Evaluación de los efectos mutagénicos/antimutagénicos de Luehea divaricata en ratones / Evaluation of the mutagenic / antimutagenic effects of Luehea divaricata in mice

La Luehea divaricata Mart. conocida popularmente en el Paraguay como "ka´aovetî o ka´aovetîpytâ", es una planta nativa de la región utilizada como fitoterápico. El objetivo de este trabajo fue el de investigar el efecto protector del extracto acuoso de las hojas de L. divaricata frente al quimioterápico ciclofosfamida, en médula ósea de ratones. Los animales fueron divididos en 4 grupos de 5 animales cada uno; el Grupo I, control negativo, que recibió 200 µL de agua, vía oral; el Grupo II, que recibió 200 µL del extracto acuoso de L. divaricata; el Grupo III, que recibió 200 µL del extracto acuoso de L. divaricata y ciclofosfamida, 50 mg/Kg de peso de animal y el Grupo IV, fue el control positivo, recibió ciclofosfamida 50mg/Kg/peso de animal. El extracto acuoso de L. divaricata fue administrado por vía oral y el tratamiento se administró por 48h, la ciclofosfamida fue administrada por vía intraperitoneal 24h antes del sacrifico del animal .Se extrajo la médula ósea de los animales y se realizó el ensayo de micronúcleo en todas las muestras. Los resultados indicaron que el extracto acuoso de L. divaricata no presentó actividad mutagénica, cuando combinado con el agente mutagénico induce una reducción de hasta 49% en la frecuencia de micronúcleos, al ser comparado con el tratamiento con ciclofosfamida, sugiriendo un potencial efecto protector frente a agentes tóxicos.

The Luehea divaricata Mart. is a plant native, popularly known in Paraguay as "ovetîka'a”or “ovetîka'aPytá" of the region used as herbal medicines. The aim of this study was toinvestigate the protective effect of aqueous extract from the leaves of L. divaricata versuscyclophosphamide chemotherapy. The animals were divided in 4 groups and each grouphave 5 animals, Group I, negative control, which received 200 uL of water, orally; Group II,which received 200 uL of aqueous extract of L. divaricata; Group III, which received 200 uLof the aqueous extract of L. divaricata and cyclophosphamide, 50 mg / kg animal weight;Group IV, positive control, cyclophosphamide received 50mg / kg / animal weight. Theaqueous extract of L. divaricata was orally administered and the treatment wasadministered for 48 hs, cyclophosphamide was administered intraperitoneal and had 24hours of treatment. Bone marrow of the animals is removed and micronucleus assay wasperformed on all samples. The results indicated that aqueous extract of L. divaricata showedno mutagenic activity and when combined with the mutagen agent induces a reduction ofup to 41% in the frequency of micronucleus, when compared to treatment withcyclophosphamide, suggesting a potential protective effect against toxic agents.

Anti-inflammatory, analgesic, and immunostimulatory effects of Luehea divaricata Mart. & Zucc. (Malvaceae) bark

Luehea divaricata (Malvaceae) is a plant widely used for treatment of various inflammatory and infectious conditions; however few reports discuss its biological properties. The aim of this study was to evaluate the anti-inflammatory and analgesic effects as well as the macrophage activity in mice treated with the hydroalcoholic crude extract of L. divaricata (CLD). Thin layer chromatography revealed presence of epicathequin, stigmasterol, lupeol and α,β-amyrin in the extract. To evaluate the anti-inflammatory and analgesic activities, animals were subjected to paw edema induced by carrageenan test, writhing, formalin and capsaicin tests. Immunomodulatory activity was evaluated by adhesion and phagocytic capacity, lysosomal volume, and reactive oxygen species (ROS) production by peritoneal macrophages, after daily treatment with CLD for 15 days. CLD promoted reduction in paw edema (36.8% and 50.2%; p<0.05 at doses of 100 and 300 mg/kg, respectively), inhibited writhing behavior at the higher dose (64.4%, p<0.05), reduced formalin reactivity (81.2% and 91.6% at doses of 100 and 300 mg/kg, respectively, p<0.05), and reduced capsaicin reactivity by 63.9% (300 mg/kg). CLD (200 mg• kg-1• day-1) increased phagocytosis capacity of macrophages (~3 fold, p<0.05), neutral red uptake (~50%, p<0.001), and ROS production (~90%, p<0.001). These data suggest that CLD possesses anti-inflammatory, analgesic and immunostimulatory properties.

Luehea divaricata (Malvaceae) é utilizada para o tratamento de várias condições patológicas, entretanto, há poucos relatos sobre sua bioatividade. O objetivo deste estudo foi avaliar o efeito anti-inflamatório e analgésico, bem como a atividade de macrófagos em camundongos tratados com extrato bruto hidroalcoólico (CLD) da planta. Cromatografia em camada delgada revelou a presença de epicatequina, estigmasterol, lupeol e α,β-amirina no material. Para avaliar a atividade anti-inflamatória e analgésica, animais foram submetidos a teste de edema de pata induzido por carragenana, teste de contorções, da formalina e da capsaicina. A atividade imunomodulatória foi avaliada pela capacidade de adesão e de fagocitose dos macrófagos, volume lisossômico e produção de espécies reativas de oxigênio (ROS), após tratamento diário com CLD por 15 dias. CLD promoveu redução do edema de pata (36,8% e 50,2%; 100 e 300 mg/kg, respectivamente; p<0,05), redução do número de contorções (64,4%; 300 mg/kg; p<0,05), redução da reatividade no teste da formalina (81,2% e 91,6%; 100 e 300 mg/kg, respectivamente; p<0,05), e no teste da capsaicina em 63,9% (300 mg/kg). CLD (200 mg• kg-1• day-1) aumentou capacidade de fagocitose dos macrófagos (~3 vezes, p<0,05), volume lisossômico (~50%, p<0,001) e produção de ROS (~90%, p<0,001). Estes dados sugerem que o CLD possui propriedades anti-inflamatórias, analgésicas e imunoestimulatórias.

Idade de segregação do lenho juvenil e adulto pela variação da massa específica de açoita-cavalo / Estimating the age of demarcation of young and mature wood in açoita-cavalo

O presente trabalho teve como objetivo estimar a idade de segregação do lenho de Luehea divaricata Mart. (açoita-cavalo) por meio da variação radial da massa específica. Para o desenvolvimento do estudo, foram coletadas dez árvores de duas regiões do Estado do Rio Grande do Sul. Foi retirado um disco por árvore, com aproximadamente 2cm de espessura a 0,10m de altura do tronco. De cada disco, retirou-se, com auxílio de uma serra-fita, uma bagueta central de 2,0cm de largura, bem orientada no sentido radial, incluindo a medula no centro, dividiu-se então a bagueta em duas amostras "A" e "B". A massa específica foi obtida da relação entre peso seco e volume úmido. O volume úmido foi determinado de acordo com a norma ASTM D 2395 - 93, 1995. A segregação dos lenhos foi encontrada por regressão linear simples dos dados de variação da massa específica no sentido medula-casca. Foi feita uma regressão linear na faixa crescente dos dados e outra na faixa decrescente. A intersecção das retas extrapoladas foi adotada como idade de segregação. Os resultados obtidos mostraram que o lenho de açoita-cavalo segrega em aproximadamente 26 anos para a região da Depressão Central e 23 anos para a região da Encosta Superior do Nordeste. Nos dados de variação de massa específica, foi feita uma análise de variância multifatorial. Constatou-se que há diferença significativa entre lenhos e entre regiões. Por outro lado, as amostras A e B de cada árvore não foram estatisticamente diferentes entre si.

This study aims to estimate the age of segregation of young and mature wood of Luehea divaricata Mart., using the radial variation of specific gravity. To the development of this study were collected ten trees from two regions of the state of Rio Grande do Sul. Discs with thickness of 2cm at 0.1m of height from the base of the trunk were used. From each disk, with the aid of a band saw, a central ribbon 2.0cm wide, and oriented in the radial direction was taken, including the pith, then splitted into two samples "A" and "B". The specific gravity was calculated as ratio between dry weight and wet volume. The wet volume was determined according to ASTM D 2395 - 93, 1997. The age of segregation was found by simple linear regression of the data of variation of specific gravity from pith to bark. The intersection of extrapolated lines was adopted as the age of segregation. The results showed that the wood segregates in 26 year for the region of Central Depression and in 23 years for the Northeast Upper Hillside. Was made a multifactorial analysis of variance in the specific gravity data, showing that there are statistical difference between regions and woods. However, there are no statistical difference between samples.

Evaluation of foliar fungal endophyte diversity and colonization of medicinal plant Luehea divaricata (Martius et Zuccarini)

Endophyte microorganisms are organisms that live inside plants without causing any apparent damage to their hosts. Since all plants exhibit endophyte microorganisms, it is believed that mutual association is of great importance in nature. Luehea divaricata (Martius & Zuccarini), known popularly in Brazil as agoita-cavalo, is a big-sized tree with a wide distribution in the country that possesses medicinal qualities for: dysentery, leucorrhea, rheumatism, blennorrhoea, tumors, bronchitis, and depuration. This research aims at isolating and molecularly characterizing fungi isolates from L. divaricata by sequence analysis of ITS1-5.8S-ITS2 rDNA. Further, the colonization of endophyte in the host plant by Light and Scanning Electron Microscopy will also be investigated. Whereas, genera Alternaria, Cochliobolus, Diaporthe, Epicoccum, Guignardia, Phoma, and Phomopsis, were identified; rDNA sequence analysis revealed intra-species variability among endophyte isolates of the genus Phomopsis sp. Light and Scanning Electron Microscopy techniques showed the presence of endophyte fungi inside L. divaricata leaves, inhabiting inter- and intra-cellular spaces. These types of extensive colonization and dissemination were reported throughout all the leaf parts in palisade parenchyma, esclerenchyma, spongy parenchyma, adaxial epidermis, and vascular bundle indicating colonization of endophytes in múltiple structural sub-niches in the host plant.

Genotoxicity of plant extracts

Aqueous extracts of seven species used in Brazilian popular medicine (Achyrocline satureoides, Iodina rhombifolia, Desmodium incanum, Baccharis anomala, Tibouchina asperior, Luehea divaricata, Maytenus ilicifolia) were screened to the presence of mutagenic activity in the Ames test (Salmonella/microsome). Positive results were obtained for A. satureoides, B anomala and L. divaricata with microsomal activation. As shown elsewhere (Vargas et al., 1990) the metabolites of A. satureoides extract also show the capacity to induce prophage and/or SOS response in microscreen phage induction assay and SOS spot chromotest.