Submitted to: Natural Product Communications
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: November 7, 2012
Publication Date: December 17, 2012
Citation: Kagan, I., Flythe, M.D. 2012. Factors affecting the separation and bioactivity of red clover (Trifolium pratense) extracts assayed against Clostridium sticklandii, a ruminal hyper ammonia-producing bacterium. Natural Product Communications. 12:1557-1690. Interpretive Summary: Red clover contains many phenolics, compounds that cause browning in cut fruit and are known for their antioxidant and medicinal properties. Crude extracts of red clover phenolics, as well as one specific phenolic, biochanin A, had been found previously to inhibit rumen bacteria that convert protein into ammonia. Because inhibiting ammonia production may result in improved weight gain for beef cattle, a study was done to determine if any compounds besides biochanin A inhibited these bacteria. Clover extracts were separated by spotting them onto flexible sheets of silica and letting different mixtures of solvents move up the sheets by capillary action. The different phenolics moved to different heights on the sheets of silica, depending on their molecular structures. When the sheets of silica were viewed under ultraviolet light, the location of different phenolics could be seen as dark bands. These bands were cut out and placed in contact with agar mixed with Clostridium sticklandii, the bovine rumen bacterium whose inhibition was of interest. If an inhibitory compound was present, bacteria did not grow in the agar that came into contact with that compound. Only biochanin A consistently prevented the bacterial growth. It was active in clover extracts that differed in biochanin A content by a factor of ten, suggesting that biochanin A could indeed account for all the inhibitory activity of the red clover phenolic extracts.
Technical Abstract: Red clover (Trifolium pratense) is rich in phenolic compounds. Both the crude phenolic extract and biochanin A, an isoflavonoid component of the extract, suppress growth of Clostridium sticklandii, a bovine, Gram positive, ruminal hyper-ammonia producing bacterium (HAB). The purpose of this study was to determine if other components of red clover extract contributed to its anti-HAB activity. Extracts of the Kenland cultivar of red clover, collected 0 h and 24 h after cutting, were separated by normal-phase thin-layer chromatography (TLC) in ethyl acetate-hexane (9:1, v/v) or ethyl acetate-methanol (4:1, v/v). Bands on TLC plates were assayed by overlaying plates with agar seeded with C. sticklandii, or by setting excised bands face-down onto plates of bacteria-seeded agar. Biochanin A inhibited C. sticklandii growth on TLC plates when as little as 8 nmol was present in the extract. An antimicrobial band, seen in a previous bioassay, was not found, suggesting that this second compound may be more labile than biochanin A.