Direct In Vitro Comparison of the Anti-Leishmanial Activity of Different Olive Oil Total Polyphenolic Fractions and Assessment of Their Combined Effects with Miltefosine.

The bioactive compounds present in the edible products of the olive tree have been extensively studied and their favorable effects on various disease risk factors have been demonstrated. The aim of this study was to perform a comparative analysis of the anti-leishmanial effects of total phenolic fractions (TPFs) derived from extra virgin olive oil with different phenolic contents and diverse quantitative patterns. Moreover, the present study investigated their association with miltefosine, a standard anti-leishmanial drug, against both extracellular promastigotes and intracellular amastigotes of a viscerotropic and a dermotropic Leishmania strain. The chemical compositions of TPFs were determined by high performance liquid chromatography with diode array detection (HPLC-DAD). Analysis of parasite growth kinetics, reactive oxygen species production and apoptotic events were determined by microscopy and flow cytometry. Our results revealed that the presence of oleacein (OLEA) and oleocanthal (OLEO) secoiridoids enhances the anti-leishmanial effect of TPF. The association between TPFs and miltefosine was suggested as being additive in Leishmania infantum and Leishmania major promastigotes, and as antagonistic in intracellular amastigotes, as was evaluated with the modified isobologram method. The obtained data verified that TPFs are bioactive dietary extracts with a strong anti-leishmanial activity and highlighted that fractions that are richer in OLEA and OLEO phenolic compounds possess stronger inhibitory effects against parasites. This study may contribute to improving the therapeutic approaches against leishmaniasis.

Exploring the Immunotherapeutic Potential of Oleocanthal against Murine Cutaneous Leishmaniasis.

Leishmaniasis is a major tropical disease with increasing global incidence. Due to limited therapeutic options with severe drawbacks, the discovery of alternative treatments based on natural bioactive compounds is important. In our previous studies we have pointed out the antileishmanial activities of olive tree-derived molecules. In this study, we aimed to investigate the in vitro and in vivo antileishmanial as well as the in vivo immunomodulatory effects of oleocanthal, a molecule that has recently gained increasing scientific attention. Pure oleocanthal was isolated from extra virgin olive oil through extraction and chromatography techniques. The in vitro antileishmanial effects of oleocanthal were examined with a resazurin-based assay, while its in vivo efficacy was evaluated in Leishmania major-infected BALB/c mice by determining footpad induration, parasite load in popliteal lymph nodes, histopathological outcome, antibody production, cytokine profile of stimulated splenocytes and immune gene expression, at three weeks after the termination of treatment. Oleocanthal demonstrated in vitro antileishmanial effect against both L. major promastigotes and intracellular amastigotes. This effect was further documented in vivo as demonstrated by the suppressed footpad thickness, the decreased parasite load and the inflammatory cell influx at the infection site. Oleocanthal treatment led to the dominance of a Th1-type immunity linked with resistance against the disease. This study establishes strong scientific evidence for olive tree-derived natural products as possible antileishmanial agents and provides an adding value to the scientific research of oleocanthal.

Total Phenolic Fraction (TPF) from Extra Virgin Olive Oil: Induction of apoptotic-like cell death in Leishmania spp. promastigotes and in vivo potential of therapeutic immunomodulation.

BACKGROUND: Leishmaniasis is a serious multifactorial parasitic disease with limited treatment options. Current chemotherapy is mainly consisted of drugs with serious drawbacks such as toxicity, variable efficacy and resistance. Alternative bioactive phytocompounds may provide a promising source for discovering new anti-leishmanial drugs. Extra Virgin Olive Oil (EVOO), a key-product in the Mediterranean diet, is rich in phenols which are associated with anti-inflammatory, anti-cancer and anti-microbial effects. In this study, we investigate the anti-leishmanial effect of Total Phenolic Fraction (TPF) derived from EVOO in both in vitro and in vivo systems by investigating the contributing mechanism of action. METHODOLOGY/PRINCIPAL FINDINGS: We tested the ability of TPF to cause apoptotic-like programmed cell death in L. infantum and L. major exponential-phase promastigotes by evaluating several apoptotic indices, such as reduction of proliferation rate, sub-G0/G1 phase cell cycle arrest, phosphatidylserine externalization, mitochondrial transmembrane potential disruption and increased ROS production, by using flow cytometry and microscopy techniques. Moreover, we assessed the therapeutic effect of TPF in L. major-infected BALB/c mice by determining skin lesions, parasite burden in popliteal lymph nodes, Leishmania-specific antibodies and biomarkers of tissue site cellular immune response, five weeks post-treatment termination. Our results show that TPF triggers cell-cycle arrest and apoptotic-like changes in Leishmania spp. promastigotes. Moreover, TPF treatment induces significant reduction of parasite burden in draining lymph nodes together with an antibody profile indicative of the polarization of Th1/Th2 immune balance towards the protective Th1-type response, characterized by the presence of IFN-γ-producing CD4+ T-cells and increased Tbx21/GATA-3 gene expression ratio in splenocytes. CONCLUSIONS/SIGNIFICANCE: TPF exhibits chemotherapeutic anti-leishmanial activity by inducing programmed cell death on cell-free promastigotes and immunomodulatory properties that induce in vivo T cell-mediated responses towards the protective Th1 response in experimental cutaneous leishmaniasis. These findings enable deeper understanding of TPF's dual mode of action that encourages further studies.

NMR-Based Metabolic Profiling of Edible Olives-Determination of Quality Parameters.

Edible olive drupes (from Olea europaea L.) are a high-value food commodity with an increasing production trend over the past two decades. In an attempt to prevent fraud issues and ensure quality, the International Olive Council (IOC) issued guidelines for their sensory evaluation. However, certain varieties, geographical origins and processing parameters are omitted. The aim of the present study was the development of a method for the quality assessment of edible olives from the Konservolia, Kalamon and Chalkidikis cultivars from different areas of Greece processed with the Spanish or Greek method. A rapid NMR-based untargeted metabolic profiling method was developed along with multivariate analysis (MVA) and applied for the first time in edible olives' analysis complemented by the aid of statistical total correlation spectroscopy (STOCSY). Specific biomarkers, related to the classification of olives based on different treatments, cultivars and geographical origin, were identified. STOCSY proved to be a valuable aid towards the assignment of biomarkers, a bottleneck in untargeted metabolomic approaches.