Anti-Viral Activity of Polyherbal Siddha Formulation “Notchi kudineer” Using Immuno-Fluorescence Assay and In-Silico Binding Studies of its Phytocompounds Against Various NSPs of SARS-CoV-2

Global health has suffered greatly because of the coronavirus disease 2019 (COVID-19) pandemic; as of late 2021, there had been over 230 million confirmed cases of illness and over 4.7 million deaths. Despite the existence of vaccines, COVID-19 is more aggressive in claiming millions of lives worldwide which is mainly due to the emergence of new variants of SARS-CoV-2 and rapid transmission rate. Thus, the world needs screening and developing new drugs with the potential of affecting multitarget through which the virulence of the virus can be largely restrained. ‘Notchi Kudineer’ (NK) a polyherbal formulation from the Indian Siddha system of medicine, has proven therapeutic effect in various viral infections such as dengue and chikungunya and is used in the management of COVID-19. To further elucidate the efficacy and antiviral activity of NK, we carried out in-vitro and in-silico analyses. Initially, the cytotoxic assay was performed in the Vero E6 cell line to study the cytocompatibility nature of the formulation by SRB assay, and later antiviral activity was evaluated by immunofluorescence assay. Results of the SRB assay showed that NK has no cytotoxic effect. Indirect immunofluorescence assay shows a decrease in fluorescence intensity of SARS-CoV-2 infected cells which shows the reduced antigen of SARS-CoV-2. This reveals the significant inhibitory effect of NK extract on the virus at 200μg/ml concentration. An in-silico study was performed with the phytochemicals present in Vitex negundo against the Non-Structural Proteins (NSPs) of SARS-CoV-2 viz., nsp1, nsp3, nsp9, nsp15 and nsp10-16. A molecular docking study of the phytocomponents of Vitex negundo with the nsp sheds light on the inhibitory mechanisms over the different nsp. Thus, revealing the multiple actions of NK over the inhibition of the viral protein. These results showed the efficacy of NK and paved the way for further drug development for the treatment of COVID-19. Major Findings: NK extract is non-toxic to Vero E6 cells (SRB assay) and it inhibits viral replication by reducing antigen expression. NK phytochemicals strongly bind SARS-CoV-2 NSPs, among these Negundin-B shows the highest binding to nsp10-16 (-8.94 kcal/mol), suggesting antiviral potential.