Generation of Highly Antioxidant Submicron Particles from Myrtus communis Leaf Extract by Supercritical Antisolvent Extraction Process

Abstract

Submicron particles have been produced from an ethanolic extract of Myrtus communnis leaves using supercritical carbon dioxide technology, hereinafter referred to as Supercritical Antisolvent Extraction (SAE). The influence of pressure (9-20 MPa), temperature (308 and 328 K) and injection rate (3 and 8 mL/min) on the particles' precipitation has been investigated, and it has been confirmed that increases in pressure and temperature led to smaller particle sizes. The obtained particles had a quasi-spherical shape with sizes ranging from 0.42 to 1.32 μm. Moreover, the bioactivity of the generated particles was assessed and large contents of phenolic compounds with a high antioxidant activity were measured. The particles were also subjected to in vitro studies against oxidative stress. The myrtle particles demonstrated cytoprotective properties when applied at low concentrations (1 μM) to macrophage cell lines.

Keywords: Myrtus communis; ROS; carbon dioxide; microparticles; supercritical antisolvent.

Figure 1

Schematic diagram of precipitation equipment.

Figure 2

SEM images of the particles precipitated from myrtle leaf extracts.

Figure 3

Precipitated powder obtained on a vessel by means of the SAE of myrtle leaf extract.

Figure 4

Pareto diagram representing the effect of different parameters on the particle size. “+” (positive effect) “−” (negative effect).

Figure 5

Pareto diagram representing the effect of different parameters on the TPC. “+” (positive effect) “−” (negative effect).

Figure 6

Mass spectra for the identification of compounds in run 8.

Figure 7

Antioxidant activity of processed particles.

Figure 8

Cytoprotective and antioxidant role of myrtle particles in murine macrophage cell lines. (A) Viability of the cells treated with 250 µM tBHP for 1 h according to MTT assay. Cytoprotective effect of the different concentrations of myrtle particles ranging from 1 to 50 µM. Antioxidant properties at a concentration of 1 µM myrtle particles (B) Advanced Oxidation Protein Products (AOPPs). qPCR assays for genes encoding free radical scavenging enzymes: SOD1 (C), CAT (D) and GPX (E). The expression values represent four biological replicates and are shown relative to the β-Actin expression as a housekeeping gene. The mRNA levels of the control group for each gene were set at 1. Activity of the antioxidant enzymes superoxide dismutase (F), catalase (G) and glutathione peroxidase (H). (I) Reduced glutathione content. mRNA levels of the catalytic (J) and modifier (K) subunits of the Glutamate-cysteine ligase. The expression values represent four biological replicates and are shown relative to the β-Actin expression as a housekeeping gene. The mRNA levels of the control group for each gene were set at 1. The results are represented as the mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. “+” (compound added) “–” (not added). Circles: control; squares: 250 µM tBHP added; up triangle: co-treatment with tBHP + particle; down triangle: only particle treatment.