Elizandra Aparecida Britta

Silver sulfadiazine loaded chitosan/chondroitin sulfate films for a potential wound dressing application

Silver sulfadiazine (AgSD) loaded chitosan/chondroitin sulfate (CHI/CS) films were formed to be applied as a potential wound dressing material. The liquid uptake capacity of both, CHI/CS and CHI/CS/AgSD, films exhibited a pH-dependent behavior. Tensile tests showed that the amount of CS used to form the films and the further incorporation of AgSD affect the mechanical properties of the films. In vitro AgSD-release assays showed that the CHI/CS mass ratio influences the AgSD release rate. All the investigated CHI/CS/AgSD films sustain the AgSD release up to 96h at physiological pH. Antibacterial activity and cell viability assays showed that all the CHI/CS/AgSD films have activity against Pseudomonas aeruginosa and Staphylococcus aureus but they were not toxic to Vero cells. The results presented in this work indicate that the CHI/CS/AgSD exhibits potential to be applied as a wound dressing material.

Antimicrobial and cytotoxic activities of medicinal plants of the Brazilian cerrado, using Brazilian cachaça as extractor liquid

ETHNOPHARMACOLOGICAL IMPORTANCE Many species of plants in the Brazilian cerrado (savanna) are widely used in ethnomedicine. However, the safety and effectiveness of medicinal plants used in communities with little or no access to manufactured drugs should be evaluated. AIM OF THE STUDY Evaluate the antimicrobial and cytotoxic activities of extracts from eight plant species, obtained using Brazilian cachaça as the extractor liquid. MATERIALS AND METHODS The extracts were tested against Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Candida albicans, Candida parapsilosis, promastigote forms of Leishmania amazonensis, and poliovirus. In addition, cytotoxic activity was assayed in Vero cells and in human erythrocytes. RESULTS The plant species Curatella americana, Sclerolobium aureum, and Plathymenia reticulata showed the best activity against yeasts, especially the crude extract of C. americana and its ethyl-acetate fraction. Kielmeyera lathrophyton showed a minimum inhibitory concentration of 250 μg/ml against S. aureus, and was inactive against gram-negative bacteria. The extract obtained from Annona coriacea showed the best activity against the promastigote forms of Leishmania amazonensis (IC(50)=175 μg/ml). Only C. americana showed potential for antipoliovirus activity. The concentrations of the crude extracts that showed toxicity to VERO cells had CC(50) between 31 and 470 μg/ml, and the lyophilized Brazilian cachaça showed a CC(50) of 307 μg/ml. None of the extracts showed toxicity against human erythrocytes. CONCLUSIONS Among the plant species studied, C. americana proved to be effective against microorganisms, especially as an antifungal. The results will help in the search for alternative drugs to be used in pharmacotherapy, and will contribute to establish safe and effective use of phytomedicines in the treatment of infectious diseases.

4-Acetoxydolastane Diterpene from the Brazilian Brown Alga Canistrocarpus cervicornis as Antileishmanial Agent

Natural marine products have shown an interesting array of diverse and novel chemical structures with potent biological activities. Our study reports the antiproliferative assays of crude extracts, fraction and pure compound (4R,9S,14S)-4α-acetoxy-9β,14α-dihydroxydolast-1(15),7-diene (1) obtained from brown alga Canistrocarpus cervicornis showing the antileishmanial activity. We showed that 1 had a dose-dependent activity during 72 h of treatment, exhibiting IC50 of 2.0 μg/mL, 12.0 μg/mL, and 4.0 μg/mL for promastigote, axenic amastigote and intracellular amastigote forms of Leishmania amazonensis, respectively. A cytotoxicity assay showed that the action of the isolated compound 1 was 93.0 times less toxic to the macrophage than to the protozoan. Additionally, compound 1 induced ultrastructural changes, including extensive mitochondrial damage; decrease in Rh123 fluorescence, suggesting interference with the mitochondrial membrane potential; and lipid peroxidation in parasite cells. The use of 1 from C. cervicornis against L. amazonensis parasites might be of great interest as a future alternative to the development of new antileishmanial drugs.

Cell death and ultrastructural alterations in Leishmania amazonensis caused by new compound 4-Nitrobenzaldehyde thiosemicarbazone derived from S -limonene

BackgroundThe treatment of leishmaniasis with pentavalent antimonials is problematic because of their toxicity. Investigations of potentially active molecules are important to discover less toxic drugs that are viable economic alternatives for the treatment of leishmaniasis. Thiosemicarbazones are a group of molecules that are known for their wide versatility and biological activity. In the present study, we examined the antileishmania activity, mechanism of action, and biochemical alterations produced by a novel molecule, 4-nitrobenzaldehyde thiosemicarbazone (BZTS), derived from S-limonene against Leishmania amazonensis.ResultsBZTS inhibited the growth of the promastigote and axenic amastigote forms, with an IC50 of 3.8 and 8.0 μM, respectively. Intracellular amastigotes were inhibited by the compound with an IC50 of 7.7 μM. BZTS also had a CC50 of 88.8 μM for the macrophage strain J774A1. BZTS altered the shape, size, and ultrastructure of the parasites, including damage to mitochondria, reflected by extensive swelling and disorganization of the inner mitochondrial membrane, intense cytoplasmic vacuolization, and the presence of concentric membrane structures inside the organelle. Cytoplasmic lipid bodies, vesicles inside vacuoles in the flagellar pocket, and enlargement were also observed. BZTS did not induce alterations in the plasma membrane or increase annexin-V fluorescence intensity, indicating no phosphatidylserine exposure. However, it induced the production of mitochondrial superoxide anion radicals.ConclusionsThe present results indicate that BZTS induced dramatic effects on the ultrastructure of L. amazonensis, which might be associated with mitochondrial dysfunction and oxidative damage, leading to parasite death.

Benzaldehyde Thiosemicarbazone Derived from Limonene Complexed with Copper Induced Mitochondrial Dysfunction in Leishmania amazonensis

Background Leishmaniasis is a major health problem that affects more than 12 million people. Treatment presents several problems, including high toxicity and many adverse effects, leading to the discontinuation of treatment and emergence of resistant strains. Methodology/Principal Findings We evaluated the in vitro antileishmanial activity of benzaldehyde thiosemicarbazone derived from limonene complexed with copper, termed BenzCo, against Leishmania amazonensis. BenzCo inhibited the growth of the promastigote and axenic amastigote forms, with IC50 concentrations of 3.8 and 9.5 µM, respectively, with 72 h of incubation. Intracellular amastigotes were inhibited by the compound, with an IC50 of 10.7 µM. BenzCo altered the shape, size, and ultrastructure of the parasites. Mitochondrial membrane depolarization was observed in protozoa treated with BenzCo but caused no alterations in the plasma membrane. Additionally, BenzCo induced lipoperoxidation and the production of mitochondrial superoxide anion radicals in promastigotes and axenic amastigotes of Leishmania amazonensis. Conclusion/Significance Our studies indicated that the antileishmania activity of BenzCo might be associated with mitochondrial dysfunction and oxidative damage, leading to parasite death.

Sulfated glycosaminoglycan-based block copolymer: preparation of biocompatible chondroitin sulfate-b-poly(lactic acid) micelles.

Despite a growing interest in amphiphilic polysaccharide-based diblock copolymers as functional polymeric drug delivery nanosystems, biologically relevant sulfated glycosaminoglycan systems were not yet investigated. Here, we report the synthesis and the self-assembly properties in water of chondroitin sulfate-b-poly(lactic acid) (CS-b-PLA(n)). The CS-b-PLA(n) were synthesized using click-grafting onto method implying reducing-end alkynation of low-molecular weight depolymerized CS (M(w) = 5000 g·mol(-1)) and azide-terminated functionalization of PLAn (M(w) = 6500 g·mol(-1) (n = 46) and M(w) = 1700 g·mol(-1) (n = 20)). The diblock copolymer self-assembled in water giving rise to spherical micelles that were characterized in solution using dynamic/static light scattering and at dry state by TEM technique. In vitro assays on healthy cells showed that at high concentrations, up to 10 μg·mL(-1), CS-b-PLA(n) were noncytotoxic. Those preliminary studies are promising in the perspective to use them as biocompatible nanovehicles for anticancer drug delivery.

In vitro and in vivo antileishmania activity of sesquiterpene lactone-rich dichloromethane fraction obtained from Tanacetum parthenium (L.) Schultz-Bip

The discovery of new treatments for neglected diseases, including leishmaniasis, is a substantial challenge for scientific research. Plant extracts have shown potential in the selective treatment of tropical diseases. The present study evaluated the in vitro and in vivo antileishmania effects of a sesquiterpene lactone-rich dichloromethane fraction (DF) obtained from the aerial parts of Tanacetum parthenium (L.) Schultz-Bip. In vitro studies of the DF indicated an IC50 of 2.40±0.76 μg mL(-1) against the promastigote form and 1.76±0.25 μg mL(-1) against the axenic amastigote form of Leishmania amazonensis. In vivo intramuscular treatment with DF decreased the growth and size of footpad lesions in mice. The DF also significantly decreased the parasite population compared with animals that were treated with the reference drug. Plasma malondialdehyde levels were increased slightly by the DF, attributable to its parthenolide-rich composition that causes cellular apoptosis, compared with the control group, demonstrating treatment efficacy without toxicity or genotoxicity. Because the isolation and purification of plant compounds are costly and time-consuming and generate low yields, extract fractions, such as the DF studied herein, represent a promising alternative for the treatment of leishmaniasis.

Induction of Early Autophagic Process on Leishmania amazonensis by Synergistic Effect of Miltefosine and Innovative Semi-synthetic Thiosemicarbazone

Drug combination therapy is a current trend to treat complex diseases. Many benefits are expected from this strategy, such as cytotoxicity decrease, retardation of resistant strains development, and activity increment. This study evaluated in vitro combination between an innovative thiosemicarbazone molecule – BZTS with miltefosine, a drug already consolidated in the leishmaniasis treatment, against Leishmania amazonensis. Cytotoxicity effects were also evaluated on macrophages and erythrocytes. Synergistic antileishmania effect and antagonist cytotoxicity were revealed from this combination therapy. Mechanisms of action assays were performed in order to investigate the main cell pathways induced by this treatment. Mitochondrial dysfunction generated a significant increase of reactive oxygen and nitrogen species production, causing severe cell injuries and promoting intense autophagy process and consequent apoptosis cell death. However, this phenomenon was not strong enough to promote dead in mammalian cell, providing the potential selective effect of the tested combination for the protozoa. Thus, the results confirmed that drugs involved in distinct metabolic routes are promising agents for drug combination therapy, promoting a synergistic effect.

Generating nanoparticles containing a new 4-nitrobenzaldehyde thiosemicarbazone compound with antileishmanial activity

Thiosemicarbazones are an important class of compounds that have been extensively studied in recent years, mainly because of their broad profile of pharmacological activity. A new 4-nitrobenzaldehyde thiosemicarbazone compound (BZTS) that was derived from S-limonene has been demonstrated to have significant antiprotozoan activity. However, the hydrophobic characteristic of BZTS limits its administration and results in low oral bioavailability. In the present study, we proposed the synthesis of nanoparticle-based block copolymers that can encapsulate BZTS, with morphological evaluation of the nanoparticle suspensions being performed by transmission and cryo-transmission electronic microscopy. The mean particle sizes of the nanoparticle suspensions were determined by static light and dynamic light scattering (SLS/DLS), and the hydrodynamic radius (Rh) was determined using the Stokes-Einstein equation. The zeta potential (ζ) and polydispersity index (PDI) were also determined. The entrapment encapsulation efficiency of the BZTS nanoparticles was measured by ultraviolet spectrophotometry. In vitro activity of BZTS nanoparticle suspensions against intracellular amastigotes of Leishmania amazonensis and cytotoxic activity were also evaluated. The results showed the production of spherical nanoparticles with varied sizes depending on the hydrophobic portion of the amphiphilic diblock copolymers used. Significant concentration-dependent inhibitory activity against intracellular amastigotes was observed, and low cytotoxic activity was demonstrated against macrophages.

Effect of the marine brown alga Canistrocarpus cervicornis on promastigote forms of Leishmania (L.) amazonensis

Leishmaniasis is a disease resulting from infection by protozoan parasites of the genus Leishmania. Pentavalent antimonials, used clinically for more than 50 years, are still the first-choice drugs for the treatment of leishmaniasis, but they are toxic, require long-term treatment, and are prone to stimulate drug resistance [1]. Marine brown algae (Phaeophyceae) belonging to the order Dictyotales have emerged as an exceptionally rich source of diterpenoids, which form part of a defensive strategy against herbivores in the marine environment [2]. We have investigated the activity of crude extracts, a fraction, and an isolated compound (4R, 9S, 14S)-4α-Acetoxy-9β,14α-dihydroxydolast-1(15),7-diene of the brown alga Canistrocarpus cervicornis against promastigote forms of Leishmania amazonensis. The antiproliferative assays showed a dose-dependent effect against promastigotes with IC50 values in the range between 20.0 and 80.0µg/mL for crude extracts, 5.0µg/mL for the fraction and 2.0µg/mL for the isolated compound from C. cervicornis. We also investigated targets in the parasite by means of electron microscopy. Ultrastructural alterations were mainly observed in the mitochondrion of parasites treated with the isolated compound. Based on the current study, compounds from C. cervicornis appear to be an alternative for the development of new antiparasitic chemotherapies. However, further in vitro and in vivo studies are necessary to elucidate the mechanism of action of this compound. Acknowledgements: CNPq, FINEP, PRONEX/Fundacao Araucaria. References: [1] Croft, S.L. et al. (2006) Indian J. Med. Res. 123:399–410. [2] Garcia, D.G. et al. (2009) Phytother. Res., in press.