The alkaloids of Papac-cr somanif crun L
- G. FAIRBAIIRiN J.W. A-ND
Ricinine-3,5-"C (N-methyl-3-cyano-4-methoxy-2-pyridone) administered to senescent leaves of Ricinus communis L. was translocated to all other tissues of the plant. Developing fruit and especially seeds were found to be labeled the most rapidly. Young growing leaves and other developing tissues of the plant imported ricinine from the senescent leaves much more quickly than mature leaves. Relative intensities of the radioactive ricinine imported and deposited in various tissues indicate a possible functional role of ricinine in the castor bean plant. Data on N-demethyl ricinine presented here may stimulate interest in the possible physiological role of the ricinine to Ndemethyl ricinine interconversion. A biological function for alkaloids in plants is not yet well established in spite of the extensive knowledge of their biosynthetic pathways (4, 5, 7, 11). The increasing evidence for catabolic reactions of alkaloids (8, 9, 12) indicates that some cannot be considered merely as final products of nitrogen metabolism. Information on the role of a particular alkaloid in the plant can be obtained by studying its catabolic reactions, translocation, and distribution in the parent plant. Only a few reports on the translocation of alkaloids in plants have appeared (1, 5). Intensive catabolism of ricinine in Ricinus cominunis L. has been proven (9, 12). The appearance in immature seeds of radioactive ricinine which had been administered to the plant before it bloomed (12) indicated translocation of the alkaloid into the seed from other tissues. Rapid demethylation of ricinine in senescent leaves (9) posed the question of a possible role of N-demethyl ricinine in the translocation of ricinine. Experiments described in this paper provide information on the translocation of ricinine within the body of the plant, on the relative extent of its deposition in various plant tissues, and on the significance of the N-demethyl ricinine to ricinine interconversion. MATERIALS AND METHODS Plant Material. Castor bean plants (Ricinus coinmiiunis L.) of the Cimarron variety were used. The feeding experiments 1 Journal Article No. 2500 of the Agricultural Experiment Station, Oklahoma State University, Stillwater, Okla. This research was supported in part by research grant GM-08624 from the National Institutes of Health, Bethesda, Md. 2Present address: Department of Biochemistry, Faculty of Science, University J. E. Purkyne, Brno, Czechoslovakia. were conducted on field-grown plants 6 months of age, about 1.2 m high which possessed flower buds, immature and mature fruit, and leaves of various ages. Another experimental plant was grown in the same type of soil in the greenhouse under a nutrient-deficient condition in a small pot in order to develop senescent yellow leaves. At the time this experiment was performed the plant was approximately 2 months old, 45 cm high, and bore no fruit or flowers. Labeled Compound. Ricinine-3, 5-`4C was chemically synthesized (14) and had the specific radioactivity given in "Results." Immediately before use it was purified, using both preparative thin layer and paper chromatography (9, 14) and only one radioactive spot was observed in each system. Administration of Labeled Compound. Labeled ricinine was administered by injecting an aqueous solution of the compound into the veins and petioles of senescent leaves using a 50-,ud syringe (9). Isolation Procedure. Fresh parts of the plants were stored at -18 C and extracted by repeated homogenization of the frozen material with acetone at room temperature using a Sorvall Omnimixer until the residue was completely free of chlorophyll. At this stage the extraction of ricinine was 90 to 95% complete. The debris was weighed and extracted three more times with 80% (v/v) ethanol which resulted in an extraction efficiency greater than 98% for ricinine and the more polar compounds. The pooled acetone extracts were evaporated to dryness under reduced pressure. After addition of a small amount of water to the residue, the chlorophyll and lipids were removed by extraction with ether; the colored ether layer was re-extracted with water to minimize possible loss of ricinine. The combined aqueous phases were evaporated under reduced pressure, combined with the ethanol extract mentioned above, and used for chromatographic separations. Acetone extraction preceded the ethanol treatment, since it prevented the chlorophyll from transesterification, which would occur in ethanol, producing artifact pigments of higher polarity which would interfere with the chromatographic separation of ricinine and more polar compounds. This isolation procedure followed by the chromatographic separation (9) supplied ricinine and N-demethyl ricinine in a purity satisfactory for quantitative analysis by UV. For specific radioactivity determination the compounds were further purified by preparative thin layer chromatography. Analytical Procedures. The methods used for detection and for measuring radioactivity on thin layer chromatographic plates have been previously described (9). Examining the thin layer chromatograms of the plant extracts by the strip counter always revealed about 5 to 10% of total radioactivity at an area where very polar substances accumulated (with RF less than 0.1). This polar radioactive fraction was not examined further. The concentration of ricinine and N-demethyl ricinine isolated by chromatography were determined spectrophotometrically (9) by absorption at 307 and 304 nm, respectively.