Contribuição ao estudo farmacognóstico da Piper ovatum Vahl / Contribution to the pharmacognostic study of Piper ovatum Vahl

RESUMO: O presente estudo descreve as características morfo-anatômicas dos órgãos vegetativos e do pó da Piper ovatum Vahl de modo que os dados obtidos possam ser utilizados como referência em análises de controle de qualidade de amostras de fármacos, a fim de verificar sua autenticidade. As raízes, caules, pecíolos e folhas foram fixadas, seccionadas à mão livre e coradas, as secções transversais e paradérmicas foram analisadas por microscopia óptica e a superfície do limbo foi observada, também, por microscopia eletrônica de varredura (MEV). Os órgãos vegetativos da P. ovatum apresentam morfologia e anatomia similar às outras espécies de Piper. No entanto, não foram observadas inclusões celulares nas folhas de P. ovatum. Análises por MEV mostraram a presença de tricomas glandulares constituídos de pedúnculo unicelular e porção secretora globóide igualmente unicelular recoberto por cutícula, na epiderme abaxial das folhas. Também foi observada a presença de uma cutícula espessa e que origina crostas no limite entre uma célula e outra, em ambas as superfícies foliares. No mesófilo foi observada a presença de idioblastos oleíferos característica marcante de outras espécies de Piperaceae. Além disso, na microscopia do pó foram observados hipoderme e idioblastos oleíferos em fragmentos do limbo, fragmentos de fibras esclerenquimáticas do caule, além de células esclerosas isoladas ou em grupos no pecíolo. O perfil cromatográfico do extrato hidroetanólico das folhas de P. ovatum foi obtido por cromatografia líquida de alta eficiência (CLAE). Nas análises por CLAE foram identificados como substâncias majoritárias do extrato as amidas piperovatina e piperlonguminina nos tempos de retenção de 10,25 e 10,81 min., respectivamente.

ABSTRACT: The present study describes the morphological and anatomical characteristics of vegetative organs and powder of the Piper ovatum Vahl, in order to use the obtained data as reference in the quality control tests of pharmaceutical samples, investigating their authenticity. The roots, stems, petioles and leaves were fixed, freehand sectioned and stained according to usual microtechniques. The transverse and paradermal sections were analyzed by optical microscopy and the leaf surface was also observed by scanning electron microscopy (SEM). The vegetative organs of the P. ovatum show morphology and anatomy similar to other species of Piper. However, cellular inclusions were not observed in the P. ovatum leaves. The SEM analysis showed the presence of glandular trichomes consisting of a unicellular stalk and globular secretory portion covered by cuticle on the abaxial surface of the leaves. The SEM also had shown one thick cuticle forming crusts in the limit of the epidermal cells, on both leaf surfaces. In the mesophyll, we observed oil idioblasts, which are typical features of other species of Piperaceae. Moreover, in the powder of the P. ovatum we observed hypodermis and oil idioblasts in leaf fragments, fragments of sclerenchyma fibers from the stem and isolated sclereids or in petiole groups. The chromatographic profile of the hydroethanolic extract of the P. ovatum leaves was obtained by high performance liquid chromatography (HPLC). In this analysis, we identified the amides piperovatine and piperlonguminine in the retention times of 10.25 and 10.81 min., respectively, as majority compounds present in the extract.

Effect of a topical formulation containing Calophyllum brasiliense Camb. extract on cutaneous wound healing in rats.

This study evaluated the wound healing effects of topical application of an emulsion containing the HPLC-standardised extract from Calophyllum brasiliense Cambess (Clusiaceae) leaves in rats. The macroscopic analysis demonstrated that the wounds treated with the C. brasiliense emulsion healed earlier than the wounds treated with emulsion base and Dersani®. The percentage of wound healing in the group treated with the C. brasiliense emulsion was significantly higher than in the other groups at 7 and 14 days. On day 14, the animals treated with the C. brasiliense emulsion exhibited a 90.67% reduction of the wound areas. The histological evaluation revealed that on day 21, the group treated with the C. brasiliense emulsion exhibited a significant increase in fibroblasts compared with the other groups. Thus, the C. brasiliense emulsion had healing properties in the topical treatment of wounds and accelerated the healing process.

Effects of (-) mammea A/BB isolated from Calophyllum brasiliense leaves and derivatives on mitochondrial membrane of Leishmania amazonensis.

We have previously demonstrated antileishmanial activity on Leishmania amazonensis of the natural (1-2), synthetic (7) and derivatives of coumarin (-) mammea A/BB (3-6) isolated from the dichloromethane extract of Calophyllum brasiliense leaves. The aim of the present study was to evaluate morphological and ultrastructural alterations in Leishmania amazonensis induced by these compounds. In promastigote forms, all seven compounds produced significant morphological and ultrastructural alterations, as revealed by scanning and transmission electron microscopy. The compound 5,7-dihydroxy-8-(2-methylbutanoyl)-6-(3-methylbutyl)-4-phenyl-chroman-2-one (3), the most active antileishmanial with LD50 of 0.9 µM), induced cell shrinkage and a rounded appearance of the cells. Parasites incubated in the presence of compound (3) showed ultrastructural changes, such as the appearance of mitochondrial swelling with a reduction in the density of the mitochondrial matrix and the presence of vesicles inside the mitochondrion, indicating damage and significant change in this organelle; abnormal chromatin condensation, alterations in the nuclear envelope, intense atypical cytoplasmic vacuolization, and the appearance of autophagic vacuoles were also observed. In addition, the compound (3) may be acting to depolarize the mitochondrial membrane potential of the cells, leading to death of the parasite.

Efficacy of components from leaves of Calophyllum brasiliense against Leishmania (Leishmania) amazonensis.

Leishmanicide potential of Calophyllum brasiliense leaves on promastigote and amastigote of Leishmania (Leishmania) amazonensis is evaluated. The LD(50) of dichloromethane extract and hexane fraction for promastigotes was respectively 40 microg/ml and 20 microg/ml. In mouse peritoneal macrophages infected with Leishmania amastigotes the Infection Index decreased respectively 100% and 84.2% in 80 microg/ml and 40 microg/ml concentrations of dichloromethane extract. Hexane fraction decreased infection index respectively by 98.7% and 91.3% within the same concentrations. It was found that pretreatment with dichloromethane extract or with hexane fraction of experimentally infected BALB/c mice decrease the volume of the lesions by L. (L.) amazonensis. Moreover, animals treated topically also revealed healing lesions. Besides, the parasite load in the animals' popliteal lymph nodes was significantly reduced in treated animals, showing that plant components actually control infection. Results show that crude extract and hexane fraction of C. brasiliense reveal a significant in vitro and in vivo leishmanicide activity.