Anti-dengue viral activity of Carica Papaya leaves extract

Abstract

Dengue fever is an infectious tropical disease, which is considered a worldwide public health problem. However, until today no licensed vaccine is approved for dengue virus. The effort to develop dengue vaccine is made complicated by the four closely related dengue virus serotypes. Nowadays, the search for anti-dengue viral activities in natural plant products has been increasing. Thus, the development of plant-based antiviral agent will have the potential to fight against dengue fever. This study aimed to investigate the anti-dengue activity of Carica papaya leaves extracts and its fraction as well as determination of possible active compound and mechanism involved. The analyze of polysaccharide, glycosaponin and total protein were carried out by standard methods. Viability of Vero cells treated with aqueous extract of Carica papaya leaves was estimated by neutral red uptake assay. The microscopic observation on cytopathic effect was observed to determine the highest tolerable dose of Carica papaya leaves extract in Vero cells. The cytotoxicity of Carica papaya extracts on Vero cells was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method. Antiviral assays of aqueous extract of Carica papaya leaves were performed in three different stages, pre-, post- and continuous treatment on Vero cells against DENV-2 by foci forming unit reduction assay. Further antiviral assays of Carica papaya leaves crude extract and its fraction was investigated. Foci reduction on DENV-2 infected cells treated with the extracts for post- and continuous treatment were determined. Number of RNA reduction of DENV-2 was investigated by SYBR® green quantitative reverse transcription polymerase chain reaction method. Finally, the possible active compounds in the extracts was carried out using high performance liquid chromatography (HPLC) analysis. In this study, the results showed that the methanol matured leaves contained the highest concentration of total polysaccharide than aqueous matured leaves with 0.23 mgmL-1 and 0.22 mgmL-1 respectively. In addition, glycosaponin content in methanol matured leaves was the highest followed by methanol young leaves with 82% and 75% respectively, as well as in total protein content of 35.9% and 29.6% respectively. The extract did not reveal cytotoxic effects on Vero and C6/36 cells at all tested concentrations as well as its fractions. However, the methanol and ethanol extracts were found to be more toxic than the water extract. Anti-adsorption effect was shown at IC50 = 435.3 µgmL-1 with SI = 7.6. The extract also inhibited DENV-2 replication in Vero cells with IC50 = 1413 µgmL-1 and SI = 2.3 when added after adsorption to the cells. The IC50 against DENV-2 was 137.6 µgmL-1 and SI = 23.9 when cells were treated 5 hours before virus infection and continuously up to four-day post-infection. From the HPLC analyze, the possible active compound of catechin, quercetin and cinnamic acid were found in the water extract of Carica papaya leaves. In conclusion, the Carica papaya leaves extract showed slight inhibition of DENV-2 replication by decreasing foci number and size. The aqueous extract of Carica papaya leaves possesses the ability of inhibiting the activity of DENV-2. Thus, this extract is worth to be further investigated and might be advantageous as a primary source in the treatment of dengue and as a potential element for drugs formulation.