Vathasura Kudineer, an Andrographis Based Polyherbal Formulation Exhibits Immunomodulation and Inhibits Chikungunya Virus (CHIKV) under in Vitro Conditions

Ethnopharmacological relevance: Chikungunya disease (CHIKD) is caused by the alphavirus, chikungunya virus (CHIKV) and is characterized by acute fever and joint inflammation; the inflammation continues even after clearance of the virus from the system, persisting for several months to years. Currently, there are no modern medicines/vaccines available for its treatment and use of over-the-counter anti-inflammatory generic medicines to relieve symptoms is generally practiced. In India, Indian traditional medicines hold a lot of promise to treat this infection and are routinely used during outbreaks.Aim of the study: In the present study, we characterized the phytochemical and physicochemical properties of aqueous and ethanol extracts of the Vathasura Kudineer (VSK), a Andrographis based Siddha polyherbal formulation. Additionally, we evaluated its immunomodulatory and antiviral potential using an in vitro system. Materials and methods: Aqueous and ethanolic extracts of VSK were prepared and their physico and phyto-chemical properties were obtained by biochemical and biophysical assays, HPTLC and FTIR. The aqueous ex-tracts of VSK and several of its ingredients were evaluated for their cytotoxicity in Vero cells and using the maximum non-toxic concentration (MNTC), were processed further for evaluating their ability to inhibit CHIKV infection in Vero cells. We performed the co-treatment assay with ethanol extract of VSK and several of its in-gredients to assess the antiviral activity against chikungunya virus on Vero cells and through pre-treatment assay (anti-adhesive effect), co-incubation assay (virucidal effect) and post-treatment assay (post-entry effect) were evaluated. Further, we tested the aqueous extract of VSK along with some of its ingredients for their immuno-modulatory properties. We performed antioxidant and anti-inflammatory assays using LPS-simulated RAW 264.7 cells. For antioxidant capacity of extracts, we performed extra-cellular ABTS radical scavenging activity and intra-cellular effects on ROS generation and SOD activity. We assessed the effect on most important in-flammatory mediators like Nitric oxide (NO) and Prostaglandin E2 (PGE2) and pro-inflammatory cytokines like interleukin-1 beta (IL-113) and tumor necrosis factor alpha (TNF alpha).Results: We provided the fingerprint of the phytochemicals of both ethanol and aqueous extracts of VSK that can be used for identification. We observed that ethanol extract was able to inhibit CHIKV infection at MNTC with 48 h of treatment on Vero cells. Its ingredient VSKI-As (Anethum sowa) found to be most effective to show virucidal effect while VSKI-Cs (Clerodendrum serratum) and VSKI-Pn (Pipper nigrum) found to be effective in post-entry effect. VSK was able to show ABTS radical scavenging activity, reduce ROS generation, inhibit the inflamma-tory mediators (NO and PGE2) and pro-inflammatory cytokines (IL-113 and TNF alpha) production in LPS-stimulated RAW 264.7 cells.Conclusions: We provided the evidence that VSK has both immunomodulatory as well as antiviral potential. It shows virucidal as well as post-entry effects on chikungunya virus. VSK can inhibit pro-inflammatory cytokines, IL-113 and TNF alpha production by suppressing the inflammatory mediators, NO and PGE2.