activity falls into a neat cluster of fractions, then one has valuable information on how to begin the purification process. This information will facilitate the resolution of the hit and lead to greater efficiency of the entire isolation and purification process.

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