Extracellular vesicles (EVs) are nanometric membranous structures secreted from almost every cell and present in biofluids. Because EV composition reflects the state of its parental tissue, EVs possess an enormous diagnostic/prognostic potential to reveal pathophysiological conditions. However, a prerequisite for such usage of EVs is their detailed characterisation, including visualisation which is mainly achieved by atomic force microscopy (AFM) and electron microscopy (EM). Here we summarise the EV preparation protocols for AFM and EM bringing out the main challenges in the imaging of EVs, both in their natural environment as biofluid constituents and in a saline solution after EV isolation. In addition, we discuss approaches for EV imaging and identify the potential benefits and disadvantages when different AFM and EM methods are applied, including numerous factors that influence the morphological characterisation, standardisation, or formation of artefacts. We also demonstrate the effects of some of these factors by using cerebrospinal fluid as an example of human biofluid with a simpler composition. Here presented comparison of approaches to EV imaging should help to estimate the current state in morphology research of EVs from human biofluids and to identify the most efficient pathways towards the standardisation of sample preparation and microscopy modes.

Pomegranate (Punica granatum L.) is a rich source of constituents with confirmed strong biological activities. However, pomegranate peel, which encompasses approximately 30–40% of its weight, is treated as a biological waste. The aim of this paper was to evaluate the potential of pomegranate peel extracts and to propose its functional properties that can be used for development of functional products. Eight ethanol extracts of pomegranate peels (PPEs) were characterized by use of direct infusion quadrupole-time of flight (Q-TOF), and afterwards tested on their antioxidant, antibacterial and antiproliferative activities. Mass spectrometry analysis revealed that the most prevalent compounds in pomegranate peels were punicalagin, granatin and their derivatives. Analysed extracts had high total phenolic contents that ranged from 5766.44 to 10599.43 mg GAE/100 g, and strong antioxidant activity (7551.31–7875.42 and 100.25–176.60 μmol TE/100 g for DPPH and FRAP assays, respectively). The results of biological activity assays showed that all PPEs possessed antibacterial activity, and that S. aureus was the most sensitive specie with minimum inhibitory concentration and minimum bactericidal concentrations ranging from 0.8 to 6.4 mg/mL. Additionally, the analysis of antiproliferative activity revealed high potency of PPEs, as the IC50 values ranged from 0.132 mg/mL to 0.396 mg/mL. Multivariate analysis pointed out the most discriminative metabolites for antioxidant or antiproliferative activity. Overall, the pomegranate peel confirmed to be a highly valuable source of bioactive compounds that could be used to improve the food functional characteristics.

Headspace solid-phase microextraction (HS-SPME; PDMS/DVB fibre) and ultrasonic solvent extraction (USE; solvent A: pentane and diethyl ether (1:2 v/v), solvent B: dichloromethane) followed by gas chromatography and mass spectrometry (GC, GC-MS) were used for the analysis of Prunus mahaleb L. honey samples. Screening was focused toward chemical composition of natural organic volatiles to determine if it is useful as a method of determining honey-sourcing. A total of 34 compounds were identified in the headspace and 49 in the extracts that included terpenes, norisoprenoids and benzene derivatives, followed by minor percentages of aliphatic compounds and furan derivatives. High vomifoliol percentages (10.7%–24.2%) in both extracts (dominant in solvent B) and coumarin (0.3%–2.4%) from the extracts (more abundant in solvent A) and headspace (0.9%–1.8%) were considered characteristic for P. mahaleb honey and highlighted as potential nonspecific biomarkers of the honey’s botanical origin. In addition, comparison with P. mahaleb flowers, leaves, bark and wood volatiles from our previous research revealed common compounds among norisoprenoids and benzene derivatives.

In the present paper, chemical composition, in vitro antimicrobial and antiproliferative activity of the essential oil from the flowers and leaves of Helichrysum italicum (Roth) G. Don (Central Dalmatia, Croatia) as a potential replacement for standard antibiotics and chemotherapeutic agents was analyzed. Essential oil was isolated by steam distillation and analyzed by GC/MS. Antimicrobial activity was carried out by agar-well diffusion and microdilution assays with Gram (+), Gram (-) bacteria and one yeast. Antiproliferative effect, apoptosis induction and cell death on cancer cell lines: HeLa, MCF-7, SW620, CFPAC-1 and MIA PaCa-2 were analyzed by cell viability and Annexin-V assay as well as by flow cytometric analysis. In essential oil, α-pinene, γ-curcumene and neryl acetate was found as major compounds. The antimicrobial assays, showed that essential oil had weak to moderate antimicrobial potential with S. aureus and S. epidermidis as the most sensitive bacterial strains. Essential oil treatment possessed moderate antiproliferative impact on MCF-7 and HeLa cell lines, while analyzing cell cycle treatment had no significant effect on tested cells except on MIA PaCa-2 with the highest cells increase in sub G1 phase cell cycle. However, treatment caused significantly induction of apoptosis in MCF-7 and HeLa cells, but not in MIA PaCa-2 cells. In this cell line, multiple cell death mechanism existed with involving apoptosis, senescence or necrosis. Results of the study provide a promising basis for the evaluation of the potential use of essential oil from H. italicum (Roth) G. Don as a source of alternative nature antibiotics or anticancer agents for the prevention or treatment of different diseases.