Schinus terebinthifolius Raddi (Brazilian pepper) leaves extract: in vitro and in vivo evidence of anti-inflammatory and antioxidant properties.

The aim of this work was to evaluate the anti-inflammatory and antioxidant effects of ethyl acetate extract obtained from the leaves of Brazilian peppertree Schinus terebinthifolius Raddi (EAELSt). Total phenols and flavonoids, chemical constituents, in vitro antioxidant activity (DPPH and lipoperoxidation assays), and cytotoxicity in L929 fibroblasts were determined. In vivo anti-inflammatory and antioxidant properties were evaluated using TPA-induced ear inflammation model in mice. Phenol and flavonoid contents were 19.2 ± 0.4 and 93.8 ± 5.2 of gallic acid or quercetin equivalents/g, respectively. LC-MS analysis identified 43 compounds, of which myricetin-O-pentoside and quercetin-O-rhamnoside were major peaks of chromatogram. Incubation with EAELSt decreased the amount of DPPH radical (EC50 of 54.5 ± 2.4 µg/mL) and lipoperoxidation at 200-500 µg/mL. The incubation with EAELSt did not change fibroblast viability up to 100 µg/mL. Topical treatment with EAELSt significantly reduced edema and myeloperoxidase activity at 0.3, 1, and 3 mg/ear when compared to the vehicle-treated group. In addition, EAELSt decreased IL-6 and TNF-α levels and increased IL-10 levels. Besides, it modulated markers of oxidative stress (reduced total hydroperoxides and increased sulfhydryl contents and ferrium reduction potential) and increased the activity of catalase and superoxide dismutase, without altering GPx activity.

Reduced cutaneous inflammation associated with antioxidant action after topical application of the aqueous extract of Annona muricata leaves.

Annona muricata L. is used in folk medicine for treatment of diseases related to inflammatory and oxidative processes. This study investigated the effect of the aqueous extract of A. muricata leaves (AEAM) on TPA-induced ear inflammation and antioxidant capacity, both in vitro and in vivo. The in vitro antioxidant capacity of AEAM was measured by the 2,2-diphenyl-1-picrylhydrazyl (DPPH), ferric reducing/antioxidant power (FRAP) and lipoperoxidation assays. Cytotoxicity and reactive oxygen species (ROS) release were evaluated in the L929 fibroblasts. Swiss mice were submitted to TPA application and were topically treated with AEAM (0.3, 1 or 3 mg/ear). After 6 h, inflammatory and oxidative parameters were evaluated. Quercetin 3-glucoside, rutin, chlorogenic acid, catechin and gallic acid were identified in AEAM. It also presented antioxidant activity in all in vitro assays used. Incubation with AEAM did not cause cell cytotoxicity but reduced ROS release from fibroblasts. Compared with the control group, treatment with AEAM significantly reduced ear oedema and mieloperoxidase activity in inflamed ears, as well as histological parameters of inflammation. These results were associated with the reduction of total hydroperoxides and modulation of catalase, but not superoxide dismutase activity. These findings show the anti-inflammatory effect of AEAM is associated with antioxidant capacity.

Ethanolic extract of Croton blanchetianus Ball induces mitochondrial defects in Leishmania amazonensis promastigotes.

Leishmaniasis is a neglected disease caused by Leishmania. Chemotherapy remains the mainstay for leishmaniasis control; however, available drugs fail to provide a parasitological cure, and are associated with high toxicity. Natural products are promising leads for the development of novel chemotherapeutics against leishmaniasis. This work investigated the leishmanicidal properties of ethanolic extract of Croton blanchetianus (EECb) on Leishmania infantum and Leishmania amazonensis, and found that EECb, rich in terpenic compounds, was active against promastigote and amastigote forms of both Leishmania species. Leishmania infantum promastigotes and amastigotes presented IC50 values of 208.6 and 8.8 µg/mL, respectively, whereas Leishmania amazonensis promastigotes and amastigotes presented IC50 values of 73.6 and 3.1 µg/mL, respectively. Promastigotes exposed to EECb (100 µg/mL) had their body cellular volume reduced and altered to a round shape, and the flagellum was duplicated, suggesting that EECb may interfere with the process of cytokinesis, which could be the cause of the decline in the parasite multiplication rate. Regarding possible EECb targets, a marked depolarization of the mitochondrial membrane potential was observed. No cytotoxic effects of EECb were observed in murine macrophages at concentrations below 60 µg/mL, and the CC50 obtained was 83.8 µg/mL. Thus, the present results indicated that EECb had effective and selective effects against Leishmania infantum and Leishmania amazonensis, and that these effects appeared to be mediated by mitochondrial dysfunction.

Praziquantel-Solid Lipid Nanoparticles Produced by Supercritical Carbon Dioxide Extraction: Physicochemical Characterization, Release Profile, and Cytotoxicity.

Solid lipid nanoparticles (SLNs) can be produced by various methods, but most of them are difficult to scale up. Supercritical fluid (SCF) is an important tool to produce micro/nanoparticles with a narrow size distribution and high encapsulation efficiency. The aim of this work was to produce cetyl palmitate SLNs using SCF to be loaded with praziquantel (PZQ) as an insoluble model drug. The mean particle size (nm), polydispersity index (PdI), zeta potential, and encapsulation efficiency (EE) were determined on the freshly prepared samples, which were also subject of Differential Scanning Calorimetry (DSC), Fourier-Transform Infrared Spectroscopy (FTIR), drug release profile, and in vitro cytotoxicity analyses. PZQ-SLN exhibited a mean size of ~25 nm, PdI ~ 0.5, zeta potential ~-28 mV, and EE 88.37%. The DSC analysis demonstrated that SCF reduced the crystallinity of cetyl palmitate and favored the loading of PZQ into the lipid matrices. No chemical interaction between the PZQ and cetyl palmitate was revealed by FTIR analysis, while the release or PZQ from SLN followed the Weibull model. PZQ-SLN showed low cytotoxicity against fibroblasts cell lines. This study demonstrates that SCF may be a suitable scale-up procedure for the production of SLN, which have shown to be an appropriate carrier for PZQ.

Increased thiol levels in antimony-resistant Leishmania infantum isolated from treatment-refractory visceral leishmaniasis in Brazil.

BACKGROUND Treatment-refractory visceral leishmaniasis (VL) has become an important problem in many countries. OBJECTIVES We evaluated the antimony-resistance mechanisms of Leishmania infantum isolated from VL patients refractory or responsive to treatment with pentavalent antimony. METHODS Strains isolated from antimony-refractory patients (in vitro antimony-resistant isolates) and antimony-responsive patients (in vitro antimony-sensitive isolates) were examined. Morphological changes were evaluated by transmission electron microscopy after trivalent antimony exposure. P-glycoprotein (P-gp) efflux pump activity was evaluated using the pump-specific inhibitor verapamil hydrochloride, and the role of thiol in trivalent antimony resistance was investigated using the enzymatic inhibitor L-buthionine sulfoximine. FINDINGS Antimony treatment induced fewer alterations in the cellular structure of L. infantum resistant isolates than in that of sensitive isolates. P-gp efflux activity was not involved in antimony resistance in these isolates. Importantly, the resistant isolates contained higher levels of thiol compared to the sensitive isolates, and inhibition of thiol synthesis in the resistant isolates recovered their sensitivity to trivalent antimony treatment, and enhanced the production of reactive oxygen species in promastigotes exposed to the drug. MAIN CONCLUSIONS Our results demonstrate that isolates from patients with antimony-refractory VL exhibited higher thiol levels than antimony-sensitive isolates. This indicates that redox metabolism plays an important role in the antimony-resistance of New World VL isolates.

Leishmanicidal Activity and Structure-Activity Relationships of Essential Oil Constituents.

Several constituents of essential oils have been shown to be active against pathogens such as bacteria, fungi, and protozoa. This study demonstrated the in vitro action of ten compounds present in essential oils against Leishmania amazonensis promastigotes. With the exception of p-cymene, all evaluated compounds presented leishmanicidal activity, exhibiting IC50 between 25.4 and 568.1 µg mL-1. Compounds with the best leishmanicidal activity presented a phenolic moiety (IC50 between 25.4 and 82.9 µg mL-1). Alicyclic alcohols ((-)-menthol and isoborneol) and ketones ((-)-carvone) promoted similar activity against the parasite (IC50 between 190.2 and 198.9 µg mL-1). Most of the compounds showed low cytotoxicity in L929 fibroblasts. Analysis of the structure-activity relationship of these compounds showed the importance of the phenolic structure for the biological action against the promastigote forms of the parasite.

Myrtenol Reduces Orofacial Nociception and Inflammation in Mice Through p38-MAPK and Cytokine Inhibition.

Orofacial pain is one of the commonest and most complex complaints in dentistry, greatly impairing life quality. Preclinical studies using monoterpenes have shown pharmacological potential to treat painful conditions, but the reports of the effects of myrtenol on orofacial pain and inflammation are scarce. The aim of this study was to evaluate the effect of myrtenol in experimental models of orofacial pain and inflammation. Orofacial nociceptive behavior and the immunoreactivity of the phosphorylated p38 (P-p38)-MAPK in trigeminal ganglia (TG) and spinal trigeminal subnucleus caudalis (STSC) were determined after the injection of formalin in the upper lip of male Swiss mice pretreated with myrtenol (12.5 and 25 mg/kg, i.p.) or vehicle. Orofacial inflammation was induced by the injection of carrageenan (CGN) in the masseter muscle of mice pretreated with myrtenol (25 and 50 mg/kg, i.p.) or its vehicle (0.02% Tween 80 in saline). Myeloperoxidase (MPO) activity and histopathological changes in the masseter muscle and interleukin (IL)-1ß levels in the TG and STSC were measured. The increase in face-rubbing behavior time induced by formalin and P-p38-MAPK immunostaining in trigeminal ganglia were significantly reduced by myrtenol treatment (12.5 and 25 mg/kg). Likewise, increased MPO activity and inflammatory histological scores in masseter muscle, as well as augmented levels of IL-1ß in the TG AND STSC, observed after CGN injection, were significantly decreased by myrtenol (25 and 50 mg/kg). Myrtenol has potential to treat orofacial inflammation and pain, which is partially related to IL-1ß levels in the trigeminal pathway and p38-MAPK modulation in trigeminal ganglia.

Gastroprotective action of the ethanol extract of Leonurus sibiricus L. (Lamiaceae) in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Leonurus sibiricus L. (Lamiaceae) is a medicinal plant known in Brazil as "rubim" or "erva de macaé". It is used for various purposes, including stomach disorders. AIM OF THE STUDY: To evaluate the effect of the ethanol extract of the aerial parts of L. sibiricus (EELs) in models of gastric damage in mice. MATERIAL AND METHODS: The effect of EELs (50, 100 and 300 mg/kg, p.o., 1 h before induction) was tested on acidified ethanol (ACEt)-induced gastric ulcers. Additionally, we tested the effect of EELs (by intraduodenal administration) in the pylorus ligation (PL) model. RESULTS: Pretreatment with EELs, at 300 mg/kg, but not 50 and 100 mg/kg, reduced the relative area of gastric ulcers induced by ACEt (p < 0.01) and lipoperoxidation (p < 0.001), and increased the sulfhydryl content (p < 0.01) in the stomach in comparison with the vehicle group. Pretreatment with N-ethylmaleimide (a blocker of non-protein sulfhydryl groups, 10 mg/kg, i.p.) or glibenclamide (a KATP channel blocker, 10 mg/kg, i.p.) inhibited the gastroprotective response caused by EELs (300 mg/kg; p < 0.001), but there were no alterations due to pretreatments with inhibitors of the synthesis of prostaglandins (indomethacin, 10 mg/kg), nitric oxide (L-NAME, 70 mg/kg) or hydrogen sulfide (DL-propargylglycine, 10 mg/kg). Treatment with EELs (300 mg/kg) reduced mucus production (p < 0.001) and the volume of gastric secretion (p < 0.001) after PL without affecting gastric acidity or pH. CONCLUSIONS: These results provide evidence that EELs exerts gastroprotective action in mice, with the participation of oxidative stress and mediation of NP-SH, KATP channels and mucus production.

Skin permeation, biocompatibility and antitumor effect of chloroaluminum phthalocyanine associated to oleic acid in lipid nanoparticles.

The objective of this study was to develop and characterize lipid nanoparticles (LNs) containing chloroaluminum phthalocyanine (ClAlPc) to reduce the aggregation of the drug and improve its skin penetration and its antitumor effect. LNs were prepared and characterized by using stearic acid (SA) as solid lipid and oleic acid (OA) as liquid lipid in different proportions. in vitro and in vivo skin penetration was evaluated using modified Franz diffusion cells and fluorescence microscopy, respectively. in vitro biocompatibility and Photodynamic Therapy (PDT) were performed using L929-fibroblasts cell line and A549 cancer cell line and melanoma BF16-F10, respectively. OA promoted the increase in the encapsulation efficiency and drug loading, reaching values of 95.8% and 4%, respectively. The formulation with 40% OA (NLC 40) showed a significantly higher (p < 0.01) amount of drug retained in the skin compared to other formulations. All formulations developed were considered biocompatible. PDT evidenced the antitumor efficacy of NLC 40 with reduced cell viability for approximately 10% of cancer cells, demonstrating that the presence of OA in the NLC seems to potentialize this antitumor effect. PDT in BF16-F10 melanoma using NLC 40 resulted in a reduction in mean cell viability of approximately 99%. According to the results obtained, the systems developed may be promising for the incorporation of ClAlPc in the treatment of skin cancer by photodynamic therapy.

The inclusion complex of carvacrol and ß-cyclodextrin reduces acute skeletal muscle inflammation and nociception in rats.

BACKGROUND: Skeletal muscle inflammation is strongly associated with pain and may impair regeneration and functional recovery after injury. Since anti-inflammatory and antinociceptive effects have been described for the inclusion complex of carvacrol and ß-cyclodextrin (ßCD-carvacrol), this study investigated the effects of ßCD-carvacrol in a model of acute skeletal muscle inflammation. METHODS: Muscle injury was induced in male Wistar rats by injection of 3% carrageenan in the gastrocnemius muscle. Rats were orally pretreated with saline (vehicle) or ßCD-carvacrol (20, 40, 80 and 180 mg/kg) one hour before administration of carrageenan. RESULTS: The injection of carrageenan in the gastrocnemius muscle increased tissue myeloperoxidase (MPO) activity (p < 0.001), edema (p < 0.001) and the levels of tumoral necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-6, macrophage inflammatory protein (MIP-2), but not IL-10 levels. Also, it increased mechanical hyperalgesia and decreased the grip force of animals. Pretreatment with ßCD-carvacrol (80 or 160 mg/kg) significantly decreased muscle MPO activity and edema 24 h after injury in comparison to vehicle-pretreated group. Animals pretreated with ßCD-carvacrol (160 mg/kg) presented significantly lower levels of IL-1ß, IL-6 and MIP-2 and higher levels of IL-10 six hours after induction and lower levels of TNF-α and MIP-2 after 24 h when compared to the vehicle group. Pretreatment with ßCD-carvacrol also reduced mechanical hyperalgesia and limited the decrease of grip force (80 or 160 mg/kg; p < 0.001) 6 and 24 h after injury. CONCLUSION: These results show that ßCD-carvacrol reduces inflammation and nociception in a model of acute injury to skeletal muscles.

Leishmania infantum Induces the Release of sTREM-1 in Visceral Leishmaniasis.

Visceral leishmaniasis (VL) is a systemic transmissible disease that remains to be a major global health problem. The inflammatory response during VL is characterized by the release of several cytokines and other pro-inflammatory mediators. Triggering Receptor Expressed on Myeloid Cells (TREM) are a group of evolutionarily conserved membrane-bound surface receptors expressed on neutrophils and monocytes. Engagement of TREM-1 directs intracellular signaling events that drive cytokine production, degranulation, and phagocytosis. In certain inflammatory-associated diseases, TREM-1 can also be found as a soluble form (sTREM-1), which can negatively regulate TREM-1 receptor signaling. In these studies, we now find that high levels of circulating sTREM-1 correlate directly with VL disease severity. In particular, high levels of sTREM-1 were observed in non-survivor VL patients. Furthermore, these levels of sTREM-1 positively correlated with liver size and negatively correlated with leukocyte counts and hemoglobin concentration. Moreover, we found that neutrophils exposure in vitro to Leishmania infantum modulates TREM-1, DAP12, and IL-8 gene expression, while also increasing release of sTREM-1. Finally, results revealed that higher sTREM-1 levels are associated with increasing parasite ratio. Taken together, these studies suggest that L. infantum may modulate TREM-1 in neutrophils and high levels of this molecule is associated with severe VL.

Increased thiol levels in antimony-resistant Leishmania infantum isolated from treatment-refractory visceral leishmaniasis in Brazil

BACKGROUND Treatment-refractory visceral leishmaniasis (VL) has become an important problem in many countries. OBJECTIVES We evaluated the antimony-resistance mechanisms of Leishmania infantum isolated from VL patients refractory or responsive to treatment with pentavalent antimony. METHODS Strains isolated from antimony-refractory patients (in vitro antimony-resistant isolates) and antimony-responsive patients (in vitro antimony-sensitive isolates) were examined. Morphological changes were evaluated by transmission electron microscopy after trivalent antimony exposure. P-glycoprotein (P-gp) efflux pump activity was evaluated using the pump-specific inhibitor verapamil hydrochloride, and the role of thiol in trivalent antimony resistance was investigated using the enzymatic inhibitor L-buthionine sulfoximine. FINDINGS Antimony treatment induced fewer alterations in the cellular structure of L. infantum resistant isolates than in that of sensitive isolates. P-gp efflux activity was not involved in antimony resistance in these isolates. Importantly, the resistant isolates contained higher levels of thiol compared to the sensitive isolates, and inhibition of thiol synthesis in the resistant isolates recovered their sensitivity to trivalent antimony treatment, and enhanced the production of reactive oxygen species in promastigotes exposed to the drug. MAIN CONCLUSIONS Our results demonstrate that isolates from patients with antimony-refractory VL exhibited higher thiol levels than antimony-sensitive isolates. This indicates that redox metabolism plays an important role in the antimony-resistance of New World VL isolates.