Soluble phenolic compounds in different cultivars of red clover and alfalfa, and their implication for protection against proteolysis and ammonia production in ruminants

Authors: Kagan, Isabelle; GOFF, BEN - University Of Kentucky; Flythe, Michael

Submitted to: Natural Product Communications
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/26/2015
Publication Date: 7/1/2015
Citation: Kagan, I., Goff, B.M., Flythe, M.D. 2015. Soluble phenolic compounds in different cultivars of red clover and alfalfa, and their implication for protection against proteolysis and ammonia production in ruminants. Natural Product Communications. 10(7):1263-1267.

Interpretive Summary: Red clover is considered a good source of protein for ruminants because the plant proteins do not break down as quickly in the rumen as they do in other legumes, such as alfalfa. Consequently, more protein is absorbed by the animal. This trait of red clover is thought to be due to its abundance of phenolic compounds, plant natural products that can bind to proteins and interfere with breakdown. Also, at least one red clover phenolic compound inhibits the growth of ruminal hyper ammonia-producing bacteria (HAB), rumen bacteria that interfere with protein absorption by converting amino acids (protein building blocks) into ammonia. Alfalfa has been found to have smaller amounts of phenolics, but amounts have been examined in relatively few cultivars of red clover or alfalfa. In this study, nine red clover and 27 alfalfa cultivars were extracted, and the phenolic content was analyzed by a colorimetric assay. In that assay, the average alfalfa phenolic content was about 40% of the average red clover phenolic content. All red clover cultivars, and 11 of the alfalfa cultivars, were separated by chromatographic techniques to be able to measure individual phenolics and their relative contributions to the total. All red clover cultivars had similar compounds, in similar amounts. The alfalfa cultivars were similar to each other in terms of the compounds present, but these were different from the compounds in red clover, and they were not identified. Total phenolic content of alfalfa was only 5% of the phenolic content of red clover when determined by chromatography. Extracts of two red clover cultivars were separated by thin-layer chromatography and tested for the presence of phenolics that could inhibit HAB. One compound was active. Inhibitory amounts confirmed that four red clover cultivars would likely be suitable sources of anti-HAB activity.

Technical Abstract: Red clover contains phenolic compounds with roles in inhibiting proteolysis and loss of amino acids as ammonia. Alfalfa has been found to have lower concentrations of phenolic compounds, but few alfalfa and red clover cultivars have been compared for phenolic content. Total soluble phenolic compounds were quantified by a Folin-Ciocalteu colorimetric assay in nine red clover (Trifolium pratense) and 27 alfalfa (Medicago sativa) cultivars. The mean soluble phenolic contents of red clover and alfalfa, in caffeic acid equivalents, were 203 ± 25 µmol/gdw and 87 ± 8 µmol/gdw, respectively. Because different standards had different response factors in the colorimetric assay, the red clover and 11 alfalfa cultivars were analyzed by HPLC to determine if the differences in total soluble phenolic content reflected differences in the amounts of phenolics present or in the responses of the dominant phenolics to the colorimetric assay. Phenolic profiles were similar among the red clover cultivars. Alfalfa produced different phenolic compounds from red clover, at 5% of the concentration of red clover phenolics. Extracts of two red clover cultivars were separated by thin-layer chromatography (TLC), and the bands were assayed for activity against Clostridium sticklandii, a bovine ruminal hyper ammonia-producing bacterium (HAB). Only biochanin A had anti-HAB activity. Inhibitory amounts confirmed that four red clover cultivars would likely be suitable sources of anti-HAB activity.