Leishmanicidal and immunomodulatory properties of Brazilian green propolis extract (EPP-AF®) and a gel formulation in a pre-clinical model.

Leishmaniasis is a widespread group of neglected vector-borne tropical diseases that possess serious therapeutic limitations. Propolis has been extensively used in traditional medical applications due to its range of biological effects, including activity against infectious agents. Here we evaluated the leishmanicidal and immunomodulatory properties of Brazilian green propolis extract (EPP-AF®) and a gel formulation incorporating EPP-AF®, in both in vitro and in vivo models of Leishmania amazonensis infection. Propolis extract, obtained from a standardized blend following hydroalcoholic extraction, showed the characteristic fingerprint of Brazilian green propolis as confirmed by HPLC/DAD. A carbopol 940 gel formulation was obtained containing propolis glycolic extract at 3.6% w/w. The release profile, assessed using the Franz diffusion cell protocol, demonstrated a gradual and prolonged release of p-coumaric acid and artepillin C from the carbomer gel matrix. Quantification of p-coumaric acid and artepillin C in the gel formulation over time revealed that p-coumaric acid followed the Higuchi model, dependent on the disintegration of the pharmaceutical preparation, while artepillin C followed a zero-order profile with sustained release. In vitro analysis revealed the ability of EPP-AF® to reduce the infection index of infected macrophages (p < 0.05), while also modulating the production of inflammatory biomarkers. Decreases in nitric oxide and prostaglandin E2 levels were observed (p < 0.01), suggesting low iNOS and COX-2 activity. Furthermore, EPP-AF® treatment was found to induce heme oxygenase-1 antioxidant enzyme expression in both uninfected and L. amazonensis-infected cells, as well as inhibit IL-1ß production in infected cells (p < 0.01). ERK-1/2 phosphorylation was positively correlated with TNF-α production (p < 0.05), yet no impact on parasite load was detected. In vivo analysis indicated the effectiveness of topical treatment with EPP-AF® gel alone (p < 0.05 and p < 0.01), or in combination with pentavalent antimony (p < 0.05 and p < 0.001), in the reduction of lesion size in the ears of L. amazonensis-infected BALB/c mice after seven or 3 weeks of treatment, respectively. Taken together, the present results reinforce the leishmanicidal and immunomodulatory effects of Brazilian green propolis, and demonstrate promising potential for the EPP-AF® propolis gel formulation as a candidate for adjuvant therapy in the treatment of Cutaneous Leishmaniasis.

Effects of Standardized Brazilian Green Propolis Extract (EPP-AF®) on Inflammation in Haemodialysis Patients: A Clinical Trial.

Background: Patients on haemodialysis (HD) present a significant inflammatory status, which has a pronounced negative impact on their outcomes. Propolis is a natural resin with anti-inflammatory and immunomodulatory properties. We assessed the safety and impact of a standardized Brazilian green propolis extract (EPP-AF®) on the inflammatory status in patients under conventional HD. Methods: Patients were assigned to receive 200 mg/day of EPP-AF® for 4 weeks followed by 4 weeks without the drug, and changes in plasma levels of interleukins (ILs), interferon gamma (IFN-γ), tumour necrosis factor-alpha (TNF-α), and high-sensitivityc-reactive protein (HsCRP) were measured. A heatmap was used to illustrate trends in data variation. Results: In total, 37 patients were included in the final analysis. Patients presented an exacerbated inflammatory state at baseline. During EPP-AF® use, there was a significant reduction in IFN-γ (p=0.005), IL-13 (p=0.04 2), IL-17 (p=0.039), IL-1ra (p=0.008), IL-8 (p=0.009), and TNF-α (p < 0.001) levels compared to baseline, and significant changes were found in Hs-CRP levels. The heatmap demonstrated a pattern of pronounced proinflammatory status at baseline, especially in patients with primary glomerulopathies, and a clear reduction in this pattern during the use of EPP-AF®. There was a tendency to maintain this reduction after suspension of EPP-AF®. No significant side effects were observed. Conclusion: Patients under haemodialysis presented a pronounced inflammatory status, and EPP-AF® was demonstrated to be safe and associated with a significant and maintained reduction in proinflammatory cytokines in this population. This trial is registered with Clinicaltrials.gov NCT04072341.

In vitro leishmanicidal effect of Yangambin and Epi-yangambin lignans isolated from Ocotea fasciculata (Nees) Mez.

Introduction: Yangambin and epi-yangambin are the main lignans found in Louro-de-Cheiro [Ocotea fasciculata (Nees) Mez], a tree native to the Atlantic forests of northeastern Brazil whose leaves and bark are widely used in folk medicine. The present study investigated the leishmanicidal and immunomodulatory effects of both lignans in in vitro models of infection by Leishmania amazonensis or Leishmania braziliensis, both etiological agents of Cutaneous Leishmaniasis in Brazil. Methods: Bone marrow-derived mouse macrophages were infected with L. amazonensis or L. braziliensis and then treated for 48 h at varying concentrations of yangambin or epi-yangambin. Results: Yangambin and epi-yangambin were found to reduce the intracellular viability of either Leishmania species in a concentration-dependent manner, with respective IC50 values of: 43.9 ± 5 and 22.6 ± 4.9 µM for L. amazonensis, compared to IC50 values of 76 ± 17 and 74.4 ± 9.8 µM for L. braziliensis. In this context, epi-yangambin proved more selective and effective against in vitro infection by L. amazonensis. However, both lignans were found to distinctly modulate the production of inflammatory mediators and other cytokines by macrophages infected by either of the Leishmania species evaluated. While yangambin increased the production of IL-10 by L. braziliensis-infected macrophages, both compounds were observed to lower the production of NO, PGE2, IL-6 and TNF-α in both Leishmania species. Discussion: The present results serve to encourage the development of novel studies aimed at screening natural bioactive compounds with the hope of discovering new therapeutic options for the treatment of Cutaneous Leishmaniasis.

Evaluation of in vitro and in vivo Efficacy of a Novel Amphotericin B-Loaded Nanostructured Lipid Carrier in the Treatment of Leishmania braziliensis Infection.

BACKGROUND: Leishmaniasis is a neglected disease, and the current therapeutic arsenal for its treatment is seriously limited by high cost and toxicity. Nanostructured lipid carriers (NLCs) represent a promising approach due to high drug loading capacity, controlled drug release profiles and superior stability. Here, we explore the efficacy of a unique pH-sensitive amphotericin B-loaded NLC (AmB-NLC) in Leishmania braziliensis infection in vitro and in vivo. METHODS AND RESULTS: AmB-NLC was assessed by dynamic light scattering and atomic force microscopy assays. The carrier showed a spherical shape with a nanometric size of 242.0 ± 18.3 nm. Zeta potential was suggestive of high carrier stability (-42.5 ± 1.5 mV), and the NLC showed ~99% drug encapsulation efficiency (EE%). In biological assays, AmB-NLC presented a similar IC50 as free AmB and conventional AmB deoxycholate (AmB-D) (11.7 ± 1.73; 5.3 ± 0.55 and 13 ± 0.57 ng/mL, respectively), while also presenting higher selectivity index and lower toxicity to host cells, with no observed production of nitric oxide or TNF-α by in vitro assay. Confocal microscopy revealed the rapid uptake of AmB-NLC by infected macrophages after 1h, which, in association with more rapid disruption of AmB-NLC at acidic pH levels, may directly affect intracellular parasites. Leishmanicidal effects were evaluated in vivo in BALB/c mice infected in the ear dermis with L. braziliensis and treated with a pentavalent antimonial (Sb5+), liposomal AmB (AmB-L) or AmB-NLC. After 6 weeks of infection, AmB-NLC treatment resulted in smaller ear lesion size in all treated mice, indicating the efficacy of the novel formulation. CONCLUSION: Here, we preliminarily demonstrate the effectiveness of an innovative and cost-effective AmB-NLC formulation in promoting the killing of intracellular L. braziliensis. This novel carrier system could be a promising alternative for the future treatment of cutaneous leishmaniasis.

Heightened Plasma Levels of Transforming Growth Factor Beta (TGF-ß) and Increased Degree of Systemic Biochemical Perturbation Characterizes Hepatic Steatosis in Overweight Pediatric Patients: A Cross-Sectional Study.

Nonalcoholic Fatty Liver Disease (NAFLD) is a common cause of chronic liver disease in childhood and strongly associated with obesity. Routine biochemical non-invasive tests remain with low accuracy for diagnosis of NAFLD. We performed a cross-sectional study to examine potential associations between anthropometric and biochemical parameters, specially TGF-ß, a prognosis marker for hepatic steatosis (HS). Between May and October 2019, seventy-two overweight adolescents were enrolled, of which 36 had hepatic steatosis. Hepatic, lipidic and glycemic profiles, and levels of vitamin D, ferritin and TGF-ß were analyzed. Hierarchical cluster and a discriminant model using canonical correlations were employed to depict the overall expression profile of biochemical markers and the biochemical degree of perturbation. Median values of alanine aminotransferase (ALT), gamma glutamyl transpeptidase (GGT), and TGF-ß were higher in the adolescents with HS. Values of body mass index (BMI)/age and ALT, but not of TGF-ß, were gradually increased proportionally to augmentation of steatosis severity. In a multivariate analysis, TGF-ß plasma concentrations were associated with occurrence of hepatic steatosis independent of other covariates. Discriminant analysis confirmed that TGF-ß concentrations can identify HS cases. Our data reveal that HS patients exhibit a distinct biosignature of biochemical parameters and imply TGF-ß as an important biomarker to evaluate risk of steatosis development.

Chemical characterization and biological activity of six different extracts of propolis through conventional methods and supercritical extraction.

Propolis is a natural product with many demonstrated biological activities and propolis extract has been used in the food, pharmaceutical and cosmetics industries. Different works have showed the variations in the chemical composition, and consequently, on the biological activity of the propolis that are associated with its type and geographic origin. Due to this study evaluated propolis extracts obtained through supercritical extraction and ethanolic extraction (conventional) in three samples of different types of propolis (red, green and brown), collected from different regions in Brazil (state of Bahia). Analyses were performed to determine the humidity, water activity, the content of total ash, proteins, lipids and fiber in raw propolis samples. The content of phenolic compounds, flavonoids, in vitro antioxidant activity (DPPH), catechin, ferulic acid and luteolin and antimicrobial activity against two bacteria (Staphylococcus aureus and Escherichia coli) were determined for all extracts. For the green and red ethanolic extracts the anti-leishmanicidal potential was also evaluated. The physicochemical profiles showed agreement in relation to the literature. The results identified significant differences among the extracts (p>0.05), which are in conformity with their extraction method, as well as with type and botanical origin of the samples. The extraction with supercritical fluid was not efficient to obtain extracts with the highest contents of antioxidants compounds, when compared with the ethanolic extracts. The best results were shown for the extracts obtained through the conventional extraction method (ethanolic) indicating a higher selectivity for the extraction of antioxidants compounds. The red variety showed the largest biological potential, which included the content of antioxidants compounds. The results found in this study confirm the influence of the type of the raw material on the composition and characteristics of the extracts. The parameters analysis were important to characterize and evaluate the quality of the different Brazilian propolis extracts based on the increased use of propolis by the natural products industry.

Parasite Killing of Leishmania (V) braziliensis by Standardized Propolis Extracts.

Treatments based on antimonials to cutaneous leishmaniasis (CL) entail a range of toxic side effects. Propolis, a natural compound widely used in traditional medical applications, exhibits a range of biological effects, including activity against infectious agents. The aim of this study was to test the potential leishmanicidal effects of different propolis extracts against Leishmania (Viannia) braziliensis promastigotes and intracellular amastigotes in vitro. Stationary-phase L. (V) braziliensis promastigotes were incubated with medium alone or treated with dry, alcoholic, or glycolic propolis extract (10, 50, or 100 µg/mL) for 96 h. Our data showed that all extracts exhibited a dose-dependent effect on the viability of L. (V) braziliensis promastigotes, while controlling the parasite burden inside infected macrophages. Dry propolis extract significantly modified the inflammatory profile of murine macrophages by downmodulating TGF-ß and IL-10 production, while upmodulating TNF-α. All three types of propolis extract were found to reduce nitric oxide and superoxide levels in activated L. braziliensis-infected macrophages. Altogether, our results showed that propolis extracts exhibited a leishmanicidal effect against both stages of L. (V) braziliensis. The low cell toxicity and efficient microbicidal effect of alcoholic or glycolic propolis extracts make them candidates to an additive treatment for cutaneous leishmaniasis.