Anxiolytic-like activity of Aloysia virgatavar. platyphylla leaves extract in mice / Actividad ansiolítica del extracto de hojas de Aloysia virgatavar. platyphylla en ratones

Background: Medicinal plants are part of traditional medicine and should be considered a therapeutic alternative for mental diseases. Several plants belonging to the Verbenaceae family have proved useful in treating general anxiety disorders, the most prevalent psychiatric disorders. Objective: This research aimed to verify the extract's safety, the effect on general behavior, and the effect on sleeping time, as well as to evaluate the anxiolytic-like effect of the methanol extract of Aloysia virgata var. platyphylla (Avp), in mice. Methodology: The toxicity test was done according to the OECD guide (mice groups n=5), and general behavior was observed during the assay. Sleeping time was assessed using the pentobarbital-induced hypnosis method (n=8). Male Swiss albino mice (n=6) were treated with 50 to 400 mg/kg of Avp extract and diazepam as a control. The anxiolytic-like effect was tested through the hole board and elevated plus-maze test. Results: The Avp extract has no side effects in tested doses, and no central nervous system depressant activity was noted. A. virgatavar. platyphyllaincreased exploration (number and time) in the hole board. In the elevated plus-maze, increased number and time into open arms were evidenced compared to the control group. Conclusion: With all these results, we concluded that the Avp extract is safe and has a potential anxiolytic-like activity in the animal model used

Antecedentes: Las plantas medicinales forman parte de la medicina tradicional y deben ser consideradas una alternativa terapéutica para las enfermedades mentales. Varias plantas pertenecientes a la familia Verbenaceae han demostrado su utilidad en el tratamiento de los trastornos de ansiedad, uno de los trastornos psiquiátricos más prevalentes. Objetivo: Esta investigación tuvo como objetivo verificar la seguridad del extracto, el efecto sobre el comportamiento general y el efecto sobre el tiempo de sueño, así como evaluar el efecto tipo ansiolítico del extracto metanólico de Aloysia virgata var. platyphylla(Avp), en ratones. Metodología: La prueba de toxicidad se realizó de acuerdo con la guía de la OCDE (grupos de ratones n=5), y se observó el comportamiento general durante el ensayo. El tiempo de sueño se evaluó mediante el método de hipnosis inducida por pentobarbital (n=8). Se trataron ratones albinos suizos macho (n=6) con 50 a 400 mg/kg de extracto de Avp y diazepam como control. El efecto ansiolítico se probó a través de la placa perforada y prueba del laberinto en cruz elevado. Resultados: El extracto de Avp no tiene efectos secundarios en las dosis probadas y no se observó actividad depresora del sistema nervioso central. A. virgata var. platyphylla aumentó la exploración (número y tiempo) en el tablero de agujeros. En el laberinto en cruz elevado, se evidenció un mayor número y tiempo en los brazos abiertos en comparación con el grupo de control. Conclusión: Con todos estos resultados, concluimos que el extracto de Avp es seguro y tiene una potencial actividad ansiolítica en el modelo animal utilizado

Caracterización in situ de poblaciones de Aloysia gratissima var. gratissima recolectadas del noreste de la provincia de San Luis, Argentina / In situ characterization of populations of Aloysia gratissima var. gratissima collected from the northeast of the province of San Luis, Argentina

Abstract: The aerial part of Aloysia gratissima (Gil lies & Hook. ex Hook.) Tronc., "Usillo", is used as aromatic and medicinal. It is a shrub of up to 3 meters, distributed in North America, from the south of the USA to the north of Mexico, and in South America up to the 37th parallel. As in other native sp ecies, the demand is covered by harvesting in wild populations, which brings about the deterioration of the resource and lack of homogeneity of the harvested product. The variability was characterized in nine populations of A. gratissima var. gratissima fr om the northeast of San Luis, Argentina, based on morphological and phytochemical characters. It was found that the species in the region presents considerable heterogeneity. Diversity was detected in the chemical characteristics of the essential oils anal yzed and the prevalence of mono and sesquiterpenes was related to the olfactory identities identified. The main components were the sesquiterpene spatulenol and the monoterpene 1,8 cineole

Resumen: La parte aérea de Aloysia gratissima (Gillies & Hook. ex Hook.) Tronc., "usillo", es utilizada como aromática y medicinal. Es un arbusto de hasta 3 metros, distribuido en Norteamerica, desde e l sur de EEUU hasta el norte de México, y en Sudamerica hasta el paralelo 37°. Al igual que en otras especies nativas, la demanda es cubierta por recolección en poblaciones silvestres, lo cual trae aparejado el deterioro del recurso y falta de homogeneidad del producto cosechado. Se caracterizó la variabilidad en nueve poblaciones de A. gratissima var. gratissima del noreste de San Luis, Argentina, en base a caracteres morfológicos y fitoquímicos. Se encontró que la especie en la región presenta una conside rable heterogeneidad. Se detectó diversidad en las características químicas de los aceites esenciales analizados y se relacionó la prevalencia de mono y sesquiterpenos con las identidades olfativas identificadas. Los componentes mayoritarios fueron el sesq uiterpeno espatulenol y el monoterpeno 1,8 cineol.

Inter and intra population phenotypic variability of Lippia integrifolia (Verbenaceae) and its natural situation in the west-center of Argentina / Variabilidad fenotípica inter e intra poblacional de Lippia integrifolia (Verbenaceae) y su situación natural en el centro-oeste de Argentina

The species Lippia integrifolia is an aromatic, sub-woody shrub, distributed from Northwest and Central Argentina to Bolivia. It is among the most important native aromatic species. It presents medicinal properties, some of which have been scientifically proven. As an alternative to harvesting, the process of domestication of the species was initiated. The objective of this work was to assess the phenotypic variability based on botanical-taxonomic and morpho-agronomic descriptors and the use of indicators to evaluate its natural situation in the west-center of Argentina. We worked in five wild populations, registering morphological variables, phenology, natural regeneration, herbivory and presence of diseases. L. integrifoliapresents a wide morphological variability, which can be explained with three morphological descriptors. Natural regeneration ́s rate is very low to zero, in four of the sites. The results obtained are of importance to make decisions related to the sustainable use in situ, and to begin a program of domestication of the species.

La especie Lippia integrifolia es un arbusto aromático, sub-leñoso, distribuido desde el noroeste y centro de Argentina hasta Bolivia. Es una de las especies aromáticas nativas más importantes. Presenta propiedades medicinales, algunas de las cuales han sido científicamente probadas. Como alternativa a la cosecha, se inició el proceso de domesticación de la especie. El objetivo de este trabajo fue evaluar la variabilidad fenotípica basada en descriptores botánico-taxonómicos y morfo-agronómicos y el uso de indicadores para evaluar su situación natural en el centro-oeste de Argentina. Trabajamos en cinco poblaciones silvestres, registrando variables morfológicas, fenología, regeneración natural, herviboría y presencia de enfermedades. L. integrifolia presenta una amplia variabilidad morfológica, que puede explicarse con tres descriptores morfológicos. La tasa de regeneración natural es muy baja a cero, en cuatro de los sitios. Los resultados obtenidos son importantes para tomar decisiones relacionadas con el uso sostenible in situ y para comenzar un programa de domesticación de la especie.

Efecto del extracto hidroalcohólico de la planta Lampaya medicinalis Phil. (Verbenaceae) sobre la esteatosis inducida in vitro en hepatocitos humanos / Effect of the hydroalcoholic extract of the plant Lampaya medicinalis Phil. (Verbenaceae) on in vitro-induced steatosis in human hepatocytes

INTRODUCCIÓN: La enfermedad del hígado graso no alcohólico (EHGNA) es la forma más común de enfermedad hepática. A nivel celular se caracteriza por la acumulación de triglicéridos (TG) en forma de gotas lipídicas (GL) dando lugar a esteatosis e inflamación. Entre los factores relevantes para la síntesis de TG se encuentran las enzimas DGAT1/2 que catalizan la etapa final de la síntesis de TG, y la proteína FABP4 que transporta lípidos intracelulares y se expresa en modelos de enfermedad hepática dependiente de obesidad. Por otra parte, TNF-α es una reconocida citoquina involucrada en el proceso inflamatorio en la EHGNA. La medicina popular del norte de Chile ha utilizado la planta Lampaya medicinalis Phil. (Verbenaceae) para el tratamiento de algunas enfermedades inflamatorias. OBJETIVO: Evaluar el efecto de un extracto hidroalcóholico de lampaya (EHL) sobre la esteatosis y expresión de marcadores de inflamación en hepatocitos tratados con ácidos grasos. Diseño experimental: Estudio in vitro en cultivos de la línea celular humana HepG2 tratadas con ácido oleico (AO) y ácido palmítico (AP). MÉTODOS: Se incubó hepatocitos HepG2 con AO/AP por 24 horas en presencia o no de EHL. Se evaluó la presencia de GL y el contenido de TG intracelulares por Oil Red O y Nile Red, respectivamente. La expresión de DGAT1/2, FABP4 y TNF-α fue evaluada por qPCR. RESULTADOS: Los hepatocitos tratados con AO/AP mostraron un aumento en las GL y TG, así como una mayor expresión de DGAT2 en comparación al control. El cotratamiento con EHL revirtió los efectos inducidos por AO/AP. CONCLUSIONES: EHL revierte el incremento en las GL, TG y en la expresión de DGAT2 inducido por AO/AP en células HepG2. Estos hallazgos sugieren un efecto hepatoprotector de la Lampaya contra la esteatosis, y apoyarían su uso complementario en el tratamiento de patologías con componente inflamatorio como la EHGNA.

Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease. At the cellular level, it is characterized by the accumulation of triglycerides (TG) in the form of lipid droplets (LD), which leads to steatosis and inflammation. Among relevant factors for TG synthesis are the enzymes DGAT1/2 catalyzing the final stage of TG synthesis, and the protein FABP4 which transports intracellular lipids and is expressed in cell models of obesity-dependent liver disease. Additionally, TNF-α is a cytokine involved in the inflammatory process associated to NAFDL. Lampaya medicinalis Phil. (Verbenaceae) is a plant used in folk medicine in northern Chile to treat some inflammatory diseases. OBJECTIVE: To evaluate the effect of the hydroalcoholic extract of lampaya (HEL) on steatosis and the expression of inflammatory markers in hepatocytes treated with fatty acids. Study design: In vitro study in cultures of the human HepG2 cell line treated with oleic acid (OA) and palmitic acid (PA). METHODS: HepG2 hepatocytes were incubated with OA/PA for 24 hours in the presence and absence of HEL. The formation of LD and the accumulation of intracellular TG were assessed by Oil Red O and Nile Red, respectively. The expression of DGAT1/2, FABP4 and TNF-α was assessed by qPCR. RESULTS: The treatment with OA/PA increased the levels of LD and TG as well as the expression of DGAT2 in HepG2 hepatocytes compared to control cells. HEL cotreatment counteracted OA/PA-induced effects. CONCLUSIONS: HEL prevents the increase in LD and TG levels and DGAT2 expression induced by OA/PA in HepG2 cells. These findings suggest that lampaya may have a protective effect against hepatic steatosis, which would support its complementary use in the treatment of pathologies associated with inflammation, such as NAFLD.

Actividad gastroprotectora, tamizaje fitoquímico y actividad atrapadora de radicales libres de Aloysia Gratissima (Verbenaceae) / Phytochemical screning, gastroprotective activity and free radical scavenging activity of Aloysia gratissima (Verbenaceae)

Se evaluó la actividad gastroprotectora de la infusión proveniente de las hojas de Aloysia gratissima (Verbenaceae), especie nativa de interés medicinal que se desarrolla en el sudoeste bonaerense, utilizando un modelo de inducción de úlceras gástricas con etanol en ratones. Se realizó un tamizaje fitoquímico para detectar la presencia de compuestos que podrían ser responsables de la actividad gastroprotectora de la planta. Se determinó el contenido de fenoles totales y la capacidad atrapadora de radicales libres mediante el método del Folin-Ciocalteu y del 2,2'-difenil-1-picrilhidrazilo (DPPH), respectivamente. Los ensayos demostraron que la infusión de la planta, administrada por vía oral en dosis de 100, 500 y 1000 mg/kg, ejerció una gastroprotección significativa frente a la inducción de úlceras. Se detectó una actividad atrapadora de radicales libres de 47,5%, similar a la sustancia de referencia (BHT). El estudio fitoquímico detectó la presencia de flavonoides y otros polifenoles, sustancias con reconocida capacidad antioxidante. Estos metabolitos ejercen efectos protectores en diferentes modelos experimentales de inducción de úlceras mediante mecanismos que pueden involucrar la neutralización de radicales libres, lo que podría explicar la actividad gastroprotectora de la planta. Estos hallazgos requieren estudios adicionales de A. gratissima como una posible terapia frente a la úlcera gástrica. (AU)

The gastroprotective activity of the infusion from the leaves of Aloysia gratissima (Verbenaceae), a native species of medicinal interest growing in South West Buenos Aires, was evaluated in an ethanol-induced gastric ulcer model in mice. Phytochemical screening was carried out in order to determine the presence of compounds that could be responsible for the pharmacological effects of the plant. Total phenolic content and the free radical scavenging activity of the plant were determined using the Folin-Ciocalteu and the 2,2'-diphenyl1-picrylhydrazyl (DPPH) method, respectively. Assays demonstrated that the infusion, orally administered at 100, 500, and 1000 mg/kg doses, exerted a significant gastroprotection effect against ulcer induction (P<0,05). A free radical scavenging activity of 47.5% -similar to the reference substance (BHT)- was detected. Phytochemical screening revealed the presence of antioxidant compounds such as flavonoids and other phenolic compounds. These compounds exert protective effects in different experimental models of ulcer induction that could involve free radical neutralization, which could explain the gastroprotective activity of the plant. These promising results support additional studies of A. gratissima as a potential therapy against gastric ulcer. (AU)

Content and chemical composition of the essential oil of Lippia gracilis Schauer accessions in different drying times / Teor e composição química do óleo essencial de acessos de Lippia gracilis Schauer em diferentes tempos de secagem

Lippia gracilis, popularly known in Brazil as 'alecrim-de-tabuleiro', is used for many purposes, especially as antimicrobial and antiseptic. The drying process of aromatic and medicinal plants aims to minimize the loss of active principles and slow their deterioration, which may greatly influence the yield and chemical composition of some species. The objective of this study was to evaluate the effect of drying times (0, 2, 4, and 8 days) on the content and chemical composition of the essential oil of L. gracilis accessions LGRA-106, LGRA-109, and LGRA-201. The leaves were dried at 40 oC, and essential oil was extracted by hydrodistillation. Chemical analysis was performed by GC/MS. The experiment was carried out in a completely randomized design with three replications. The accessions of L. gracilis LGRA-106, LGRA-109, and LGRA-201 presented higher essential oil at four days of drying time. The accession LGRA-201 showed the highest essential oil yields at four and eight days of drying, with mean values of 0.038 and 0.029 mL g-1, respectively. The drying time did not influence the contents of thymol, methyl-thymol, γ-terpinene, and carvacrol in the essential oils of L. gracilis, but affected the contents of ß-caryophyllene, p-cymene, and carvacrol acetate. The essential oils of the three accessions analyzed in this study revealed different chemical profiles.

Lippia gracilis, conhecida popularmente como alecrim-de-tabuleiro é usada para muitos efeitos, especialmente como antimicrobiano e antisséptico. O processo de secagem das plantas aromáticas e medicinais visa minimizar a perda de princípios ativos e retardar a sua deterioração os mesmos podem afetar sobremaneira o rendimento e a composição química de algumas plantas. O objetivo deste trabalho foiavaliar o efeito de diferentes tempos de secagem (0, 2, 4 e 8 dias) no teor e na composição química do óleo essencial dos acessos LGRA-106, LGRA-109 e LGRA-201 de L. gracilis.As folhas foram secas a 40 oC e a extração do óleo essencial foi por hidrodestilação. A análise química foi feita através CG/EM. O ensaio foi implantado em delineamento inteiramente casualizado com três repetições. Os acessos de L. gracilis LGRA-106, LGRA- 109 e LGRA-201 apresentaram maiores rendimentos de óleo essencial no tempo de secagem de quatro dias. O acesso LGRA-201 apresentou os maiores teores de óleo essencial aos quatro e oito dias de secagem, com valores médios de 0,038 e 0,029 mL g-1, respectivamente. O tempo de secagem não influenciou os teores de timol, metil-timol, terpineno e carvacrol nos óleos essenciais de L. gracilis, porém afetou os teores de ß-cariofileno, ρ-cimeno, and acetato de carvacrol. Os óleos essenciais dos três acessos analisados apresentaram perfis químicos diferentes entre si.

Effect of meteorological elements on the content and composition of Aloysia triphylla (L´Hérit) Britton essential oil / Efecto de los elementos meteorológicos sobre el contenido y la composición del aceite esencial de Aloysia triphylla (L´Hérit) Britton

The concentration and composition of an essential oil can vary according to environmental variations and seasonal periods. The objective of this study was to correlate meteorological elements with the content and concentration of components of Aloysia triphylla essential oil, for the four seasons of the year. The experiment was conducted in a plastic greenhouse of the UFSM, Frederico Westphalen campus ­ RS, Brazil, in a complete randomized blocks design during the four seasons, with three repetitions. The meteorological data were collected with the aid of a compact meteorological station, in order to characterize the environment in which the species was conducted. The evaluations were carried out at the middle date of each season. It was observed a correlation between the meteorological variables with the essential oil production and its components. According to the analysis, there influence of the temperature on the essential oil contents, considering that the maximum temperature shows a direct positive influence on the concentration of limonene and negative for spathulenol.

El contenido y la composición de un aceite esencial pueden variar según las variaciones ambientales y los períodos estacionales. El objetivo de este estudio fue correlacionar los elementos meteorológicos con el contenido y la concentración de los componentes del aceite esencial de Aloysia triphylla, para las cuatro estaciones del año. El experimento se realizó en un invernadero de plástico de la UFSM, campus de Frederico Westphalen - RS, Brasil, en un diseño completo de bloques al azar durante las cuatro estaciones, con tres repeticiones. Los datos meteorológicos se recopilaron con la ayuda de una estación meteorológica compacta, para caracterizar el entorno en el que se realizó la especie. Las evaluaciones se llevaron a cabo en la fecha media de cada temporada. Se observó una correlación entre las variables meteorológicas con la producción de aceites esenciales y sus componentes. De acuerdo con el análisis, existe una influencia de la temperatura en los contenidos de aceites esenciales, considerando que la temperatura máxima muestra una influencia positiva directa sobre la concentración de limoneno y negativa para el espatulenol.

Essential oil composition in natural population of Lippia origanoides (Verbenaceae) during dry and rainy seasons / Composición de los aceites esenciales en poblaciones naturales de Lippia origanoides (Verbenaceae) durante las estaciones seca y lluviosa

Abstract Lippia origanoides Kunth. is a medicinal shrub native to some countries in South America, Central America and the Caribbean. This study aims to investigate the chemical composition of the essential oil and the genetic diversity of a natural population of Lippia origanoides in two seasons. Leaf samples were collected from 30 individuals in the dry and rainy seasons. The essential oil was extracted into a Clevenger apparatus and analyzed by gas chromatography coupled to mass spectrometry (GC-MS). The relative quantification of thymol and carvacrol was performed by gas chromatography coupled to a flame ionization detector (GC-FID). Multivariate analysis was used to evaluate the genetic divergence between plants. Carvacrol was the major compound found in most plants for both seasons. In the rainy season, plant 15 presented thymol as the major compound, and plants 16, 27, 28 and 29 presented camphor as the major compound. In the dry season, thymol remained the main compound of plant 15 and camphor remained the main compound of plants 16, 28 and 29; however, plant 27 presented carvacrol as the main compound in this season. After carvacrol, the compounds with the highest content were γ-terpinene, p-cymene and methyl-ether-thymol. Of the 30 plants studied, only five differed in their chemical composition, showing some degree of stability in relation to the dry and rainy seasons. There was no variation in the main compounds between the two seasons, but there was chemical diversity among the main compounds. Tocher grouping revealed five distinct groups, with group 1 including most of the plants in both dry and rainy seasons, indicating that the time of year evaluated did not significantly interfere with the essential oil composition of most plants.(AU)

Resumen Lippia origanoides Kunth. es un arbusto medicinal nativo de algunos países de Centro y Sur América y el Caribe. El objetivo de este estudio fue investigar la composición química de los aceites esenciales y la diversidad genética de una población natural de L. origanoides en dos estaciones. El aceite esencial se extrajo en un equipo de Clevenger y se analizó por cromatografía de gases acoplado a espectrometría de masas (CG-EM). La cuantificación relativa de timol y carvacrol se realizó por cromatografía de gases acoplado a un detector de ionización de llamas (CG-DIL). Se utilizó un análisis multivariado para evaluar la divergencia genética entre plantas. El carvacrol fue el compuesto principal encontrado en la mayoría de las plantas para ambas estaciones. En la estación lluviosa, la planta 15 presentó timol como el compuesto principal y las plantas 16, 27, 28 y 29 presentaron alcanfor como el compuesto principal. En la estación seca, el timol se mantuvo como el principal compuesto en la planta 15, de igual manera el alcanfor se mantuvo como el principal en las plantas 16, 28 y 29. Sin embargo, la planta 27 presentó carvacrol como el principal compuesto en esta estación. Después del carvacrol, los compuestos con el mayor contenido fueron γ-terpineno, p-cimeno and metil-eter-timol. De las 30 plantas estudiadas, solo cinco difirieron en su composición química, mostrando algún grado de estabilidad en relación a la estación seca y lluviosa. No hubo variación en los compuestos principales entre las dos estaciones, pero hubo diversidad química entre los compuestos principales. El agrupamiento de Tocher mostró cinco grupos distintos, con el grupo 1 incluyendo la mayoría de las plantas en ambas estaciones, lo cual indica que el periodo del año evaluado no interfiere significativamente con la composición de aceites esenciales en la mayoría de plantas.(AU)

Biocidal effect of a hexane-soluble extract of Lippia graveolens Kunth (Verbenaceae) / Efecto biocida del extracto hexánico de Lippia graveolens Kunth (Verbenaceae)

Lippia graveolens Kunth (Verbenaceae) is an economically important shrub known in Mexico as Oregano. In this work, the biocidal effect of the hexane extract of L. graveolens leaves was evaluated on two crop pests. Thus, larvae of Spodoptera frugiperda were fed with mixtures of extract and artificial diet. The nematicidal activity was evaluated on juveniles of Meloydogine javanica. Regarding S. frugiperda, quantitative differences between treatments and control were observed in dead pupae, surviving adults, and deformed adults (P < 0.05). All the surviving adults from the extract treatments were deformed. Nematicidal effect was registered, the LC50 and LC90 were 0.672 (0.654-0.690) and 0.965 (0.937-0.998) mg/mL respectively. The extract was characterized by NMR and GC-MS, being thymol the most abundant component (70.6%) in addition to carvacrol (22.8%). The results suggest the consideration of the hexane extract of L. graveolens leaves within the alternatives for the biological control of pests.

Lippia graveolens Kunth (Verbenaceae) es un arbusto con importancia económica conocido en México como Orégano. En éste trabajo se evaluó el efecto biocida del extracto hexánico de hojas L. graveolens sobre dos plagas agrícolas. Así, larvas de S. frugiperda fueron alimentadas con mezclas de dieta artificial y extracto. La actividad nematicida fue evaluada en juveniles de Meloydogine javanica, Respecto a S. frugiperda, se observaron diferencias cuantitativas entre tratamiento y control en cuanto a pupas muertas, adultos sobrevivientes y adultos deformes (P < 0.05). Todos los adultos provenientes de tratamientos con extracto estuvieron malformados. Hubo efecto nematicida, calculándose CL50 y CL90 de 0.672 (0.654-0.690) y 0.965 (0.937-0.998) mg/mL respectivamente. El extracto se caracterizó por RMN y CG-EM. Los compuestos más abundantes fueron timol (70.6%), ademas del carvacrol (22.8%). Los resultados sugieren considerar al extracto hexánico de hojas de L. graveolens dentro de las alternativas para el control biológico de plagas.

Foliar phenolic compounds of ten wild species of Verbenacea as antioxidants and specific chemomarkers / Compostos fenólicos das folhas de dez especies selvagem de Verbenaceae como antioxidantes e quimiomarcadores específicos

Abstract The family Verbenaceae hosts important species used in traditional medicine of many countries. The taxonomic controversies concerning the specific delimitation of several of its species make it difficult to guarantee the botanical origin of herbal preparations based on species of this family. To contribute to the development of both specific chemomarkers and a quality control tool to authenticate the botanical origin of herbal preparations of Verbenacea species, we determined the foliar HPLC-DAD phenolic profiles and the antioxidant properties of 10 wild species of this family occurring in Mexico. The contents of phenols and flavonoids varied significantly among species. Priva mexicana showed the highest levels of total phenolics (53.4 mg g-1 dry tissue) and Verbena carolina had the highest levels of flavonoids (17.89 mg g-1 dry tissue). Relevant antioxidant properties revealed by antiradical and reducing power were found for the analyzed species. These properties varied significantly in a species-dependent manner. The phenolic compounds accumulated were flavones and phenolic acids. Flavones were the only type of flavonoids found. The results of a cluster analysis showed that the compounds were accumulated in species-specific profiles. The phenolic profiles are proposed as valuable chemomarkers that can become a useful tool for the quality control concerning the botanical origin of herbal medicinal preparations based on the species analyzed. In addition, phenolic profiles could contribute importantly to solve the taxonomic controversies concerning species delimitation in the family Verbenaceae.

Resumo A família Verbenaceae compreende importantes espécies utilizadas na medicina popular de muitos países. As dificuldades taxonômicas relativas à delimitação específica de muitas das suas espécies face difícil a verificar a origem botânico das preparações herbales baseadas nas espécies desta família. Para fazer uma contribuição ao desenvolvimento de indicadores taxonômicos e dum método de controle de qualidade para verificar a origem botânico de preparações herbales das espécies de Verbenaceae, os perfis fenólicos, obtidos pares HPLC-DAD, e as atividades antioxidantes das folhas de 10 espécies selvagens Mexicanas desta família foram determinados. Os conteúdos dos compostos fenólicos totais e dos flavonoides foram significativamente diferentes entre as espécies. Priva mexicana apresentou a maior quantidade de compostos fenólicos totais (53.4 mg g-1 amostra seca) e Verbena carolina apresentou a maior quantidade de flavonoides (17.89 mg g-1 amostra seca). Verifica-se importantes propriedades antioxidantes, como os resultados dos ensaios da capacidade antiradical e do poder redutor indicaram. As propriedades antioxidantes foram significativamente diferentes entre as espécies. Verificou-se que os compostos fenólicos conteúdos nas folhas das espécies analisadas foram só flavonas e ácidos fenólicos. Os resultados das análises de agrupamento provarãn que os perfiles fenólicos foram espécie-específicos. Estes perfis podem ser considerados como indicadores químicos da qualidade relativa à origem botânico de preparações medicinais baseadas nas espécies analisadas e podem fazer importantes contribuições para a delimitação específica na família Verbenaceae.

Pharmaceutical development of tablets containing a spray-dried optimized extract from Lippia origanoides H. B. K.: influence of excipients and toxicological assessment

ABSTRACT Lippia origanoides is a honey shrub which has showed hypotensive potential assessed by in vivo studies. The aim of this work is the development of a pharmaceutical formulation composed by an optimized extract obtained from aerial parts of L. origanoides. The quantification of the naringenin marker in the dry extract and tablets developed was performed, as well as the assessment of the oral acute toxicity in rats. The hydroalcoholic extract of L. origanoides was spray-dried with the addition of colloidal silicon dioxide (Lo-HAE/CSD), and then applied in the preparation of eight different lots of tablets. The influence of the diluent (cellulose or babassu mesocarp), the presence of binder, and the percentage of lubricant, as well as organoleptic and physicochemical characteristics were screened. For the quantification of the marker content both in Lo-HAE/CSD and in the tablets, an analytical curve of the naringenin standard was fitted, and the samples were then analyzed in UFLC. The toxicological assessment was performed in female Wistar rats according to the Acute Toxic Class Method from OECD. The developed tablets produced meet acceptable macroscopic characteristics, and the presence of babassu as diluent provided improved physicochemical properties. The best content of Lo-HAE/CSD in the tablet (100.27%) was identified for the lot containing babassu, composed by 1.0% magnesium stearate, without PVP binder in its formulation. Moreover, Lo-HAE/CSD showed no signs of toxicity. Therefore, the babassu mesocarp powder is a promising pharmaceutical excipient for the development of herbal tablets containing the Lippia origanoides extract.

Analysis of Essential oil, Quantification of Six Glycosides, and Nitric Oxide Synthase Inhibition Activity in Caryopteris incana

Caryopteris incana (Verbenaceae) has been used to treat cough, arthritis, and eczema in Oriental medicine. The two fractions (CHCl₃- and BuOH fractions) and the essential oil of the plant material were subjected to the inducible nitric oxide synthase (iNOS) assay. The IC₅₀ of the CHCl₃ fraction and the essential oil on LPS-induced macrophage RAW 264.7 cells were 16.4 µg/mL and 23.08 µg/mL, respectively. On gas chromatography (GC)-mass spectroscopy (MS) analysis, twenty-five components representing 85.5% amount of total essential oil were identified. On the chromatogram, three main substances, trans-pinocarveol, cis-citral, and pinocarvone, occupied 18.8%, 13.5% and 18.37% of total peak area. Furthermore, by HPLC-UV analysis, six compounds including one iridoid (8-O-acetylharpagide)- and five phenylethanoid glycosides (caryopteroside, acteoside, phlinoside A, 6-O-caffeoylphlinoside, and leucosceptoside A) isolated from the BuOH fraction were quantified. The content of six compounds were shown as the following order: caryopteroside (162.35 mg/g) > 8-O-acetylharpagide (93.28 mg/g) > 6-O-caffeoylphlinoside (28.15mg/g) > phlinoside (22.60mg/g) > leucosceptoside A (16.87 mg) > acteoside (7.05 mg/g).

Anesthesia of Epinephelus marginatus with essential oil of Aloysia polystachya: an approach on blood parameters

ABSTRACT This study investigated the anesthetic potential of the essential oil (EO) of Aloysia polystachya in juveniles of dusky grouper (Epinephelus marginatus). Fish were exposed to different concentrations of EO of A. polystachya to evaluate time of induction and recovery from anesthesia. In the second experiment, fish were divided into four groups: control, ethanol and 50 or 300 µL L−1 EO of A. polystachya, and each group was submitted to induction for 3.5 min and recovery for 5 or 10 min. The blood gases and glucose levels showed alterations as a function of the recovery times, but Na+ and K+ levels did not show any alteration. In conclusion, the EO from leaves of A. polystachya is an effective anesthetic for dusky grouper, because anesthesia was reached within the recommended time at EO concentrations of 300 and 400 µL L−1. However, most evaluated blood parameters showed compensatory responses due to EO exposure.

In vitro conservation and leaf anatomy of different chemotypes of Lippia alba (Mill.) N. E. BR / Conservação in vitro e anatomia foliar de diferentes quimiotipios de Lippia alba (Mill.) N. E. BR

The aim of this work was to develop an in vitro conservation protocol for Lippia alba that involved mineral oil and to study the leaf anatomy of L. alba genotypes in in vitro and ex vitro environments. In vitro conservation involved five genotypes; LA-13 and LA-57 (carvone chemotype); LA-22 (linalool chemotype); and LA-29 and LA-44 (citral chemotype). Each genotype was treated with mineral oil, with water, and a control at 18°C and 23°C. Survival (%), shoot height (cm) and leaf color were assessed at 180 and 270 days. The genotypes with the best in vitro conservation outcomes (LA-13 and LA-57) were used to study the anatomy of the leaves. Midrib thickness, leaf blade thickness, adaxial and abaxial cuticles and the number of glandular and tector trichomes were assessed. A temperature of 18°C was optimal for the conservation of genotypes of the carvone chemotypes. The explants were short and had green leaves. This was especially true for LA-57, whose height did not exceed 2.0 cm. At 270 days, the LA-13 and LA-57 genotypes continued to have green and viable leaves, especially LA-57, which had the lowest mean height. Field plants of the carvone chemotype had thicker midribs, leaf blades, and cuticles and had more trichomes. The LA-57 genotype had the most glandular and tector trichomes.

O objetivo deste estudo foi desenvolver um protocolo de conservação in vitro com óleo mineral e estudar a anatomia foliar de genótipos de L. alba nos ambientes in vitro e ex vitro. A conservação in vitro constou de cinco genótipos LA-13 e LA-57 (quimiotipo carvona); LA-22 (quimiotipo linalol); LA-29 e LA-44 (quimiotipo citral), três tratamentos (com óleo mineral, com água e controle) e duas temperaturas 18 e 23°C. Aos 180 e 270 dias, avaliou-se a sobrevivência (%), altura da parte aérea (cm) e coloração das folhas. Os genótipos com os melhores resultados da conservação in vitro (LA-13 e LA-57) foram utilizados para estudos de anatomia. Avaliou-se espessura da nervura central, espessura do limbo foliar, cutículas adaxial e abaxial e quantidade de tricomas glandulares e tectores. A temperatura a 18°C foi a ideal para a conservação dos genótipos do quimiotipo carvona, com folhas verdes e baixos valores para altura, destacando-se LA-57 cuja altura não ultrapassou 2,0 cm. Aos 270 dias LA-13 e LA-57 permaneceram com folhas verdes e viáveis, com destaque para LA-57 com menores médias de altura. A anatomia do quimiotipo carvona mostrou que as plantas de campo apresentaram maiores espessuras da nervura, limbo e cutículas, e maiores quantidades de tricomas. O genótipo LA-57 obteve a maior quantidade de tricomas glandulares e tectores.

Antibacterial activity of essential oils of aloysia polystachya and lippia turbinata (Verbenaceae) / Actividad antibacteriana de los aceites esenciales de aloysia polystachya y lippia turbinata (Verbenaceae)

Aloysia polystachya and Lippia turbinata are medicinal and aromatic plants. Nevertheless, there are few reports in literature concerning the biological properties of species that grow in northeastern Argentina. The antibacterial activity and the chemical composition of both essential oils were evaluated in this work. The extraction was performed by steam distillation and their volatile compounds were determined by gas chromatography/mass spectrometry. The antibacterial activity was evaluated by disc diffusion and broth microdilution assay. The main compounds were carvone (78.9 percent) and limonene (14.2 percent) in A. polystachya and carvone (80.77 percent), limonene (8.73 percent), beta- caryophyllene (2.13 percent) and 1,8-cineole (1.70 percent) in L. turbinata. Both essential oils were bactericide against Escherichia coli ATCC 35218 and clinical isolates of Enterobacter cloacae and Klebsiella pneumoniae. Essential oil of A. polystachya was also bactericide against Staphylococcus aureus ATCC 29212, S. aureus ATCC 25923 and clinical strain of S. aureus methicillin susceptible.

Aloysia polystachya y Lippia turbinata son plantas medicinales y aromáticas. Hay pocos informes en la literatura sobre las propiedades biológicas de especies que crecen en el nordeste de Argentina. La actividad antibacteriana y la composición química de ambas especies se evaluaron en este trabajo. La extracción se realizó por destilación con vapor y sus compuestos se determinaron por cromatografía gaseosa/espectrometría de masa. La actividad antibacteriana fue evaluada por difusión en discos y microdilución en caldo. Los principales compuestos fueron carvona (78.9 por ciento) y limoneno (14.2 por ciento) en A. polystachya y carvona (80.77 por ciento), limoneno (8.73 por ciento), beta-cariofileno (2.13 por ciento) y 1,8-cineol (1.70 por ciento) en L. turbinata. Ambos aceites esenciales fueron activos contra Escherichia coli ATCC 35218 y aislamientos clínicos de Enterobacter cloacae y Klebsiella pneumoniae. El aceite esencial de A. polystachya fue bactericida contra Staphylococcus aureus ATCC 29212, S. aureus ATCC 25923 y aislamientos clínicos de S. aureus sensible a meticilina.

Variabilidad química del “tomillo silvestre” (acantholippia seriphioides, verbenaceae) en la meseta Patagónica / Chemical Variability of “tomillo silvestre” (acantholippia seriphioides, verbenaceae) in the Patagonian plateau

El tomillo silvestre es una planta aromática y medicinal de amplio uso en varias regiones de Argentina como medicinal y saborizante. Se estudiaron poblaciones de Acantholippia seriphioides en dos zonas de la meseta chubutense, los parajes El Pajarito y La Rueda. Las partes aéreas oreadas fueron extraídas por hidrodestilación y los aceites esenciales se analizaron por GC-FID-MS. Los rendimientos en promedio de aceites esenciales fueron de 5.2 ml/kg y 3.2 ml/kg, para La Rueda y El Pajarito respectivamente. Los tipos químicos encontrados fueron: ipsilon-terpineno/timol/carvacrol en La Rueda y mirtenol/geraniol/carvona en El Pajarito. El probable quimiotipo de este último sitio difiere marcadamente de los reportados en trabajos previos para esta especie y presenta además importante variabilidad cuantitativa, según el estado fenológico.

“Tomillo silvestre” is a widespread medicinal and aromatic plant with medicinal and flavoring uses in numerous regions of Argentina. Acantholippia seriphioides populations from two areas of the Chubut’s plateau: El Pajarito and La Rueda, have been studied. The dried aerial parts were extracted by hydrodistillation and the essential oils were analyzed by GC/FID/MS. The average yields of oils were 5.2 ml/kg and 3.2 ml/kg for La Rueda and El Pajarito, respectively. The chemical profiles found were: ypsilon-terpinene/thymol/carvacrol in La Rueda and myrtenol/geraniol/carvone in El Pajarito. The feasible chemotype from the latter area is significantly different from those previously reported for this species, and it also showed an important quantitative variation according to the phenological stages.

Potencial do óleo essencial de erva-luísa (Aloysia citriodora Palau) no controle de Fusarium sp. in vitro / Essential oil potential herb-luisa (Aloysia citriodora Palau) in the control of Fusarium sp. in vitro

RESUMO O presente trabalho teve como objetivo principal avaliar o potencial do uso do óleo essencial de Aloysia citriodora no controle in vitro de Fusarium sp., isolado de plântulas de beterraba infectadas com o mesmo. O trabalho foi realizado por meio de dois experimentos: um sobre o efeito do óleo essencial no crescimento micelial, e outro sobre o efeito na germinação de conídios do fungo. No primeiro trabalho, avaliou-se em delineamento inteiramente ao acaso, o efeito das concentrações 0,0155%; 0,0315%; 0,0625%; 0,125%; 0,250% e 0,500% do óleo essencial de A. citriodora em placas de Petri® com meio de cultivo BDA, mais a testemunha, com meio BDA puro. Cada placa foi considerada uma repetição, as quais foram incubadas a 24ºC±1ºC e submetidas a fotoperíodo de doze horas. Avaliou-se o crescimento radial do patógeno em função do crescimento micelial do patógeno quando este atingia as bordas da primeira placa. No segundo experimento, as mesmas concentrações foram testadas, nas mesmas condições de incubação, no entanto, em lâminas de microscopia com meio BD. Utilizou-se delineamento inteiramente ao acaso, sendo considerada como unidade experimental cada lâmina utilizada. Avaliou-se 24 horas após a incubação, em microscópio óptico, a germinação dos primeiros vinte conídios visualizados a partir do canto esquerdo superior para o direito. Os resultados do segundo experimento foram expressos em porcentagem de germinação de conídios. Os resultados obtidos permitiram concluir que este óleo essencial possui efeito fungistático e fungicida sobre o crescimento micelial e na germinação de conídios de Fusarium sp.. Além disso este efeito é maior em função do aumento da concentração do óleo essencial.

ABSTRACT This study aimed to evaluate the potential use of Aloysia citriodora essential oilisolated from beet seedlings infected with it. The study was performed by conducting two experiments evaluating the effect of the essential oil on mycelial growth and fungus conidia germination. In the first study, in a completely randomized design, the effect concentrations (0.0155%; 0.0315%; 0.0625%; 0.125%; 0.250% and 0.500%) of essential oil of A. citriodorawas evaluated on Petri® dish with a PDA cultivation medium, plus the control, with half pure PDA. Each plate was taken as a repeat, and incubated at 24°C±1°C and a photoperiod of twelve hours. The radial growth of the pathogen, when the first plate was hit by the mycelial growth of the pathogen on its edges was evaluated. In the second experiment, the same concentrations were tested under the same incubationconditions, however, on microscope slides with half PD medium. The design was used completely randomized, each microscope slide used was considered as one experimental unit. Germination of the first 2 conidia strains, viewed from the upper left to the right was evaluated24 hours after incubation, using an optical microscope. The results of the second experiment were expressed as a percentage of conidia germination. The results obtained showed that this essential oil has fungistatic and fungicidal effect on the mycelial growth and at the conidia germination of Fusarium sp., which has a greater effect with increasing concentrations.

Evidences that human disturbance simplify the ant fauna associated a Stachytarpheta glabra Cham. (Verbenaceae) compromising the benefits of ant-plant mutualism / Evidências de que os impactos antrópicos simplificam a fauna de formiga associada a Stachytarpheta glabra (Verbenaceae) comprometendo os benefícios do mutualismo formiga-planta

Interaction among species, like ants and plants through extrafloral nectaries (EFNs), are important components of ecological communities’ evolution. However, the effect of human disturbance on such specific interactions and its ecological consequences is poorly understood. This study evaluated the outcomes of mutualism between ants and the EFN-bearing plant Stachytarpheta glabra under anthropogenic disturbance. We compared the arthropod fauna composition between two groups of twenty plant individuals, one in an area disturbed by human activities and one in a preserved area. We also check the plant investment in herbivory defense and the consequential leaf damage by herbivore. Our results indicate that such disturbances cause simplification of the associated fauna and lack of proper ant mutualist. This led to four times more herbivory on plants of disturbed areas, despite the equal amount of EFN and ant visitors and low abundance of herbivores. The high pressure of herbivory may difficult the re-establishment of S. glabra, an important pioneer species in ferruginous fields, therefore it may affect resilience of this fragile ecological community.

As interações entre espécies, como por exemplo formigas e plantas através de nectários extraflorais (NEFs), são importantes componentes na evolução das comunidades. Entretanto, pouco é conhecido sobre os efeitos dos impactos antrópicos em interações específicas e suas consequências ecológicas. Este estudo avaliou os resultados do mutualismo entre formigas e NEF em Stachytarpheta glabra em área impactada pela atividade humana. Nós comparamos a composição e estrutura da fauna de artrópodes, em quarenta plantas de dois grupos, um impactado por atividades humanas e o outro preservado. Nós também avaliamos o investimento da planta em defesas contra herbívoros e os danos foliares causados por herbívoros. Nossos resultados indicam que os distúrbios causam a simplificação da fauna associada e a ausência de uma formiga mutualista anti-herbívoros. Isto leva a quatro vezes mais herbivoria nas plantas da área impactada, a despeito da mesma quantidade encontrada para NEF e formigas visitantes. A grande pressão de herbivoria pode dificultar o reestabelecimento de S. glabra, uma espécie pioneira importante nos campos ferruginosos, dessa forma, afetando a resiliência dessa comunidade ecológica ameaçada.

Growth inhibition and morphological alterations of Staphylococcus aureus caused by the essential oil of Aloysia triphylla / Inhibición del crecimiento y alteraciones morfológicas de Staphylococcus aureus causadas por el aceite esencial de Aloysia triphylla

Aloysia triphylla essential oil (EO) has antimicrobial activity on Staphylococcus aureus which is a pathogen responsible for severe infections and food contamination. The target of EO is mainly in the cytoplasmic membrane. In this work the mechanisms of action of the EO of A. triphylla on S. aureus were investigated. A. triphylla was collected from La Paz. The oil was analyzed by GC-MS. The antimicrobial effects were evaluated by MIC, MBC, killing time and TEM. MIC values were 23 μg/mL for 6.3 x 104 CFU/mL, 92 μg/mL for 5.71 x 105 CFU/mL and 180 μg/mL for 9 x 106 CFU/mL. The MBC was 5920 μg/mL for all cellular concentrations and it was necessary more time to kill bigger cell populations. Multilamellar and mesosome-like structures on the membrane were seen by TEM. A. triphylla oil is an antibacterial compound against S. aureus which main mechanism of action seems to be the cytoplasmic membrane disruption.

El aceite esencial (AE) de Aloysia triphylla posee actividad antimicrobiana contra Staphylococcus aureus, patógeno responsable de infecciones nosocomiales e alimenticias. El blanco de acción de los AE es la membrana citoplasmática. El mecanismo de acción del AE de A. triphylla sobre S. aureus fue investigado. El AE de A. triphylla (La Paz, Argentina) fue analizado por CG-EM. Se evaluó́ el efecto antimicrobiano por CIM, CBM, tiempo de muerte y MET. Los valores de CIM fueron 23 μg/mL para 6.3 x 104 UFC/mL, 92 μg/mL para 5.71 x 105 UFC/mL y 180 μg/mL para 9 x 106 UFC/mL. La CBM fue 5920 μg mL para todas las concentraciones celulares estudiadas. El tiempo de muerte fue determinado; necesitando mayor tiempo para matar una población celular elevada. Fueron observados por MET estructuras multilamerales y semejantes a mesosomas. El AE de A. triphylla es un potencial compuesto antibacteriano contra S. aureus donde su principal mecanismo de acción es por disrupción de la membrana citoplasmática.