Archangelolide: A sesquiterpene lactone with immunobiological potential from Laserpitium archangelica

Sesquiterpene lactones are secondary plant metabolites with sundry biological effects. In plants, they are synthesized, among others, for pesticidal and antimicrobial effects. Two such compounds, archangelolide and trilobolide of the guaianolide type, are structurally similar to the well-known and clinically tested lactone thapsigargin. While trilobolide has already been studied by us and others, there are only scarce reports on the biological activity of archangelolide. Here we present the preparation of its fluorescent derivative based on a dansyl moiety using azide–alkyne Huisgen cycloaddition having obtained the two sesquiterpene lactones from the seeds of Laserpitium archangelica Wulfen using supercritical CO2 extraction. We show that dansyl-archangelolide localizes in the endoplasmic reticulum of living cells similarly to trilobolide; localization in mitochondria was also detected. This led us to a more detailed study of the anticancer potential of archangelolide. Interestingly, we found that neither archangelolide nor its dansyl conjugate did exhibit cytotoxic effects in contrast to the structurally closely related counterparts trilobolide and thapsigargin. We explain this observation by a molecular dynamics simulation, in which, in contrast to trilobolide, archangelolide did not bind into the sarco/endoplasmic reticular calcium ATPase cavity utilized by thapsigargin. Last, but not least, archangelolide exhibited anti-inflammatory activity, which makes it promising compound for medicinal purposes.


General techniques and apparatus
For thin layer chromatography (TLC), we used plates coated by silica gel bound with starch for detection in UV light (TLC Silica gel 60 F254, Merck). For visualization, diluted sulfuric acid in MeOH was used and the plates were then heated. For column chromatography, silica gel (30-60 μm, SiliTech, MP Biomedicals) was used.  Table S1. For column chromatography, silica gel (30-60 μm, SiliTech, MP Biomedicals) was used. The click reactions were carried out in a microwave reactor Biotage Initiator Classic. Table S1: 13 C and 1 H NMR data of compounds 1, 3, 4 and 5 in CDCl3. Data for a substituent in the position 11 in compounds 4 and 5 are shown in Figure S1.   Figure S1: Assignments of the 1 H and 13 C signals for the azidopentanoate and "dansylated" residues.

IR spectroscopy, optical rotations
Archangelolide ( For isolation of archangelolide, fine ground seeds of L. archangelica were used ( Figure S2).

HPLC Analyses of the tested compounds
HPLC analyses were performed using C18 column (0.5 × 280 mm) with UV detection. We used gradient elution with a flow rate of 0.8 mL·min −1 with the following systems: system A -100 % water and system B 100 % MeOH. The method was optimized for 15 min with the following gradient:

Fluorescence properties of compound 5
The fluorescence spectra were recorded using a Cary Eclipse fluorescence spectrometer. The sample was dissolved in MeOH to a 10 −4 M solution, which was further diluted with MeOH or 0.01 M phosphate-buffered saline (PBS) to 10 −5 M. The PBS sample contained 10% of MeOH.
An excitation wavelength of 340 nm was used.

Super critical extraction method -why did we use it?
There are multiple advantages to supercritical CO2 extraction over extraction with organic solvents and the procedure is extensively used in extraction of natural compounds [2]. The diffusivity is increased in supercritical CO2 compared to solvent extraction and consequently the duration of the process is reduced [3]. Supercritical CO2 is a good solvent for the rather nonpolar compounds we were extracting and was used in the past to extract other SLs, such as parthenolide [4][5][6]. Furthermore, the extraction can be finetuned by varying pressure as well as S19 temperature, which is favorable for thermally unstable compounds and particularly natural products. Also, the use of organic solvent is significantly reduced during supercritical CO2 extraction compared to conventional solvent extraction [3]. Finally, a slight disadvantage of this procedure may come forward when plant material rich in water is used for extraction [3].
However, this was not the case here, as we used seeds, which generally have low water content.
We admit that we may have not used the method to its full potential. As we know from the literature, the procedure could be optimized to enrich for the desired compound [3], which we did not attempt. However, this does not in any way diminish the fact that supercritical CO2 extraction is a facile and rapid method by means of which we reached our objective, that is, the isolation of archangelolide.