Ecdysteroids represent a particularly versatile group of natural products due to their chemical variability and the broad range of bioactivities they can exert. They are best known as analogues of the insect-molting hormone 20-hydroxyecdysone (20E). Their polar, polyhydroxylated character hinders the absorption of typical phytoecdysteroids through the cuticle of insects; in contrast, they need to be consumed to function as insect hormones, which prevents their use as sprays in pest management. Nevertheless, these compounds serve as models for the rational design of synthetic analogues, (1,2) rendering the study of their structure–activity relationships important. Ecdysteroids are also bioactive in mammals; some of their representatives, including 20E and its metabolite poststerone, (3,4) act as nonhormonal, green anabolic agents and adaptogens and offer a wide range of metabolic benefits. As a result, their consumption is typically considered “healthy”. This has led to the production and worldwide marketing of ecdysteroid-containing herbal extracts (5) for various purposes, particularly as anabolic food supplements for athletes. A simple Internet search revealed that ecdysteroid-containing extracts typically prepared from the roots of Cyanotis arachnoidea C.B. Clarke (Commelinaceae) are available for online purchase up to a scale of several metric tons per month at highly competitive prices; depending on the purity, some companies offer extracts at 1 USD/kg. The roots of C. arachnoidea are indeed very rich in ecdysteroids, containing as much as 2–3% of these compounds. Several studies reported the isolation of minor phytoecdysteroids from this plant, comprising a total of 22 compounds. (6−10)
Over the past few decades, attempts to translate the chemical complexity of ecdysteroids into possible pharmacological use(s) have been performed in two major directions, namely, (i) the isolation and bioactivity evaluation of new natural compounds and (ii) the extension of the chemical space of these compounds by performing semisynthetic modifications to improve/optimize certain bioactivities and achieve new ones. In the context of this latter strategy, our group has investigated certain structure–activity relationships to explore the effect of these compounds on the drug resistance of cancer cell lines. (11−14)
Unfortunately, the study of the bioactivity of ecdysteroids has been limited by their availability in sufficient amounts. Despite their very high structural diversity, which has led to the discovery of 526 natural ecdysteroids as of November 2020, (15) the ecdysteroid composition of plants is always dominated by a few major compounds. Among these, 20E is by far the most abundant, and other analogues are present in much lower amounts. Therefore, much research effort has been devoted to the preparation of rare phytoecdysteroids from 20E via semisynthesis. In the present work, our aim was to initiate a large-scale phytochemical investigation into the potential of commercial Cyanotis extracts as valuable and plentiful raw materials of ecdysteroids. Although this is a rather unorthodox way to initiate a phytochemical study because the truly biosynthetic or artifactual origin of any compound isolated from such a preprocessed raw material is unknown, these studies are of importance because of the large amounts of Cyanotis extracts that are consumed by humans worldwide. Further, the industrial-scale availability of these extracts provides a stable background for a large-scale production and further development of new bioactive compounds for their use as pure substances. As a starting point of our in-depth evaluation of the biological value of compounds present in commercial Cyanotis extracts, we first aimed to test newly isolated ecdysteroids for their insect hormone activity, which could pave the way toward the development of new biological and green synthetic plant-protecting agents.
Inspired by an unexpected outcome of a previous study conducted by our group, in which the chromatographic processing of only 5 g of a C. arachnoidea-containing food supplement led to the discovery of two new ecdysteroids, (5) it was decided to initiate an extensive preparative work on a much larger scale (several kilograms) to search for new, minor bioactive ecdysteroid derivatives. It needs to be stressed that in this work the starting material was not a ground plant but an industrial extract purchased online. Nevertheless, the starting material showed a qualitative minor constituent fingerprint using high-performance liquid chromatography (HPLC) photodiode array (PDA), which conformed with that of other C. arachnoidea extracts that were independently purchased and used in previous related studies. This supported the manufacturer’s declaration on the botanical identity of the source plant.
Because of the extremely rich ecdysteroid composition of the starting material, an extensive, multistep chromatographic purification was required to obtain the minor compounds. It is worth mentioning that this procedure led to the isolation of many ecdysteroids that are out of the scope of this contribution. Here, we report and discuss 10 compounds (1–10) that were successfully obtained in this study. For the structural elucidation, we performed a comprehensive one- and two-dimensional (1D and 2D, respectively) NMR analysis, (16,17) achieving a complete 1 H and 13 C NMR signal assignment for all the investigated compounds. Because of the molecular mass of the compounds (∼500 Da), the signal/noise value of the selective rotating-frame Overhauser effect (ROE) experiments strongly exceeded those of the selective nuclear Overhauser effects (NOEs). The 1 H and 13 C NMR chemical shifts of compounds 1–10 are compiled in Tables 1–3. Characteristic NMR spectra of these compounds, along with their stereostructures, 1 H and 13 C assignments, and characteristic HMBC correlations and steric proximities, are presented in Figures S1–S69, Supporting Information.
Run in DMSO-d 6. Run in MeOH-d 4.
