Flavonol Robinobiosides and Rutinosides from Alternanthera brasiliana ( Amaranthaceae ) and their Effects on Lymphocyte Proliferation In Vitro

Claudia de O. Brochado, Ana P. de Almeida, Beatriz P. Barreto, Leandro P. Costa, Luciene S. Ribeiro, Rachel L. da C. Pereira, Vera L. Gonçalves Koatz b and Sonia S. Costa* a Núcleo de Pesquisas de Produtos Naturais, Universidade Federal do Rio de Janeiro, 21941-590 Rio de Janeiro RJ, Brazil b Departamento de Bioquímica Médica, Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, 21941-590 Rio de Janeiro RJ, Brazil


Introduction
Alternanthera brasiliana Kuntze (Amaranthaceae) is a herbaceous plant used against inflammation, cough and diarrhoea in Brazilian popular medicine. 1In a search for some action of this plant against inflammatory cells of the immune system, it was shown that aqueous or ethanolic A. brasiliana leaf extracts are able to block human mitogeninduced lymphocyte proliferation, without any toxic effect. 2Different biological activities have been established for A. brasiliana species; [2][3][4] however, this is the first chemical study of its secondary metabolites.

Results and Discussion
Bioassay-guided fractionation of a water-soluble phase of an ethanolic leaf extract (EE) was carried out with the aim of identifying compounds responsible for the antiproliferative effect of the crude extract on lymphocytes.
were described from Blutaparon portulacoides, another medicinal species of Amaranthaceae. 14lavonoids 1-6 are reported here for the first time for Alternanthera.Kaempferol 3-O-robinobioside (4) and 3-O-rutinoside (6) inhibited lymphocyte proliferation to a greater extent (IC 50 ≅ 25 µg mL -1 ) and were twice as active as the crude extract (Table 1).The anti-inflammatory effects 15 observed in vivo can be attributed to the effects of these flavonoids on T-cell function, thereby accounting for the medicinal properties of A. brasiliana.( 1 H-, 400 MHz; 13 C-, 100 MHz).[α] D was measured in a Perkin-Elmer 243B polarimeter; and melting points were obtained using a Kofler apparatus.Reversed-phase chromatography was performed on RP-2 or RP-8 silanized silica (Merck) and size exclusion chromatography on Sephadex LH-20 (Sigma).Eluates were monitored by thinlayer chromatography on Silica 60 F 254 (Merck) using butanol-acetic acid-water (8:1:1) for development and ceric sulfate solution as detection reagent.

Plant material
A. brasiliana was collected out of flowering season on the University (UFRJ) campus (Ilha do Fundão).A voucher specimen (RFA-25 052) was deposited in the herbarium of the Department of Botany (Instituto de Biologia, UFRJ, Brazil).

Lymphocyte proliferation assay
Human peripheral blood mononuclear cells (PBMC) were obtained from healthy volunteers by Ficoll-Hypaque density gradient centrifugation.Cells (10 6 cells mL -1 ) were cultivated in the presence of 5 µg mL -1 phytohemagglutinin (PHA) at 37 °C and 5% CO 2 .Dose-response curves were obtained by adding different concentrations of extract, flavonoids and azathioprine to triplicate samples, and inhibition was expressed as IC 50 .Proliferation was measured by [ 3 H] thymidine incorporation into cellular DNA: 0.5 µCi/well was added 6 h before the end of the culture period

Table 1 .
Inhibition of the in vitro proliferative response of human T-cells by A. brasiliana extract (WE) and its flavonoids 1-6 a positive control.