Fresh sample preparation and extraction. Mature fruit and leaves used in this study were collected in late December at the Citrus Variety Collection at the University of California, Riverside. Fresh samples were stored frozen in sealed clear polyethylene plastic bags (168 mm × 150 mm) (Glad-Lock zipper sandwich bags, First Brands Corp., Danbury, CT*) at 20 °C for up to 3 months until they could be processed for extraction. Comparison of the chromatographic profiles of extracts prepared from fresh samples and from the same samples that had been stored frozen for over one year showed there was little degradation of the phenolic content during freezing.
About 200 mg of thawed flavedo and albedo tissues, 100 mg of thawed leaf tissue, or 500 mg of thawed juice vesicles were ground or cut into small pieces, weighed, and placed in a 1.5-mL plastic microfuge tube. One mL of a dimethylsulfoxide:methanol solution (1:1) was added to each tube. The sample was thoroughly mashed with a spatula, then allowed to soak at 25 °C for 2 hr or longer. The sample was remashed with the spatula every 30 min during the 2-hr soaking period. The extracts were then processed for HPLC analysis as outlined below or returned to 20 °C storage until they could be processed.
Dried leaf sample preparation and extraction. Collected leaves were oven-dried at 60 °C in brown paper bags for at least 24 hr. The dried leaves were then ground into a fine powder by one of two methods. Samples of less than 10 leaves were ground to a fine powder with a mortar and pestle with the addition of a small amount of sand. Larger samples of more than 10 leaves were ground to a powder in a small electrically driven coffee bean mill. The dried, powdered samples could be stored indefinitely in a refrigerator at 4 °C until time of extraction. Samples (approximately 250 mg) were extracted with 1 mL of a 1:1 dimethylsulfoxide:methanol mixture at 50 °C. Each extraction was allowed to soak for at least 1 hour. After centrifugation, the supernatent was decanted, the pellet re-extracted with an additional 1 mL of dimethylsulfoxide:methanol, centrifuged, decanted, and extracted a third time. The three extractions of each sample were combined and prepared for HPLC analysis.
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Original posting: April 1, 1999.