A rapid method for the measurement of Leucaena spp Proanthocyanidins by the proanthocyanidin (butanol/HCl) assay

Proanthocyanidin (PA) extraction, sample preparation and proanthocyanidin assay (butanol/HCl) reaction conditions were evaluated for measuring PA in Leucaena spp leaf material. The optimal conditions for extracting PA from leaf tissue are described, with short sequential sonications in 70% aqueous acetone being as efficient as prolonged sequential mechanical agitation. In methanol-based extracts, after back extraction to remove pigments, increasing the water content of the reagent/sample matrix suppressed colour development. The addition of low concentrations of Fe3+ to the butanol/HCl reagent enhanced colour yield, but higher Fe3+ concentrations suppressed colour development. The presence of ascorbic acid in the sample extract was shown to increase colour development. Varying the reagent: sample extract ratio from 4:1 to 6:1 significantly decreased colour yield, but neither ratio was different from 5:1. Optimum conditions for the PA assay were as follows: a water content of 8%, the omission of Fe3+, a reagent: sample extract ratio of 5:1 and the addition of ascorbic acid to the stock PA standard solution to match that contributed by the extract in the final mixture. Sample preparation procedures, using back extraction to remove pigments and non-PA phenolics with diethyl ether and ethyl acetate, respectively, were time-consuming and subject to PA losses. The measurement of PA directly in the 70% aqueous acetone extract eliminated these PA losses, but the PA assay required additional optimisation for direct analysis of crude acetone extracts. In the final optimised procedure, PA was extracted by sequential sonication with 70% aqueous acetone containing 5.26 mM sodium metabisulphite as the antioxidant. These extracts were directly analysed by the butanol/HCl reaction using a reagent: sample extract ratio of 5:1, the omission of Fe3+ from the butanol/HCl reagent and the addition of sodium metabisulphite to match that contributed by the extract. This produced consistent linear calibration curves over the range 25-1000 microgram PA with an average recovery of 101%.