Preparation, Physicochemical Characterization, and Stability Study of Lippia origanoides Essential Oil-Based Nanoemulsion as a Topical Delivery System.

INTRODUCTION: Fungal diseases are a priority in research, development, and health care, according to the WHO, mainly due to Candida spp. Essential oils (EOs) of the genus Lippia have demonstrated broad antimicrobial biological activity. Previous studies identified the anti-Candida potential of a thymol/p-cymene chemotype EO from Lippia origanoides H.B.K coded "0018". Nanoemulsions favor the biological activity of EOs and overcome limitations such as low solubility, instability against oxidizing agents, pH, light, and low permeability. To develop, characterize, and adjust a prototype of an O/W nanoemulsion containing the "0018" EO from Lippia origanoides for its evaluation in an In vitro permeability study. METHOD: Nanoemulsions were obtained using a high energy high shear method. Their particle size distribution, Z potential, viscosity, pH, encapsulation efficiency (EE), thermodynamic stability and the Turbiscan Stability Index (TSI) were evaluated. The nanoemulsion prototype was adjusted to improve performance characteristics and microbiological efficacy. Thymol was used as an analyte in the EO quantification using UHPLC-DAD. RESULTS: An O/W nanoemulsion with hydrodynamic diameter <200 nm and polydispersity index <0.3, EE >95%, with TSI < 1.5, anti-Candida albicans efficiency >95% was obtained; permeable with a flow of 6.0264 µg/cm2/h and permeability coefficient of 1.3170x10-3 cm/h. CONCLUSION: A pharmaceutical formulation prototype is obtained that maintains the physical and physicochemical characteristics over time. Permeability is verified in an in-vitro model. It is proposed to evaluate its antifungal activity in preclinical or clinical studies as a contribution to the treatment of topical fungal diseases caused by Candida spp., through the use of biological resources and Colombian biodiversity.

Antifungal susceptibility profile of Trichosporon inkin: About three cases of White Piedra.

Trichosporon spp. usually cause systemic or superficial infections. Three cases of White Piedra produced by Trichosporon inkin are described. The in vitro antifungal activity to fluconazole, amphotericin B, ketoconazole and caspofungin against the three clinical isolates were evaluated. Sensitivity to fluconazole and ketoconazole was evidenced. However, the treatment of this mycosis is still a challenge.

Lippia origanoides derivatives in vitro evaluation on polymicrobial biofilms: Streptococcus mutans, Lactobacillus rhamnosus and Candida albicans.

OBJECTIVE: This work evaluated the Lippia origanoides derivatives in vitro effect on polymicrobial biofilms of Streptococcus mutans, Lactobacillus rhamnosus and Candida albicans. Additionally, the cytotoxic effect of the oils on human skin keratinocytes (HaCaT) and fibroblasts of the periodontal ligament (FLP) cell lines was evaluated. DESIGN: The minimum inhibitory concentration, the inhibitory activity on monomicrobial (S. mutans) and polymicrobial biofilm (S. mutans, L. rhamnosus and C. albicans) of L. origanoides four essential oils and terpenes (thymol and carvacrol) were evaluated. The cytotoxic effect of each one of the compounds was measured, and all the tests were compared against chlorhexidine. RESULTS: All the evaluated compounds reached an inhibition percentage of S. mutans monomicrobial biofilms formation of 100 % at 600 µg/mL (p < 0.0001). The highest concentration (2 MIC) eradicated 100 % of S. mutans-preformed biofilms after 5 min L. origanoides carvacrol + thymol and thymol chemotypes showed marked reductions in topography, the number of microbial cells and extracellular matrix on polymicrobial biofilm. The cytotoxic effect of the compounds was very similar to chlorhexidine. CONCLUSIONS: L. origanoides essential oils have an inhibitory effect on mono and polymicrobial biofilms. The oils present a similar cytotoxic effect to chlorhexidine on HaCaT and FLP cell lines. However, including these compounds in formulations for clinical use is an exciting proposal yet to be investigated.

Onychomycosis caused by the environmental mold Neoscytalidium dimidiatum in Colombia, and in vitro antifungal susceptibility evaluation.

Neoscytalidium dimidiatum is a plant pathogen, but can also cause onychomycosis. We compared clinical and epidemiological data of cases of onychomycosis caused by N. dimidiatum and Trichophyton rubrum. We also evaluated the in vitro antifungal susceptibility of N. dimidiatum clinical isolates. It was not possible to establish any statistical differences between groups, except the place of residence and the number of affected nails. The results suggest that onychomycosis caused by N. dimidiatum is clinically similar to that caused by T. rubrum; besides, N. dimidiatum has been shown to have low sensitivity to itraconazole, but high to terbinafine. LAY SUMMARY: Cases of onychomycosis caused by Neoscytalidium dimidiatum were studied and compared to cases of onychomycosis caused by T. rubrum. The individuals affected were adults, and the clinical characteristics were not different between groups; accordingly, mycological diagnosis is mandatory.

Mechanisms of skin aging / Mecanismos de envejecimiento de la piel / Mecanismos de envelhecimento da pele

SUMMARY Skin aging is an inevitable biological phenomenon of human life that results from either the age-dependent decline of cell function (intrinsic aging) or from cumulative exposure to external harmful influences (extrinsic aging). Intrinsic and extrinsic factors act synergistically to induce skin changes that manifest clinically as burns, erythema, hyperpigmentation, telangiectasia, skin dryness or sagging, coarse wrinkles, skin texture changes or eventually as skin cancer. The molecular mechanisms of both types of skin aging are similar. This review focuses on intrinsic and extrinsic mechanisms of skin aging, and on current and new perspectives for prevention and treatment options extracted from natural products.

RESUMEN El envejecimiento cutáneo es un fenómeno biológico inevitable y puede ser el resultado de la disminución de la función celular como consecuencia del proceso normal de envejecimiento (envejecimiento intrínseco) o de la acumulación de los efectos dañinos debido a la exposición a factores nocivos externos (envejecimiento extrínseco). Los factores intrínsecos y extrínsecos actúan sinérgicamente para inducir cambios en la piel que se manifiestan clínicamente como quemaduras, eritema, hiperpigmentación, telangiectasias, sequedad de la piel o flacidez, arrugas profundas, cambios de textura o cáncer de piel. Los mecanismos moleculares de ambos tipos de envejecimiento de la piel son similares. Esta revisión se centra en una descripción general de los principales mecanismos intrínsecos y extrínsecos del envejecimiento de la piel así como en las medidas actuales de fotoprotección y en las nuevas perspectivas en cuanto a la prevención y al tratamiento basados en productos naturales.

RESUMO O envelhecimento cutâneo e um fenômeno biológico inevitável e pode ser o resultado da diminuição da função celular como consequência do processo normal do envelhecimento (envelhecimento intrínseco) ou do cúmulo dos efeitos daninhos devido à exposição com fatores nocivos externos (envelhecimento extrínseco). Os fatores intrínsecos e extrínsecos atuam sinergicamente para induzir mudanças na pele, que clinicamente manifestam-se como queimaduras, eritema, hiperpigmentação, telangiectasias, pele seca, rugas profundas, perda da tonalidade natural ou câncer de pele. Os mecanismos moleculares dos dois tipos de envelhecimento são similares. Esta revisão, centra-se na descrição geral dos principais mecanismos intrínsecos e extrínsecos do envelhecimento da pele, assim como nas medidas atuais de foto proteção e nas perspectivas referentes à prevenção e tratamento baseados no uso de produtos naturais.

Role of catalase overproduction in drug resistance and virulence in Candida albicans.

AIM: To investigate the role of Cat1 overproduction in Candida albicans. MATERIALS & METHODS: Strains overproducing the CAT1 gene were constructed. RESULTS: Cells overproducing CAT1 were found to be more resistant to some oxidants and mammalian phagocytic cells. They also showed reduced intracellular reactive oxygen species generated by amphotericin B or ciclopirox olamine. CAT1 overproduction did not change the minimum inhibitory concentration of fungal cells to fungistatic or fungicidal azoles nor to amphotericin B although increased twofold the minimum inhibitory concentration to caspofungin. The role of Cat1 overproduction in virulence and colonization was also analyzed in mouse models. CONCLUSION: The overproduction of Cat1 protects against oxidants, phagocytes and certain antifungals at subinhibitory concentration but does not increase virulence in a systemic infection mouse model.

Impact of Resistance to Fluconazole on Virulence and Morphological Aspects of Cryptococcus neoformans and Cryptococcus gattii Isolates.

Cryptococcus sp. are responsible for around 1 million cases of meningitis every year. Fluconazole (FLU) is commonly used in the treatment of cryptococcosis, mainly in immunocompromised patients and the resistance is usually reported after long periods of treatment. In this study, the morphological characterization and virulence profile of FLU-susceptible and FLU-resistant clinical and environmental isolates of C. neoformans and C. gattii were performed both in vitro and in vivo using the Galleria mellonella model. FLU-susceptible isolates from C. neoformans were significantly more virulent than the FLU-resistant isolates. FLU-susceptible C. gattii isolates showed a different virulence profile from C. neoformans isolates where only the environmental isolate, CL, was more virulent compared with the resistant isolates. Cell morphology and capsule size were analyzed and the FLU-resistant isolates did not change significantly compared with the most sensitive isolates. Growth at 37°C was also evaluated and in both species, the resistant isolates showed a reduced growth at this temperature, indicating that FLU resistance can affect their growth. Based on the results obtained is possible suggest that FLU resistance can influence the morphology of the isolates and consequently changed the virulence profiles. The most evident results were observed for C. neoformans showing that the adaptation of isolates to antifungal selective pressure influenced the loss of virulence.

The production of reactive oxygen species is a universal action mechanism of Amphotericin B against pathogenic yeasts and contributes to the fungicidal effect of this drug.

Amphotericin B (AMB) is an antifungal drug that binds to ergosterol and forms pores at the cell membrane, causing the loss of ions. In addition, AMB induces the accumulation of reactive oxygen species (ROS), and although these molecules have multiple deleterious effects on fungal cells, their specific role in the action mechanism of AMB remains unknown. In this work, we studied the role of ROS in the action mechanism of AMB. We determined the intracellular induction of ROS in 44 isolates of different pathogenic yeast species (Candida albicans, Candida parapsilosis, Candida glabrata, Candida tropicalis, Candida krusei, Cryptococcus neoformans, and Cryptococcus gattii). We also characterized the production of ROS in AMB-resistant isolates. We found that AMB induces the formation of ROS in all the species tested. The inhibition of the mitochondrial respiratory chain by rotenone blocked the induction of ROS by AMB and provided protection from the killing action of the antifungal. Moreover, this phenomenon was absent in strains that displayed resistance to AMB. These strains showed an alteration in the respiration rate and mitochondrial membrane potential and also had higher catalase activity than that of the AMB-susceptible strains. Consistently, AMB failed to induce protein carbonylation in the resistant strains. Our data demonstrate that the production of ROS by AMB is a universal and important action mechanism that is correlated with the fungicidal effect and might explain the low rate of resistance to the molecule. Finally, these data provide an opportunity to design new strategies to improve the efficacy of this antifungal.

Comparison of the ATB® Fungus 2 with the AFST-EUCAST for in vitro susceptibility testing of Candida spp

The increase of diseases caused by Candida spp., and the treatment failures, has underscored the need for testing the susceptibilities to antifungal agents. The commercial panel ATB® Fungus 2 was compared with the reference testing method of the European Subcommittee on Antifungal Susceptibility Testing of the Committee on Antimicrobial Susceptibility Testing (AFST-EUCAST) for the evaluation of the susceptibility of isolates of Candida spp. to three agents. The percentage of agreement was calculated based on the minimum inhibitory concentrations. There was a high correlation for AMB (100% қ = 1.0 Bhapkar coefficient p = 1.0); while it was lower with azoles (85%, қ = 0.41, p = Bhapkar coefficient 0.02 and 83.0%, қ = 0.15, Bhapkar coefficient p = 0.0006, respectively). The ATB® Fungus 2 and AFST-EUCAST are fully comparable methods for testing the susceptibility to AMB and to lesser extend comparable for ITR and FCA.

El aumento de infecciones por Candida spp. y de las fallas en los tratamientos, suscitan la necesidad de pruebas de susceptibilidad. Se comparó la marca comercial ATB® Fungus 2 con la técnica estándar del Subcomité para las Pruebas de Sensibilidad Antifúngica de la Unión Europea, de la Sociedad de Microbiología Clínica y Enfermedades Infecciosas (AFST-EUCAST) para evaluar la susceptibilidad de aislamientos de Candida spp. a tres antifúngicos. Con base en las concentraciones inhibitorias mínimas se calculó el porcentaje de acuerdo. La concordancia para anfotericina B (AMB) fue alta (100% қ = 1.0, Coeficiente de Bhapkarp = 1.0); para itraconazol (ITR) y fluconazol (FCA) fue inferior (85% қ = 0.41, Coeficiente de Bhapkarp =0.02 y 83.0 %, қ = 0.15, Coeficiente de Bhapkarp = 0.0006, respectivamente). Por lo tanto, ambas técnicas son comparables para la evaluación de la susceptibilidad a AMB; con los azoles el porcentaje de acuerdo es menor.

Antifungal efficacy during Candida krusei infection in non-conventional models correlates with the yeast in vitro susceptibility profile.

The incidence of opportunistic fungal infections has increased in recent decades due to the growing proportion of immunocompromised patients in our society. Candida krusei has been described as a causative agent of disseminated fungal infections in susceptible patients. Although its prevalence remains low among yeast infections (2-5%), its intrinsic resistance to fluconazole makes this yeast important from epidemiologic aspects. Non mammalian organisms are feasible models to study fungal virulence and drug efficacy. In this work we have used the lepidopteran Galleria mellonella and the nematode Caenorhabditis elegans as models to assess antifungal efficacy during infection by C. krusei. This yeast killed G. mellonella at 25, 30 and 37°C and reduced haemocytic density. Infected larvae melanized in a dose-dependent manner. Fluconazole did not protect against C. krusei infection, in contrast to amphotericin B, voriconazole or caspofungin. However, the doses of these antifungals required to obtain larvae protection were always higher during C. krusei infection than during C. albicans infection. Similar results were found in the model host C. elegans. Our work demonstrates that non mammalian models are useful tools to investigate in vivo antifungal efficacy and virulence of C. krusei.

The non-mammalian host Galleria mellonella can be used to study the virulence of the fungal pathogen Candida tropicalis and the efficacy of antifungal drugs during infection by this pathogenic yeast.

Although Candida tropicalis is a frequent cause of invasive fungal diseases, its interaction with the host remains poorly studied. Galleria mellonella is a Lepidoptera model which offers a useful tool to study virulence of different microorganisms and drug efficacy. In this work we investigated the virulence of C. tropicalis in G. mellonella at different temperatures and the efficacy of antifungal drugs in this infection model. When larvae were infected with yeast inocula suspensions of different concentrations (4 × 10(6), 2 × 10(6), 10(6) and 5 × 10(5) cells/larva), we observed a dose-dependent effect on the killing of the insect (50% survival ranging from 1.4 ± 0.8 to 8.8 ± 1.2 days with the higher and lower inocula, respectively). Candida tropicalis killed G. mellonella larvae at both 30°C and 37°C, although at 37°C the virulence was more evident. Haemocytes phagocytosed C. tropicalis cells after 2 hours of infection, although the phagocytosis rate was lower when compared with other fungal pathogens, such as Cryptococcus neoformans. Moreover, the haemocyte density in the haemolymph decreased during infection and the yeast formed pseudohyphae in G. mellonella. The efficacy of amphotericin B, caspofungin, fluconazole and voriconazole was tested at different concentrations, and a protective effect was observed with all the drugs at concentrations equivalent to therapeutic dose. Fungal burden increased in infected larvae during time of infection and amphotericin B and fluconazole reduced the number of colony-forming units in the worms. Moreover, antifungal treatment was associated with the presence of cell aggregates around infected areas. We conclude that G. mellonella offers a simple and feasible model to study C. tropicalis virulence and drug efficacy.

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.

Antifungal, cytotoxic and chemical analyses of essential oils of Lippia origanoides H.B.K grown in Colombia / Actividad antifúngica, citotó;xica y composición química de aceites esenciales de Lippia origanoides H.B.K recolectadas en Colombia

Introduction: Aspergillus fumigatus is most commonly associated to invasive aspergillosis. Strong antifungal activity against A. fumigatus of L. origanoides essential oil gives a new added value to this natural product from Boyacá-Colombia. Aims: The increase in fungal infections, the development of resistance and toxicity of wide-spectrum antifungals have led to a constant search for therapeutic alternatives. The chemical composition, antifungal and cytotoxic activity of nine essential oils obtained from L. origanoides were evaluated and the relationship between the antifungal activities of the oil and of its major components were explored. Methods and Results: Antifungal activity was determined following the protocols AFST-EUCAST for Candida krusei and C. parapsilosis, and CLSI-M38A for Aspergillus fumigatus and A. flavus. The GC-MS analysis identified three chemotypes: thymol, carvacrol and p-cymene/trans-beta-caryophyllene. The essential oil of the thymol chemotype was the most active in antifungal assays with MIC values of 157.5, 198.4, 125 and 31 μg ml-1 against C. parapsilosis, C. krusei, A. flavus and A. fumigatus, respectively. The major components carvacrol and thymol were not active against A. fumigatus at concentrations below 157.5 μg ml-1. In general, the oils were not cytotoxic. Conclusions: The essential oil of the thymol chemotype of L. origanoides from the region of Boyacá- Colombia showed the highest antifungal activity against A. fumigatus among all the oils and major components tested. Salud UIS 2011; 43 (2): 141-148.

Introducción: La infección por el hongo Aspergillus fumigatus está más comúnmente asociada a la aspergilosis invasiva. La fuerte actividad antimicótica del aceite esencial de L. origanoides contra A. fumigatus ha dado un nuevo valor agregado a este producto natural de Boyacá-Colombia. Objetivo: El aumento de las infecciones por hongos, el desarrollo de la resistencia y la toxicidad de los antifúngicos de amplio espectro han llevado a una constante búsqueda de alternativas terapéuticas. En este estudio fueron evaluados la composición química, la actividad antifúngica y citotóxica de nueve aceites esenciales obtenidos de L. origanoides; y la relación entre la actividad antifúngica de los aceites con respecto a la presencia de sus principales componentes. Métodos y Resultados: La actividad antifúngica se determinó siguiendo los protocolos AFST-EUCAST para Candida krusei y C. parapsilosis; y CLSI M38A para Aspergillus fumigatus y A. flavus. El análisis por GC-MS identificó tres quimiotipos: carvacrol timol y p-cymene/trans-beta-caryophyllene. El aceite esencial del quimiotipo timol fue el más activo en los ensayos antifúngicos con valores de MIC de 157,5, 198,4, 125 y 31 mg ml-1 frente a C. parapsilosis, C. krusei, A. flavus y A. fumigatus, respectivamente. El carvacrol y el timol, los principales componentes, no fueron activos frente a A. fumigatus en concentraciones inferiores a 157,5 g / ml-1. En general, los aceites no fueron citotóxicos. Conclusiones: El aceite esencial de L origanoides, quimiotipo timol, de la región de Boyacá-Colombia presentó la mayor actividad antifúngica frente a A. fumigatus entre todos los aceites evaluados; igualmente, sus principales componentes fueron los más activos en comparación a los otros quimiotipos. Salud UIS 2011; 43 (2): 141-148.

Amphotericin B mediates killing in Cryptococcus neoformans through the induction of a strong oxidative burst.

We studied the effects of Amphotericin B (AmB) on Cryptococcus neoformans using different viability methods (CFUs enumeration, XTT assay and propidium iodide permeability). After 1h of incubation, there were no viable colonies when the cells were exposed to AmB concentrations ≥ 1 mg/L. In the same conditions, the cells did not become permeable to propidium iodide, a phenomenon that was not observed until 3h of incubation. When viability was measured in parallel using XTT assay, a result consistent with the CFUs was obtained, although we also observed a paradoxical effect in which at high AmB concentrations, a higher XTT reduction was measured than at intermediate AmB concentrations. This paradoxical effect was not observed after 3h of incubation with AmB, and lack of XTT reduction was observed at AmB concentrations higher than 1mg/L. When stained with dihydrofluorescein, AmB induced a strong intracellular oxidative burst. Consistent with oxidative damage, AmB induced protein carbonylation. Our results indicate that in C. neoformans, Amphotericin B causes intracellular damage mediated through the production of free radicals before damage on the cell membrane, measured by propidium iodide uptake.

Actividad antimicótica y citotóxica de aceites esenciales de plantas de la familia Asteraceae / Antifungal activity, cytotoxicity and composition of essential oils from the Asteraceae plant family

Antecedentes En la medicina tradicional de Colombia, las plantas de la familia Asteraceae se han utilizado con fines medicinales. Objetivo Evaluar la actividad antimicótica y el efecto citotóxico de 15 aceites esenciales de plantas de la familia Asteraceae. Métodos La actividad antimicótica se evaluó con las cepas Candida parapsilosis ATCC 22019, Candida krusei ATCC 6258, Aspergillus flavus ATCC 204304 y Aspergillus fumigatus ATCC 204305 de acuerdo con las técnicas EUCAST y CLSI M38-A para levaduras y hongos filamentosos, respectivamente. La actividad citotóxica se evaluó mediante la técnica del MTT en la línea celular Vero. Resultados Los aceites de las plantas Achyrocline alata y Baccharis latifolia fueron los únicos activos contra A. fumigatus (media geométrica de la concentración mínima inhibitoria=78,7 y 157,4mg/ml, respectivamente). En contraste, no se evidenció actividad de los aceites contra especies de Candida. Además, estos aceites no fueron citotóxicos en las células Vero. Conclusiones Los aceites de A. alata y B. latifolia podrían ser candidatos para la desinfección de ambientes hospitalarios y para la inhibición de formación de biopelículas por A. fumigatus(AU)

Background The plants of the Asteraceae family have been used for medicinal purposes,in traditional Colombian medicine. Aim To evaluate the antifungal activity and the cytotoxic effects of 15 essential oils from plants of the Asteraceae family. Methods Antifungal activity was evaluated against Candida parapsilosis ATCC 22019, Candida krusei ATCC 6258, Aspergillus flavus ATCC 204304 and Aspergillus fumigatus ATCC 204305 following EUCAST and CLSI M38-A standard methods, for yeast and filamentous fungi, respectively. Cytotoxic effect was evaluated on Vero cell line by MTT assay. Results The oils from the plants Achyrocline alata and Baccharis latifolia were the only ones active against A. fumigatus (GM-MIC=78.7 and 157.4mg/ml, respectively). In contrast, there was no evidence of oils active against Candida species. In addition, these oils were not cytotoxic on Vero cells. The oils of A. alata and Baccharis latifolia could be candidates for disinfecting hospital environments and for inhibiting biofilm formation by A. fumigatus Conclusions The oils of A. alata and B. latifolia could be candidates for disinfecting hospital environments and for inhibiting biofilm formation by A. fumigatus(AU)

[Antifungal activity, cytotoxicity and composition of essential oils from the Asteraceae plant family]. / Actividad antimicótica y citotóxica de aceites esenciales de plantas de la familia Asteraceae.

BACKGROUND: The plants of the Asteraceae family have been used for medicinal purposes,in traditional Colombian medicine. AIM: To evaluate the antifungal activity and the cytotoxic effects of 15 essential oils from plants of the Asteraceae family. METHODS: Antifungal activity was evaluated against Candida parapsilosis ATCC 22019, Candida krusei ATCC 6258, Aspergillus flavus ATCC 204304 and Aspergillus fumigatus ATCC 204305 following EUCAST and CLSI M38-A standard methods, for yeast and filamentous fungi, respectively. Cytotoxic effect was evaluated on Vero cell line by MTT assay. RESULTS: The oils from the plants Achyrocline alata and Baccharislatifolia were the only ones active against A. fumigatus (GM-MIC=78.7 and 157.4 microg/ml, respectively). In contrast, there was no evidence of oils active against Candida species. In addition, these oils were not cytotoxic on Vero cells. The oils of A. alata and Baccharis latifolia could be candidates for disinfecting hospital environments and for inhibiting biofilm formation by A. fumigatus CONCLUSIONS: The oils of A. alata and B. latifolia could be candidates for disinfecting hospital environments and for inhibiting biofilm formation by A. fumigatus.

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 biológica de aceites esenciales de Calycolpus moritzianus recolectado en el Norte de Santander, Colombia / Chemical composition and biological activity of essential oils calycolpus moritzianus collectd in the Norte de Santander, Colombia

Introducción: actualmente, se están llevando a cabo estudios sobre la actividad biológica de AEs de plantas de la familia Myrtaceae. El objetivo de esta investigación fue la caracterización química y la evaluación de la actividad antimicótica, antitumoral y antiviral de 40 aceites extraídos de plantas de la especie Calycolpus moritzianus, comúnmente llamada “arrayán ó cínaro”, recolectadas en varias zonas del Norte de Santander, Colombia. Materiales y métodos: la actividad antimicótica se evaluó contra: Candida krusei, C. parapsilosis, Aspergillus fumigatus y A. flavus, siguiendo las técnicas estándar EUCAST y CLSI M38-A, respectivamente, para levaduras y hongos filamentosos. La citotoxicidad se evaluó en las líneas celulares tumorales (HeLa, Jurkat) y no tumorales (Vero) mediante la técnica fotocolorimétrica del MTT. La actividad antiviral in vitro, contra el virus Herpes simplex tipo HSV-1, se realizó mediante la “técnica de titulación del punto final (EPTT)”. Resultados y Discusión: los aceites X38 y X39, de muestras recolectadas en los municipios de Chinácota y Salazar, mostraron actividad contra C. krusei con valores de concentraciones inhibitorias mínimas de 500 µg/mL. Ninguno de los aceites fue citotóxico en células no tumorales Vero. Los aceites X9 y X10, procedentes de los municipios de Salazar y Pamplonita, fueron los más citotóxicos sobre células HeLa, a concentraciones de 37,8 ± 2.4 y 40,6 ± 2,6 µg/mL, respectivamente. La reducción de la carga viral en los cultivos celulares sólo fue lograda por el aceite X11. La actividad antimicótica y antitumoral de los aceites fue correlacionada con la composición de los componentes mayoritarios, evidenciándose la actividad del Terpinen-4-ol, (-) Limoneno, α –Pineno y Linalol.

Background: Essential oils of plants of the family Myrtaceae have been evaluated for different biological activities. The aim of this research was the chemical characterization and evaluation of antifungal, antiviral and cytotoxic activity of 40 essential oils extracted from Calycolpus moritzianus, commonly called “arrayán” or “cínaro”, collected in several areas of Norte de Santander, Colombia. Methods: antifungal activity was determined following the protocols AFST-EUCAST for Candida krusei and C. parasilopsis, and CLSI-M38A for Aspergillus fumigatus and A. flavus. Cytotoxicity was evaluated on tumoral (HeLa and Jurkat), and non tumoral (Vero) cell lines by the MTT photo-colorimetric technique The in vitro antiviral activity against Herpes simplex virus HSV-1 was determined by the end-point titration technique (EPTT). Results and Discussion: X38 and X39 oils of plants collected in regions of Chinácota and Salazar regions showed activity against C. krusei with minimal inhibitory concentration values of 500 µg/mL. None of the oils was cytotoxic in no-tumor (Vero) cells . Oils X9 and X10 of plants from regions of Salazar and Pamplonita were the most cytotoxic on HeLa cells at concentrations of 37,8 ± 2,4 and 40,6 ± 2,6 µg /mL, respectively. The reduction of viral load in cell cultures only was showed by X11 oil. Antifungal and antitumor activity of these oils was correlated with the composition of the major components showing activity for Terpinen-4-ol, (-) Limonene, Linalool and α-Pinene.

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 microg/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 microg/mL for A. fumigatus and 39.7 microg/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 microg/mL for C. krusei and 176.8 microg/mL for A. fumigatus.

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.