Natural Dibenzo[b,f]oxepines, Pacharin and Bauhiniastatin-1, Isolated from Bauhinia acuruana Induce Apoptosis on Breast Cancer Cells via MCL-1 Protein Reduction.

Herein, we describe the antiproliferative effects of two natural dibenzo [b,f]oxepines, pacharin and bauhiniastatin-1, isolated from Bauhinia acuruana on a breast cancer cell line and the mode of action underlying the cytotoxicity. Both compounds were cytotoxic in a panel of six tumor lines analyzed by the MTT assay, and IC50 values ranged from 7.8 to 45.1 µM, including human breast adenocarcinoma (MCF-7) cells. In contrast, none of the compounds were cytotoxic on normal human peripheral blood mononuclear cells (IC50 > 100 µM). Human breast adenocarcinoma (MCF-7) cells treated with pacharin or bauhiniastatin-1 20 µM for 24 h presented a reduction in cell volume and intensification of chromatin condensation, DNA fragmentation, and apoptotic cells. These findings became more evident after 48 h of exposure. Antiapoptotic B-cell lymphoma-2 family members, such as myeloid cell leukemia-1 and B-cell lymphoma-extra large, are important targets in cancer cells since their overexpression confers resistance to cancer treatments. A significant reduction of the myeloid cell leukemia-1 protein levels in human breast adenocarcinoma (MCF-7) cells after 24 h of treatment with pacharin or bauhiniastatin-1 at 20 µM was observed, while the B-cell lymphoma-extra large protein content was reduced in bauhiniastatin-1-treated cells at 40 µM only. The cytotoxic effects of pacharin and bauhiniastatin-1 are likely linked to myeloid cell leukemia-1 inhibition, which leads to the apoptosis of breast adenocarcinoma cells.

Antioxidant, anti-inflammatory and healing potential of ethyl acetate fraction of Bauhinia ungulata L. (Fabaceae) on in vitro and in vivo wound model.

The present work aimed to investigate the antioxidant, anti-inflammatory and wound healing potential of ethyl acetate fraction from Bauhinia ungulata L. (FABU) on in vitro and in vivo models. Wound healing assay using human lung adenocarcinoma A549 cell line was employed to evaluate the ability of FABU in modulating cell migration. In addition, a surgical wound model in C57BL/6 mice was used to study the healing potential of FABU incorporated into gel carbomer 940 (Carbopol®). Evaluation of lipid peroxidation, inflammatory and anti-inflammatory mediator gene expression, rate of wound closure, and histological analysis were done. FABU significantly reduced the gap area in in vitro wound healing assay, 24 h after treatment. In the animal model, FABU at 0.5% topically applied once-daily for 5 days to the surgical wounds significantly reduced the lesion area. Moreover, it significantly decreased the levels of lipid peroxidation in the lesions and decreased the relative gene expression levels of IL-1ß and TNF-α in the injured region. In conclusion, our study suggests that Bauhinia ungulata can effectively promote the wound healing, probably by regulating the inflammatory environment during the early stages of the process.

Essential oil from Myrcia ovata: chemical composition, antinociceptive and anti-inflammatory properties in mice.

The leaves of Myrcia ovata, popularly known as "laranjinha do mato", are frequently used as an infusion in folk medicine. The essential oil obtained from these leaves is rich in citral, a mixture of neral and geranial isomers, known for its analgesic effect. Male Swiss mice (20-22 g) were tested in models of acute pain (acetic acid-induced abdominal writhing, tail flick, and formalin tests) and acute inflammation (paw oedema and air pouch tests) as well as in a model for evaluation of spontaneous motor performance (open-field test). The essential oil from M. ovata was administered orally at doses of 50-300 mg/kg. In addition, water, vehicle, morphine (5.01 mg/kg for evaluation of pain and motor performance), acetyl salicylic acid (200 mg/kg in the formalin test), and dexamethasone (2.25 mg/kg for evaluation of oedema formation, leukocyte extravasation, and quantification of cytokines) were administered. The essential oil showed a significant effect at doses of 200 and 300 mg/kg in the acute pain and acute inflammation tests. The effect of the essential oil was reduced by pretreatment with naloxone. The essential oil did not induce motor impairment. The extract was not toxic after oral administration (LD50 > 3000 mg/kg). These data provide initial evidence that the traditional use of M. ovata can be effective in reducing pain and inflammation.

Larvicidal activity against Aedes aegypti of pacharin from Bauhinia acuruana.

The aim of the present study was to evaluate the activity of pacharin isolated from the ethanol extract from roots of Bauhinia acuruana on third-instar larvae of Aedes aegypti Linn. (Diptera: Culicidae). The crude ethanol extract showed larvicidal activity at the concentration of 500 µg/mL. Given this larvicidal activity, this extract was submitted to chromatographic fractionation on a silica gel column eluted with n-hexane, dichloromethane, ethyl ether, ethyl acetate, and methanol in order to isolate the active compound(s). Pacharin, obtained in pure form from fraction eluted with ethyl ether, was evaluated for their larvicidal effects against A. aegypti. In these bioassays, the larvae were exposed at concentrations of 500, 250, 100, 50, and 25 µg/mL of the crude ethanol extract or pacharin. After 24 h, the number of dead larvae was counted and the LC50 values for larval mortality were calculated. Pacharin showed LC50 value of 78.9 ± 1.8 µg/mL. The structure of isolated compound was identified on the basis of their spectral data (IR, 1D- and 2D-NMR) and by comparison with literature spectral data. The results indicate pacharin as a potential natural larvicide.

Chemical composition and acaricidal activity of essential oil from Lippia sidoides on larvae of Dermacentor nitens (Acari: Ixodidae) and larvae and engorged females of Rhipicephalus microplus (Acari: Ixodidae).

The aim of this work was to identify the compounds and to investigate the acaricidal activity of the essential oil from the leaves of Lippia sidoides on Rhipicephalus microplus and Dermacentor nitens. The oil was obtained by hydrodistillation and analyzed by gas chromatography (GC/FID) and gas chromatography/mass spectrometry. In total, 15 compounds comprising 99.97 % of the total peak area were identified. The main constituent of the essential oil was thymol (67.60 %). The acaricidal activity was assessed by the modified larval packet test, with oil concentrations of 2.5, 5.0, 10.0, 15.0, and 20.0 µl/ml, and by the female immersion test with concentrations of 10.0, 20.0, 40.0, 60.0, and 80.0 µl/ml. The mortality of the R. microplus and D. nitens larvae was greater than 95 % starting at concentrations of 10.0 and 20.0 µl/ml, respectively. In the test with the engorged females, the L. sidoides essential oil starting at a concentration of 40.0 µl/ml caused a significant reduction (p < 0.05) in the values of the egg mass weight and egg production index. The viability of the eggs was affected in all the treated groups, with significantly lower hatching rates (p < 0.05) in relation to the control group. The control percentages at concentrations of 10.0, 20.0, and 30.0 µl/ml were 54, 57, and 72 %, and reached 100 % at the highest two concentrations (60.0 and 80.0 µl/ml). Therefore, it can be concluded that the essential oil from the leaves of L. sidoides has acaricidal activity on R. microplus and D. nitens.

Bauhinia pulchella: chemical constituents, antioxidant and alpha-glucosidase inhibitory activities.

Phytochemical investigation of the stems of B. pulchella led to the isolation of the known compounds identified as a mixture of taraxerone (1) and ß-amirenone (2), a mixture of sitosterol (3) and stigmasterol (4), 2-hydroxy-3,5-dimethoxybibenzyl (5), 3',4'-dihydroxyphenyl-chroman-7-ol (6), fisetinidol (7), epicatechin (8), guibourtinidol (9), vanillic acid (10), 6'-O-vanilloylisotachioside (11) and 6'-O-syringoylisotachioside (12). The structures of these compounds were elucidated on the basis of their NMR spectroscopic data. The antioxidant activity of compound 7 has been investigated using DPPH° and ABTS°+ assays and the results showed inhibition in the both models. The compounds 6, 7 and 9 showed strong alpha-glucosidase inhibitory activities, being more active than acarbose, the positive control. In addition, all the compounds were isolated from B. pulchella for the first time, and among them, compounds 11 and 12 have not been reported previously from this genus.

Ferulic acid derivatives inhibiting Staphylococcus aureus tetK and MsrA efflux pumps.

Background: Bacterial resistance to multiple drugs has recently emerged as a serious health problem. Concomitantly, the characterization of new substances with potential antimicrobial activity has been less frequent in the drug development industry. The overexpression of genes encoding efflux pumps that expel antimicrobial drugs from the intracellular environment, lowering these to subinhibitory concentrations, are among the resistance mechanisms predisposing microorganisms to high drug resistance. Staphylococcus aureus is a bacterium found in the normal microbiota of the skin and mucous membranes, and is an opportunistic microorganism capable of causing infections with high rates of morbidity and mortality. TetK is an efflux pump characterized by its ability to provide bacterial resistance to antibiotics from the tetracycline class. This study aimed to evaluate the inhibitory effect of ferulic acid and four of its esterified derivatives against resistant Staphylococcus aureus strains. Method: Ferulic acid derivatives were obtained by esterification and then characterized by hydrogen and carbon-13 nuclear magnetic resonance analysis. The minimum inhibitory concentrations (MIC) of ferulic acid and its esterified derivatives, ethidium bromide, and antibiotics were obtained using the microdilution test, while the efflux pump inhibition test was conducted by examining reduction in the MICs. Results: Propylferulate was seen to reduce the minimum inhibitory concentration (MIC) of both the control substance ethidium bromide and the tested antibiotic, indicating that this compound is promising for the use of efflux pump inhibition of IS-58 strains. Conclusions: This study provides strong evidence that the molecular basis for this activity is potentially due to the MsrA and TetK efflux pumps. However, further investigations are necessary to prove this hypothesis and elucidate the potentiating mechanism of the modulatory effect.

Galactosyl prodrug of palmitoylethanolamide: synthesis, stability, cell permeation and cytoprotective activity.

N-Palmitoylethanolamide (PEA) is emerging as a novel therapeutic agent in the treatment of neuropathic pain and neurodegenerative diseases. Unfortunately, PEA poorly reaches the central nervous system (CNS), after peripheral administration, since it is inactivated through intracellular hydrolysis by lipid amidases. Since prodrug approach is one of the most popular methods used to increase cell permeability, the aim of this paper consists in the synthesis of a new galactosyl prodrug of PEA, the palmitoylethanolamide-succinamyl-D-galactos-6'-yl ester (PEAGAL). Biological experiments both in neuroblastoma and in C6 glioma cells, together with quantitative analyses performed through a LC-MS-MS technique, demonstrate the better efficacy of PEAGAL compared to PEA and its higher cell permeation. Our results encourage further experiments in animal models of neuropathic pain and of neurological disorders and/or neurodegenerative diseases, in order to promote a more effective peripherally administrated derivative of PEA.

In vitro and in vivo leishmanicidal activity of Astronium fraxinifolium (Schott) and Plectranthus amboinicus (Lour.) Spreng against Leishmania (Viannia) braziliensis.

The aim of the present work was to evaluate antileishmanial activity of Astronium fraxinifolium and Plectranthus amboinicus. For the in vitro tests, essential oil of P. amboinicus (OEPA) and ethanolic extracts from A. fraxinifolium (EEAF) were incubated with 10(6) promastigotes of L. (Viannia) braziliensis. The OEPA was able to reduce the parasite growth after 48 h; nonetheless, all the EEAFs could totally abolish the parasite growth. For the in vivo studies, BALB/c mice were infected subcutaneously (s.c.) with 10(7) L. braziliensis promastigotes. Treatment was done by administering OEPA intralesionally (i.l.) for 14 days. No difference was found in lesion thickness when those animals were compared with the untreated animals. Further, golden hamsters were infected s.c. with 10(6) L. braziliensis promastigotes. The first protocol of treatment consisted of ethanolic leaf extract from A. fraxinifolium (ELEAF) administered i.l. for 4 days and a booster dose at the 7th day. The animals showed a significant reduction of lesion thickness in the 6th week, but it was not comparable to the animals treated with Glucantime. The second protocol consisted of 15 daily intralesional injections. The profiles of lesion thickness were similar to the standard treatment. In conclusion, in vivo studies showed a high efficacy when the infected animals were intralesionally treated with leaf ethanolic extract from A. fraxinifolium.

Efficacy of Plectranthus amboinicus (Lour.) Spreng in a Murine Model of Methicillin-Resistant Staphylococcus aureus Skin Abscesses.

The present work aimed to evaluate the effectiveness of Plectranthus amboinicus (Lour.) Spreng against MRSA clinical isolates. The in vitro antimicrobial activity of the hydroalcoholic extract (HE), the ethyl acetate (EA) fraction and its subfractions were determined by broth microdilution and bioautography against MRSA clinical isolates. The microdilution checkerboard method was used to assess in vitro drug combination studies. To induce abscess formation, bacterial suspensions were added to Citodex and inoculated subcutaneously into male Swiss mice. The treatment protocol consisted of 2 doses of HE, the EA fraction or vancomycin introduced intraperitoneally into mice 3 and 12 h after infection. The EA fraction and its subfractions presented the lowest minimal inhibitory concentrations (MIC, 0.25 to 0.5 mg/mL). The plant samples were bacteriostatic at 2x and 4x MIC and bactericidal at 100 mg/mL. The EA fraction presented synergism with vancomycin and an additive effect with ciprofloxacin. A significant reduction of abscess volume, bacterial cell counts in abscess slurries, and inflammatory scores was observed in the HE and EA fraction-treated groups. The samples were effective in treating the animals in a dose-dependent fashion. The present study proved the effectiveness of P. amboinicus fractions against MRSA using in vitro and in vivo assays.

Phytochemical study guided by the myorelaxant activity of the crude extract, fractions and constituent from stem bark of Hymenaea courbaril L.

ETHNOPHARMACOLOGICAL RELEVANCE: Hymenaea courbaril L. (Caesalpinoideae) is used in Brazilian folk medicine to treat anemia, kidney problems, sore throat and other dysfunctions of the respiratory system, such as bronchitis and asthma, although such properties are yet to be scientifically validated. AIM OF THE STUDY: In order to give a scientific basis to support the traditional use of Hymenaea courbaril, this study was designed to evaluate antioxidant, myorelaxant and anti-inflammatory properties of the ethanol extract from stem bark and its fractions. The myorelaxant effect of astilbin, a flavonoid isolated from the bioactive ethyl acetate fraction (EAF), has also been evaluated. MATERIAL AND METHODS: In the present study ethanol extract from stem bark (EEHC) and fractions were analyzed using bioassay-guided fractionation. The following activities were investigated: antioxidant by 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, myorelaxant on rat tracheal smooth muscle, and anti-inflammatory using ovalbumin-induced leukocytosis and airway hyperresponsiveness in rats. RESULTS: The results of the present investigation show that the whole extract of Hymenaea courbaril and some of its fractions strongly scavenged DPPH radical. The extract showed myorelaxant activity on rat trachea, being EAF its highest efficient fraction. Bio-guided study allowed the isolation of astilbin, a well-known flavonoid. The activity induced by this compound indicates that it may be partly responsible for the myorelaxant effect of EAF. EAF reduced contractions that depended on divalent cation inflow through voltage-operated Ca(2+) channels (VOCCs) or receptor-operated Ca(2+) channels (ROCCs), but it was more potent to inhibit VOCC- than ROCC-dependent contraction induced by Ca(2+) addition in ACh-enriched Ca(2+)-free medium. Oral pretreatment of antigen-challenged animals with EAF prevented airway hyperresponsiveness on KCl-induced contraction and reduced the number of total white cells, particularly eosinophils and neutrophils in bronchoalveolar lavage. CONCLUSIONS: This study provided scientific basis that Hymenaea courbaril presents potential antioxidant, myorelaxant and anti-inflammatory actions, which support its use in folk medicine to treat inflammatory airway diseases.