Factors affecting the separation and bioactivity of red clover (Trifolium pratense) extracts assayed against...

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 either ethyl acetate-hexanes (9:1, v/v) or ethyl acetate-methanol (4:1, v/v). Bands on TLC plates were assayed by either overlaying the plates with agar seeded with C. sticklandii, or setting the 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.