Effects of pH, sample size, and solvent partitioning on recovery of soluble phenolic acids and isoflavonoids in red clover (Trifolium pratense cv. Kenland)

Authors: Kagan, Isabelle

Submitted to: Natural Product Communications
Publication Type: Research Notes
Publication Acceptance Date: 9/26/2011
Publication Date: 11/16/2011
Citation: Kagan, I. 2011. Effects of pH, sample size, and solvent partitioning on recovery of soluble phenolic acids and isoflavonoids in red clover (Trifolium pratense cv. Kenland). Natural Product Communications. 6:1567-1660.

Interpretive Summary: Red clover is a protein-rich forage crop that is also rich in phenolics, phytochemicals that have many different effects on human and animal health. In order to determine how to extract an optimal amount of phenolics from the Kenland cultivar of red clover, samples were extracted in several different ways, varying the extraction conditions one at a time and comparing the effects of those variations on the types and amounts of phenolics recovered. Soluble phenolics (those stored by plants in the vacuole of the cells) and bound phenolics (part of the cell wall) were extracted and analyzed. Adding an additional extraction step for soluble phenolics (taking an acidic extract and recovering phenolics in a neutral extract) did not change the overall profiles of the extract, but the yields of phenolics were lower and more variable than they were when quantified in the original extract. Therefore, although this step is necessary for some procedures, it should be avoided when trying to determine exact amounts of phenolics in a sample. Bound phenolics did not respond uniformly to changes in extraction conditions (some compounds were extracted more efficiently by smaller volumes of solvent, while others were extracted less efficiently), and the amount of information gained from the bound phenolic extracts was minimal. Extracting from less tissue reduced the amounts of all soluble phenolics extracted, by an amount proportional to the change in tissue mass.

Technical Abstract: Several extraction parameters were tested to determine optimal conditions for extracting phenolics from red clover (Trifolium pratense L. cv. Kenland). HPLC-UV profiles were compared before and after partitioning a methanolic extract of soluble phenolics with ethyl acetate-ether (1:1, v/v). The effect of extract pH on the partitioning of phenolics into the ethyl acetate-ether phase was evaluated as well. Solvent volumes were varied in extraction of bound phenolics to assess the effects on recovery, and tissue mass was varied to assess the effects on HPLC profiles of soluble and bound phenolics. HPLC profiles of soluble phenolics were similar in the methanolic extracts and the partitioned ethyl acetate-ether extracts, but peak areas in the latter were more variable. Recoveries in the ethyl acetate-ether extracts were generally higher if the pH of the methanolic extract was lowered to 2 before partitioning. Bound phenolic profiles changed in variable ways in response to smaller extraction volumes, and the amount of information gained from the profiles was minimal. In methanolic extracts of soluble phenolics, peak areas were directly proportional to the amount of tissue extracted.