Biophytis, Sorbonne Université, Paris, France.
Paris-Seine Biology Institute (BIOSIPE), CNRS, Sorbonne Université, Paris, France.
Phytochem Anal. 2020 Sep;31(5):643-661. doi: 10.1002/pca.2930. Epub 2020 Mar 17.
Phytoecdysteroids are analogues of arthropod steroids occurring in plants. They contribute to invertebrate deterrence. A wide diversity of ecdysteroids occurs in phytoecdysteroid-containing plant species, sometimes in high amounts. Ecdysteroids demonstrate potentially useful pharmaceutical actions in mammals.
Establish reversed-phase high-performance liquid chromatography with tandem mass spectrometry (RP-HPLC-MS/MS) and RP-HPLC-DAD-MS (diode array detector mass spectrometry) methods for the separation, identification and quantification of ecdysteroids to screen for species containing significant amounts of 20-hydroxyecdysone (20E) and other useful ecdysteroids.
Micro-extracts of seed samples (ca. 30 mg) in 50% ethanol were subjected to RP-SPE (solid-phase extraction) purification prior to analysis by RP-HPLC-MS/MS and RP-HPLC-DAD-MS. The method was initially applied to genera (Amaranthus, Centaurea, Lychnis, Ourisia, Serratula, Silene and Trollius) where high-accumulating species had been previously encountered. Seeds of 160 randomly selected species, many of which have not previously been assessed, were then analysed. HPLC-MS/MS with a short analysis time initially identifies ecdysteroid-positive extracts and quantifies 20E. The positive extracts (20 ng 20E) are then analysed by HPLC-MS/MS with a longer analysis time to identify and quantify 17 common phytoecdysteroids and, finally, HPLC-DAD-MS (0.1-0.25 μg 20E) is used to obtain UV- and MS-spectra to confirm identifications or as a basis for characterisation of partially identified or novel analogues.
Lychnis coronaria, Silene fimbriata and Silene hookeri ecdysteroids are characterised for the first time and those of Cucubalus baccifer and Ipheion uniflorum are more extensively characterised.
The procedure provides a rapid/sensitive method for screening small plant samples for the presence, quantification and identification of ecdysteroids. It permits ready dereplication of samples, identifying extracts containing large amounts or novel analogues.
植物源性蜕皮甾酮是节肢动物类固醇的类似物，存在于植物中。它们有助于阻止无脊椎动物的侵害。含有植物源性蜕皮甾酮的植物物种中存在着广泛多样的蜕皮甾酮，有时含量很高。蜕皮甾酮在哺乳动物中表现出潜在的有用的药物作用。
建立反相高效液相色谱-串联质谱（RP-HPLC-MS/MS）和反相高效液相色谱-二极管阵列检测器-质谱（RP-HPLC-DAD-MS）方法，用于分离、鉴定和定量蜕皮甾酮，以筛选含有大量 20-羟基蜕皮甾酮（20E）和其他有用蜕皮甾酮的物种。
将约 30mg 的种子样品的微提取物置于 50%乙醇中，然后进行反相固相萃取（SPE）纯化，再通过 RP-HPLC-MS/MS 和 RP-HPLC-DAD-MS 进行分析。该方法最初应用于先前遇到高积累物种的属（苋属、矢车菊属、雀麦属、Ourisia 属、Serratula 属、Silene 属和 Trollius 属）。然后分析了 160 种随机选择的物种的种子，其中许多物种以前没有进行过评估。使用短分析时间的 HPLC-MS/MS 最初可以鉴定出含有蜕皮甾酮的提取物，并定量 20E。然后对阳性提取物（20ng 20E）进行更长分析时间的 HPLC-MS/MS 分析，以鉴定和定量 17 种常见的植物源性蜕皮甾酮，最后使用 HPLC-DAD-MS（0.1-0.25μg 20E）获得 UV 和 MS 光谱，以确认鉴定结果，或作为部分鉴定或新型类似物的特征的基础。
首次对 Lychnis coronaria、Silene fimbriata 和 Silene hookeri 的蜕皮甾酮进行了表征，并且对 Cucubalus baccifer 和 Ipheion uniflorum 的蜕皮甾酮进行了更广泛的表征。
该程序提供了一种快速/敏感的方法，用于筛选小植物样品中蜕皮甾酮的存在、定量和鉴定。它允许对样品进行快速重复鉴定，鉴定含有大量或新型类似物的提取物。
