Spectrophotometric Determination of Unsaturatedi Pyrrolizidine Alkaloids A. R. Mattocks Toxicology Aesearck Unit, Medical Research Council Laboratories, Woodmansterne Road, Carshalton, Surrey, Engfand A color reaction is described with which, for the first time, certain pyrrolizidine alkaloids can be detected and determined in quantities down to a few micrograms. The reaction is specific for alkaloids(and other compounds) having an unsaturated basic moiety (Aapyrroline ring), and hence it is useful for all the markedly hepatotoxic alkaloids, which fall in this class. Procedures are described by which the alkaloids can be distinguished from their N-oxides and from interfering compounds whiich also give a color. Application to the estimation of pyrrolizidine alkaloids in biological materials is briefly discussed.
PYRROLIZIDINE ALKALOIDS occur in widely distributed plant species, especially of the genera Senecio, Crotalaria, Heliotropium, Erechtites, and Trichodesma. Many of these alkaloids are of considerable vlzterinary and medical interest and economic importance, because of their toxic action on grazing animals, and probably on human populations in underdeveloped regions where they may find their way into foods, herbal medicines, or bush teas. Watt and Breyer-Brandwijk ( I ) give many examples, The toxic action takes the form of chronic liver damage, and is caused only by alkaloids which have inter a h , an allylic ester group, as exemplified by retrorsine (I), lasiocarpine (III), and indicine (IV). Alkaloids which do not possess this group are not hepatotoxic--e.g., retronecine (V), strigosine (VI) and rosmarinine (VII). N-Oxides of hepatotoxic alkaloids--e.g., retrorsine N-oxide
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(isatidine, 11)-which usually occur together with the free bases in the plant, show similar toxicity, though to a lesser extent. Chemical, and especially toxicological, investigations of these alkaloids have hitherto been hindered by the lack of a sensitive and specific method for their detection and estimation. Such a method is now described. The method depends on a Polonovsky reaction ( 2 ) as applied to the 3-pyrroline ring system (3), whereby the base (VIII) is oxidized to the N-oxide (IX) which, when heated with acetic anhydride, is dehydrogenated to a pyrrole. (This was employed by Dann (4) as a means of detecting pyrrolizidine alkaloid N-oxides on paper chromatograms, the fluorescence of the pyrroles being observed.) The pyrrole is then coupled with 4-dimethylaminobenzaldehyde (Ehrlich reagent) to give a color which is measured spectrophotometrically.
(1) J. M. Watt and M. G. Breyer-Brandwijk, “Medicinal and Poisonous Plants of Southern and Eastern Africa,” 2nd ed., E. & S. Livingstone, Ltd., Edinburgh & London, 1962, pp. 257-89 and 571-90. (2) M. Polonovsky and M. Polonovsky, Bull. SOC.Chim. France, 41,1190(1927). (3) R . Kreher and H. Pawelczyk, Angew. Chem. Ititertr. Ed. Engl., 3,510 (1964). (4) A. T. Dam, Nature, 186,1051 (1960).
YHaOH C- OH
YHzOH
CH3CH=C-CH2-CH-C-OH I I YHzOCO COY
CHaCH=C-CHz-CH-
(io
t I
0
I1 CH3 CH3 \/ C-OH
HC - CH3
II
CH3-C-COO
I
CHzOCOq-VHCH3
