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.

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.

Biomassa e teor de óleo essencial em Aloysia triphylla (l'hérit) Britton submetida a diferentes níveis de reposição hídrica e à variação sazonal das condições ambientais / Biomass and essential oil content in Aloysia triphylla (L'Herit) Britton subjected to different levels of water reposition and season alchanges of environmental conditions

RESUMOA variação nos elementos meteorológicos ao longo do ano, configurando as quatro estações, proporciona diferentes condições de crescimento e desenvolvimento das plantas, alterando o metabolismo e a constituição dos tecidos vegetais. Assim, o presente trabalho teve o objetivo de determinar a influência da sazonalidade em parâmetros fenométricos e na produção de óleo essencial em Aloysia triphylla (L'Hérit) Britton, submetida a diferentes níveis de reposição hídrica cultivada em ambiente protegido. As avaliações foram realizadas no dia que marcou a metade de cada estação do ano (verão, outono, inverno e primavera), sendo coletado todo o material vegetativo 20 cm acima do solo. A reposição hídrica foi baseada na evapotranspiração de referência (ETo) para o ambiente externo em função dos dados de entrada da equação de Penman-Monteith a qual apresenta parametrizações para dados obtidos em estações meteorológicas em padrões oficiais, sendo assim aplicados 125, 100, 75 e 50% da ETo em turno de rega de dois dias. A produção de massa fresca, seca e de parte aérea, bem como a área foliar, apresentaram maiores valores na estação primavera, sendo o inverno e o verão responsáveis pelos menores valores. O maior teor de óleo essencial foi observado nas estações de menor produção de biomassa, ou seja, verão e inverno. A reposição hídrica que proporcionou maior produção de material vegetativo foi a de 125% da ETo; no entanto, o maior teor de óleo foi observado nas menores reposições hídricas (75 e 50 % da ETo, respectivamente). A produção de óleo foi maior na primavera, a qual não sofreu influência da reposição hídrica ao longo do ano.

ABSTRACTThe variation in weather elements throughout the year, setting the four seasons, provides different conditions for growth and development of plants, altering the metabolism and the structure of plants` tissues. This study aimed to determine the influence of seasonality on fenometrics parameters and on the oil production in Aloysia triphylla (L'Herit) Britton, subjected to different levels of water availability cultivated in greenhouse conditions. The evaluations were made on the day that marked the middle of each season (summer, autumn, winter and spring), and all plant material were collected 20 cm above ground. The water reposition was based on the evapotranspiration reference (ETo) to the external environment being applied 125, 100, 75 and 50% of ETo in an irrigation interval of two days. The production of fresh, dry and aerial part mass, as well as leaf areamass, showed higher values in the spring season, while winter and summer were responsible for lower values. The highest content of essential oil was observed at seasons with lower biomass production. The water availability that provided higher production of plant material was the one with 125% of ETo, however, the highest oil content was observed at lower water availability (75% and 50% of ETo, respectively). The oil production was higher in the spring and was not affected throughout the year by the water availability.

Uso popular de espécies medicinais da família Verbenaceae no Brasil / Popular use of medicinal species of the Verbenaceae family in Brazil

RESUMO Com o presente trabalho objetivou-se realizar um levantamento bibliográfico acerca das espécies da família Verbenaceae utilizadas na medicina popular nas diversas regiões do Brasil. Os trabalhos acadêmicos utilizados para elaboração desta pesquisa foram extraídos de revistas indexadas junto ao Scielo publicadas no período de 2000 a 2012, sendo também realizada busca no Google acadêmico. Os dados foram compilados e organizados em ordem alfabética por espécies, seguidas do nome popular, forma de uso, parte usada, indicações e área de estudo. Foram encontrados um total de 85 publicações indicando 55 espécies distribuídas em oito gêneros. A espécie com maior número de citações foi Lippia alba (Mill.) N.E.Br., com 56 registros. Das plantas descritas na literatura, a folha foi o órgão mais citado como parte usada e o chá, o modo de preparo mais utilizado. Dentre as categorias de indicações medicinais, as plantas que mereceram destaque foram às relacionadas ao tratamento das doenças do sistema respiratório e digestório. Constatou-se que o Rio de Janeiro foi o estado que contou com o maior número de publicações na área. O uso de plantas medicinais está cada vez mais presente entre as diversas comunidades. Com isso, as informações contidas neste trabalho servirão para auxiliar na elaboração de banco de dados sobre o modo de uso das plantas pertencentes à família Verbenaceae com potencial medicinal encontradas nos Estados brasileiros.

ABSTRACT The current work aimed to conduct a literature review about the Verbenaceae family species used in popular medicine in different regions of Brazil. The academic papers employed for the preparation of this research were taken from journals indexed by the Scielo published from 2000 to 2012, and a research in Google scholar tool was also performed. The data were compiled and organized alphabetically by species, followed by the popular name, manner of use, employed part, indications and study area. A total of 85 publications indicating 55 species were found and distributed into eight genera. The species with the highest number of citations was the Lippia alba (Mill.) N.E.Br., with 56 records. From the plants described in the literature, the leaf was the most cited part of the vegetables, and the tea was the most widely used preparation method. Among the categories of medicinal recommendations, the highlighted plants were the ones related to the treatment of diseases of the respiratory and the digestive systems. It was observed that Rio de Janeiro was the state in which the largest number of publications in the area was concentrated. The use of medicinal plants is increasingly present among several communities. Thus, the information in this work will be applied to assist in database development on how to use the plants belonging to the family Verbenaceae with medicinal potential found in Brazilian states.

Respuesta de la regeneración natural en manglares del Golfo de Urabá (Colombia) a la variabilidad ambiental y climática intra-anual / Natural regeneration response in mangroves of the Gulf of Urabá (Colombia) to the environmental and intra-annual climate variability

The natural regeneration process allows the mangrove forests remain over time. Both, biological and physical factors can affect the establishment and early stages along the development of trees. This study examined the response of natural regeneration of mangroves in the Turbo River delta and El Uno bay (Urabá Gulf, Colombia) to intra-annual environmental variability. We quantified mortality, survival and recruitment of seedlings of three mangrove species, seasonally during one year, in 72 semi-perma-nent sub-plots of 1m². In the sub-plots, the total height and the diameter at the base of the stem of all mangrove seedlings with basal diameter less than 2.5cm were measured. Damage by herbivores was also recorded to each seedling. While Laguncularia racemosa recorded the highest rates of mortality, Rhizophora mangle showed the highest survival rate during the study period, although Avicennia germinans dominated the natural regeneration. Through a Redundancy Analysis these processes were associated to environmental variables such as: Direct Site Factor-DSF (sunlight), sediments input rate, herbivory, distances from mangroves to the river, to inner lakes, and to the coastline. These variables explained 43% of the natural regeneration variation; sedimentation rate was the most important variable, while light was very representative for the R. mangle survival. Based on historical records of precipitation, Turbo River flow rate and associated sediment loads, it was established that during the highest precipitation peak, the survival of all species decreases and during the dry season, when the conditions of flooding and sediments input were lower, it was improved. The results indicated that the sediments input rates and sunlight play an important role in the survival of natural regeneration of evaluated mangrove species.

En los bosques de mangle, tanto factores biológicos como físicos, afectan el establecimiento y los estados tempranos de desarrollo de los árboles y determinan la distribución espacial de las especies. Se evaluó la respuesta de la regeneración natural de los manglares del delta del río Turbo y la bahía El Uno a la variabilidad ambiental y climática intra-anual. Se cuantificaron la mortalidad, la sobrevivencia y el reclutamiento de las plántulas de tres especies, estacionalmente durante un año. Avicennia germinans dominó la regeneración natural, Rhizophora mangle presentó la mayor la supervivencia y Laguncularia racemosa la mayor mortalidad. Estos procesos se asociaron principalmente con el aporte de sedimentos aluviales, la disponibilidad de luz, la herbivoría, la distancia al río, a las lagunas internas y a la línea de costa, las cuales explicaron el 43% de la variación en la regeneración. La sedimentación fue la variable más importante para el reclutamiento de A. germinans, mientras la luz fue muy representativa para la supervivencia de R. mangle. Con base en registros históricos de precipitación, caudales y carga de sedimentos, se estableció que en la época con mayor precipitación se presenta un descenso en la supervivencia, y en la época seca se favorece el balance entre el reclutamiento y la mortalidad. Los resultados indicaron que la precipitación, el aporte de sedimentos y la luz desempeñan un papel importante en el proceso de la regeneración de las especies evaluadas.

Chemical composition and antimicrobial activity of the essential oil from the leaves and flowers of Aloysia gratissima / Composição química e atividade antimicrobiana do óleo essencial de folhas e flores de Aloysia gratissima

Volatile oils from leaves and flowers of Aloysia gratissima were investigated for their chemical composition and antimicrobial activity against the bacteria Bacilus subtilis, Staphylococcus aureus, Salmonella choleraesuis, Pseudomonas aeruginosa, Streptococcus pneumoniae and the Candida albicans yeast. The minimum inhibitory concentrations (MIC) of the oils were determined by the micro-dilution method, while the chemical composition was determined by GC-MS (gas chromatography mass spectrometry). The fresh leaves and inflorescence were subjected to hydrodistillation for 120 min using a Clevenger-type apparatus, and the essential oil was tested against microorganisms. High concentrations of sesquiterpenes were observed for the inflorescence, and monoterpenes were observed for the leaves. The main compounds of the inflorescence essential oil were E-caryophyllene, germacrene B, guaiol and bulnesol, while in the leaves the main compounds were trans-pinocamphone, trans-pinocarveyl acetate, and guaiol. The essential oil from the leaves showed an effect against P. aeruginosa and S. pneumonia, and the essential oil of the inflorescence showed an effect against P. aeruginosa, S. pneumonia, and Candida albicans.

O óleo essencial de folhas e de flores de Aloysia gratissima foi avaliado quanto à composição química e ação antimicrobiana contra as bactérias Bacilus subtilis, Staphylococcus aureus, Salmonella choleraesuis, Pseudomonas aeruginosa, Streptococcus pneumoniae, e a levedura Candida albicans. A concentração mínima inibitória (MIC) dos óleos essenciais foi determinada pelo método da microdiluição e a composição química determinada por CG-EM (Cromatografia Gasosa acoplada a Espectrômetro de Massas). Folhas e inflorescências frescas foram hidrodestiladas por 120 minutos em aparelho Clevenger sendo o óleo essencial testado contra microorganismos. Para as flores foi observada maior concentração de sesquiterpenos, enquanto que as folhas apresentaram maior concentração de monoterpenos. Os principais constituintes do óleo essencial da flor foram: E-cariofileno, germacreno B, guaiol e bulnesol; e das folhas foram: trans-pinocamfona, acetato de trans-pinocarveol e guaiol. O óleo essencial da folha mostrou atividade contra P. aeruginosa e S. pneumoniae, e o óleo essencial da flor mostrou atividade contra P. aeruginosa, S. pneumoniae e Candida albicans.

Growth rate and ring width variability of teak, Tectona grandis (Verbenaceae) in an unmanaged forest in East Timor

Teak (Tectona grandis) is one of the most valuable timbers in international trade and an important species for tropical forestry. Teak is found on the island of East Timor but no information is available on teak growth from this region. A pure stand planted in 1940-50 in the North of East Timor and left unmanaged was studied. Fifteen trees were sampled in October-November 2003 and stem discs taken at three height levels of its height (1.7m, 9.5m and 18.7m), and cores were collected at DBH. Transverse surfaces of the discs and cores were polished for ring identification. Core cross sections were first digitized and disc cross sections were observed under the microscope. Three randomly selected radii were analyzed in each disc. Ring width measurement and ring counting were done using image analysis software. The distinction between heartwood and sapwood was performed macroscopically by colour difference, and heartwood radius and sapwood width were measured. The relationship between stem and heartwood radius was studied for each disc and heartwood percentage by radius was determined. Radial ring width curves are presented for the different axial positions within the stem, and ring width variability was analyzed. Growth rates were calculated and age-radius relationships were estimated using cumulative growth curves. Growth rings were large and well defined in the juvenile phase, reflecting the specie’s fast-growing character. The year-to-year variation of ring width showed a similar pattern among trees. Mean ring width ranged between 4.3-7.3mm for the first 20 years and 3.3-5.1mm for 30 to 45 years. Pith eccentricity was evident in the lower part of the stem and ring wedging occurred. On average, heartwood represented 84% of the radius and sapwood contained 6 to 11 rings. The age-related variation of ring width and the occurrence in the lower part of the tree stems of eccentricity and wedging rings, highlights the importance of appropriate stand management, particularly regarding basal density distribution over time, whenever optimized timber production is envisaged.

La madera de teca (Tectona grandis L.f.) es una de las más valiosas en el comercio internacional y una especie importante para la silvicultura tropical. La teca se encuentra en la isla de Timor Leste, pero no existe información disponible sobre su crecimiento en esta región. Se estudió una plantación pura no manejada que fue establecida entre 1940 y 1950 en el Norte de Timor Leste. Entre Octubre- Noviembre 2003 se cosecharon 15 árboles y al tronco se les extrajeron discos a tres niveles de altura (1.7m, 9.5m y 18.7 m), y se recolectaron núcleos a la altura del pecho (DAP). El conteo y la medición de los anillos se realizaron mediante el software de análisis de imagen. La distinción entre el duramen y la albura se realizó macroscópicamente por la diferencia de color, y se midieron el radio del duramen y el ancho de la albura. Para cada disco se estudió la relación entre el tallo y radio del duramen y el porcentaje del duramen. Las curvas de variación del ancho de los anillos se presentan para las diferentes posiciones axiales dentro del tronco y se analizó la variabilidad del ancho de los anillos. Las tasas de crecimiento fueron calculadas y las relaciones de radio con la edad se calcularon usando las curvas de crecimiento acumulado. Los anillos de crecimiento eran grandes y bien definidos en la fase juvenil, lo que refleja el carácter de crecimiento rápido de esta especie. La variación en la anchura de los anillos año a año mostró un patrón similar entre los árboles. La anchura media del anillo osciló entre 4.3-7.3mm para los primeros 20 años y 3.3-5.1mm para los de 30 a 45 años. La excentricidad de la médula fue evidente en la parte inferior del tallo y se observó la formación de anillos sobrepuestos. En promedio, el duramen representó el 84% del radio y la albura mostró entre 6 y 11 anillos. La variación del ancho de los anillos con la edad, la presencia de excentricidad en la parte inferior del árbol y los anillos sobrepuestos pone de relieve la importancia del manejo forestal con particular atención a la distribución de densidad basal en el tiempo, cuando se pretende la producción optimizada de la madera.

Efeito da época de colheita na produção de fitomassa e rendimento de óleo essencial de alecrim-pimenta (Lippia sidoides Cham. ) / Effect of harvesting time on phytomass production and essential oil yield in "alecrim-pimenta" (Lippia sidoides Cham. )

O objetivo do estudo foi avaliar a massa fresca, massa seca e rendimento de óleo essencial de Lippia sidoides cultivada no Norte de Minas. O delineamento foi em blocos ao acaso, com quatro tratamentos e cinco repetições. Os tratamentos foram constituídos por intervalos de colheita: 120, 180, 240, 300 e 360 dias após o transplantio. Em cada colheita, foram determinados a altura das plantas, o diâmetro da base do caule, o rendimento de matéria fresca de folhas, caule, parte aérea e o rendimento de matéria seca da parte aérea, além do teor e rendimento de óleo essencial por planta. O teor máximo de óleo essencial foi detectado aos 180 dias, enquanto que a produção máxima de matéria seca ocorreu aos 329 dias. A produção de óleo essencial por planta foi 2,34±1,15 g. A produtividade de óleo essencial foi de 124,8 kg ha-1, sendo recomendada a colheita aos 180 dias.

The aim of this study was to evaluate fresh and dry mass, as well as essential oil yield, in Lippia sidoides cultivated in the north of Minas Gerais State, Brazil. The experimental design was in randomized blocks, with five treatments and four replicates. Treatments consisted of the following harvesting intervals: 120, 180, 240, 300 and 360 days after transplanting. Measurements on each harvesting included plant height, stem base diameter, leaf, stem and shoot fresh matter yield, and shoot dry matter yield, besides essential oil content and yield per plant. The highest essential oil content was detected at 180 days, whereas the highest dry matter production was observed at 390 days. Essential oil yield per plant was 2.34±1.15 g and productivity was 124.8 kg ha-1. Thus, harvesting at 180 days is recommended.