Hallazgo de diclofenaco en un producto fitoterapéutico a base de caléndula comercializado en Colombia / Finding diclofenac in a marigold-based phytotherapeutic product marketed in Colombia

Resumen Introducción: la consulta de un particular que trajo un producto fitoterapéutico a base de caléndula cuyo consumo le causó fuertes reacciones adversas, originó esta investigación sobre la composición de este producto. Objetivo: caracterizar la composición química de muestras de lotes diferentes de un producto comercial denominado fitoterapéutico a base de caléndula (Calendula officinalis) (PFC) comercializado en Colombia. Metodología: se analizaron tabletas de ocho cajas del PFC de cuatro lotes diferentes de producción (2017 y 2018). Se llevó a cabo el análisis de espacio de cabeza (HS) de tabletas por microextracción en fase sólida (SPME), con una fibra PDMS/DVB (65 µm), expuesta al HS de la muestra durante 30 min a 50 °C. Las fracciones volátiles se analizaron por cromatografía de gases acoplada a espectrometría de masas (GC/MS). Los extractos de tabletas obtenidos con mezcla de metanol:agua (1:1, v/v) se analizaron por cromatografía líquida (LC) de alta (HPLC) y ultra-alta eficiencia (UHPLC), con detectores de arreglo de diodos (DAD) y espectrometría de masas de alta resolución (HRMS), respectivamente; la cuantificación de diclofenaco se hizo por calibración con patrón externo y por adición de estándar. Los espectros de masas de baja y alta resolución y patrones de fragmentación de las sustancias detectadas se estudiaron, usando GC/HRMS y LC/HRMS-Orbitrap. Resultados: en tabletas analizadas por HSSPME, se encontraron monoterpenoides y sesquiterpenoides de origen vegetal, ftalatos, residuos de solventes (2-cloroetanol, etilenglicol) y sustancias químicas intermediarias en la síntesis de diclofenaco (2,6-dicloroanilina y 2,6-cloro-N-fenil-bencenamina). En los cromatogramas, obtenidos por GC/MS de los extractos de tabletas obtenidos con diclorometano, se detectaron diclofenaco, sus impurezas A, B y C, los ésteres de diclofenaco y algunas otras impurezas. Diclofenaco en cantidad ca. 40 mg (7-8%) se cuantificó por HPLC en tabletas (> 70 analizadas) escogidas al azar de ocho cajas del PFC, adquirido en el mercado local de Bucaramanga (Colombia). Conclusión: en cada tableta analizada se determinaron alrededor de 40 mg del compuesto sintético diclofenaco (sustancia no declarada en la etiqueta del producto) y en ninguna se detectaron ésteres de los triterpenoides oleanano o faradiol, constituyentes del extracto de caléndula que poseen actividad antiinflamatoria; se encontraron algunos flavonoides comunes a muchas plantas, en cantidades mil veces menores que la de diclofenaco.

Abstract Introduction: The consultation of a person who brought a marigold-based phytotherapeutic product whose consumption caused strong adverse reactions, originated this investigation of the composition of this product. Objective: to characterize the chemical composition of samples of different lots of a commercial product called calendula-based phytotherapeutic product (Calendula officinalis) (PFC) commercialized in Colombia. Methodology: Tablets of eight packs of the phytotherapeutic product from four different production batches (2017 and 2018) were analyzed. Headspace analysis (HS) of tablets by solid phase microextraction (SPME) was carried out with a PDMS/ DVB fiber (65 µm), exposed to the HS of the sample for 30 min at 50 °C. Volatile fractions were analyzed by gas chromatography coupled to mass spectrometry (GC/MS). Tablet extracts obtained with methanol:water mixture (1:1, v / v) were analyzed by liquid chromatography (LC) of high (HPLC) and ultra-high performance (UHPLC) with diode array (DAD) and high-resolution mass spectrometric (HRMS) detectors, respectively; diclofenac was quantified by external calibration and standard addition. Low- and high-resolution mass spectra (MS, HRMS) and fragmentation patterns of detected substances were studied, using GC/HRTOF-MS and LC/HRMS-Orbitrap. Results: in tablets analyzed by HS-SPME, monoterpenoids and sesquiterpenoids of plant origin, phthalates, solvent residues (2-chloroethanol, ethylene glycol) and intermediary chemicals in diclofenac synthesis (2,6-dichloroaniline and 2,6- chloro-N-phenyl-benzenamine) were found. In the chromatograms (GC/MS) of the extracts of tablets obtained with organic solvent (dichloromethane), diclofenac, its impurities A, B and C, diclofenac esters, and some other compounds were detected; diclofenac quantification by HPLC found amounts of ca. 40 mg (7 - 8%) in tablets (> 70 analyzed) chosen at random from eight packs of the calendula-based phytotherapeutic product, purchased in the local market in Bucaramanga (Colombia). Conclusion: each analyzed tablet contained around 40 mg of the synthetic compound diclofenac (substance not declared in the product's label) and no tablet contained detectable amounts of esters of the triterpenoids oleanane or faradiol, which are calendula extract constituents that possess antiinflammatory activity; a few flavonoids that are common to many plants were found in amounts a thousand times smaller than that of diclofenac.

Odontogenic cell culture in PEGDA hydrogel scaffolds for use in tooth regeneration protocols

In order to obtain a tooth-like structure, embryonic oral ectoderm cells (EOE) and bone marrow-derived stem cells (BMSC) were stratified within a synthetic hydrogel matrix (PEGDA) and implanted in the ileal mesentery of adult male Lewis rats. Wholemount in situ hybridization was used to evaluate the expression of Pitx2, Shh and Wnt10a signals indicative of tooth initiation. In rats, expression of the three markers was present in the oral ectoderm starting at embryonic stage E12.5, which was therefore selected for cell harvesting. Embryos were obtained by controlled service of young female Lewis rats in which estrus was detected by impedance reading. At E12.5, pregnant rats were humanely euthanized and embryos were collected. The mandibular segment of the first branchial arch was dissected and the mesenchyme separated from the ectoderm by enzymatic digestion with pancreatin trypsin solution. BMSCs were collected by flushing the marrow of tibiae and femurs of adult Lewis rats with a-MEM and cultured in a-MEM in 25 cm2 flasks. Second passage BMSC's were recombined with competent oral ectoderm (E12.5-E13) stratifying them within a 3D PEGDA scaffold polymerized by exposure to UV (365nm) inside a pyramidal polypropylene mold. Constructs were incubated from 24 to 48 hrs in a-MEM and then implanted for four to six weeks in the mesentery of adult male (3- 6 month old) Lewis rats. 76 constructs were implanted (37 experimental, 27 negative controls and 12 positive controls). Upon maturation, constructs were harvested, fixed in buffered formalin, processed and stained with hematoxylin eosin (HE). Histological evaluation of the experimental and negative constructs showed that BMSCs underwent an apoptotic process due to lack of matrix interactions, known as anoikis, and were thus incapable of interacting with the competent ectoderm. In contrast, embryonic oral ectoderm was able to proliferate during the mesenteric implantation. In conclusion, PEGDA scaffolds are incompatible with BMSCs, therefore it is essential to continue the search for an ideal scaffold that allows proper tissue interactions.

Para lograr la formacion de una estructura similar a un diente se estratificaron celulas de ectodermo oral embrionario (EOE) con celulas troncales de medula osea (BMSC) dentro de una matriz de diacrilato de polietilenglicol (PEGDA) que se implanto en el mesenterio ileal de machos adultos de ratas Lewis. Mediante hibridizacion in situ de bloque completo se evaluo la expresion de tres genes iniciadores putativos de la formacion dental (Pitx2, Shh y Wnt10a), estableciendo que en ratas las senales iniciadoras de dentogenesis aparecen entre E12.5 y E13. El tejido ectodermico embrionario se obtuvo haciendo cruces controlados de hembras a las que se les detecto el estro mediante impedanciometria. En E12.5 las hembras se sacrificaron y se extrajeron los embriones. Se diseco la porcion mandibular del primer arco branquial y el ectodermo se separo del mesenquima mediante disociacion enzimatica con una solucion de pancreatina tripsina. Las BMSC se extrajeron de los huesos largos de las extremidades inferiores de ratas mediante lavado con a-MEM y se cultivaron en cajas de 25cm2 hasta un segundo pasaje. Las BMSC fueron recombinadas con ectodermo embrionario competente (E12.5 - E13) estratificandolas en un soporte tridimensional de PEGDA, polimerizado con luz ultravioleta (365nm) dentro de un molde piramidal de polipropileno (PP). Los constructos se cultivaron entre 48 y 72 horas en a-MEM y posteriormente fueron implantados en el mesenterio ileal de machos adultos (3 a 6 meses de edad) por un periodo de cuatro a seis semanas. Se implantaron 76 constructos (37 experimentales, 27 controles negativos y 12 controles positivos). En la fecha determinada los animales se sacrificaron mediante asfixia con una mezcla de CO2 y aire recuperando los constructos que se fijaron en formalina tamponada para luego procesarlos y tenirlos con hematoxilina eosina (HE). La evaluacion histologica de los constructos experimentales, positivos y negativos mostro que las BMSC incluidas en el hidrogel sufrieron un proceso de apoptosis conocido como anoikis que impidio su interaccion con las celulas ectodermicas. En contraste el EOE prolifero durante el periodo de implantacion. A futuro se debe buscar la matriz portadora ideal que permita el confinamiento de los dos grupos celulares y que brinde el soporte estructural necesario para la proliferacion de las BMSC facilitando su interaccion con las celulas inductoras de origen ectodermico.

Anti-Candida albicans activity, cytotoxicity and interaction with antifungal drugs of essential oils and extracts from aromatic and medicinal plants / Actividad contra Candida albicans, citotoxicidad e interacción con antifúngicos de aceites esenciales y extractos de plantas medicinales y aromáticas

Objective: To determine anti-Candida albicans activity, cytotoxicity and drug interaction of essential oils and extracts from plants collected in Colombia. Materials and methods: The antifungal activity was evaluated following the AFST-EUCAST protocol. With most active samples, the inhibition of the formation of germ tubes and budding, the in vitro pharmacodynamics, using time-kill assays, and the interaction with itraconazole and amphotericin B following the chequerboard technique were evaluated. The cytotoxicity assay for all samples was done using MTT. Results: Strong activity in 17.57% of the samples was found. The lowest MIC values were obtained with Piper bredemeyeri Jacq and Lippia origanoides Kunth (B) oils and Morinda royoc L extract. The three samples inhibited the formation of germ tubes and budding. P. bredemeyeri Jacq oil and M. royoc L extract samples showed fungicidal activity at 2xMIC. A synergistic effect was obtained with the combination of P. bredemeyeri Jacq oil and itraconazole, but not for the combination with amphotericin B. Active samples against C. albicans were not cytotoxic on Vero cells ATCC CCL-81, excluding P. bredemeyeri Jacq oil. Conclusions: The results of this study suggest that Colombian medicinal and aromatic plants represent an untapped source of compounds with anti-C. albicans activity that could be a resource in the development of new therapeutic natural products.

Objetivo. Determinar la actividad anti-Candida albicans, la citotoxicidad y la interacción con antifúngicos de aceites y extractos de plantas recolectadas en Colombia. Materiales y métodos. La actividad antifúngica fue evaluada siguiendo el protocolo Antifungal Susceptibility Testing Subcommittee of the European Committee on Antimicrobial Susceptibility Testing (AFST-EUCAST). Con las muestras más activas se evaluó la inhibición de la formación de tubo germinal y la gemación, la farmacodinamia mediante curvas de tiempo muerte y la interacción con itraconazol y anfotericina B. Se determinó la citotoxicidad mediante la técnica MTT. Resultados. Se encontró actividad en 17,57 % de las muestras. La mayor actividad se obtuvo con los aceites de Piper bredemeyeri Jacq y Lippia origanoides Kunth (B) y el extracto de Morinda royoc L. Las tres muestras inhibieron la formación de tubo germinal y la gemación. El aceite de P. bredemeyeri Jacq y el extracto de M. royoc L mostraron actividad fungicida con dos veces la concentración inhibitoria mínima. Se encontró un efecto sinérgico por la combinación del aceite de P. bredemeyeri Jacq e itraconazol, pero no con anfotericina B. Las muestras activas no fueron citotóxicas, excepto el aceite de P. bredemeyeri Jacq. Conclusión. Los resultados de este estudio sugieren que las plantas de Colombia son una fuente no explorada de compuestos con actividad anti-C. albicans, útiles para el desarrollo de nuevos productos terapéuticos.

In vitro antifungal activity and cytotoxic effect of essential oils and extracts of medicinal and aromatic plants against Candida krusei and Aspergillus fumigatus / Atividade antifúngica in vitro e os efeitos citotóxicos de óleos essenciais e extratos de plantas medicinais e aromáticas contra Candida krusei e Aspergillus fumigatus

The plants are usually used in traditional medicine as antimicrobial agents and their essential oils and extracts have been known to possess antifungal activity. The aim of this study was to evaluate in vitro the activity of 32 essential oils and 29 extracts against C. krusei and A. fumigatus as well as the cytotoxic effect on Vero cells. Time-kill curve and interaction between antifungals and the most active sample against C. krusei, was also evaluated. The oils from C. ambrosioides and the extract of M. cucullata showed antifungal activity against C. krusei (GM-MIC 7.82 and 31.25 µg/mL, respectively). L. citriodora was actives against C. krusei and A. fumigates (GM-MIC = 99.21 µg/mL and 62.5 µg/mL respectively). Time-kill assays done with C. ambrosioides oil showed fungicidal activity at 4x MIC. The interaction of C. ambrosioides oil with itraconazole and amphotericin B was tested following the chequerboard technique. No interaction was detected for the combination of C. ambrosioides oil with amphotericin B and itraconazole (FICI range = 1.03-1.06 and 1.03-1.00, respectively). Cytotoxicity assays for all samples were carried out with MTT. Only the oil from Hedyosmun sp. and L. dulcis were cytotoxic.

As plantas são geralmente utilizadas na medicina tradicional como agentes antimicrobianos e seus óleos essenciais e extratos foram conhecidos por possuir atividade antifúngica. O objetivo deste estudo foi avaliar in vitro a atividade de 32 óleos essenciais e 29 extratos contra Candida krusei e Aspergillus fumigatus, bem como o efeito citotóxico em células Vero. A curva do tempo-morte e a interação entre antifúngicos e Chenopodium ambrosioidese do extrato de Myrcia cucullata mostraram atividade antifúngica contra C. krusei (geometric means of the minimal inhibitory concentration [GM-MIC] 7,82 e 31,25 µg/mL, respectivamente). Lippia citriodora foi ativa contra C. krusei e A. fumigatus (GM-CIM = 99,21 µg/mL e 62,5 µg/mL, respectivamente). Os testes de tempo-morte feitos com óleo de C. ambrosioides mostraram atividade fungicida em 4x MIC. A interação do óleo C. ambrosioides com itraconazol e anfotericina B foi testada pela técnica de xadrez. Nenhuma interação foi detectada pela combinação do óleo C. ambrosioides com anfotericina B e itraconazol (intervalo fractional inhibitory index [FICI] = 1,03-1,06 e 1,03-1,00, respectivamente). Os ensaios de citotoxicidade para todas as amostras foram realizadas com MTT. Apenas os óleos Hedyosmun sp. e L. dulcis foram citotóxicos.

Actividad citotóxica de aceites esenciales de Lippia origanoides H.B.K. y componentes mayoritarios / Cytotoxic activity of essencial oils of Lippia origanoides H.B.K and its major constituents

Introducción: Lippia origanoides H.B.K. (Verbenacea), es una planta aromática conocida comúnmente como “orégano”. Los aceites esenciales de 8 muestras de L. origanoides y algunos de sus componentes mayoritarios fueron evaluados in vitro sobre la línea tumoral HeLa y la línea no tumoral Vero para identificar su potencial citotóxico. Materiales y métodos: la concentración inhibitoria cincuenta (IC50) se determinó mediante la técnica fotocolorimétrica del MTT (3-(4,5-Dimetiltiazol-2-il)-2,5-bromuro difeniltetrazolio) y los valores de IC50 se obtuvieron por análisis estadístico mediante regresión lineal simple. El índice de selectividad (IS), definido como la IC50 en células Vero sobre IC50 en células HeLa, fue calculado con el fin de encontrar aceites o componentes con potencial citotóxico selectivo hacia líneas celulares tumorales. Resultados y conclusiones: se determinó por cromatografía de gases y espectrometría de masas GC/MS la composición química de los aceites más citotóxicos. El aceite de L. origanoides que presentó la mayor actividad citotóxica sobre células HeLa con un valor de IC50 de 9.1 ± 1 µg/mL e índice de selectividad de 7,1, fue identificado como quimiotipo trans-β-cariofileno/r-cimeno. Los componentes mayoritarios del aceite quimiotipo trans-β-cariofileno/r-cimeno fueron: trans-β-cariofileno (11.3%), r-cimeno (11,2%), α-felandreno (9,9%), limoneno (7,2%), 1,8-cineol (6,5%) y α-humuleno (6,0%). Los componentes mayoritarios evaluados no mostraron actividad citotóxica relevante sobre células HeLa, sólo el limoneno y β-mirceno presentaron valores de IS, respectivamente, de 6,97 y 3,01. Sin embargo, los valores de IC50 fueron más altos que el del aceite activo. Estos resultados sugieren que la actividad citotóxica de los aceites no se debe sólo a sus componentes mayoritarios, sino a un sinergismo entre sus componentes.

Introduction: Lippia origanoides H.B.K. (Verbenaceae) is an aromatic plant commonly called as "oregano". Eight essential oils of L. origanoides and some of their main components were evaluated in vitro on tumor cell line HeLa and non-tumor cell line Vero to identify tumoural cytotoxic potential. Materials and methods: Inhibition 50% of cell population (IC50) was determined using the photo-colorimeter technique MTT (3 - (4.5-dimethylthiazol-2-yl) -2,5-difeniltetrazolium bromide). IC50 values were obtained by linear regression analysis. The selectivity index (SI), defined as Vero IC50 on HeLa IC50, it was calculated in order to find oil or major components with selective tumor cytotoxic potential. Results and conclusions: the chemical composition of the oil most cytotoxic was determined by gas chromatography and mass spectrometry GC/MS. The L. origanoides oil identified as chemotype trans-β- caryophyllene/p-cymene showed the highest cytotoxic activity on HeLa cells with IC50 value of 9.1 ± 1 µg/mL and selectivity index of 7.1. The main components were: trans-β-caryophyllene (11.3 p-cymene (11.2%), α-phellandrene (9.9%), limonene (7.2%), 1.8-cineol (6.5%) and α-humulene (6.0%). The most of the major components did not show cytotoxic activity on HeLa cells, only limonene and β-myrcene showed IS values of 6.97 and 3.01, respectively. However, the IC50 values were higher than active oil. These results suggest that cytotoxic activities of the oils are not only due to their main components, but to a synergism among its components.

Actividad antimicótica, citotoxicidad y composición de aceites esenciales de plantas de la familia Labiatae / Antifungal activity, cytotoxicity and composition of essential oils from Asteraceae family plants

Introducción: Candida spp. y Aspergillus spp. son causa importante de infecciones a nivel mundial. Considerando la resistencia de estos patógenos a algunos de los antimicóticos disponibles, es necesaria la búsqueda de nuevos agentes antimicóticos. Diferentes aceites esenciales y extractos de plantas han mostrado actividad antimicótica in vitro. El objetivo de este estudio fue evaluar la actividad antimicótica, citotóxica y la composición química de aceites esenciales de la familia Labiatae. Materiales y métodos: Se evaluó la actividad antimicótica de 22 aceites de plantas de la familia Labiatae contra C. parapsilosis ATCC 22019, C. krusei ATCC 6258, A. flavus ATCC 204304 y A. fumigatus ATCC 204305, siguiendo las técnicas estándar EUCAST y CLSI M38-A para levaduras y hongos filamentosos, respectivamente. Adicionalmente la actividad citotóxica se evaluó en la línea celular Vero mediante la técnica colorimétrica del MTT. La caracterización de los aceites esenciales se llevó a cabo por cromatografía de gases acoplada a masas. Resultados: El aceite esencial mas activo fue el de Minthostachys mollis frente a todas las cepas evaluadas con rangos concentraciones mínimas inhibitorias (CMIs) entre 250 y 375 µg/mL. El aceite de la planta Hyptis mutabilis mostró actividad frente a A. fumigatus (CMI = 396.8 µg/mL). Estos aceites esenciales no fueron citotóxicos sobre las células Vero. Los componentes principales de los aceites de las plantas M. mollis y H. mutabillis fueron epóxido de cis-piperitona y 1,8-cineol, respectivamente. Conclusiones: Los aceites esenciales de las plantas M. mollis y H. mutabillis mostraron actividad antimicótica y no fueron citotóxicos en células Vero.

Introduction: Aspergillus spp. and Candida spp. are important cause of infections worldwide. Considering the resistance of these pathogens to some antifungal agents, there is greater need to search for new antifungal agents. Many extracts and essential oils isolated from plants have shown to exert antifungal effects in vitro. The aim of this study was to evaluate the antifungal, cytotoxic effect, and chemical composition of essential oils of family Labiatae. Materials and methods: Antifungal activity of twenty two essential oils from Labiatae family was evaluated against C. parapsilosis ATCC 22019, C. krusei ATCC 6258, A. flavus ATCC 204304 y A. fumigatus ATCC 204305, following EUCAST and M38-A standard protocols for yeast and filamentous fungi, respectively. Additionally, cytotoxic activity was evaluated on Vero cell line by colorimetric assay MTT. Essential oils was characterized by gas chromatography coupled to mass spectrometry. Results: The most active oil with all strains was obtained of Minthostachys mollis (MIC range 250 - 375 µg/mL). The essential oil from Hyptis mutabillis showed activity against A. fumigatus (GM-MIC = 396.8 µg/mL). These essential oils were not cytotoxic on Vero cells. The major components of essential oils from M. mollis and H. mutabillis were cis-piperitone epoxide and 1,8-cineol, respectively. Conclusions: Essential oils of H. mutabillis and M. mollis showed antifungal activity and they were not cytotoxic on Vero cells.

Composición química y actividad anti-tripanosomal de aceites esenciales obtenidos de Tagetes (Fam. Asteraceae), recolectados en Colombia / Chemical composition and anti-tripanosomal activity of essential oils from Tagetes (Asteraceae Fam) grown in Colombia

Introducción: La quimioterapia actual para enfermedad de Chagas es precaria con solo dos opciones de tratamiento: nifurtimox y benznidazol. Las plantas representan una fuente inmensa de moléculas potencialmente activas contra agentes infecciosos. Objetivo: Determinar la composición química y evaluar la actividad de aceites esenciales de Tagetes, recolectados en Colombia, contra Trypanosoma cruzi y su célula de mamífero hospedera. Materiales y métodos: Los aceites esenciales se obtuvieron de plantas colectadas en diversas regiones de Colombia; se extrajeron por hidrodestilación asistida por la radiación de microondas y se caracterizaron por cromatografía de gases acoplada a espectrometría de masas. La actividad de siete (7) aceites esenciales se determinó en epimastigotes, amastigotes intracelulares de T. cruzi y en células Vero. Los resultados fueron expresando como la concentración que inhibe (CI50, CI90) o destruye (CC50, CC90) 50 ó 90 % de parásitos o células. Resultados: Los componentes mayoritarios de los aceites fueron estragol, dihidrotagetona y cis-tagetona con diferencias de composición entre las especies de Tagetes evaluadas. Todos los aceites esenciales fueron activos en epimastigotes de T. cruzi. El aceite de T. heterocarpha fue activo en amastigotes intracelulares (CI50:41.35 µg/mL). Los aceites de T. caracasana y T. heterocarpha fueron tóxicos para las células Vero. Conclusiones: Los aceites esenciales obtenidos de T. heterocarpha, T. caracasana y T. zipaquirensis mostraron capacidad para inhibir el crecimiento de T. cruzi. Estudios complementarios de sus componentes mayoritarios se realizan actualmente.

Introduction: The current chemotheraphy of Chagas diseases is poor, with only two treatment options: nifurtimox and benznidazole. The plants represent an immense source of potentially active molecules against infectious agents. Aim: To determine the chemical composition and biological activity of Colombian Tagetes essential oils against Trypanosoma cruzi and its mammalian host cell. Materials and methods: Plants were collected in various region of Colombia and essential oils were extracted by microwave-assisted hidrodistillation and characterized by gas chromatography coupled with mass spectrometry. The activities of seven (7) essentials oils were determinate in epimastigotes and amastigotes of T. cruzi and on Vero cells. The result were expressed as the concentration to inhibit (IC50 IC90) or destroy (CC50 CC9) 50 or 90 % of parasites or cells. Results: Estragole, dihidrotagetone and cis-tagetone were the main components of essential oils, with quantitative differences between the evaluated Tagetes species. All essential oils were active in epimastigotes of T. cruzi. T. heterocarpha essential oil was active in intracellular amastigotes (IC50:41.35 µg/mL). The oils from T. caracasana y T. heterocarpha were toxic to Vero cells. Conclusions: The essential oils obtained from T. heterocarpha, T. caracasana and T. zipaquirensis inhibit the growth of T. cruzi. Additional studies on the major components activities against parasites are now under study.

Citral and carvone chemotypes from the essential oils of Colombian Lippia alba (Mill.) N.E. Brown: composition, cytotoxicity and antifungal activity

Two essential oils of Lippia alba (Mill.) N.E. Brown (Verbenacea), the carvone and citral chemotypes and 15 of their compounds were evaluated to determine cytotoxicity and antifungal activity. Cytotoxicity assays for both the citral and carvone chemotypes were carried out with tetrazolium-dye, which showed a dose-dependent cytotoxic effect against HeLa cells. Interestingly, this effect on the evaluated cells (HeLa and the non-tumoural cell line, Vero) was lower than that of commercial citral alone. Commercial citral showed the highest cytotoxic activity on HeLa cells. The antifungal activity was evaluated against Candida parapsilosis, Candida krusei, Aspergillus flavus and Aspergillus fumigatus strains following the standard protocols, Antifungal Susceptibility Testing Subcommittee of the European Committee on Antibiotic Susceptibility Testing and CLSI M38-A. Results demonstrated that the most active essential oil was the citral chemotype, with geometric means-minimal inhibitory concentration (GM-MIC) values of 78.7 and 270.8 μg/mL for A. fumigatus and C. krusei, respectively. Commercial citral showed an antifungal activity similar to that of the citral chemotype (GM-MIC values of 62.5 μg/mL for A. fumigatus and 39.7 μg/mL for C. krusei). Although the citronellal and geraniol were found in lower concentrations in the citral chemotype, they had significant antifungal activity, with GM-MIC values of 49.6 μg/mL for C. krusei and 176.8 μg/mL for A. fumigatus.

Adhesion of salivary components to Streptococcus mutans peptides

Streptococcus mutans es el principal microorganismo asociadoa la caries dental, esta bacteria se une al esmalte a través de su interacción con las proteínas de la película adquirida yla proteína de superficie celular comúnmente denominada PAc. Por lo menos dos sitios de PAc interactúan in vitro con los receptores salivales, uno está dentro de la región más conservadade esta proteína que comprende los residuos de 816-1213 y el otro dentro de la secuencia rica en Alanina, residuos186-469. El objetivo del presente trabajo fue establecer similitudes o diferencias en la interacción de péptidos de PAc con los componentes salivales de individuos con y sin experienciade caries, para lo cual se tomaron muestras de saliva por salivación espontánea de 20 individuos con caries y 20 sin caries. A partir de las muestras de saliva se extrajeron las proteínas de la película adquirida (PA) utilizando hidroxilapatita sintética y fueron sometidas a la interacción con trespéptidos sintéticos de los segmentos de unión de PAc con los componentes salivales: PAc (301-319), PAc (365-377) y PAc (1024-1044). Los resultados muestran una baja interacciónentre los componentes de la PA y los péptidos en todos los individuos, sugiriendo que con base en las similitudes entre los individuos sanos y los individuos con la enfermedad lospéptidos de PAc estudiados no son relevantes en la adhesión

Streptococcus mutans is the main microorganism associated to dental caries; it adheres to the dental enamel by interacting with the acquired film’s proteins and the cell surface adhesin, called variously antigen PAc. At least two distinct sites in PAc interact with salivary receptors in vitro, these are within residues 816-1213, the most conserved portion of PAc, and within residues 186-469, the alanine-rich sequence. Our purpose was to establish differences or similarities in PAc’s peptides interactions with the salivary components of individuals with and without previous caries experience. 40 saliva samples were obtained from patients with (n=20) and without (n=20) caries. The acquired film’s proteins were extracted using hydroxyapatite, and subjected to interaction with three synthetic PAc peptides (PAc (301-319), PAc (365-377), and PAc (1025-1044)) synthesized from PAc’s bonding sites to the salivary components. The results show low interaction between the acquired pellicle components and the peptides in all patients. This suggests that the examined PAc’s are not relevant as far as the initial adhesion of Streptococcus mutans to the tooth’s surface is concerned, as defined by the similarities in the results for healthy and affected individuals.

Análise comparativa da resistência de fêmures de cães após a confecção de janelas ósseas circular e quadrada / Comparative analysis of dog femur resistance after receiving circular and square holes

Com o objetivo de avaliar o enfraquecimento causado pela confecção de janela óssea cortical, os autores confeccionaram uma janela circular no osso cortical da diáfise de oito fêmures de carcaças de cães e uma janela quadrada nos oito pares destes fêmures, com diagonal semelhante ao diâmetro da janela circular contralateral. As peças anatômicas foram submetidas a teste de tensão torcional em uma máquina de ensaios mecânicos; obtendo-se o torque máximo e a rigidez à torção. Os resultados mostraram que, para o fêmur com janela circular, o torque máximo médio foi de 13,65 ± 5,12 Nm, e a rigidez média foi de 1,18 ± 0,45 Nm/grau, enquanto que para a janela quadrada o torque máximo médio foi de 13, 39 ± 5,23 Nm, e a rigidez média foi de 1,05 ± 0,41 Nm/grau. A resistência nos ossos com janela circular e quadrada submetidos à tensão torcional foi praticamente igual, fato este corroborado pela análise estatística que não revelou diferença significante (p = 0,05).