Screening of anti-dengue activity in methanolic extracts of medicinal plants

<p>Abstract</p> <p>Background</p> <p>Dengue fever regardless of its serotypes has been the most prevalent arthropod-borne viral diseases among the world population. The development of a dengue vaccine is complicated by the antibody-dependent enhancement effect. Thus, the development of a plant-based antiviral preparation promises a more potential alternative in combating dengue disease.</p> <p>Methods</p> <p>Present studies investigated the antiviral effects of standardised methanolic extracts of <it>Andrographis paniculata, Citrus limon, Cymbopogon citratus, Momordica charantia, Ocimum sanctum </it>and <it>Pelargonium citrosum </it>on dengue virus serotype 1 (DENV-1).</p> <p>Results</p> <p><it>O. sanctum </it>contained 88.6% of total flavonoids content, an amount that was the highest among all the six plants tested while the least was detected in <it>M. charantia</it>. In this study, the maximum non-toxic dose (MNTD) of the six medicinal plants was determined by testing the methanolic extracts against Vero E6 cells <it>in vitro</it>. Studies also determined that the MNTD of methanolic extract was in the decreasing order of <it>M. charantia </it>><it>C. limon </it>><it>P. citrosum, O. sanctum </it>><it>A. paniculata </it>><it>C. citratus</it>. Antiviral assay based on cytopathic effects (CPE) denoted by degree of inhibition upon treating DENV1-infected Vero E6 cells with MNTD of six medicinal plants showed that <it>A. paniculata </it>has the most antiviral inhibitory effects followed by <it>M. charantia</it>. These results were further verified with an <it>in vitro </it>inhibition assay using MTT, in which 113.0% and 98.0% of cell viability were recorded as opposed to 44.6% in DENV-1 infected cells. Although methanolic extracts of <it>O. sanctum </it>and <it>C. citratus </it>showed slight inhibition effect based on CPE, a significant inhibition was not reflected in MTT assay. Methanolic extracts of <it>C. limon </it>and <it>P. citrosum </it>did not prevent cytopathic effects or cell death from DENV-1.</p> <p>Conclusions</p> <p>The methanol extracts of <it>A. paniculata </it>and <it>M. charantia </it>possess the ability of inhibiting the activity of DENV-1 in <it>in vitro </it>assays. Both of these plants are worth to be further investigated and might be advantageous as an alternative for dengue treatment.</p

Investigating the role of protein folding and assembly in cell-type dependent expression of alpha7 nicotinic receptors using a green fluorescent protein chimera

To test the hypothesis that cell-dependent expression of α7 receptors is due to differences in protein folding or assembly, we constructed a chimeric rat α7 subunit with green fluorescent protein (GFP) at the receptor C-terminal. Expression of α7-GFP in Xenopus oocytes resulted in currents that were indistinguishable from wild type receptors but were only 33% of control. 125I-α-bungarotoxin (αBGT) binding at the oocyte surface was reduced to 23% of wild type. Transfection of α7-GFP into GH4C1 cells produced fluorescence that was less intense than GFP alone, but showed significant α-BGT binding compared to transfection with GFP. In contrast, α7-GFP transfection in SH-EP1, HEK293 and CHO-CAR cells produced fluorescence without αBGT binding. Flow cytometry of cells transfected with α7-GFP indicated fluorescence in both SH-EP1 and GH4C1 cells, but surface toxin binding sites and sites immunoprecipitated using anti-GFP antibodies were undetectable in SH-EP1 cells, suggesting a problem in folding/assembly rather than trafficking. Surprisingly, integrated fluorescence intensities in GH4C1 cells transfected with α7-GFP did not correlate with amounts of cell surface or immunoprecipitable αBGT binding. Therefore, GFP folding at the C-terminal of the α7-GFP chimera is cell-line independent, but toxin binding is highly cell-line dependent, suggesting that if altered protein folding is involved in the cell-type dependence of α7 receptor expression, the phenomenon is restricted to specific protein domains. Further, C-terminal GFP-labeled α7 receptors decreased the efficiency of folding/assembly not only of chimeric subunits, but also wild-type subunits, suggesting that the C-terminal is an important domain for α7 receptor assembly.Supported by the Ministry of Education and Science of Spain and FEDER (SAF2005-00534 and SAF2005-02045), by NIH NS22472, and by an RSDF grant from Northeastern University.Peer Reviewe