SCREENING NATURAL-PRODUCTS - BIOASSAY-DIRECTED ISOLATION OF ACTIVE COMPONENTS BY CENTRIFUGAL PARTITION CHROMATOGRAPHY

A Centrifugal Partition Chromatograph (CPC), Model LLN by Sanki, was used on routine basis as a primary tool for over 200 assay-directed fractionations in search of active principles from extracts of natural products. Various extracts were found in several receptor and enzymatic assays incorporated into a high capacity screening system targeting discovery of new antiiflammatory, immunomodulating and antiviral agents. Centrifugal partition chromatography is well suited for performing the assay-directed fractionations, since like other countercurrent techniques it does not involve solid phase adsorbents and thus is inherently less destructive. The instrument was found reliable, simple to operate and applicable to the entire range of polarity of natural products. It can handle milligram as well as multigram quantities. This method alone was applied to a mixture of saponins from an extract of Alysicarpus sp. Isolation and subsequent identification of four new saponins, Alysicarpins A, B, C, and D allowed a necessary in vitro evaluation of their therapeutic potential. An extract of Psychotria acuminata, a tropical forest plant, gave inhibitory response in another assay. Two crucial fractionation steps carried out by the CPC increased the concentration of the active components over 100 times. These steps greatly facilitated isolation of two plant pigments, Pheophorbide A and Pyropheophobide A, each present in the dry plant material at a concentration of 0.5 ppm. The high resolving power of the 1000E (prep.) cartridges was determined by a direct comparison with the 250W (analyt.) cartridges. The 1000E cartridges were applied to a scale up isolation of a minor (50 ppm) bioactive component.