Nematostatic activity of isoprenylated guanidine alkaloids from Pterogyne nitens and their interaction with acetylcholinesterase.

Although new nematicides have appeared, the demand for new products less toxic and more efficient for the control of plant-parasitic nematodes are still high. Consequently, studies on natural secondary metabolites from plants, to develop new nematicides, have increased. In this work, nineteen extracts from eleven Brazilian plant species were screened for activity against Meloidogyne incognita. Among them, the extracts of Piterogyne nitens showed a potent nematostatic activity. The alkaloid fraction obtained from the ethanol extract of leaves of P. nitens was more active than the coming extract. Due to the promising activity from the alkaloid fraction, three isoprenylated guanidine alkaloids isolated from this fraction, galegine (1), pterogynidine (2), and pterogynine (3) were tested, showing similar activity to the alkaloid fraction, which was comparable to that of the positive control Temik at 250 µg/mL. At lower concentrations (125-50 µg/mL), compound 2 showed to be the most active one. As several nematicides act through inhibition of acetylcholinesterase (AChE), the guanidine alkaloids were also employed in two in vitro AChE assays. In both cases, compound 2 was more active than compounds 1 and 3. Its activity was considered moderated compared to the control (physostigmine). Compound 2 was selected for an in silico study with the electric eel (Electrophorus electricus) AChE, showing to bind mostly to the same site of physostigmine in the AChEs, pointing out that this could be the mechanism of action for this compound. These results suggested that the guanidine alkaloids 1,2 and 3 from P. nitens are promising for the development of new products to control M. incognita, especially guanidine 2, and encourage new investigations to confirm the mechanism of action, as well as to determine the structure-activity relationship of the guanidine alkaloids.

Mycobacterium tuberculosis and Paracoccidioides brasiliensis Formation and Treatment of Mixed Biofilm In Vitro.

Co-infection of Mycobacterium tuberculosis and Paracoccidioides brasiliensis, present in 20% in Latin America, is a public health problem due to a lack of adequate diagnosis. These microorganisms are capable of forming biofilms, mainly in immunocompromised patients, which can lead to death due to the lack of effective treatment for both diseases. The present research aims to show for the first time the formation of mixed biofilms of M. tuberculosis and P. brasiliensis (Pb18) in vitro, as well as to evaluate the action of 3'hydroxychalcone (3'chalc) -loaded nanoemulsion (NE) (NE3'chalc) against monospecies and mixed biofilms, the formation of mixed biofilms of M. tuberculosis H37Rv (ATCC 27294), 40Rv (clinical strains) and P. brasiliensis (Pb18) (ATCC 32069), and the first condition of formation (H37Rv +Pb18) and (40Rv + Pb18) and second condition of formation (Pb18 + H37Rv) with 45 days of total formation time under both conditions. The results of mixed biofilms (H37Rv + Pb18) and (40Rv + Pb18), showed an organized network of M. tuberculosis bacilli in which P. brasiliensis yeasts are connected with a highly extracellular polysaccharide matrix. The (Pb18 + H37Rv) showed a dense biofilm with an apparent predominance of P. brasiliensis and fragments of M. tuberculosis. PCR assays confirmed the presence of the microorganisms involved in this formation. The characterization of NE and NE3'chalc displayed sizes from 145.00 ± 1.05 and 151.25 ± 0.60, a polydispersity index (PDI) from 0.20± 0.01 to 0.16± 0.01, and zeta potential -58.20 ± 0.92 mV and -56.10 ± 0.71 mV, respectively. The atomic force microscopy (AFM) results showed lamellar structures characteristic of NE. The minimum inhibitory concentration (MIC) values of 3'hidroxychalcone (3'chalc) range from 0.97- 7.8 µg/mL and NE3'chalc from 0.24 - 3.9 µg/mL improved the antibacterial activity when compared with 3'chalc-free, no cytotoxicity. Antibiofilm assays proved the efficacy of 3'chalc-free incorporation in NE. These findings contribute to a greater understanding of the formation of M. tuberculosis and P. brasiliensis in the mixed biofilm. In addition, the findings present a new possible NE3'chalc treatment alternative for the mixed biofilms of these microorganisms, with a high degree of relevance due to the lack of other treatments for these comorbidities.

Antibiofilm and cytotoxic effect of 3,3'-dihydroxycurcumin (DHC) as photosensitizer agent in antimicrobial photodynamic therapy for endodontic purposes.

BACKGROUND: Curcuminoids have been designed not only to improve chemical and metabolic stability of curcumin (CUR), but also to increase its antimicrobial activity, without effecting its ability as photosensitizer agent in antimicrobial photodynamic therapy (aPDT) with light emitting diode (LED). This study evaluated the antimicrobial and antibiofilm action of curcumin analog DHC (or 3,3'-dihydroxycurcumin), submitted or not to LED irradiation, on microorganisms of endodontic importance and its influence on fibroblasts viability. METHODS: DHC was synthetized by modified Pablon's methodology and the experiments were conducted under irradiation or not with indium gallium nitride-based LED (440-480nm, 100 mW/cm2, 0.78 cm2,60 s). The antimicrobial activity of CUR and DHC were determined by the Minimum Inhibitory and Bactericidal Concentration assays against Gram-positive and Gram-negative bacteria and the effect of both compounds on fibroblast viability was tested using colorimetric assays. They were also evaluated on 72h and 7days single-species biofilms and on 14 days multispecies biofilms formed inside dentin tubules by bacterial colonies counts and confocal microscopy, respectively. Data were analyzed statistically considering p<0.05. RESULTS: DHC had bactericidal effect against all bacteria tested higher than CUR, in planktonic conditions. CUR and DHC (at 39 and 19 µg/mL, respectively) were cytocompatible and LED irradiation reduced fibroblast viability, regardless of compound. CUR and DHC reduced the growth of single-species biofilms and the effect of aPDT was bacteria dependent. DHC reduced more than 70% of microorganisms from multispecies biofilms, superior to CUR effect. CONCLUSIONS: DHC showed low cytotoxicity and antibiofilm effect similar to curcumin, when submitted or not to aPDT, and could be further explored as a bioactive compound for endodontic purposes.

Diacetylcurcumin: a new photosensitizer for antimicrobial photodynamic therapy in Streptococcus mutans biofilms.

This study evaluated the effect of antimicrobial photodynamic therapy (aPDT) on S. mutans using diacetylcurcumin (DAC) and verified DAC toxicity. In vitro, S. mutans biofilms were exposed to curcumin (CUR) and DAC and were light-irradiated. Biofilms were collected, plated and incubated for colony counts. DAC and CUR toxicity assays were conducted with Human Gingival Fibroblast cells (HGF). In vivo, G. mellonella larvae were injected with S. mutans and treated with DAC, CUR and aPDT. The hemolymph was plated and incubated for colony counts. Significant reductions were observed when DAC and CUR alone were used and when aPDT was applied. HGF assays demonstrated no differences in cell viability for most groups. DAC and CUR reduced the S. mutans load in G. mellonella larvae both alone and with aPDT. Systematic toxicity assays on G. mellonella demonstrated no effect of DAC and CUR or aPDT on the survival curve.

Nitensidine B affects proteins of the glycolytic pathway and induces apoptosis in cervical carcinoma cells immortalized by HPV16.

BACKGROUND: Cervical cancer, the fourth most common type of cancer among women worldwide, accounts for approximately 12% of all types of malignancies that affect women. Natural products have contributed significantly to the development of modern therapies; approximately 70% of the drugs available for chemotherapy are naturally based products. PURPOSE: The purpose of this study was to examine the biological activities of nitensidine B (NTB), a guanidinic alkaloid isolated from the leaves of Pterogyne nitens Tul. (Fabaceae) in a cervical cancer cell line. METHODS: In vitro experiments were performed using cervical carcinoma cells immortalized by human papillomavirus type 16 (HPV16, SiHa cells), since epidemiological and molecular studies have demonstrated robust associations between the etiologies of cervical cancer and HPV infection. Cytotoxicity as well as the effect of NTB treatment on intracellular signals of apoptosis, fragmentation of internucleosomal DNA via terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), and levels of apoptosis effectors (Caspase 3/7) were evaluated. In addition, differential proteomic analysis (iTRAQ) and protein validation using western blot were performed. RESULTS: The cytotoxicity of NTB treatment in the SiHa cell line was concentration-dependent, with the minimum inhibitory concentration of 50% of the cells of 40.98 µM. In the TUNEL assay, SiHa cell apoptosis with 3/7 caspase activation was reported at 12 h following treatment. Differential proteomic analysis by iTRAQ demonstrated that proteins of the glycolytic pathway, aldolase A, alpha-enolase, pyruvate kinase, and glyceraldehyde 3-phosphate dehydrogenase were underexpressed. CONCLUSION: These results indicated that NTB could play a role in decreasing glycolysis . Since tumor cells prefer the glycolytic pathway to generate energy, these findings suggest that NTB may be a reliable model for the study of human cervical cancer cell lines immortalized by HPV16, however more experiments can be performed.

Trypanosoma cruzi: analysis of two different strains after piplartine treatment.

The hemoflagellate protozoan, Trypanosoma cruzi, mainly transmitted by triatomine insects through blood transfusion or from mother-to-child, causes Chagas' disease. This is a serious parasitic disease that occurs in Latin America, with considerable social and economic impact. Nifurtimox and benznidazole, drugs indicated for treating infected persons, are effective in the acute phase, but poorly effective during the chronic phase. Therefore, it is extremely urgent to find innovative chemotherapeutic agents and/or effective vaccines. Since piplartine has several biological activities, including trypanocidal activity, the present study aimed to evaluate it on two T. cruzi strains proteome. Considerable changes in the expression of some important enzymes involved in parasite protection against oxidative stress, such as tryparedoxin peroxidase (TXNPx) and methionine sulfoxide reductase (MSR) was observed in both strains. These findings suggest that blocking the expression of the two enzymes could be potential targets for therapeutic studies.

Trypanosoma cruzi: analysis of two different strains after piplartine treatment

ABSTRACT The hemoflagellate protozoan, Trypanosoma cruzi, mainly transmitted by triatomine insects through blood transfusion or from mother-to-child, causes Chagas' disease. This is a serious parasitic disease that occurs in Latin America, with considerable social and economic impact. Nifurtimox and benznidazole, drugs indicated for treating infected persons, are effective in the acute phase, but poorly effective during the chronic phase. Therefore, it is extremely urgent to find innovative chemotherapeutic agents and/or effective vaccines. Since piplartine has several biological activities, including trypanocidal activity, the present study aimed to evaluate it on two T. cruzi strains proteome. Considerable changes in the expression of some important enzymes involved in parasite protection against oxidative stress, such as tryparedoxin peroxidase (TXNPx) and methionine sulfoxide reductase (MSR) was observed in both strains. These findings suggest that blocking the expression of the two enzymes could be potential targets for therapeutic studies.

Antifungal Activity of Decyl Gallate against Several Species of Pathogenic Fungi.

This work aims to demonstrate that the gallic acid structure modification to the decyl gallate (G14) compound contributed to increase the antifungal activity against several species of pathogenic fungi, mainly, Candida spp., Cryptococcus spp., Paracoccidioides spp., and Histoplasma capsulatum, according to standardized microdilution method described by Clinical Laboratory Standard Institute (CLSI) documents. Moreover this compound has a particularly good selectivity index value, which makes it an excellent candidate for broad-spectrum antifungal prototype and encourages the continuation of subsequent studies for the discovery of its mechanism of action.

Anti-trichophyton activity of protocatechuates and their synergism with fluconazole.

Dermatophytosis and superficial mycosis are a major global public health problem that affects 20-25% of the world's population. The increase in fungal resistance to the commercially available antifungal agents, in conjunction with the limited spectrum of action of such drugs, emphasises the need to develop new antifungal agents. Natural products are attractive prototypes for antifungal agents due to their broad spectrum of biological activities. This study aimed to verify the antifungal activity of protocatechuic acid, 3,4-diacetoxybenzoic, and fourteen alkyl protocatechuates (3,4-dihydroxybenzoates) against Trichophyton rubrum and Trichophyton mentagrophytes and to further assess their activities when combined with fluconazole. Susceptibility and synergism assays were conducted as described in M38-A2 (CLSI), with modifications. Three strains of Trichophyton rubrum and three strains of Trichophyton mentagrophytes were used in this work. The pentyl, hexyl, heptyl, octyl, nonyl, and decyl protocatechuates showed great fungicidal effects, with minimum inhibitory concentration (MIC) values ranging from 0.97 to 7.8 mg/L. Heptyl showed a synergistic activity (FIC index = 0.49), reducing the MIC of fluconazole by fourfold. All substances tested were safe, especially the hexyl, heptyl, octyl, and nonyl compounds, all of which showed a high selectivity index, particularly in combination with fluconazole. These ester associations with fluconazole may represent a promising source of prototypes in the search for anti-Trichophyton therapeutic agents.

Nitensidine A, a guanidine alkaloid from Pterogyne nitens, is a novel substrate for human ABC transporter ABCB1.

The Pterogyne nitens (Fabaceae) tree, native to South America, has been found to produce guanidine alkaloids as well as bioactive flavonols such as kaempferol, quercetin, and rutin. In the present study, we examined the possibility of interaction between human ATP-binding cassette (ABC) transporter ABCB1 and four guanidine alkaloids isolated from P. nitens (i.e., galegine, nitensidine A, pterogynidine, and pterogynine) using human T cell lymphoblast-like leukemia cell line CCRF-CEM and its multi-drug resistant (MDR) counterpart CEM/ADR5000. In XTT assays, CEM/ADR5000 cells were resistant to the four guanidine alkaloids compared to CCRF-CEM cells, although the four guanidine alkaloids exhibited some level of cytotoxicity against both CCRF-CEM and CEM/ADR5000 cells. In ATPase assays, three of the four guanidine alkaloids were found to stimulate the ATPase activity of ABCB1. Notably, nitensidine A was clearly found to stimulate the ATPase activity of ABCB1 as strongly as the control drug, verapamil. Furthermore, the cytotoxic effect of nitensidine A on CEM/ADR5000 cells was synergistically enhanced by verapamil. Nitensidine A inhibited the extrusion of calcein by ABCB1. In the present study, the possibility of interaction between ABCB1 and two synthetic nitensidine A analogs (nitensidine AT and AU) were examined to gain insight into the mechanism by which nitensidine A stimulates the ATPase activity of ABCB1. The ABCB1-dependent ATPase activity stimulated by nitensidine A was greatly reduced by substituting sulfur (S) or oxygen (O) for the imino nitrogen atom (N) in nitensidine A. Molecular docking studies on human ABCB1 showed that, guanidine alkaloids from P. nitens dock to the same binding pocket as verapamil. Nitensidine A and its analogs exhibit similar binding energies to verapamil. Taken together, this research clearly indicates that nitensidine A is a novel substrate for ABCB1. The present results also suggest that the number, binding site, and polymerization degree of the isoprenyl moiety in the guanidine alkaloids and the imino nitrogen atom cooperatively contribute to their stimulation of ABCB1's ATPase activity.

Alkaloids from stems of Esenbeckia leiocarpa Engl. (Rutaceae) as potential treatment for Alzheimer disease.

Esenbeckia leiocarpa Engl. (Rutaceae), popularly known as guarantã, goiabeira, is a native tree from Brazil. Bioactivity-guided fractionation of the ethanol stems extract afforded the isolation of six alkaloids: leiokinine A, leptomerine, kokusaginine, skimmianine, maculine and flindersiamine. All isolated compounds were tested for acetyl cholinesterase inhibition, in vitro and displayed anticholinesterasic activity. The alkaloid leptomerine showed the highest activity (IC50 = 2.5 mM), similar to that of the reference compound galanthamine (IC50 = 1.7 mM). The results showed for the first time the presence of alkaloids leptomerine and skimmianine in E. leiocarpa (Engl.) with potent anticholinesterasic activity.

Alkaloids extracted from Pterogyne nitens induce apoptosis in malignant breast cell line.

In the present study, two alkaloids isolated from Pterogyne nitens, a plant native to Brazil, have been shown to induce apoptosis in human breast cancer cells. These compounds, pterogynine (PGN) and pterogynidine (PGD), were tested for their effect on a human infiltrating ductal carcinoma cell line (ZR-7531). The cell line was treated with each alkaloid at several concentrations. Time-dependence (with or without recuperation time) and concentration-dependence (in the range 0.25-10 mM) were investigated in cytotoxicity and apoptosis assays. The annexin assay indicated an apparently higher percentage of death by necrosis of malignant cells after 24 h exposure to both P. nitens extracts than the Hoechst assay. Thus, our results in the two tests demonstrated that the Hoechst assay can discriminate between late apoptotic cells and necrosis, whereas the flow cytometry-based annexin V assay cannot. We concluded that PGN and PGD have effective antineoplastic activity against human breast cancer cells in vitro, by inducing programmed cell death.

In vitro activity of compounds isolated from Piper crassinervium against Trypanosoma cruzi.

This study describes the antichagasic potential of five compounds isolated from leaves of Piper crassinervium (Piperaceae). Two prenylated benzoic acid derivatives, one prenylated hydroquinone and two flavanones, were evaluated. The in vitro trypanocidal activity was determined against epimastigote forms of Trypanosoma cruzi (Y strain), the etiologic agent of Chagas disease. The most active compound was the prenylated hydroquinone [1,4-dihydroxy-2-(3(0),7(0)-dimethyl-1(0)-oxo-2(0)-E,6(0)-octadienyl)benzene] with an IC(50) value of 6.10 microg mL(-1), which was in the same order of activity if compared with the positive control benznidazole (IC(50) = 1.60 microg mL(-1)). This is the first report of trypanocidal activity for prenylated hydroquinone and benzoic acid derivatives.

Flavonols from Pterogyne nitens and their evaluation as myeloperoxidase inhibitors.

A myeloperoxidase inhibitory kaempferol derivative, namely pterogynoside (1), was isolated from fruits of Pterogyne nitens, along with six known flavonols, kaempferol, afzelin, kaempferitrin, quercetin, isoquercetrin and rutin. The structures of all compounds were elucidated primarily from 1D and 2D NMR spectroscopic analyses, as well as by high resolution mass spectrometry. All flavonols were screened to identify secondary metabolites as potential myeloperoxidase (MPO) inhibitors, and at concentrations of 0.50-50nM, quercetin (5), isoquercitrin (6) and rutin (7) exhibited strong inhibitory effects with IC50 values of 1.22+/-0.01, 3.75+/-0.02 and 3.60+/-0.02, respectively. The MPO activity detected for the new derivative 1 was markedly decreased (IC(50) 10.3+/-0.03) when compared with known flavonols 5-7, and interestingly increased when tested against ABTS scavenging activity.

Evaluation of micronuclei frequency in Tradescantia pallida pollen mother cells treated with ethanolic extracts isolated from Cryptocarya mandioccana, Cryptocarya moschata and Pterogyne nitens

Apesar de extratos vegetais possuírem uma série de compostos com atividade farmacológica, eles podem também apresentar substâncias com potencial mutagênico. O objetivo do atual estudo é avaliar a mutagenicidade do extrato etanólico das plantas: Cryptocarya mandioccana, Cryptocarya moschta e Pterogyne nitens utilizando o ensaio do micronúcleo em células mãe de grão de pólen (tétrades) de Tradescantia pallida (Trad-MCN). Inflorescências de T. pallida foram tratadas com diferentes concentrações do extrato etanólico das plantas selecionadas. Para C. mandioccana, C. moschata e P. nitens o ensaio Trad-MCN foi executado os tratamentos, controle positivo (formaldeido 10000 ppm), controle negativo (solução de Hoagland), e controle de veículo (Tween 20 por cento ou DMSO 3 por cento). Os micronúcleos foram quantificados em 300 tétrades/inflorescência, a média e o erro padrão foram estabelecidos no mínimo para 10 inflorescências/tratamento. Os extratos avaliados demonstraram efeito clastogênico dose resposta, respectivamente: C. mandioccana (0.5, 1.0 e 2.0 mg/mL) e P. nitens (1.0 and 2.0 mg/mL). Entretanto, C. moschata não demonstrou atividade clastogênica/aneugênica nas concentrações avaliadas no presente estudo. A partir desses resultados foi possível concluir que os extratos de C. mandioccana and P. nitens apresentaram atividade clastogênica/aneugênica nas maiores concentrações enquanto o extrato C. moschata não apresentou o mesmo efeito.

Although herbal extracts contain several classes of compounds with pharmacological activity, they also present toxic substances with mutagenic effects. The aim of the present study was to verify the mutagenicity of Cryptocarya moschata, Cryptocarya mandioccana and Pterogyne nitens using micronucleus assay in pollen mother cells (tetrads) in Tradescantia pallida (Trad-MCN). T. pallida inflorescences were treated with different concentrations of ethanolic extracts from the selected plant species. For C. mandioccana C. moschata and P. nitens, Trad-MCN assays were carried out simultaneoulsly, followed by positive control (formaldehyde 10000 ppm), negative control (Hoagland's solution), and vehicle control (Tween 20 20 percent or DMSO 3 percent). MCN present in tetrads were quantified in 300 tetrads/inflorescence and the mean(percent) and standard error (SE) were established for at least 10 inflorescences per treatment. The extracts demonstrated dose response mutagenicity (clastogenic/aneugenic effects), respectively, C. mandioccana (0.5, 1.0 and 2.0 mg/mL) and P. nitens (1.0 and 2.0 mg/mL) However, no mutagenic effect was observed to C. moschata at the concentrations evaluated in the present study. We can conclude that the C. mandioccana and P. nitens extracts demonstrated clastogenic/aneugenic effects in highest concentrations whereas C. moschata extract did not demonstrate the same effect.