MULTIFUNCTIONAL TETRANORTRITERPENOIDS FOR IPM ISOLATED FROM NEEM SEEDS (AZADIRACHTA INDICA A. JUSS. AND A. EXCELSA JACK): A CHALLENGE FOR THE CHEMICAL ECOLOGISTS MET BY MLCCC AT THE MILLIGRAM TO MULTIGRAM SCALE1

摘要

Multi-Layer Counter Current Chromatography (MLCCC) is a high resolution liquid/liquid chromatographic technique with a number of distinctive features. These are most useful for chemical ecologists in their quest of obtaining milligram but also semi-preparative quantities of pure natural products of complicated tetranortriterpenoid structures. It is important to isolate enough material for elemental analysis, spectroscopic identification, and characterization by bioassays as insect antifeedants and modifiers of insect development.Crude, defatted extracts (Schmutterer, 1995, 2002) from neem seeds served as starting material for various purification attempts on the following MLCCC centrifuges suitable for the analytical to the semipre-parative scale: 1. monocolumn, P.C. Inc. 315 mlcoil volume, 2. Triplet CCC-1000, 180 ml volume, and 3. Triplet CPC, 850 ml volume. Biphasic solvent systems composed of hexane, ethyl acetate (or alternatively methyl- tertiary butyl ether), methanol, and water in the approx. 4:5:4:5 ratio were spun at800 to 1000 rpm, with a low pressure pump supplying the mobile phase transporting the mobile solvent phase through the rotating coil column containing the stationary phase. Online detections typically use UV- and/or refractive index-methods. Simultaneous online mass spectrometric analysis with special electrospray inlets is also possible. Azadirachtin (C35H44O16) and structurally closely related marrangin (C35H44O15) structures differ by only 16 daltons and are difficult to separate otherwise. With MLCCC, separations at the ten-gram scale were achieved on centrifuge 3: MLCCC separations typically can be accomplished within one working day with automatic solvent withdrawal in vacuo overnight (Hummel et al., 1997; Hein & Hummel, 1998). Thus, MLCCC has an advantage over repeated and wasteful fractionated adsorption column chromatography runs in which several members of the azadirachtin (aza) group, marrangin, nimbin and salannin -the main components of biological interest - have a tendency to irreversibly stick to solid adsorbents. Under unfavourable conditions they also undergo partial hydrolysis and rearrangement of functional groups. MLCCC works in a purely liquid/liquid partition mode. Its principles have been described earlier (Ito & Zhang, 1988A/B/C; Ito et al., 1989; Conway, 1990; Foucault, 1995; Ito & Conway, 1996).