Supercritical Carbon Dioxide Extraction of Coumarins from the Aerial Parts of Pterocaulon polystachyum

Abstract

Pterocaulon polystachyum is a species of pharmacological interest for providing volatile and non-volatile extracts with antifungal and amebicidal properties. The biological activities of non-volatile extracts may be related to the presence of coumarins, a promising group of secondary metabolites. In the present study, leaves and inflorescences previously used for the extraction of essential oils instead of being disposed of were subjected to extraction with supercritical CO₂ after pretreatment with microwaves. An experimental design was followed to seek the best extraction condition with the objective function being the maximum total extract. Pressure and temperature were statistically significant factors, and the optimal extraction condition was 240 bar, 60 °C, and pretreatment at 30 °C. The applied mathematical models showed good adherence to the experimental data. The extracts obtained by supercritical CO₂ were analyzed and the presence of coumarins was confirmed. The extract investigated for cytotoxicity against bladder tumor cells (T24) exhibited significant reduction in cell viability at concentrations between 6 and 12 μg/mL. The introduction of green technology, supercritical extraction, in the exploration of P. polystachyum as a source of coumarins represents a paradigm shift with regard to previous studies carried out with this species, which used organic solvents. Furthermore, the concept of circular bioeconomy was applied, i.e., the raw material used was the residue of a steam-distillation process. Therefore, the approach used here is in line with the sustainable exploitation of native plants to obtain extracts rich in coumarins with cytotoxic potential against cancer cells.

Keywords: Pterocaulon polystachyum; cell viability assay; coumarin; mathematical modeling; supercritical fluid extraction.

Figure 1

Response surfaces for effects of two independent variables on the global yield of the extract obtained by supercritical extraction: (a) extraction pressure (P) and pretreatment temperature (TP); (b) extraction temperature (TE) and extraction pressure (P); (c) pretreatment temperature (TP) and extraction temperature (TE).

Figure 2

Curve of the optimal condition for supercritical fluid extraction: experimental data and adjustment of mathematical models.

Figure 3

TLC of the 13 different extracts under UV light (365 nm).

Figure 4

(A) UFLC chromatogram profile of the extract obtained from plant material subjected only to SFE, (B) SFE extract obtained from plant material previously subjected to steam distillation and (C) SFE extract obtained from plant material previously subjected to steam distillation and subsequently exposed to microwaves.

Figure 5

UV absorption spectra (327 nm) of the compounds indicated by peaks 1–5 in the chromatograms shown in Figure 4A–C.

Figure 6

Coumarins characterized shown in Figure 4.

Figure 7

Cytotoxic effect of extract obtained by supercritical CO₂ on the viability of T24 cell line. At 80-90% of confluence, T24 cells were treated with 6, 12, 25, 50, 100, and 200 µg.mL⁻¹ of extract obtained by supercritical CO₂ for 24 h. The data were analyzed for statistical significance using one-way ANOVA, followed by Tukey’s post hoc test. C+ is the control with the vehicle (ethanol) in which the extract was solubilized. **** p < 0.0001.