<i>Cissampelos pareira</i> Linn: Natural Source of Potent Antiviral Activity against All Four Dengue Virus Serotypes

<div><p>Background</p><p>Dengue, a mosquito-borne viral disease, poses a significant global public health risk. In tropical countries such as India where periodic dengue outbreaks can be correlated to the high prevalence of the mosquito vector, circulation of all four dengue viruses (DENVs) and the high population density, a drug for dengue is being increasingly recognized as an unmet public health need.</p><p>Methodology/Principal findings</p><p>Using the knowledge of traditional Indian medicine, Ayurveda, we developed a systematic bioassay-guided screening approach to explore the indigenous herbal bio-resource to identify plants with pan-DENV inhibitory activity. Our results show that the alcoholic extract of <i>Cissampelos pariera</i> Linn (<i>Cipa</i> extract) was a potent inhibitor of all four DENVs in cell-based assays, assessed in terms of viral NS1 antigen secretion using ELISA, as well as viral replication, based on plaque assays. Virus yield reduction assays showed that <i>Cipa</i> extract could decrease viral titers by an order of magnitude. The extract conferred statistically significant protection against DENV infection using the AG129 mouse model. A preliminary evaluation of the clinical relevance of <i>Cipa</i> extract showed that it had no adverse effects on platelet counts and RBC viability. In addition to inherent antipyretic activity in Wistar rats, it possessed the ability to down-regulate the production of TNF-α, a cytokine implicated in severe dengue disease. Importantly, it showed no evidence of toxicity in Wistar rats, when administered at doses as high as 2g/Kg body weight for up to 1 week.</p><p>Conclusions/Significance</p><p>Our findings above, taken in the context of the human safety of <i>Cipa</i>, based on its use in Indian traditional medicine, warrant further work to explore <i>Cipa</i> as a source for the development of an inexpensive herbal formulation for dengue therapy. This may be of practical relevance to a dengue-endemic resource-poor country such as India.</p></div

Analysis of interaction between paracetamol and <i>Cipa</i> extract.

<p>(A) DENV-3 (50 PFU) was pre-incubated with <i>Cipa</i> extract in the absence (solid black diamonds) or presence of 1 μg/ml (solid blue diamonds), 10 μg/ml (solid red circles), or 100 μg/ml (solid green circles) paracetamol overnight at 4°C, followed by analysis of viral inhibition in a type-1 assay. (B) Febrile Wistar rats were mock-treated (empty red circles), or treated with paracetamol (solid blue circles), <i>Cipa</i> extract (empty green squares) or a combination of both (solid black squares), followed by monitoring of rectal temperature for 3 hours post-treatment at regular intervals. Rectal temperatures between the control (mock-treated) and treatment groups were compared using one-way ANOVA followed by Dunnett’s multiple comparison test (the single and double stars indicate significant differences in the treatment groups with respect to the control group, corresponding to <i>p</i> values of ≤0.05 and ≤0.01, respectively).</p

Effect of <i>Cipa</i> extract on platelets.

<p>(A) Freshly collected human blood was incubated with saline (white bars) or <i>Cipa</i> extract (at 2 μg/ml: blue bars; or 10 μg/ml: red bars) for up to 4 hours. Aliquots were drawn at the indicated times for determination of platelet counts. (B) Wistar rats were orally administered 0.25% methyl cellulose containing <i>Cipa</i> extract ranging from 0–1000 mg/Kg body weight. Fresh blood collected from these rats at 0 (white bars), 1 (blue bars) and 4 (red bars) hours post-administration, were analysed for platelet counts. For both panels, data shown are mean values (<i>n</i> = 5); the vertical bars represent standard deviation, SD.</p

Evaluation of protective efficacy of <i>Cipa</i> extract <i>in vivo</i>.

<p>AG129 mice (9–12 weeks old) were injected i.p. with 10⁶ PFU brain-passaged DENV-2. Infected mice were treated orally with 0.25% methyl cellulose (solid red squares) or <i>Cipa</i> extract at 125 mg (empty blue circles) and 250 mg/kg body weight (solid blue circles). Treatment was twice daily for the first 5 days. A sham control group that was not virus-infected, but which received the higher dose of <i>Cipa</i> extract orally (solid green squares), was tested in parallel. The mice were monitored daily for mortality and the resultant data plotted as Kaplan-Meier survival curves. The <i>p</i> values to assess the statistical significance in the survival rates on day 35 between the <i>Cipa</i> extract-treated and placebo-treated (0.25% methyl cellulose) groups were determined using the Log-rank test.</p

Effect of pre-incubation time on antiviral activity of <i>Cipa</i> extract.

<p>DENV-3 (50 PFU) was pre-incubated with <i>Cipa</i> extract at 11 μg/ml (filled red circles), 33 μg/ml (empty blue squares) and 100 μg/ml (filled black squares) for different durations (2–24 hours) followed by assay of antiviral activity in a type-1 assay.</p

Schematic representation of the antiviral screening assays.

<p>An outline of the three types of screening assays (indicated by Arabic numbers 1, 2 and 3) is shown. The multi-coloured sphere represents DENV and the Eppendorf tube with green liquid represents the herbal extract. These two were pre-incubated (1) before addition to the monolayer or added sequentially (2, 3) to the monolayer. In assays 1 and 2, the treated-monolayers were overlaid with methyl cellulose-containing growth medium. Shown at the bottom are the possible outcomes of the type 1 and 2 assays. The ‘x’ mark denotes failure of entry into cells. In assay 3, liquid growth medium was added instead of the methyl cellulose overlay, and followed by analysis of NS1 and virus released into the culture supernatant.</p

Inhibition of DENV antigen and virus production by <i>Cipa</i> extract treatment.

<p>Vero cells were used to test the effect of <i>Cipa</i> extract on DENV-3 in a type-3 assay format. The figure depicts the kinetics of NS1 antigen (A) and infectious virus (B) released into the culture supernatant in the absence (empty black circles) and presence of <i>Cipa</i> extract at 22 μg/ml (filled blue circles), 66 μg/ml (empty red squares) and 200 μg/ml (filled green squares) concentrations.</p

Effect of <i>Cipa</i> extract on RBCs.

<p>(A) Erythrocytes from freshly collected human blood were incubated for 1 hour at 37°C, with varying concentrations of <i>Cipa</i> extract (0–400 μg/ml), followed by measurement of haemolysis at 576 nm. TX-100 represents a control in which an equivalent aliquot of erythrocytes were treated with Triton X-100 to achieve complete lysis. (B) Fresh blood collected from the <i>Cipa</i> extract-treated Wistar rats (described in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004255#pntd.0004255.g005" target="_blank">Fig 5B</a>) at 0 (white bars), 1 (blue bars) and 4 (red bars) hours post-administration, were analysed for RBC counts. Data shown are mean values (<i>n</i> = 5); the vertical bars represent SD.</p