In vitro Safety Assessment of Extracts and Compounds From Plants as Sunscreen Ingredients.

This work investigated the safety of extracts obtained from plants growing in Colombia, which have previously shown UV-filter/antigenotoxic properties. The compounds in plant extracts obtained by the supercritical fluid (CO2) extraction method were identified using gas chromatography coupled to mass spectrometry (GC/MS) analysis. Cytotoxicity measured as cytotoxic concentration 50% (CC50) and genotoxicity of the plant extracts and some compounds were studied in human fibroblasts using the trypan blue exclusion assay and the Comet assay, respectively. The extracts from Pipper eriopodon and Salvia aratocensis species and the compound trans-ß-caryophyllene were clearly cytotoxic to human fibroblasts. Conversely, Achyrocline satureioides, Chromolaena pellia, and Lippia origanoides extracts were relatively less cytotoxic with CC50 values of 173, 184, and 89 µg/mL, respectively. The C. pellia and L. origanoides extracts produced some degree of DNA breaks at cytotoxic concentrations. The cytotoxicity of the studied compounds was as follows, with lower CC50 values representing the most cytotoxic compounds: resveratrol (91 µM) > pinocembrin (144 µM) > quercetin (222 µM) > titanium dioxide (704 µM). Quercetin was unique among the compounds assayed in being genotoxic to human fibroblasts. Our work indicates that phytochemicals can be cytotoxic and genotoxic, demonstrating the need to establish safe concentrations of these extracts for their potential use in cosmetics.

Essential oil of Cordia curassavica (Jacq) Roem. & Schult: in vitro and in silico evaluation of effects on dengue virus replication and cytokines production / Aceite esencial de Cordia curassavica (Jacq) Roem. & Schult: evaluación in vitro e in silico del efecto sobre la replicación del virus dengue y la producción de citoquinas

The lack of effective conventional therapie s against dengue has created an interest in herbal preparations as alternative therapies. In the present study, in vitro effects of Cordia curassavica essential oil (EO) on both dengue virus replication and cytokine production were examined. Predictions of molecular interactions between EO compounds and virus and cell proteins were performed with AutoDock Vina. The EO inhibited replication of dengue virus serotypes at IC 50 < 30 µg/mL, and it reduced 87% TNF - α, 67% IL - 8 and 46% IFN - α in LPS - stimulated PBMCs. The main EO compounds were trans - ß - caryophyllene (21.4%), germacrene D (17.8%), α - copaene (16.5%), trans - ß - guaiene (8.2%), and α - pinene (6.0%). The first two compounds, δ - cadinene, α - muurolene, α - cubebene and ß - burbonene were coupled to proteins involved in the TLR - 4 cytokine effector pathway. 3,7 - Guaiadiene was coupled to the viral E and C proteins. This study demonstrates the potential of C. curassavica EO as a starting point for discovering novel therapeutic for dengue.

La falta de terapias eficaces para el dengue ha suscitado interés por preparados herbales como terapias alternativas. En el presente estudio se examinaron efectos in vitro del aceite e sencial (AE) de Cordia curassavica sobre la replicación del virus dengue y producción de citoquinas. Se realizaron predicciones de interacciones moleculares entre los compuestos del AE y proteínas virales y celulares con AutoDock Vina. El AE inhibió la rep licación de serotipos del virus a CI 50 < 30 µg/mL y redujo 87% TNF - α, 67% IL - 8 y 46% IFN - α en MNCP. Los principales compuestos del AE fueron trans - ß - cariofileno, germacreno D, α - copaeno, trans - ß - guaieno y α - pineno. Los dos primeros compuestos, el δ - cadineno, el α - muuroleno, el α - cubebeno y el ß - burboneno se acoplaron a proteínas implicadas en la vía efectora de citoquinas TLR - 4. El 3,7 - guaiadiene se acopló a las proteínas virales E y C. Este estudio demuestra el potencial del AE de C. curassavica como punto de partida para descubrir nuevas tera pias para el dengue.

Comparative Virucidal Activities of Essential Oils and Alcohol-Based Solutions against Enveloped Virus Surrogates: In Vitro and In Silico Analyses.

The large-scale use of alcohol (OH)-based disinfectants to control pathogenic viruses is of great concern because of their side effects on humans and harmful impact on the environment. There is an urgent need to develop safe and environmentally friendly disinfectants. Essential oils (EOs) are generally recognized as safe (GRAS) by the FDA, and many exhibit strong antiviral efficacy against pathogenic human enveloped viruses. The present study investigated the virucidal disinfectant activity of solutions containing EO and OH against DENV-2 and CHIKV, which were used as surrogate viruses for human pathogenic enveloped viruses. The quantitative suspension test was used. A solution containing 12% EO + 10% OH reduced > 4.0 log10 TCID50 (100% reduction) of both viruses within 1 min of exposure. In addition, solutions containing 12% EO and 3% EO without OH reduced > 4.0 log10 TCID50 of both viruses after 10 min and 30 min of exposure, respectively. The binding affinities of 42 EO compounds and viral envelope proteins were investigated through docking analyses. Sesquiterpene showed the highest binding affinities (from -6.7 to -8.0 kcal/mol) with DENV-2 E and CHIKV E1-E2-E3 proteins. The data provide a first step toward defining the potential of EOs as disinfectants.

Essential Oils from Colombian Plants: Antiviral Potential against Dengue Virus Based on Chemical Composition, In Vitro and In Silico Analyses.

Currently, there are no therapies to prevent severe dengue disease. Essential oils (EOs) can serve as primary sources for research and the discovery of phytomedicines for alternative therapy. Fourteen EOs samples were obtained by distillation from six plants used in Colombian folk medicine. GC/MS analysis identified 125 terpenes. Cytopathic effect (CPE) reduction assays revealed differences in antiviral activity. EOs of Lippia alba, citral chemotype and carvone-rich fraction; Lippia origanoides, phellandrene chemotype; and Turnera diffusa, exhibited strong antiviral activity (IC50: 29 to 82 µg/mL; SI: 5.5 to 14.3). EOs of Piper aduncum, Ocimum basilicum, and L. origanoides, carvacrol, and thymol chemotypes, exhibited weak antiviral activity (32 to 53% DENV-CPE reduction at 100 µg/mL; SI > 5.0). Cluster and one-way ANOVA analyses suggest that the strong antiviral activity of EOs could be attributed to increased amounts of non-phenolic oxygenated monoterpenes and sesquiterpene hydrocarbons. Docking analyses (AutoDock Vina) predicted binding affinity between the DENV-2 E protein and terpenes: twenty sesquiterpene hydrocarbons (−8.73 to −6.91 kcal/mol), eight oxygenated monoterpenes (−7.52 to −6.98 kcal/mol), and seven monoterpene hydrocarbons (−7.60 to −6.99 kcal/mol). This study reports for the first time differences in the antiviral activity of EOs against DENV, corresponding to their composition of monoterpenes and sesquiterpenes.

Flower Extracts from Ornamental Plants as Sources of Sunscreen Ingredients: Determination by In Vitro Methods of Photoprotective Efficacy, Antigenotoxicity and Safety.

Plants are sources of sunscreen ingredients that prevent cellular mutations involved in skin cancer and aging. This study investigated the sunscreen properties of the extracts from some ornamental plants growing in Colombia. The UV filter capability of the flower extracts obtained from Rosa centifolia L., Posoqueria latifolia (Rudge) Schult, and Ipomoea horsfalliae Hook. was examined. Photoprotection efficacies were evaluated using in vitro indices such as sun protection factor and critical wavelength. UVB antigenotoxicity estimates measured with the SOS Chromotest were also obtained. Extract cytotoxicity and genotoxicity were studied in human fibroblasts using the trypan blue exclusion and Comet assays, respectively. Major compounds of the promising flower extracts were identified by UHPLC-ESI+-Orbitrap-MS. The studied extracts showed high photoprotection efficacy and antigenotoxicity against UVB radiation, but only the P. latifolia extract showed broad-spectrum photoprotection at non-cytotoxic concentrations. The P. latifolia extract appeared to be safer for human fibroblast cells and the R. centifolia extract was shown to be moderately cytotoxic and genotoxic at the highest assayed concentrations. The I. horsfalliae extract was unequivocally cytotoxic and genotoxic. The major constituents of the promising extracts were as follows: chlorogenic acid, ecdysterone 20E, rhamnetin-rutinoside, cis-resveratrol-diglucoside, trans-resveratrol-diglucoside in P. latifolia; quercetin, quercetin-glucoside, quercetin-3-rhamnoside, kaempferol, kaempferol-3-glucoside, and kaempferol-rhamnoside in R. centifolia. The potential of the ornamental plants as sources of sunscreen ingredients was discussed.

Dengue in Santander State, Colombia: fluctuations in the prevalence of virus serotypes are linked to dengue incidence and genetic diversity of the circulating viruses.

OBJECTIVE: To investigate the link between fluctuations in the prevalence of dengue virus (DENV) serotypes and the number of dengue cases in the metropolitan area of Bucaramanga, Santander State, Colombia, in the 2007-2010 and 2014-2017 periods. METHOD: Viruses were isolated from febrile patient samples by direct application to C6/36-HT cells and typed using monoclonal antibodies. We performed autocorrelation and cross-correlation analyses to determine whether fluctuations in the prevalence of DENV serotypes and dengue cases were correlated. Full envelope (E) gene sequences were employed to examine the genetic diversity of serotypes circulating by using a phylogenetic approach. RESULTS: All four dengue virus serotypes were detected. DENV-1 was the dominant serotype in both periods followed by DENV-3 or DENV-2 depending on the period; DENV-4 was the least prevalent virus in both periods. Cross-correlation analyses suggest a temporal relation between the fluctuations in the prevalence of DENV serotypes, which were almost simultaneous (lag = 0) or related to recent past fluctuations (lag > 1.0) in the number of dengue cases. Data suggest that a sustained predominance of DENV-1, an increase of the DENV-4 prevalence, and a switch from DENV-3 to DENV-2 could be linked to an outbreak. Circulating viruses were grouped into Genotype V, Asia/American III and II for DENV-1, -2, -3 and -4, respectively; intragenotypic diversity was detected. CONCLUSIONS: The present work highlights the need of comprehensive studies on dynamics of DENV in Colombia to understand transmission of dengue and evaluate the effectiveness of a vaccination programme.

OBJECTIF: Etudier le lien entre les fluctuations de la prévalence des sérotypes du virus de la dengue (DENV) et le nombre de cas de dengue dans la région métropolitaine de Bucaramanga, dans l'Etat de Santander, en Colombie, au cours des périodes 2007-2010 et 2014-2017. MÉTHODE: Les virus ont été isolés à partir d'échantillons de patients fébriles par application directe sur des cellules C6/36-HT et typés à l'aide d'anticorps monoclonaux. Nous avons effectué des analyses d'autocorrélation et de corrélation croisée afin de déterminer si les fluctuations de la prévalence des sérotypes du DENV et des cas de dengue étaient corrélées. Des séquences de gènes d'enveloppe complète (E) ont été utilisées pour examiner la diversité génétique des sérotypes en circulation en utilisant une approche phylogénétique. RÉSULTATS: Tous les quatre sérotypes du virus de la dengue ont été détectés. DENV-1 était le sérotype dominant dans les deux périodes, suivi de DENV-3 ou DENV-2, selon la période; le virus DENV-4 était le moins prévalent au cours des deux périodes. Les analyses de corrélation croisée suggèrent une relation temporelle entre les fluctuations de la prévalence des sérotypes de DENV, qui étaient presque simultanées (lag = 0) ou liées aux fluctuations passées récentes (lag > 1,0) du nombre de cas de dengue. Les données suggèrent qu'une prédominance durable de DENV-1, qu'une augmentation de la prévalence de DENV-4 et qu'un passage de DENV-3 à DENV-2 pourraient être liés à une éclosion. Les virus en circulation ont été regroupés dans les génotypes V, Asie/Amérique III et II pour DENV-1, -2, -3 et -4, respectivement; une diversité intra-génotypique a été détectée. CONCLUSIONS: Le présent travail souligne la nécessité d'études approfondies sur la dynamique du DENV en Colombie afin de comprendre la transmission de la dengue et évaluer l'efficacité d'un programme de vaccination.

Photoprotective and Antigenotoxic Effects of the Flavonoids Apigenin, Naringenin and Pinocembrin.

This work evaluated the photoprotective and antigenotoxic effects against ultraviolet B (UVB) radiation of flavonoid compounds apigenin, naringenin and pinocembrin. The photoprotective efficacy of these compounds was estimated using in vitro photoprotection indices, and the antigenotoxicity against UVB radiation was evaluated using the SOS chromotest and an enzymatic (proteinase K/T4 endonuclease V enzyme) comet assay in UV-treated Escherichia coli and human (HEK-293) cells, respectively. Naringenin and pinocembrin showed maximum UV-absorption peak in UVC and UVB zones, while apigenin showed UV-absorption capability from UVC to UVA range. These compounds acted as UV filters reducing UV-induced genotoxicity, both in bacteria and in human cells. The enzymatic comet assay resulted highly sensitive for detection of UVB-induced DNA damage in HEK-293 cells. In this work, the photoprotective potential of these flavonoids was widely discussed.

Evolutionary history and spatio-temporal dynamics of dengue virus serotypes in an endemic region of Colombia.

Dengue is a prevalent disease in Colombia and all dengue virus serotypes (DENV-1 to -4) co-circulate in the country since 2001. However, the relative impact of gene flow and local diversification on epidemic dynamics is unknown due to heterogeneous sampling and lack of sufficient genetic data. The region of Santander is one of the areas with the highest incidence of dengue in Colombia. To provide a better understanding of the epidemiology of dengue, we inferred DENV population dynamics using samples collected between 1998 and 2015. We used Bayesian phylogenetic analysis and included 143 new envelope gene sequences from Colombia, mainly from the region of Santander, and 235 published sequences from representative countries in the Americas. We documented one single genotype for each serotype but multiple introductions. Whereas the majority of DENV-1, DENV-2, and DENV-4 strains fell into one single lineage, DENV-3 strains fell into two distinct lineages that co-circulated. The inferred times to the most recent common ancestors for the most recent clades of DENV-1, DENV-2, and DENV-4 fell between 1977 and 1987, and for DENV-3 was around 1995. Demographic reconstructions suggested a gradual increase in viral diversity over time. A phylogeographical analysis underscored that Colombia mainly receives viral lineages and a significant diffusion route between Colombia and Venezuela. Our findings contribute to a better understanding of the viral diversity and dengue epidemiology in Colombia.

Actividad antiproliferativa de aceites esenciales de plantas cultivadas en Colombia / Antiproliferative Activity of Essential Oils from Colombian Plants

Colombia posee gran diversidad de plantas medicinales, pero pocas han sido objeto de investigación. En este trabajo se evaluó la actividad antiproliferativa de aceites esenciales obtenidos por hidrodestilación asistida por microondas. Se analizaron 15 muestras de 11 especies en ensayos del MTT en células cancerosas MCF-7, HeLa y HepG-2 y se incluyeron células normales humanas (HEK293) y de animales (Vero y B16F10) para evaluar selectividad. La composición química de muestras activas se determinó por cromatografía de gases acoplada a espectrometría de masas (GC-MS). Aceites esenciales de cuatro especies mostraron actividad antiproliferativa (CI50: 50 µg/mL) en células HeLa y/o MCF-7, en el siguiente rango (índice de selectividad en paréntesis): Pipercumanense H.B.K. (4,7) > Piper subflavum var. espejuelanum C.DC (3,9) > Salvia officinalis L. (3,6) > Piper eriopodom (Miq.) C. DC. (3,1). Ninguna muestra fue activa en células HepG-2. El análisis por CG-MS identificó ß-cariofileno, a-copaeno, ß-pineno, α-pineno y linalol como componentes mayoritarios. Los aceites esenciales activos pueden ser puntos de partida para desarrollo de medicamentos herbales para cuidado paliativo del cáncer.

Discovery of antiviral molecules for dengue: In silico search and biological evaluation.

BACKGROUND: Dengue disease is a global disease that has no effective treatment. The dengue virus (DENV) NS2B/NS3 protease complex is a target for designing specific antivirals due to its importance in viral replication and its high degree of conservation. METHODS: NS2B/NS3 protease complex structural information was employed to find small molecules that are capable of inhibiting the activity of the enzyme complex. This inhibitory activity was probed with in vitro assays using a fluorescent substrate and the complex NS2B/NS3 obtained by recombinant DNA techniques. HepG2 cells infected with dengue virus serotype 2 were used to test the activity against dengue virus replication. RESULTS: A total of 210,903 small molecules from PubChem were docked in silico to the NS2B/NS3 structure (PDB: 2FOM) to find molecules that were capable of inhibiting this protein complex. Five of the best 500 leading compounds, according to their affinity values (-11.6 and -13.5 kcal/mol), were purchased. The inhibitory protease activity on the recombinant protein and antiviral assays was tested. CONCLUSIONS: Chemicals CID 54681617, CID 54692801 and CID 54715399 were strong inhibitors of NS2B/NS3, with IC50 values (µM) and percentages of viral titer reductions of 19.9, 79.9%; 17.5, 69.8%; and 9.1, 73.9%, respectively. Multivariate methods applied to the molecular descriptors showed two compounds that were structurally different from other DENV inhibitors. GENERAL SIGNIFICANCE: This discovery opens new possibilities for obtaining drug candidates against Dengue virus.

Propuesta para seleccionar aceites esenciales de plantas de Colombia para investigación con base en su citotoxicidad / Proposal to select essential oils from colombian plants for research based on its cytoxicity

Antecedentes: Conocer la tendencia de productos de plantas a causar toxicidad en humanos es parte de la investigación orientada al descubrimiento de un medicamento natural. Las pruebas en animales son relativamente costosas, de bajo rendimiento, asociadas a sufrimiento del animal y diferencias relativas a la especie hacen difícil inferir efectos en humanos. Las pruebas en célula viva son recomendadas. Objetivo: Estudiar la tendencia a toxicidad de aceites esenciales (AE) de plantas de Colombia usando un ensayo basado en célula. Método: Los AE de 18 especies distintas de plantas fueron estudiados. Se usó el ensayo del MTT en seis líneas celulares de humano y animal derivadas de tejido normal y canceroso, las cuales se trataron antes y después de la proliferación. Los AE se organizaron en el orden de una agrupación jerárquica con base en los valores de CC50 y la sumatoria de la jerarquía ponderada en el panel de células (∑JPi) se usó como indicador de similitud. Cuanto mayor fue el valor de ∑JPi menor fue la tendencia a toxicidad. Resultados: Los AE con valores de CC50>200 µg/mL en al menos cinco condiciones experimentales presentaron valores de ∑JPi > 6,0 sugiriendo baja tendencia a toxicidad y fueron en orden descendente (∑JPi en paréntesis): Calycolpus moritzianus (O.Berg) Burret (9,7) < Psidium sartorianum (O. Berg) Nied. (8,9) < Wedelia calycina (6,5) < Lippia micromera Schauer (6,2) ≈ Piper haltonii. (6,2). AE con valores de CC50 < 100 µg/mL en cuatro o más condiciones experimentales presentaron valores ∑JPi < 4.0 sugiriendo alta tendencia a toxicidad y fueron en orden ascendente: Tagetes caracasana Kunth (2,7 ­ 2,8) > Chromolaena odorata (L.) R.M.King & H.Rob. (3,0) > Ageratina aff. popayanensis (Hieron.) R.M.King & H.Rob. (3,1) > Lantana colombiana López-Pal. (3,3) >Turnera disffusa. (3,4). AE de Tagetes caracasana presentó actividad antiproliferante (CI50: 42,2 y 47,9 µg/mL) sobre células humanas de cáncer de cérvix. Conclusión: El abordaje metodológico permitió identificar AE con baja y alta tendencia a toxicidad. Los resultados podrían tener valor para predecir actividad in vivo y priorizar muestras para futuras investigaciones.

Background: Part of the research process focused on discovering natural medicines is the study of products derived from plants, which may be toxic to humans. Animal-based test methods can be relatively expensive, low-throughput and associated with animal suffering, and differences in animal species may difficult to infer human health effects. Methods based on living cells are recommended. Objectives: To study the tendency to toxicity of essential oils (EOs) from plants of Colombia using a cell-based assay. Methods: EOs from different species (n = 18) of plants were included. The MTT assay was used on six human and animal cell lines derived from normal and cancerous organs, which were treated before and after proliferation. The EOs were arranged in the order of a hierarchical clustering based on their CC50 values, and the sum of weighted hierarchy across cell panel (∑iWH) was used as the similarity metric. The greater the value of ∑iWH lesser tendency to toxicity. Results: The EOs, which showed CC50 values > 200 µg/mL in at least five experimental conditions presented ∑iWH values > 5,0 suggesting lower tendency to toxicity, and they were in descending order (∑iWH in parentheses), as follows: Calycolpus moritzianus (O.Berg) Burret (9,7) < Psidium sartorianum (O. Berg) Nied. 1893 (8,9) < Wedelia calcycina (6,5) < Lippia micromera Schauer (6,2) ≈ Piper haltonii Jacq. (6,2) The EOs, which showed CC50 < 100 µg/mL in four or more experimental conditions presented ∑iWH values < 4.0 suggesting higher tendency to toxicity, and they were in ascending order, as follows: Tagetes caracasana Kunth (2,7 ­ 2,8) > Chromolaena odorata (L.) R.M.King & H.Rob. (3,0) > Ageratina aff. popayanensis (Hieron.) R.M.King & H.Rob. (3,1) > Lantana colombiana López-Pal. (3,3) > Turnera diffusa (3,4). EO from Tagetes caracasana Kunth presented relevant antiproliferative activity (CI50: < 50.0 µg/mL) on cells from human cervical carcinoma. Conclusions: The methodological approach allows identifying EOs with lower and higher tendency to toxicity. Data generated may be valuable for predicting in vivo toxicity and for prioritizing samples for further studies.

Comparative study on in vitro activities of citral, limonene and essential oils from Lippia citriodora and L. alba on yellow fever virus.

The aim of this study was to compare the antiviral activities in vitro of citral, limonene and essential oils (EOs) from Lippia citriodora and L. alba on the replication of yellow fever virus (YFV). Citral and EOs were active before and after virus adsorption on cells; IC50 values were between 4.3 and 25 microg/mL and SI ranged from 1.1 to 10.8. Results indicate that citral could contribute to the antiviral activity of the L. citriodora EO. Limonene was not active and seemed to play an insignificant role in the antiviral activity of the examined EOs.

Virucidal activity of Colombian Lippia essential oils on dengue virus replication in vitro.

The inhibitory effect of Lippia alba and Lippia citriodora essential oils on dengue virus serotypes replication in vitro was investigated. The cytotoxicity (CC50) was evaluated by the MTT assay and the mode of viral inhibitory effect was investigated with a plaque reduction assay. The virus was treated with the essential oil for 2 h at 37 masculineC before cell adsorption and experiments were conducted to evaluate inhibition of untreated-virus replication in the presence of oil. Antiviral activity was defined as the concentration of essential oil that caused 50% reduction of the virus plaque number (IC50). L. alba oil resulted in less cytotoxicity than L. citriodora oil (CC50: 139.5 vs. 57.6 microg/mL). Virus plaque reduction for all four dengue serotypes was observed by treatment of the virus before adsorption on cell. The IC50 values for L. alba oil were between 0.4-32.6 microg/mL and between 1.9-33.7 microg/mL for L. citriodora oil. No viral inhibitory effect was observed by addition of the essential oil after virus adsorption. The inhibitory effect of the essential oil seems to cause direct virus inactivation before adsorption on host cell.

Virucidal activity of Colombian Lippia essential oils on dengue virus replication in vitro

The inhibitory effect of Lippia alba and Lippia citriodora essential oils on dengue virus serotypes replication in vitro was investigated. The cytotoxicity (CC50) was evaluated by the MTT assay and the mode of viral inhibitory effect was investigated with a plaque reduction assay. The virus was treated with the essential oil for 2 h at 37ºC before cell adsorption and experiments were conducted to evaluate inhibition of untreated-virus replication in the presence of oil. Antiviral activity was defined as the concentration of essential oil that caused 50 percent reduction of the virus plaque number (IC50). L. alba oil resulted in less cytotoxicity than L. citriodora oil (CC50: 139.5 vs. 57.6 μg/mL). Virus plaque reduction for all four dengue serotypes was observed by treatment of the virus before adsorption on cell. The IC50 values for L. alba oil were between 0.4-32.6 μg/mL and between 1.9-33.7 μg/mL for L. citriodora oil. No viral inhibitory effect was observed by addition of the essential oil after virus adsorption. The inhibitory effect of the essential oil seems to cause direct virus inactivation before adsorption on host cell.

Aceites esenciales de plantas colombianas inactivan el virus del dengue y el virus de la fiebre amarilla / Essential oils from colombian plants inactive dengue virus and yellow fever virus

Introducción: Un antiviral contra el virus del dengue (VDEN) y el virus de la fiebre amarilla (VFA) para tratamiento de los enfermos no está disponible en el mercado, a pesar de numerosas investigaciones con compuestos sintéticos. Objetivo: Evaluar el efecto inhibitorio in vitro sobre el VDEN y el VFA del aceite esencial obtenido de plantas cultivadas en Colombia. Materiales y métodos: Los virus se incubaron con el aceite esencial (100 µg/mL) 2 h a 37oC antes de la adsorción a la célula y el efecto inhibitorio fue determinado por el método de reducción de placa. Resultados: El aceite esencial obtenido de 10 y 8 plantas redujo desde 74 hasta 100% placas del VDEN y del VFA, respectivamente. Los aceites de Lippia citriodora (verbena) y Pimenta racemosa (laurel) fueron más activos contra ambos virus reduciendo 100% las placas. La magnitud del efecto inhibitorio se relacionó con el método de extracción del aceite y la parte de la planta seleccionada. Conclusión: El aceite esencial de las plantas colombianas puede inhibir la replicación in vitro del VDEN y VFA. Se requieren más estudios para determinar la concentración mínima inhibitoria y el índice de selectividad para considerar estas plantas como fuente de compuestos antivirales.

Background: Products obtained from plants can inhibit in vitro viruses that cause human diseases. An antiviral drug against dengue virus (DENV) and yellow fever virus (YFV) does not exist despite extensive research exploring synthetic compounds. Objective: To evaluate the inhibitory effect on DENV and YFV of essential oils obtained from Colombian plants. Materials and methods: Viruses were incubated with essential oil (100 µg/mL) 2 h at 37oC before cell adsorption and the inhibitory effect was determined by plaque reduction assay. Results: The essential oil obtained from 10 and 8 plants reduced from 74 to 100% DENV and YFV plaques, respectively. Essential oils from Lippia citriodora and Pimenta racemosa were the most active against both viruses causing 100% reduction of plaques. The magnitude of the inhibitory effect was related to the method of oil extraction and part of plant used. Conclusion: Essential oils from Colombian plants can inhibit the replication in vitro of DENV and YFV. Further studies determining the minimal inhibitory concentrations and selectivity index are needed in order to consider these plants as a source of antiviral compounds.

Inhibitory effect of essential oils obtained from plants grown in Colombia on yellow fever virus replication in vitro.

BACKGROUND: An antiviral drug is needed for the treatment of patients suffering from yellow fever. Several compounds present in plants can inactive in vitro a wide spectrum of animal viruses. AIM: In the present study the inhibitory effect of essential oils of Lippia alba, Lippia origanoides, Oreganum vulgare and Artemisia vulgaris on yellow fever virus (YFV) replication was investigated. METHODS: The cytotoxicity (CC(50)) on Vero cells was evaluated by the MTT reduction method. The minimum concentration of the essential oil that inhibited virus titer by more than 50% (MIC) was determined by virus yield reduction assay. YFV was incubated 24 h at 4 degrees C with essential oil before adsorption on Vero cell, and viral replication was carried out in the absence or presence of essential oil. Vero cells were exposed to essential oil 24 h at 37 degrees C before the adsorption of untreated-virus. RESULTS: The CC(50) values were less than 100 microg/mL and the MIC values were 3.7 and 11.1 microg/mL. The CC(50)/MIC ratio was of 22.9, 26.4, 26.5 and 8.8 for L. alba, L origanoides, O. vulgare and A. vulgaris, respectively. The presence of essential oil in the culture medium enhances the antiviral effect: L. origanoides oil at 11.1 microg/mL produced a 100% reduction of virus yield, and the same result was observed with L. alba, O. vulgare and A. vulgaris oils at 100 microg/mL. No reduction of virus yield was observed when Vero cells were treated with essential oil before the adsorption of untreated-virus. CONCLUSION: The essential oils evaluated in the study showed antiviral activities against YFV. The mode of action seems to be direct virus inactivation.

Anticuerpos neutralizantes contra el virus de la fiebre amarilla 17 D en colombianos vacunados y no vacunados con inmunidad a dengue / Yellow fever virus 17D neutralising antibodies in vaccinated Colombian people and unvaccinated ones having immunity against dengue

Objetivo Determinar la frecuencia de título protector de anticuerpos neutralizantes contra el virus de la fiebre amarilla (AN-VFA a título >1:10) en colombianos vacunados con la cepa 17 D y conocer la magnitud de neutralización del VFA por anticuerpos contra dengue. Metodología Se colectó suero de 100 individuos con vacuna documentada por carné y de 116 residentes en municipios de Norte de Santander afectados por el brote en 2002-2003, quienes informaron haber sido vacunados. Se incluyeron sueros de individuos no vacunados con (n=61) y sin (n=16) anticuerpos contra dengue. Todos los sueros se analizaron por la prueba de neutralización para VFA por 75 por ciento de reducción de placa. Resultados AN-VFA a título >1:10 se encontraron en 90 por ciento de vacunados con carné y sin variación aparente en relación con edad. Al contrario, hubo correlación entre disminución de la frecuencia de título protector de anticuerpos e incremento del tiempo de inmunización (r=0,95; p=0,04). En residentes de Norte de Santander, AN-VFA a título >1:10 se encontraron en 92,6 por ciento adultos y 69 por ciento niños. El VFA fue neutralizado (52 -100 por ciento) por sueros de inmunes a dengue más eficientemente que por sueros de no inmunes (p<0.001). Conclusiones Vacunados con el virus 17 D podrían no estar protegidos contra fiebre amarilla: hasta 31 por ciento niños y 10 por ciento adultos. Anticuerpos contra dengue inhibieron el VFA y su significancia en términos de protección contra fiebre amarilla deberá ser investigada.

Objective Determining the frequency of yellow fever seroprotective antibody neutralising titres (YF-NT >1:10) in Colombians vaccinated with the 17 D virus and ascertaining the extent to which YF virus can be neutralised by dengue antibodies. Materials and Methods Serum samples were taken from 100 subjects who showed their vaccination record and from 116 residents in municipalities (Norte de Santander) affected by a wild YF outbreak in 2002-2003 who were reported to have been YF vaccinated. Sera from individuals with (n=61) and without (n=16) dengue antibodies who had never been YF vaccinated were included. All the sera were tested by 75 percent YF plaque-reduction neutralization test. Results YF-NT titres >1:10 were founded in 90 percent of subjects with vaccination recorded with minors variations in relation to age. In contrast, there was correlation between decrease of seroprotective YF-NT titres frequency and increase of immunization time (r=0.95; p=0.04). In residents in YF endemic area, YF-NT titres > 1.10 were founded in 92,6 percent adults and 69 percent children. YF 17 D virus was neutralized (52-100 percent) by dengue sera more efficiently than non-dengue immune sera (p<0.001). Conclusions Individuals immunised with YF vaccine 17 D could not be protected against YF: up to 31 percent children and 10 percent adults. Dengue antibodies inhibited YF virus and its significance in terms of YF protection must be investigated.

[Yellow fever virus 17D neutralising antibodies in vaccinated Colombian people and unvaccinated ones having immunity against dengue]. / Anticuerpos neutralizantes contra el virus de la fiebre amarilla 17 D en colombianos vacunados y no vacunados con inmunidad a dengue.

OBJECTIVE: Determining the frequency of yellow fever seroprotective antibody neutralising titres (YF-NT >or=1:10) in Colombians vaccinated with the 17 D virus and ascertaining the extent to which YF virus can be neutralised by dengue antibodies. MATERIALS AND METHODS: Serum samples were taken from 100 subjects who showed their vaccination record and from 116 residents in municipalities (Norte de Santander) affected by a wild YF outbreak in 2002-2003 who were reported to have been YF vaccinated. Sera from individuals with (n=61) and without (n=16) dengue antibodies who had never been YF vaccinated were included. All the sera were tested by 75 % YF plaque-reduction neutralization test. RESULTS: YF-NT titres >or=1:10 were founded in 90 % of subjects with vaccination recorded with minors variations in relation to age. In contrast, there was correlation between decrease of seroprotective YF-NT titres frequency and increase of immunization time (r=0.95; p=0.04). In residents in YF endemic area, YF-NT titres >or= 1.10 were founded in 92,6 % adults and 69 % children. YF 17 D virus was neutralized (52-100 %) by dengue sera more efficiently than non-dengue immune sera (p<0.001). CONCLUSIONS: Individuals immunised with YF vaccine 17 D could not be protected against YF: up to 31% children and 10 % adults. Dengue antibodies inhibited YF virus and its significance in terms of YF protection must be investigated.

[Dengue hemorrhagic fever serotype and infection pattern in a Colombian endemic area]. / Serotipo, Patrón de Infección y Dengue Hemorrágico en Area Endémica Colombiana.

OBJECTIVE: Describing the relationship between viral serotypes, infection pattern and dengue hemorrhagic fever. METHODS: 1,545 febrile patients were studied from 1998-2004 in the Santander department of Colombia. Dengue infection was confirmed by IgM ELISA and the virus was isolated in C6/36 cells. Infection pattern was established by detecting IgG antibodies in acute serum. Neutralising antibody titres were investigated in dengue cases occurring during years when less (1998) and more (2001) dengue hemorrhagic cases were reported by using PRNT. RESULTS: DEN-1 predominance in 1998 and the re-introduction of DEN-3 in 2001 coincided with an epidemic. DEN-2 infection caused more hemorrhagic cases than DEN-3 infection (24,5 % cf 11,2 %; p<0.05). DEN-2 was more associated with secondary infection than DEN-3 (56,8 % cf 15,7 %; p<0.001). An annual decrease of DHF was correlated with decreased DEN-2 dominance (r=0.95; p= 0.01), and secondary infection (r=0.9; p=0.03) and increased DEN-3 predominance (r=-0.91; p=0.03). There were no differences in neutralising antibody titres amongst analysed cases. DEN-1 neutralising antibodies presented the highest titres. CONCLUSIONS: Change in relative dengue virus serotype abundance was associated with changed infection pattern and DHF frequency. Continuing virological surveillance should become a priority for preventing dengue hemorrhagic fever in endemic areas.

Serotipo, Patrón de Infección y Dengue Hemorrágico en Area Endémica Colombiana / Dengue hemorrhagic fever serotype and infection pattern in a Colombian endemic area

Objetivo: La relación entre serotipo del virus, patrón de infección y dengue hemorrágico es presentada. Métodos: Se estudiaron 1 545 pacientes febriles de municipios del Departamento de Santander, Colombia, entre 1998-2004. El dengue se confirmó por ELISA-IgM y el aislamiento viral se hizo en células C6/36. El patrón de infección se estableció investigando anticuerpos IgG en suero agudo. El título de anticuerpos neutralizantes se determinó usando la prueba de neutralización por reducción de placa (PRNT). Resultados: Predominancia del DEN-1 en 1998 y re-introducción del DEN-3 en 2001 coincidieron con epidemias. El dengue hemorrágico fue más frecuente en infecciones por virus DEN-2 que DEN-3 (24,5 por ciento vs 11,2 por ciento; p<0,05). El DEN-2 se asoció más con infección secundaria que el DEN-3 (56,8 por ciento vs 15,7 por ciento; p< 0,001). Disminución anual del DH correlacionó con disminución de la dominancia del DEN-2 (r = 0,95, p=0,01) y de la infección secundaria (r=0,9; p=0,03) e incremento de la dominancia del DEN-3 (r=-0,91; p=0,03). No se encontraron diferencias en el título de anticuerpos neutralizantes en los casos analizados. Los anticuerpos neutralizantes del DEN-1 fueron los de mayor título. Conclusión: Cambios en la abundancia relativa de serotipos del virus se asociaron con cambios en el patrón de infección y frecuencia del dengue hemorrágico. La vigilancia virológica permanente deberá ser prioridad para la prevención del dengue hemorrágico en áreas endémicas.

Objective: Describing the relationship between viral serotypes, infection pattern and dengue hemorrhagic fever. Methods: 1 545 febrile patients were studied from 1998-2004 in the Santander department of Colombia. Dengue infection was confirmed by IgM ELISA and the virus was isolated in C6/36 cells. Infection pattern was established by detecting IgG antibodies in acute serum. Neutralising antibody titres were investigated in dengue cases occurring during years when less (1998) and more (2001) dengue hemorrhagic cases were reported by using PRNT. Results: DEN-1 predominance in 1998 and the re-introduction of DEN-3 in 2001 coincided with an epidemic. DEN-2 infection caused more hemorrhagic cases than DEN-3 infection (24,5 percent cf 11,2 percent; p<0.05). DEN-2 was more associated with secondary infection than DEN-3 (56,8 percent cf 15,7 percent; p<0.001). An annual decrease of DHF was correlated with decreased DEN-2 dominance (r=0.95; p= 0.01), and secondary infection (r=0.9; p=0.03) and increased DEN-3 predominance (r=-0.91; p=0.03). There were no differences in neutralising antibody titres amongst analysed cases. DEN-1 neutralising antibodies presented the highest titres. Conclusions: Change in relative dengue virus serotype abundance was associated with changed infection pattern and DHF frequency. Continuing virological surveillance should become a priority for preventing dengue hemorrhagic fever in endemic areas.