ABSTRACT
Uncaria tomentosa (Willd.) DC., a large woody vine native to the Amazon and Central American rainforests has been used medicinally by indigenous peoples since ancient times and has scientifically proven immunomodulating, anti-inflammatory, cytotoxic and antioxidant activities. Several inflammatory mediators that are implicated in vascular permeability and shock are produced after Dengue Virus (DENV) infection by monocytes, the primary targets for virus replication. Here we assessed the immunoregulatory and antiviral activities from U. tomentosa-derived samples, which were tested in an in vitro DENV infection model. DENV-2 infected human monocytes were incubated with U. tomentosa hydro-alcoholic extract or either its pentacyclic oxindole alkaloid-enriched or non-alkaloid fractions. The antiviral activity was determined by viral antigen (DENV-Ag) detection in monocytes by flow cytometry. Our results demonstrated an in vitro inhibitory activity by both extract and alkaloidal fraction, reducing DENV-Ag+ cell rates in treated monocytes. A multiple microbead immunoassay was applied for cytokine determination (TNF-alpha, IFN-alpha, IL-6 and IL-10) in infected monocyte culture supernatants. The alkaloidal fraction induced a strong immunomodulation: TNF-alpha and IFN-alpha levels were significantly decreased and there was a tendency towards IL-10 modulation. We conclude that the alkaloidal fraction was the most effective in reducing monocyte infection rates and cytokine levels. The antiviral and immunomodulating in vitro effects from U. tomentosa pentacyclic oxindole alkaloids displayed novel properties regarding therapeutic procedures in Dengue Fever and might be further investigated as a promising candidate for clinical application.
Subject(s)
Alkaloids/pharmacology , Antiviral Agents/pharmacology , Cat's Claw , Dengue Virus/drug effects , Immunologic Factors/pharmacology , Monocytes/drug effects , Alkaloids/analysis , Cat's Claw/chemistry , Cells, Cultured , Cytokines/biosynthesis , Humans , Monocytes/immunology , Monocytes/virologyABSTRACT
O gênero Uncaria (Rubiaceae) é representado na América do Sul e Central por duas espécies: U. tomentosa (Willd.) DC. e U. guianensis (Aubl.) Gmel., conhecidas popularmente como unha-de-gato. Ambas são trepadeiras perenes, sendo empregadas na prevenção e cura de várias doenças. Nessas plantas são encontrados alcalóides oxindólicos e indólicos, triterpenos glicosilados, taninos e flavonóides. Seis alcalóides oxindólicos pentacíclicos, considerados seus marcadores: especiofilina, mitrafilina, uncarina F, isomitrafilina, pteropodina e isopteropodina, são usados na padronização do material vegetal e fitoterápicos derivados. O presente trabalho descreve o desenvolvimento de metodologia analítica qualitativa utilizando cromatografia em camada delgada (CCD) para determinação do perfil dos seis alcalóides oxindólicos pentacíclicos marcadores das espécies. O desenvolvimento do método incluiu a comparação entre o uso do extrato metanólico bruto, e de frações enriquecidas obtidas por partição ácido-base clássica ou pelo uso de resina básica Poliamida 6. Utilizou-se gel de sílica como fase estacionária, e variaram-se alguns parâmetros como: eluentes, concentração da amostra, espaço de eluição e tipos de reveladores. O método desenvolvido em CCD mostrou-se confiável, reprodutível e seletivo para os alcalóides alvos, sendo aplicado na análise de amostras de folhas e caule das duas espécies e também de fitoterápicos comerciais à base de U. tomentosa.
The species Uncaria tomentosa (Willd.) DC. and U. guianensis Gmel. (Rubiaceae), known as cat's claw, are large woody vines occurring in the Amazon rain forest and other tropical areas of South and Central America. It has been used medicinally by indigenous peoples for at least 2,000 years for several diseases. Tetra- and pentacyclic oxindole alkaloids, triterpenoid glycosides, sterols and flavonoids are found in these plants. Among these metabolites, six pentacyclic oxindole alkaloids, speciophylline, mitraphylline, pteropodine, uncarine F, isopteropodine and isomitraphylline, are considered to be the biochemical markers and are used to standardize commercial herbal medicines. The present study describes the development of an analytical methodology to determine the profile of these alkaloid markers through thin layer chromatography (TLC). This development has also included a comparison among the use of the crude methanol extract and fractions obtained through the classical acid-base partition or by using the basic resin Polyamide 6. Silica gel was used as stationary phase with the variation of some parameters such as solvent systems, sample concentration, distance of development and detection method. The TLC method developed was shown to be reliable, reproducible and selective for the target alkaloids. It has been applied to the analysis of leaves and stems from both species as well as phytopharmaceutical derivatives based on U. tomentosa.
