RP-HPLC and LC-MS-MS determination of a bioactive artefact from Ipomoea pes-caprae extract

Abstract Solvents play important and critical role in natural product chemistry and could generate artefacts during the extraction and purification of metabolites from a biological matrix. This study aimed to correlate the chromatographic profile with biological activity of Ipomoea pes-caprae (L.) R. Br., Convolvulaceae, extracts obtained with hydroethanolic extraction. Thus, aerial parts of I. pes-caprae were extracted with different concentration of ethanol (50, 70 and 90°GL) and the obtained extracts were analysed by HPLC-UV. HPLC data were studied employing chemometrics to discriminate the samples. Moreover these samples were further characterized by using UPLC-QTOF/MS data. The extracts were also biomonitored through the paw-oedema and spontaneous nociception induced by trypsin in mice. Different chromatographic profiles were obtained and the exploratory analysis clearly revealed higher level of ethyl caffeate in extracts of lower strength of ethanol (50°GL). This compound was suggested to be an artefact formed by transesterification of caffeoylquinic acid derivatives present in the plant, once it was not observed when other solvents were employed. During the biological assay, only the extract obtained with ethanol 50°GL presented significant inhibition of inflammation (45 ± 9%) and nociception (24 ± 3%). Ethyl caffeate seems to be linked to the anti-inflammatory effect since it reduced 86 ± 5% of paw-oedema induced by trypsin. Artefacts could contribute to the biological activity of herbal preparations and consequently lead to misinterpretation of the results.

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.

Evaluation of the proliferative activity of methanol extracts from six medicinal plants in murine spleen cells

A number of natural compounds have been used as immunomodulatory agents, enabling the function of the immune system to be modified by stimulating or suppressing it. There has been increasing interest in the study of therapeutic action of plant extracts regarding their immunomodulatory activity. The aim of this study was to identify and evaluate the action of extracts of the medicinal plants Calophyllum brasiliense, Ipomoea pes-caprae, Matayba elaeagnoides, Maytenus robusta, Rubus imperialis and Vernonia scorpioides on the development of spleen cells from mice, using the in vitro cellular proliferation assay. The cells, obtained by mechanical rupture of mice spleen (5x10(4) cells/mL), were incubated with methanol extracts (10, 50, 100 and 200 µg/mL) and phytohemagglutinin (PHA, 5 µg/mL). The basal control for proliferation consisted of cells alone, while the positive control consisted of cells and PHA. The cell culture was kept at 37 ºC in 5 percent CO2 for 72 hours, and cell proliferation was revealed by the blue tetrazolium reduction assay (MTT). The results were expressed as percentage of growth and were analyzed using the Kruskal-Wallis and Mann-Whitney tests. The C. brasiliense, I. pes-caprae and M. elaeagnoides extracts showed dose-dependent induction of cell proliferation, with a significant increase in cell proliferation (p<0.03) and percentage growth of 88.2 percent, 73.1 percent and 52.7 percent, respectively, suggesting T lymphocyte stimulation. By contrast, M. robusta, R. imperialis and V. scorpioides extracts showed significance only with a negative percentage of growth, suggesting inhibition of cell proliferation (p<0.04). Further biomonitoring studies will enable the fractions and isolated substances responsible for the immunomodulatory activities to be identified.

Várias substâncias de origem natural têm sido utilizadas como agentes imunomoduladores, permitindo modificar a função do sistema imune e propiciando o estudo de atividades terapêuticas de extratos de plantas. Este trabalho objetivou identificar a atividade imunomodulatória dos extratos de seis plantas medicinais da flora brasileira, Calophyllum brasiliense, Ipomoea pes-caprae, Matayba elaeagnoides, Maytenus robusta, Rubus imperialis e Vernonia scorpioides, sobre a proliferação de células esplênicas de camundongos. As células esplênicas murinas obtidas por ruptura mecânica do baço (5x14³ células/mL) foram incubadas com os extratos metanólicos das plantas (10, 50, 100, 200 µg/mL) e fito-hemaglutinina (PHA, 5 µg/mL). O controle basal de proliferação foi constituído de células apenas e o controle positivo formado por células e PHA. O cultivo celular foi mantido a 37 ºC, 5 por cento de CO2, 72 horas, com quantificação da proliferação celular pelo ensaio de redução do azul de tetrazólio. Os resultados expressos em percentagem de crescimento foram analisados pelos testes de Kruskal-Wallis e Mann-Whitney. Os extratos de C. brasiliense, I. pes-caprae e M. elaeagnoides mostraram indução dose-dependente da proliferação celular (p<0,03), com percentagem de crescimento de, respectivamente, 88,2 por cento, 73,1 por cento e 52,7 por cento, sugerindo estímulo de linfócitos T. Contrariamente, os extratos de M. robusta, R. imperialis e V. scorpioides apresentaram significância apenas com percentagem de crescimento negativa, indicando inibição da proliferação celular (p<0,04). A continuidade no estudo biomonitorado permitirá a identificação das frações e substâncias isoladas responsáveis pelas atividades imunomoduladoras.