PubMed1985
We have recently reported that freeze-dried extracts (FDE) of certain plants form high molecular weight adducts with bovine TSH (bTSH), preventing it from binding to and stimulating adenylate cyclase in human thyroid membranes. We have now studied 34 pure compounds identical or structurally related to compounds present in FDE from Lycopus or Lithospermum, 2 of the 3 species of active plants studied previously. In studies conducted at 4 C in 20 mM Tris-HCl-0.5% BSA buffer, pH 7.45, eight 3,4-dihydroxylated compounds, all structurally related to cinnamic acid, inhibited the binding of [125I] bTSH to human thyroid membranes. Of these, 4 (caffeic, rosmarinic, chlorogenic, and ellagic acids) are present in the plants, and 4 (3,4-dihydroxyphenylacetic acid, deoxyepinephrine, adenochrome, and nordihydroguaretic acid) are structurally related thereto. These compounds were inactive when tested directly but became active when allowed to undergo auto-oxidation. With all 8 compounds, half-maximum inhibition of [125I]bTSH binding required quantities of oxidized product equivalent to 20-80 micrograms/ml (60-195 microM) of the original compound. Half-maximum inhibitory concentrations of oxidized caffeic and ellagic acids were increased 2- to 4-fold when experiments were performed at 37 C in medium containing 50 mM NaCl. Preincubation of membranes with active oxidation products in concentrations up to 100 micrograms/ml, followed by washing, had no effect on the subsequent binding of [125I]bTSH. As has been shown in the case of FDE, when [125I]bTSH was preincubated with oxidation products of caffeic and ellagic acids and was then chromatographed on Sephadex G-100, its elution pattern was advanced from an apparent mol wt of 30,000 to the void volume, and [125I]bTSH in the early eluting fractions displayed greatly reduced binding to thyroid membrane preparations. Addition of a large excess of unlabeled bTSH during preincubation prevented the shift in the elution pattern of [125I]bTSH produced by these oxidation products. To ascertain whether FDE and active compounds interact with the protein or carbohydrate moieties of bTSH, studies of their effects on the binding and chromatographic behavior of 125I-deglycosylated-bTSH (dg-bTSH) were also performed. Effects were similar to those observed for intact bTSH, suggesting that they do not interact with the carbohydrate moiety of TSH. Preincubation of both bTSH and dg-bTSH with either active FDE or oxidation products of caffeic or rosmarinic acid also greatly decreased their activity in the McKenzie mouse assay.(ABSTRACT TRUNCATED AT 400 WORDS)