Ana Carolina Pereira

Curcumin-loaded into PLGA nanoparticles Preparation and in vitro schistosomicidal activity

The incorporation of the curcumin into poly(lactic-co-glycolic)acid (PLGA) nanospheres by the nanoprecipitation technique, the characterization of the nanoparticles and the schistosomicidal activity of the curcumin-loaded into PLGA nanospheres were reported. The incorporation process occurred with high efficiency and the images of field-emission scanning electron microscopy (FESEM) revealed the production of spherically shaped particles. According to the dynamic light scattering measurements, the particles are nanometric and monodisperse. The curcumin-loaded PLGA nanoparticles (50 and 100 μM) caused the death of all worms and a separation between 50% and 100% of Schistosoma mansoni couples at concentrations from 30xa0μM. Moreover, the curcumin-loaded PLGA nanoparticles also decreased the motor activity and caused partial alterations in the tegument of adult worms. This study marks the first time that schistosomicidal activity has been reported for curcumin-loaded PLGA nanoparticles.

Effect of the dibenzylbutyrolactone lignan (-)-hinokinin on doxorubicin and methyl methanesulfonate clastogenicity in V79 Chinese hamster lung fibroblasts.

The dibenzylbutyrolactone lignan (-)-hinokinin (HK) was obtained by partial synthesis from (-)-cubebin, isolated from the dry seeds of the pepper, Piper cubeba. In view of the trypanocidal activity of HK and its potential as a lead compound for drug development, evaluation of its possible genotoxic activity is required. We have tested HK for possible genotoxicity and evaluated the compounds effect on the activity of the clastogens doxorubicin (DXR) and methyl methanesulfonate (MMS) in the micronucleus (MN) assay with Chinese hamster lung fibroblast V79 cells. HK alone did not induce MN, at concentrations up to 128microM. In combined treatments, HK reduced the frequency of MN induced by MMS. With respect to DXR, HK exerted a protective effect at lower concentrations, but at higher concentrations it potentiated DXR clastogenicity.

Furocoumarins and coumarins photoinactivate Colletotrichum acutatum and Aspergillus nidulans fungi under solar radiation

The increasing tolerance to currently-used fungicides is a major problem both in clinical and agricultural areas leading to an urgent need for the development of novel antifungal strategies. This study investigated the in vitro antimicrobial photo treatment (APT) of conidia of the plant-pathogenic fungus Colletotrichum acutatum and the ascomycete Aspergillus nidulans with the furocoumarins 8-methoxypsoralen (8-MOP) and isopimpinellin, and a mixture of two coumarins (7-methoxy coumarin and citropten). Subcellular localization of the photosensitizer 8-MOP was also determined in C. acutatum conidia. Additionally, the effects of APT on the leaves of the plant host Citrus sinensis were determined. APT with 8-MOP (50μM) led to a reduction of approximately 4 logs in the survival of the conidia of both species, and the mixture of the two coumarins (12.5mgL(-1)) resulted in a reduction of approximately 4 logs for A. nidulans and 3 logs for C. acutatum. Isopimpinellin (50μM) displayed a reduction of 4 logs for A. nidulans but less than 2 logs for C. acutatum. Washing the conidia to remove unbound photosensitizers before light exposure reduced the photodynamic inactivation of C. acutatum both with 8-MOP and the mixture of the two coumarins. The reduction was smaller for A. nidulans. 8-MOP spread throughout the cytoplasm and accumulated in structures such as lipid bodies of C. acutatum conidia. No damage to orange tree leaves was observed after APT with any of the photosensitizers.

Schistosomicidal and trypanocidal structure-activity relationships for (+/-)-licarin A and its (-)- and (+)-enantiomers

(±)-Licarin A (1) was obtained by oxidative coupling, and its enantiomers, (-)-licarin A (2) and (+)-licarin A (3), were resolved by chiral HPLC. Schistosomicidal and trypanocidal activities of these compounds were evaluated in vitro against Schistosoma mansoni adult worms and trypomastigote forms of Trypanosoma cruzi. The racemic mixture (1) displayed significant schistosomicidal activity with an LC₅₀ value of 53.57 μM and moderate trypanocidal activity with an IC₅₀ value of 127.17 μM. On the other hand, the (-)-enantiomer (2), displaying a LC₅₀ value of 91.71 μM, was more active against S. mansoni than the (+)-enantiomer (3), which did not show activity. For the trypanocidal assay, enantiomer 2 showed more significant activity (IC₅₀ of 23.46 μM) than enantiomer 3, which showed an IC₅₀ value of 87.73 μM. Therefore, these results suggest that (±)-licarin A (1) and (-)-licarin A (2) are promising compounds that could be used for the development of schistosomicidal and trypanocidal agents.

Reduction of parasitism tissue by treatment of mice chronically infected with Trypanosoma cruzi with lignano lactones

The reduction of parasitism tissue upon treatment with two lignano lactones, namely (−)- cubebin (CUB) and (−)-hinokinin (HNK), was evaluated in the chronic phase of Chagas’ disease by quantifying the enzyme β-galactosidase expressed by the CL B5 clone strain of Trypanosoma cruzi. Tissue karyometry was also performed. Treatment with the assessed lignans led to a larger reduction in parasitism tissue in all evaluated organs, compared with benznidazole (BZN). Oral treatment with CUB or HNK was more effective. Karyometry results demonstrated that the infected control animals had increased nuclear area compared with uninfected controls, indicating cellular hypertrophy. Results also revealed that use of CUB or HNK was able to significantly prevent this increase, and a slight decrease in the nuclear area was observed, compared with mice treated with BZN. Taken together, these data demonstrate that CUB and HNK could be considered as potential compounds for the development of new drugs for treatment of Chagas’ disease.

Antimycobacterial Activity of Natural and Semi-Synthetic Lignans

The antimycobacterial activity of (-)-cubebin (1), hinokinin (2), and some of their semisynthetic derivatives, namely (-)-O-acetyl-cubebin (3), (-)-O-methyl-cubebin (4), (-)-O-(N,Ndimethylamine- ethyl)-cubebin (5) and (-)-6,6´-dinitrohinokinin (6), was evaluated against Mycobacterium tuberculosis (ATCC 27294), M. kansasii (ATCC 12478), and M. avium (ATCC 15769). The MIC values ranged from 31.25 to 2000 μg/mL. Among the evaluated compounds, 2 displayed a MIC value of 62.5 μg/mL against M. tuberculosis, while 3 and 4 displayed MIC values of 62.5 and 31.25 μg/mL, respectively, against M. avium. All compounds were inactive against M. kansasii. These are promising results concerning the search for biologically active natural products, highlighting that new approaches to the prevention, treatment, and cure of tuberculosis are extremely important.

In vitro and in vivo anthelmintic activity of (-)-6,6'-dinitrohinokinin against schistosomula and juvenile and adult worms of Schistosoma mansoni.

The chemotherapy of schistosomiasis relies on the use of praziquantel. However, concerns over drug resistance have encouraged the search for new drug leads. This paper is the first report on the in vitro and in vivo activity of (-)-6,6-dinitrohinokinin (DNK) against Schistosoma mansoni. In vitro, the lethal concentrations for 50% of parasites (LC50) of DNK against adult worms were 103.9±3.6 and 102.5±4.8μM at 24 and 72h, respectively. Scanning electron microscopy images showed extensive tegumental alterations such as peeling and smaller numbers of tubercles in the spine of adult worms. DNK also elicited high mortality of schistosomula, with LC50 values of 57.4±2.3, 32.5±0.9, and 20.4±1.2μM at 24, 48, and 72h, respectively. DNK displayed moderate activity against the juvenile liver parasite, with an LC50 value of 179.5±2.3 μM at 72h. This compound reduced the total number of eggs by over 83%, and it affected the development of eggs produced by adult worms. The selectivity index showed that at 24h, DNK was 8.5 and 15.4 times more toxic to the adult worms and schistosomula than to Chinese hamster lung fibroblast cells, respectively. Treatment of infected mice with DNK moderately decreased worm burden (33.8-52.3%), egg production (40.7-60.0%), and spleen and liver weights. Together, our results indicated that DNK presents moderate in vitro and in vivo activities against S. mansoni, and it might therefore be interesting to explore the structure-activity relationship of the antischistosomal activity of this compound.

Trypanosoma cruzi: evaluation of (−)-cubebin derivatives activity in the messenger RNAs processing

No fully effective treatment has been developed since the discovery of Chagas’ disease. Since drug-resistant Trypanosoma cruzi strains are occurring and the current therapy is effective in the acute phase but with various adverse side effects, more studies are needed to characterize the susceptibility of T. cruzi to new drugs. Pre-mRNA maturation in trypanosomatids occurs through a process called trans-splicing, which is unusual RNA processing reaction, and it implies the processing of polycistronic transcription units into individual mRNAs; a short transcript spliced leader (SL RNA) is trans-spliced to the acceptor pre-mRNA, giving origin to the mature mRNA. Cubebin derivatives seem to provide treatments with less collateral effects than benznidazole and showed similar or better trypanocidal activities than benznidazole. Therefore, the cubebin derivatives ((−)-6,6′-dinitrohinokinin (DNH) and (−)-hinokinin (HQ)) interference in the mRNA processing was evaluated using T. cruzi permeable cells (Y and BOL (Bolivia) strains) following by RNase protection reaction. These substances seem to intervene in any step of the RNA transcription, promoting alterations in the RNA synthesis, even though the RNA processing mechanism still occurs. Furthermore, HQ presented better activity against the parasites than DNH, meaning that BOL strain seems to be more resistant than Y.

The lignan (-)-hinokinin displays modulatory effects on human monoamine and GABA transporter activities.

The neurotransmitter transporters of the SLC6 family play critical roles in the regulation of neurotransmission and are the primary targets of therapeutic agents used to treat clinical disorders involving compromised neurotransmitter signaling. The dopamine and norepinephrine transporters have been implicated in clinical disorders such as attention deficit hyperactivity disorder (ADHD) and substance abuse. The GABA transporters (GATs) serve as a target for anxiolytic, antidepressant, and antiepileptic therapies. In this work, the interaction with neurotransmitter transporters was characterized for a derivative of the lignan (-)-cubebin (1), namely, (-)-hinokinin (2). Using in vitro pharmacological assays, 2 selectively inhibited the human dopamine and norepinephrine transporters, in a noncompetitive manner possibly mediated by binding to a novel site within the transporters, and displayed low affinity for the serotonin transporter. Compound 2 also specifically inhibited the GAT-1 GABA transporter subtype. Compound 2 is not a substrate of the carriers as it had no effect on the efflux of either of the neurotransmitters investigated. This compound is inactive toward glutamate and glycine transporters. These results suggest that 2 may serve as a tool to develop new therapeutic drugs for ADHD and anxiety that target the DAT, NET, and GAT-1 transporters.

Inactivation of plant-pathogenic fungus Colletotrichum acutatum with natural plant-produced photosensitizers under solar radiation.

The increasing tolerance to currently used fungicides and the need for environmentally friendly antimicrobial approaches have stimulated the development of novel strategies to control plant-pathogenic fungi such as antimicrobial phototreatment (APT). We investigated the in vitro APT of the plant-pathogenic fungus Colletotrichum acutatum with furocoumarins and coumarins and solar radiation. The compounds used were: furocoumarins 8-methoxypsoralen (8-MOP) and 5,8-dimethoxypsoralen (isopimpinellin), coumarins 2H-chromen-2-one (coumarin), 7-hydroxycoumarin, 5,7-dimethoxycoumarin (citropten) and a mixture (3:1) of 7-methoxycoumarin and 5,7-dimethoxycoumarin. APT of conidia with crude extracts from Tahiti acid lime, red and white grapefruit were also performed. Pure compounds were tested at 50μM concentration and mixtures and extracts at 12.5mgL(-1). The C. acutatum conidia suspension with or without the compounds was exposed to solar radiation for 1h. In addition, the effects of APT on the leaves of the plant host Citrus sinensis were determined. APT with 8-MOP was the most effective treatment, killing 100% of the conidia followed by the mixture of two coumarins and isopimpinellin that killed 99% and 64% of the conidia, respectively. APT with the extracts killed from 20% to 70% of the conidia, and the extract from Tahiti lime was the most effective. No damage to sweet orange leaves was observed after APT with any of the compounds or extracts.