Improved method for isolation of hypoglycins A and B from fruit of Blighia sapida

ABSTRACT

This report describes an isolation procedure for obtaining á-(methylenecyclopropyl) alanine, and its ç-glutamyl peptide (hypoglycins A and B) from ackee fruit or seeds (Blighia sapida) that is less laborious than previous methos (Hassall & Ryle, 1955; Ellington, Hassall & others, 1959; West, 1968) and gives leucine-free hypoglycin. One kg of arilli from immature ackee fruit were blended in batches with a total of 2 litres of 80 percent ethanol. After filtration through muslin and overnight settling the ethanolic extract was dried under vacuum, and the portion soluble in 0.1n HCI was applied to a column (90 x 4.5 cm) of Dowex 50 (x8) resin in the (H+) form. Several column volumes of 0.1n HCI and of water were passed through the column, after which elution with 1.0M pyridine until the ninhydrin test was negative removed neutral amino-acids with hypoglycin A, as well as acidic amino-acids and hypoglycin B. The eluate was dried by evaporation under vacuum. The residue was taken up in 0.5n acetic acid and placed on a column (70 x 41/2 cm) of Dowex 1 (x8) resin in the acitate form. Elution with 0.5N acetic acid rapidly removed a mixture of neutral amino-acids with hypoglycin A as the main component. This was recovered from the eluate after removing excess solvent by evaporation under vacuum. Crystallization, repeated twice from 50 percent ethanol, routinely yeilded 0.8 to 1.2 g of nearly pure product per kg of starting material. Continued elution with 0.5N acetic acid removed a second zone of ninhydrin-positive material, well separated from the neutral amino-acids. This contained mainly hypoglycin B, present in seeds but not in arilli. This, in turn, was well separated from glutamic and aspartic acids, which appeared subsequently. The identity of hypoglycin A was checked by nmr (Millington & Sheppard, 1968), and paper and thin-layer chromatography using a variety of solvent systems. However, for establishing the level of contamination by leucine and isoleucine, which exhibit similar solubility and chromatographic properties to those of hypoglycin A, it was necessary to perform chromatography of the derivatives formed with dimethylaminonaphtalene-5-sulphonly chloride, using either filter paper (Abrahams & Kean, 1969 or polyamide layer (Woods & Wang, 1967). This technique was used to confirm that, by two or three recrystallizations of hypoglycin A derived from ackee arillus, contamination by these amino-acids could reduced to less than 1 percent. In this respect, seeds were a less desirable starting material, in that the crude product contained relatively larger amounts of leucine and other amino-acids. Hypoglycin B was identified from the products of hydrolysis (boiling with 8N acetic acid for 4 h), which were shown to be hypoglyucin A and glutamic acid. Hypoglycin A with no detectable impurity was thus obtained in yields of up to 0.04 percent (based on the fresh weight of seeds used), by passage of the concentrated hydrolysate through a column of Dowex 1 (acetate form) as described above. Identification of relevant compounds was readily achieved by thin-layer chromatography on silica gel (Eastman Chromatogram Sheets). The solvent system propanol-water (73) gave the Rf values hypoglycin A, 0.73, hypoglycin B 0.51, glutamic acid 0.30. The utility of the procedure lies in the selective elution, by dilute pyridine, of the neutral amino-acid and acidic peptide components in the extract, after adsorption on a strongly acidic cation-exchange resin. This fractionation could not be achieved with dilute ammonia. A solution of 1.0M pyridine is only weakly alkaline (pH 8.0) and this, together with the possibility of graded selectivity of the resin for differentiations, including pyridinium, might explain the retention of the basic amino-acids. Additionally, advantage was taken of the convenient fractionation of hypoglycins A (one amino and one carboxyl group free) and B (one amino and two carboxyl groups free), on a strongly basic anion-exchange resin by dilute acetic acid; this was based on the work of Hirs, Moore & Stein (1954) (AU)