The active principles of plant extracts with antithyrotropic activity: oxidation products of derivatives of 3,4-dihydroxycinnamic acid.
The active principles of plant extracts with antithyrotropic activity: oxidation products of derivatives of 3,4-dihydroxycinnamic acid.
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DOI：
10.1210/endo-116-5-1677
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发表时间：
1985-05
期刊：
影响因子：
4.8
通讯作者：
M. Auf’mkolk;S. Amir;K. Kubota;S. Ingbar
中科院分区：
文献类型：
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作者：
M. Auf’mkolk;S. Amir;K. Kubota;S. Ingbar
我们最近报道了某些植物的冻干提取物（FDE）与牛TSH （bTSH）形成高分子量加合物，阻止其结合并刺激人甲状腺膜上的腺苷酸环化酶。目前，我们已经研究了34种与番茄或石芋中FDE相同或结构相关的纯化合物，石芋和石芋是之前研究过的三种活性植物中的两种。在20 mM Tris-HCl-0.5% BSA缓冲液中，pH为7.45，在4℃下进行的研究中，8种3,4-二羟基化化合物（结构上均与肉桂酸相关）抑制了[125I] bTSH与人甲状腺膜的结合。其中，4种（咖啡酸、迷迭香酸、绿原酸和鞣花酸）存在于植物中，4种（3,4-二羟基苯基乙酸、去氧肾上腺素、腺色素和去二氢瓜苷酸）在结构上与之相关。这些化合物在直接测试时是无活性的，但在允许进行自动氧化时变得有活性。在所有8种化合物中，对[125I]bTSH结合的半最大抑制作用需要相当于原化合物20-80微克/毫升（60-195微米）的氧化产物量。在含50 mM NaCl的37℃培养基中进行实验时，氧化咖啡酸和鞣花酸的半最大抑制浓度增加了2- 4倍。用浓度高达100微克/毫升的活性氧化产物对膜进行预孵育，然后清洗，对随后的[125I]bTSH结合没有影响。正如FDE案例所显示的那样，当[125I]bTSH与咖啡酸和鞣花酸的氧化产物预孵卵，然后在Sephadex G-100上进行色谱分析时，其洗脱模式从表观摩尔重量为30,000提前到空穴体积，并且早期洗脱部分中的[125I]bTSH与甲状腺膜制品的结合大大减少。在预孵育期间加入大量未标记的bTSH，可以防止这些氧化产物产生的[125I]bTSH的洗脱模式发生变化。为了确定FDE和活性化合物是否与bTSH的蛋白质或碳水化合物部分相互作用，还研究了它们对125i -去糖基化-bTSH （dg-bTSH）的结合和色谱行为的影响。效果与完整的bTSH相似，表明它们不与TSH的碳水化合物部分相互作用。在McKenzie小鼠实验中，将bTSH和dg-bTSH与活性FDE或咖啡酸或迷迭香酸的氧化产物预先孵化也大大降低了它们的活性。（摘要删节为400字）
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)