In vitro evaluation of cytotoxic potential of essential oil extracted from leaves of Croton heliotropiifolius Kunth in human tumor cells.

Croton heliotropiifolius Kunth, popularly known as "velame," is a shrub that resides in northeastern Brazil. The essential oil of C. heliotropiifolius contains high concentrations of volatile compounds in the leaves and is widely used in folk medicine for many purposes as an antiseptic, analgesic, sedative, and anti-inflammatory agent. Due to the apparent limited amount of information, the aim of this study was to determine the cytotoxic potential of essential oil extracted from leaves of C. heliotropiifolius, utilizing different human cancer cell lines (HL-60, leukemia; HCT-116, colon; MDA-MB435, melanoma; SF295, glioblastoma) and comparison to murine fibroblast L929 cell line. The chemical characterization of the essential oil revealed the presence of large amounts of monoterpenes and sesquiterpenes, the majority of which were aristolene (22.43%), germacrene D (11.38%), ɣ-terpinene (10.85%), and limonene (10.21%). The essential oil exerted significant cytotoxicity on all cancer cells, with low activity on murine L929 fibroblasts, independent of disruption of cell membranes evidenced by absence of hemolytic activity. The cytotoxicity identified was associated with oxidative stress, which culminated in mitochondrial respiration dysfunction and direct or indirect DNA damage (strand breaks and oxidative damage), triggering cell death via apoptosis. Our findings suggest that extracts of essential oil of C. Heliotropiifolius may be considered as agents to be used therapeutically in treatment of certain cancers.

Diterpenoids from Leaves of Cultivated Plectranthus ornatus.

Two new diterpenoid derivatives 7α,12ß,17-triacetoxy-6ß,19-dihydroxy-13ß,16-spirocicloabiet-8-ene-11,14-dione ( 1: ) and 6ß-acetoxy-3ß,7α,12α-trihydroxy-13ß,16-spirocicloabiet-8-ene-11,14-dione ( 2: ) along with 11 ( 3: - 13: ) miscellaneous compounds were isolated from the leaves of Plectranthus ornatus Codd. Their structures were elucidated by spectroscopic analysis and gauge independent atomic orbitals 13C NMR calculations. The isolated compounds were screened for their effects on intestinal motility using guinea-pig ileum and duodenum and by their cytotoxicity against 4 human cancer cell lines (HCT-116, SF-295, PC-3, and HL-60). Compounds 6: and 9: were moderately cytotoxic against HL-60, whereas 6: and 13: were more active on SF-295 and HCT-116.

Involvement of anti-inflammatory, antioxidant, and BDNF up-regulating properties in the antipsychotic-like effect of the essential oil of Alpinia zerumbet in mice: a comparative study with olanzapine.

The current drug therapy for schizophrenia effectively treats acute psychosis and its recurrence; however, this mental disorder's cognitive and negative symptoms are still poorly controlled. Antipsychotics present important side effects, such as weight gain and extrapyramidal effects. The essential oil of Alpinia zerumbet (EOAZ) leaves presents potential antipsychotic properties that need further preclinical investigation. Here, we determined EAOZ effects in preventing and reversing schizophrenia-like symptoms (positive, negative, and cognitive) induced by ketamine (KET) repeated administration in mice and putative neurobiological mechanisms related to this effect. We conducted the behavioral evaluations of prepulse inhibition of the startle reflex (PPI), social interaction, and working memory (Y-maze task), and verified antioxidant (GSH, nitrite levels), anti-inflammatory [interleukin (IL)-6], and neurotrophic [brain-derived neurotrophic factor (BDNF)] effects of this oil in hippocampal tissue. The atypical antipsychotic olanzapine (OLZ) was used as standard drug therapy. EOAZ, similarly to OLZ, prevented and reversed most KET-induced schizophrenia-like behavioral alterations, i.e., sensorimotor gating deficits and social impairment. EOAZ had a modest effect on the prevention of KET-associated working memory deficit. Compared to OLZ, EOAZ showed a more favorable side effects profile, inducing less cataleptic and weight gain changes. EOAZ efficiently protected the hippocampus against KET-induced oxidative imbalance, IL-6 increments, and BDNF impairment. In conclusion, our data add more mechanistic evidence for the anti-schizophrenia effects of EOAZ, based on its antioxidant, anti-inflammatory, and BDNF up-regulating actions. The absence of significant side effects observed in current antipsychotic drug therapy seems to be an essential benefit of the oil.

Action mechanism of naphthofuranquinones against fluconazole-resistant Candida tropicalis strains evidenced by proteomic analysis: The role of increased endogenous ROS.

The increased incidence of candidemia in terciary hospitals worldwide and the cross-resistance frequency require the new therapeutic strategies development. Recently, our research group demonstrated three semi-synthetic naphthofuranquinones (NFQs) with a significant antifungal activity in a fluconazole-resistant (FLC) C. tropicalis strain. The current study aimed to investigate the action's preliminary mechanisms of NFQs by several standardized methods such as proteomic and flow cytometry analyzes, comet assay, immunohistochemistry and confocal microscopy evaluation. Our data showed C. tropicalis 24 h treated with all NFQs induced an expression's increase of proteins involved in the metabolic response to stress, energy metabolism, glycolysis, nucleosome assembly and translation process. Some aspects of proteomic analysis are in consonance with our flow cytometry analysis which indicated an augmentation of intracellular ROS, mitochondrial dysfunction and DNA strand breaks (neutral comet assay and γ-H2AX detection). In conclusion, our data highlights the great contribution of ROS as a key event, probably not the one, associated to anti-candida properties of studied NFQs.

Study of tensiometric properties, microbiological and collagen content in nile tilapia skin submitted to different sterilization methods.

Tissue bioengineering development is a global concern and different materials are studied and created to be safe, effective and with low cost. Nile Tilapia skin had shown its biological potential as covers for the burn wound. This study evaluates the tilapia skin histological, collagen properties and tensiometric resistance, after treatment by different sterilization methods. Tilapia skin samples were submitted to two sterilization processes: (1) chemical, which consisted in two 2% chlorhexidin baths, followed by sequential baths in increasing glycerol concentrations; and (2) radiation, when glycerolized skin samples were submitted to gamma radiation at 25, 30 and 50 kGy. Microscopic analyzes were performed through Haematoxylin-eosin and Picrosirius Red under polarized light. For tensiometric analysis, traction tests were performed. Glycerol treated skin presented a discrete collagen fibers disorganization within the deep dermis, while irradiated skin did not show any additional change. Throughout the steps of chemical sterilization, there was a higher proportion of collagen with red/yellow birefringence (type I) in the skin samples up to the first bath in chlorhexidin, when compared to samples after the first two glycerol baths (P < 0.005). However, there was no difference in relation to total collagen between groups. In irradiated skin, there was a larger total collagen preservation when using until 30 kGy (P < 0.005). Tensiometric evaluation did not show significant differences in relation to maximum load in the groups studied. We concluded that chemical and radiation (25 and 30 kGy) are efficient methods to sterilize Nile Tilapia skin without altering its microscopic or tensiometric characteristics.

Impact of testicular sperm extraction and testicular sperm aspiration on gonadal function in an experimental rat model.

PURPOSE: To assess the impact of sperm retrieval on the gonadal function of rats with impaired spermatogenesis by comparing testicular sperm extraction (TESE) to aspiration (TESA). The efficacy of these procedures to sperm obtainment was also compared. MATERIALS AND METHODS: A pilot study showed impaired spermatogenesis, but normal testosterone (T) production after a bilateral orchidopexy applied to 26 rats, which were randomly assigned into four groups: TESE (n=7), TESA (n=7), SHAM (n=6) and Control (n=6). The T levels were measured through comparative analysis after the orchidopexy. RESULTS: There was no statistical difference in the animal's baseline T levels after orchidopexy in comparison to the controls: the TESE and TESA groups, 6.66±4.67ng/mL; the SHAM group (orchidopexy only), 4.99±1.96ng/mL; and the Control, 4.75±1.45ng/ mL, p=0.27. Accordingly, no difference was found in the postoperative T levels: TESE, 5.35±4.65ng/mL; TESA, 3.96±0.80ng/mL; SHAM, 3.70±1.27ng/mL; p=0.4. The number of sperm cells found through TESE (41.0±7.0) was significantly larger than that found through TESA (21.3±8.1, p=0.001). Moreover, higher tissue weight was found through TESE (0.09±0.02g versus 0.04±0.04g, p=0.04). CONCLUSIONS: The testicular sperm capture performed in rats through extraction or aspiration, after orchidopexy, did not significantly decrease the T levels. The amount of sperm found through testicular sperm extraction was higher than that through testicular sperm aspiration.

In vitro anti-Candida activity of selective serotonin reuptake inhibitors against fluconazole-resistant strains and their activity against biofilm-forming isolates.

Recent research has shown broad antifungal activity of the classic antidepressants selective serotonin reuptake inhibitors (SSRIs). This fact, combined with the increased cross-resistance frequency of the genre Candida regarding the main treatment today, fluconazole, requires the development of novel therapeutic strategies. In that context, this study aimed to assess the antifungal potential of fluoxetine, sertraline, and paroxetine against fluconazole-resistant Candida spp. planktonic cells, as well as to assess the mechanism of action and the viability of biofilms treated with fluoxetine. After 24 h, the fluconazole-resistant Candida spp. strains showed minimum inhibitory concentration (MIC) in the ranges of 20-160 µg/mL for fluoxetine, 10-20 µg/mL for sertraline, and 10-100.8 µg/mL for paroxetine by the broth microdilution method (M27-A3). According to our data by flow cytometry, each of the SSRIs cause fungal death after damaging the plasma and mitochondrial membrane, which activates apoptotic signaling pathways and leads to dose-dependant cell viability loss. Regarding biofilm-forming isolates, the fluoxetine reduce mature biofilm of all the species tested. Therefore, it is concluded that SSRIs are capable of inhibit the growth in vitro of Candida spp., both in planktonic form, as biofilm, inducing cellular death by apoptosis.

Berberine Antifungal Activity in Fluconazole-Resistant Pathogenic Yeasts: Action Mechanism Evaluated by Flow Cytometry and Biofilm Growth Inhibition in Candida spp.

The incidence of fungal infections and, in particular, the incidence of fungal antibiotic resistance, which is associated with biofilm formation, have significantly increased, contributing to morbidity and mortality. Thus, new therapeutic strategies need to be developed. In this context, natural products have emerged as a major source of possible antifungal agents. Berberine is a protoberberine-type isoquinoline alkaloid isolated from the roots, rhizomes, and stem bark of natural herbs, such as Berberis aquifolium, Berberis vulgaris, Berberis aristata, and Hydrastis canadensis, and of Phellodendron amurense Berberine has been proven to have broad antibacterial and antifungal activity. In the present study, the potential antifungal effect of berberine against fluconazole-resistant Candida and Cryptococcus neoformans strains, as well as against the biofilm form of Candida spp., was assessed. The antifungal effect of berberine was determined by a broth microdilution method (the M27-A3 method of the Clinical and Laboratory Standards Institute) and flow cytometry techniques, in which the probable mechanism of action of the compound was also assessed. For biofilm assessment, a colorimetric 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was used to determine the susceptibility of sessile cells. The isolates used in the study belonged to the Laboratory of Bioprospection and Experiments in Yeast (LABEL) of the Federal University of Ceará. After 24 and 72 h, fluconazole-resistant Candida and Cryptococcus neoformans strains showed berberine MICs equal to 8 µg/ml and 16 µg/ml, respectively. Cytometric analysis showed that treatment with berberine caused alterations to the integrity of the plasma and mitochondrial membranes and DNA damage, which led to cell death, probably by apoptosis. Assessment of biofilm-forming isolates after treatment showed statistically significant reductions in biofilm cell activity (P < 0.001).

Endophytic Actinobacteria from the Brazilian Medicinal Plant Lychnophora ericoides Mart. and the Biological Potential of Their Secondary Metabolites.

Endophytic actinobacteria from the Brazilian medicinal plant Lychnophora ericoides were isolated for the first time, and the biological potential of their secondary metabolites was evaluated. A phylogenic analysis of isolated actinobacteria was accomplished with 16S rRNA gene sequencing, and the predominance of the genus Streptomyces was observed. All strains were cultured on solid rice medium, and ethanol extracts were evaluated with antimicrobial and cytotoxic assays against cancer cell lines. As a result, 92% of the extracts showed a high or moderate activity against at least one pathogenic microbial strain or cancer cell line. Based on the biological and chemical analyses of crude extracts, three endophytic strains were selected for further investigation of their chemical profiles. Sixteen compounds were isolated, and 3-hydroxy-4-methoxybenzamide (9) and 2,3-dihydro-2,2-dimethyl-4(1H)-quinazolinone (15) are reported as natural products for the first time in this study. The biological activity of the pure compounds was also assessed. Compound 15 displayed potent cytotoxic activity against all four tested cancer cell lines. Nocardamine (2) was only moderately active against two cancer cell lines but showed strong activity against Trypanosoma cruzi. Our results show that endophytic actinobacteria from L. ericoides are a promising source of bioactive compounds.

Synergistic effect of the flavonoid catechin, quercetin, or epigallocatechin gallate with fluconazole induces apoptosis in Candida tropicalis resistant to fluconazole.

Flavonoids are a class of phenolic compounds commonly found in fruits, vegetables, grains, flowers, tea, and wine. They differ in their chemical structures and characteristics. Such compounds show various biological functions and have antioxidant, antimicrobial, anti-inflammatory, and antiapoptotic properties. The aim of this study was to evaluate the in vitro interactions of flavonoids with fluconazole against Candida tropicalis strains resistant to fluconazole, investigating the mechanism of synergism. Three combinations formed by the flavonoids (+)-catechin hydrated, hydrated quercetin, and (-)-epigallocatechin gallate at a fixed concentration with fluconazole were tested. Flavonoids alone had no antifungal activity within the concentration range tested, but when they were used as a cotreatment with fluconazole, there was significant synergistic activity. From this result, we set out to evaluate the possible mechanisms of cell death involved in this synergism. Isolated flavonoids did not induce morphological changes or changes in membrane integrity in the strains tested, but when they were used as a cotreatment with fluconazole, these changes were quite significant. When evaluating mitochondrial damage and the production of reactive oxygen species (ROS) only in the cotreatment, changes were observed. Flavonoids combined with fluconazole were shown to cause a significant increase in the rate of damage and the frequency of DNA damage in the tested strains. The cotreatment also induced an increase in the externalization of phosphatidylserine, an important marker of early apoptosis. It is concluded that flavonoids, when combined with fluconazole, show activity against strains of C. tropicalis resistant to fluconazole, promoting apoptosis by exposure of phosphatidylserine in the plasma membrane and morphological changes, mitochondrial depolarization, intracellular accumulation of ROS, condensation, and DNA fragmentation.