Much research effort has been focused on investigating new compounds derived from low-cost sources, such as natural products, for treating leishmaniasis. Oleuropein derived from numerous plants, particularly from the olive tree, Olea europaea L. (Oleaceae), is a biophenol with many biological activities. Our previous findings showed that oleuropein exhibits leishmanicidal effects against three Leishmania spp. in vitro, and minimizes the parasite burden in L. donovani-infected BALB/c mice. The aim of the present study is to investigate the possible mechanism(s) that mediate this leishmanicidal activity. We determined the efficacy of oleuropein in elevating ROS and NO production in L. donovani-infected J774A.1 macrophages and in explanted splenocytes and hepatocytes obtained from L. donovani-infected BALB/c mice. We also assessed the expression of genes that are related to inflammation, T-cell polarization and antioxidant defense, in splenocytes. Finally, we determined the ratios of specific IgG2a/IgG1 antibodies and DTH reactions in L. donovani-infected BALB/c mice treated with oleuropein. Oleuropein was able to elevate ROS production in both in vitro and in vivo models of visceral leishmaniasis and raised NO production in ex vivo cultures of splenocytes and hepatocytes. The extensive oxidative stress found in oleuropein-treated mice was obviated by the upregulation of the host’s antioxidant enzyme (mGCLC) and the simultaneous downregulation of the corresponding enzyme of the parasite (LdGCLC). Moreover, oleuropein was able to mount a significant Th1 polarization characterized by the expression of immune genes (IL-12β, IL-10, TGF-β1, IFN-γ) and transcription factors (Tbx21 and GATA3). Moreover, this immunomodulatory effect was also correlated with an inhibitory effect on IL-1β gene expression, rather than with the expression of IL-1α, IL-1rn and TNF-α. Furthermore, oleuropein-treated BALB/c mice mounted a delayed-type hypersensitivity (DTH) response and an elevated Leishmania-specific IgG2a/IgG1 ratio that clearly demonstrated an in vivo protective mechanism. The ability of Oleuropein to promote a Th1 type immune response in L. donovani-infected BALB/c mice points towards the candidacy of this bioactive compound as an immunomodulatory agent that may complement therapeutic approaches to leishmaniasis.

Vaccination is the most effective tool against infectious diseases. Subunit vaccines are safer compared to live-attenuated vaccines but are less immunogenic and need to be delivered with an adjuvant. Adjuvants are essential for enhancing vaccine potency by improving humoral and cell-mediated immune responses. Only a limited number of adjuvants are licensed for human vaccines, and their mode of action is still not clear.Leishmaniaeukaryotic initiation factor (LeIF) has been described having a dual role, as a natural adjuvant and as an antigen that possesses advantageous immunomodulatory properties. In this study, we assessed the adjuvant properties of recombinantLeishmania infantumeukaryotic initiation factor (LieIF) throughin vitroandin vivoassays. LieIF was intraperitoneally administered in combination with the protein antigen ovalbumin (OVA), and the widely used alum was used as a reference adjuvant. Ourin vitrostudies using J774A.1 macrophages showed that LieIF induced stimulatory effects as demonstrated by the enhanced surface expression of CD80 and CD86 co-stimulatory molecules and the induced production of the immune mediators NO and MIP-1α. Additionally, LieIF co-administration with OVA in anin vivomurine model induced a proinflammatory environment as demonstrated by the elevated expression ofTNF-α,IL-1β, andNF-κB2genes in peritoneal exudate cells (PEC). Furthermore, PEC derived from OVA-LieIF-immunized mice exhibited elevated expression of CD80 molecule and production of NO and MIP-1αin culture supernatants. Moreover, LieIF administration in the peritoneum of mice resulted in the recruitment of neutrophils and monocytes at 24 h post-injection. Also, we showed that this immunopotentiating effect of LieIF did not depend on the induction of uric acid danger signal. These findings suggest the potential use of LieIF as adjuvant in new vaccine formulations against different infectious diseases.

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.

Natural product drug discovery faces important challenges related to efficient complex mixture analysis. To address this, dereplication strategies have been developed to enhance and accelerate the detection of bioactive compounds in plant extracts. Among these, statistical spectroscopy-based methods HeteroCovariance Approach (HetCA) and Statistical Total Correlation Spectroscopy (STOCSY) have been proposed. In this study, HetCA and STOCSY are applied to Artemisia umbelliformis subsp. eriantha extract, to discover antileishmanial compounds. Spectral data from appropriately generated extract fractions were statistically correlated with antileishmanial activity results. HetCA along with STOCSY revealed sets of NMR signals correlated to the activity and led to the identification of specific bioactive compounds. These components were purposefully isolated and tested against L. infantum promastigotes, designating that the positively correlated compounds 5-deoxy-5-hydroperoxytelekin 1 and telekin 2 exert strong antileishmanial effects, with a half-maximal effective concentration (EC₅₀) of 4.5 and 5.1 μΜ, respectively. Their toxicity was also tested on model host cells, THP-1 macrophages, yielding a 50 % cytotoxic concentration of 29.7 μΜ and 15 μΜ, respectively. For verification purposes, non-correlated compounds were also isolated and didn't show significant antileishmanial activity. Overall, the combination of HetCA and STOCSY was able to identify the most active compounds, facilitating the detection of bioactive components and opening new insights into natural product drug discovery.

Background/Objectives: The health-protective properties of olive oil phenolics, including their potential chemopreventive and anticancer effects, have attracted considerable scientific interest. However, their in vivo efficacy and mechanisms of action remain insufficiently understood. Recent advances in extraction and purification technologies have enabled large-scale production of highly purified olive oil phenols and phenolic-rich extracts, facilitating translational research. Methods: Herein, the antitumor efficacy of isolated olive oil phenols and phenolic-rich formulations was investigated in an MDA-MB-231 triple-negative breast cancer (TNBC) xenograft model. Results: Intraperitoneal administration of oleocanthal (OLC), oleuropein aglycone (OleA) or their combination reduced endpoint tumor burden, with OLC exhibiting the most pronounced effect. Oral administration of total olive oil phenolics (OOPs) achieved comparable efficacy. Pre-treatment initiated before tumor cell implantation conferred the greatest protection, consistent with a prophylactic mode of action. In contrast, delayed intervention displayed diminished or no antitumor benefit. Phenolic-rich extra virgin olive oil likewise showed an inhibition trend in tumor progression. Mechanistically, OOPs attenuated plasma protein oxidation, modulated proteasome mediated proteolysis, and reduced γH2AX levels in vivo. Furthermore, OOPs negatively affected the MDA-MB-231 cell migration in a concentration-dependent manner in vitro. Conclusions: Collectively, these findings are consistent with antitumor activities of olive oil phenolics via multiple mechanisms and support their further investigation as prophylactic agents in TNBC and as nutraceuticals.