Recent studies have shown that flavonoids such as glabranine and 7-O-Methyle glabranine exhibited significant antiviral activities against dengue virus. In vitro
treatment of infected cells with these flavonoids resulted in the reduction of intracellular replication of dengue virus by 76.9% and 75% following treatment with 25 μM of glabranine and 7-O-Methyle glabranine, respectively [22
]. Similarly, other synthetic flavonoid derivatives also showed antiviral activity in HepG2 cells [23
]. Whereas, pinostrobin was reported to inhibit DENV-2 NS2B/NS3 protease an enzyme important in dengue virus replication in an in vitro
]. These suggest that flavonoids as a group could consist of select compounds that possesses inhibitory activities against DENV. To investigate which of the many flavonoids could affect DENV infection, in the present study, we examined the potential effects of quercetin, naringin, hesperetin and daidzein on dengue virus infection of Vero cells. Unlike the previous studies which evaluated antiviral activity of flavonoids only after adsorption of virus to the cells [22
], the present study evaluated antiviral activity by different treatment procedures tailored to determine prophylactic, post adsorption, continuous treatment and direct virucidal activities of quercetin, naringin, daidzein and hesperetin.
Our findings demonstrated that quercetin was the only compound among all tested flavonoids that consistently showed significant antiviral activity against DENV-2 in Vero cells. Selectivity indices for quercetin when the infected cells were treated or when uninfected cells were treated continuously 5 h before infection until 4 days post-infection were 7.07 and 8.74, respectively. The noted differences between SI values for quercetin could be due to the intracellular accumulation of quercetin during continuous treatment. A weak effect for prophylactic activity of quercetin however, was also observed. These findings suggest that the main anti-dengue activity of quercetin is likely due to its activity against the different stages of intracellular replication of DENV-2 instead of early stages of its replication cycle such as virus attachment or entry. Although, no direct virucidal activity or anti-attachment activity of quercetin was observed in the present study, antiviral activity of quercetin against human cytomegalovirus was reported with IC50
= 3.2 μM [13
]. Quercetin was also reported to be effective against herpes viruses where it is more specific against HSV-1 with SI = 22 compared to HSV-2 with SI = 5.7 [11
The mechanisms of how quercetin exerts its antiviral effects are not known. However, the effects of other flavonoids against cellular RNA polymerases and formation of the complex with RNA have been reported suggesting that quercetin could also affect the similar replication enzymes [25
]. Sylimarin, a flavonoid found effective against hepatitis C virus (HCV), another member of flaviviridae family [27
], inhibits virus replication by inhibiting the activity of viral RNA polymerase. In our study, results from the qRT-PCR supported the findings from the viral foci reduction assays that quercetin inhibits DENV-2 replication and the significant reduction in the DENV specific RNA suggests that quercetin may also target the virus replication machinery, namely by inhibiting the RNA polymerase.
Antiviral activity of naringin has been evaluated against few herpesviruses and rotavirus but their reported antiviral activities against HSV-1 and HSV-2 are inconclusive [12
]. In addition it was reported that naringin did not exhibit any antiviral activity against another RNA virus, sindbis virus [14
]. In the present study, the only anti-dengue activity of this flavonoid was demonstrated against adsorption and attachment of virus to the Vero cells and based on its antiviral activity (IC50
= 168.2 μg mL-1
) and its related selectivity index (SI = 1.3), it may not be a good candidate for further development as anti-dengue drug. Similarly, daidzein activity against DENV-2 was not significant compared to quercetin (SI = 1.03). Continuous treatment of the infected Vero cells from 5 h before virus infection up to 4 days post infection did not improve its anti-dengue activity significantly. This compound therefore, could not be a suitable candidate for further development as anti-dengue drug. Hesperetin, the other flavonoid evaluated in our study, did not show no anti-dengue activity in any stages of virus infection and replication processes and this is despite the previously reported antiviral activity of hesperetin against sindbis virus [14
]. Therefore, hesperetin is also not recommended for further investigations for anti-dengue drug development. In all our experiments, we showed that 0.5% of DMSO, the highest concentration of solvent used in the bioflavonoid treatment did not exhibit any antiviral activity against DENV-2 and this eliminated any probable antiviral activity from DMSO.
Findings from our study, suggest that there are select flavonoids including quercetin and fisetin, which are both flavonol, that exhibited significant DENV replication inhibition properties [28
]. While the flavonoids in general share common basic molecular base structure, flavone (2-phenyl-1,4-benzopyrone), we showed here that the flavanone, hesperetin, and flavanone glycoside, naringin, showed no significant anti-DENV replication activities. In addition, we had earlier shown that naringenin [28
], another flavanone metabolized from naringin and here, daidzein, an isoflavone also had no significant DENV replication inhibition properties. While quercetin was shown here to be effective in inhibiting DENV replication, its glycoside form, rutin (quercetin-3-O-rutinoside) showed no significant inhibition properties [28
]. These suggest that while flavonol could be the basic molecule that possesses anti DENV replication properties, specific structural properties of the different flavonol derivatives would have different effects on the efficacy of the compounds against dengue.
The demonstration in vitro that flavonols including quercetin and fisetin possess anti DENV replication properties does not necessarily translate into immediate use of these compounds as antivirals against DENV. Further studies will be needed to demonstrate the antiviral activities of these compounds against different genotypes of dengue virus and in appropriate animal model. There is also a need to address the issue of the low bioavailability of quercetin especially for therapeutic use [29
]. Several strategies to increase the bioavailability of quercetin that include using lipids and emulsifiers, co-crystalization of quercetin or using ester-based precursors have been investigated [32
]. The other topic of research would be combination drug study. At its current calculated IC50
values, the antiviral efficacy of quercetin can be further improved possibly by combining it with other potential anti-dengue compounds. This is exemplified in a study that reported the synergistic effect of α- glucoside in combination with a standard antiviral drug, ribavirin is effective against dengue infection [35