Every year million tons of by-products and waste from olive and orange processing are produced by agri-food industries, thus triggering environmental and economic problems worldwide. From the perspective of a circular economy model, olive leaves and orange peels can be valorized in valuable products due to the presence of bioactive compounds such as polyphenols exhibiting beneficial effects on human health. The aqueous extracts of olive leaves and orange peels rich in phenolic compounds were prepared by ultrasound-assisted extraction. Both extracts were characterized in terms of yield of extraction, total phenolic content and antioxidant capacity; the polyphenolic profiles were deeper investigated by HPLC-MS analysis. Each extract was included in liposomes composed by a natural phospholipid, 1,2-dioleoyl-sn-glycero-3-phosphocholine,and cholesterol prepared according to the thin-layer evaporation method coupled with a sonication process. The antimicrobial activity of the extracts, free and loaded in liposomes, was investigated according to the broth macrodilution method against different strains of potential bacterial pathogenic species: Staphylococcus aureus (NCIMB 9518), Bacillus subtilis (ATCC 6051) and Enterococcus faecalis (NCIMB 775) as Gram-positive, while Escherichia coli (NCIMB 13302), Pseudomonas aeruginosa (NCIMB 9904) and Klebsiella oxytoca (NCIMB 12259) as Gram-negative. The encapsulation of olive leaves extract in liposomes enhanced its antibacterial activity against S. aureus by an order of magnitude.

Antibiotic resistance due to bacterial biofilm formation is a major global health concern that makes the search for new therapeutic approaches an urgent need. In this context,, trans-resveratrol (RSV), a polyphenolic natural substance, seems to be a good candidate for preventing and eradicating biofilm-associated infections but its mechanism of action is poorly understood. In addition, RSV suffers from low bioavailability and chemical instability in the biological media that make its encapsulation in delivery systems necessary. In this work, the anti-biofilm activity of free RSV was investigated on Staphylococcus aureus and, to highlight the possible mechanism of action, we studied the anti-adherence activity and also the cell wall damage on a MRSA strain. Free RSV activity was compared to that of RSV loaded in liposomes, specifically neutral liposomes (L = DOPC/Cholesterol) and cationic liposomes (LG = DOPC/Chol/GLT1) characterized by a galactosylated amphiphile (GLT1) that promotes the interaction with bacteria. The results indicate that RSV loaded in LG has anti-adherence and anti-biofilm activity higher than free RSV. On the other side, free RSV has a higher bacterial-growth-inhibiting effect than encapsulated RSV and it can damage cell walls by creating pores; however, this effect can not prevent bacteria from growing again. This RSV ability may underlie its bacteriostatic activity.

According to the innovative and sustainable perspective of the circular economy model, Olea europaea leaves, a solid byproduct generated every year in large amounts by the olive oil production chain, are considered a valuable source of bioactive compounds, such as polyphenols, with many potential applications. In particular, the following study aimed to valorize olive leaves in order to obtain products with potential antibacterial activity. In this study, olive leaf extracts, rich in polyphenols, were prepared by ultrasound-assisted extraction using green solvents, such as ethanol and water. The extracts were found to be rich in polyphenols up to 26.7 mg_GAE/g_leaves; in particular, hydroxytyrosol-hexose isomers (up to 6.6 mg/g_dry extract) and oleuropein (up to 324.1 mg/g_dry extract) turned out to be the most abundant polyphenolic compounds in all of the extracts. The extracts were embedded in liposomes formulated with natural phosphocholine and cholesterol, in the presence or in the absence of a synthetic galactosylated amphiphile. All liposomes, prepared according to the thin-layer evaporation method coupled with an extrusion protocol, showed a narrow size distribution with a particle diameter between 79 and 120 nm and a good polydispersity index (0.10-0.20). Furthermore, all developed liposomes exhibited a great storage stability up to 90 days at 4 °C and at different pH values, with no significant changes in their size and polydispersity index. The effect of the encapsulation in liposomes of O. europaea leaf extracts on their antimicrobial activity was examined in vitro against two strains of Staphylococcus aureus: ATCC 25923 (wild-type strain) and ATCC 33591 (methicillin-resistant S. aureus, MRSA). The extracts demonstrated good antimicrobial activity against both bacterial strains under investigation, with the minimum inhibitory concentration ranging from 140 to 240 μg_extract/mL and the minimum bactericidal concentration ranging from 180 to 310 μg_extract/mL, depending on the specific extract and the bacterium tested. Moreover, a possible synergistic effect between the bioactive compounds inside the extracts tested was highlighted. Notably, their inclusion in galactosylated liposomes highlighted comparable or slightly increased antimicrobial activity compared to the free extracts against both bacterial strains tested.

Hydroxytyrosol (HTyr), a plant-derived phenolic compound found in Olea europaea L. products and by-products, is well-known for its antioxidant activity and a wide range of biological effects, including anti-inflammatory, anticancer, antiviral, cardioprotective, neuroprotective, and antibacterial properties. However, due to its high hydrophilicity, HTyr exhibits unfavorable pharmacokinetic properties, preventing its potential therapeutic use. Various strategies can be employed to address these limitations. In this study, we evaluated the effect of two specific approaches on the HTyr antimicrobial and antioxidant activities: chemical modification of HTyr by lipophilization of the alcoholic moiety and encapsulation in liposomes. Based on our experience in the synthesis and biological activities of HTyr derivatives, the attention was focused on HTyr oleate (HTyr-OL), having a C-18 unsaturated alkylic chain responsible for an increased lipophilicity compared to HTyr. This structural feature enhanced antimicrobial activity against both tested strains of S. aureus, ATCC 25923 (wild-type strain) and ATCC 33591 (MRSA), and comparable antioxidant activity against two different radicals, Galvinoxyl radical and 1,1-diphenyl-2-picrylhydrazyl radical. Moreover, liposomes as delivery systems for HTyr and HTyr-OL were developed using both natural and synthetic amphiphiles, and the impact of encapsulation on their activities was further investigated. The experimental results showed that the antimicrobial properties of HTyr and HTyr-OL against S. aureus strains were not enhanced after encapsulation in liposomes, while the high antioxidant activity of HTyr-OL was preserved when conveyed in liposomes.