In vitro evaluation of antiviral efficacy of a Siddha Formulation against SARS-CoV-2


 Introduction: SARS-CoV-2 virus caused COVID-19 pandemic with 218 million cases and 45 million deaths world over. It has challenged the already overburdened healthcare systems and created an urgent need to investigate solutions present in other healthcare systems. In this study Kabasura Kudineer is investigated as an intervention to influence the immune response which is beneficial for the host and stop the viral replication. Methods: Kabasura Kudineer is a polyherbal formulation containing 15 herbal drugs mixed in equal quantity. It is an official Siddha formulation, used for phlegmatic fevers and flu-like symptoms. To conduct this study Vero E6 (CL1008), the African monkey kidney epithelial cell line was taken and infected with SARS-CoV-2 viral isolate. The Kabasura Kudineer was added in different concentrations; 0.5 mg/mL, 0.25 mg/mL, 0.12 mg/mL, 0.06 mg/mL and 0.03 mg/mL to the infected cells respectively. These cell plates were incubated for 3 days in 5% CO2 incubator. Remdesivir was used as a positive control. The cells were fixed with formaldehyde, stained with crystal violet and plaques were visualised. Plaques were counted as PFU/ml. Result: Kabasura Kudineer was found to exhibit good antiviral activity against SARS-CoV-2. The highest antiviral activity was 81.5% at a concentration of 0.5 mg/ml. The IC-50 value was found to be 0.2 mg/mL. Conclusion: The antiviral efficacy of Kabasura Kudineer in our study showed reduction in the viral load which supports the results of clinical studies. Kabasura Kudineer can be used widely in a clinical setting as a treatment for COVID-19.


Introduction
SARS-CoV-2 virus is responsible for the ongoing pandemic known as COVID-19. More than 218 million cases have been con rmed worldwide and the disease has claimed around 45 million lives (3 September 2021 COVID Database, WHO) The wide spread of COVID-19 has highlighted the challenges that the already overburdened healthcare systems faces. Proper healthcare is required to care for the patients. COVID positive patients' world over have shown major symptoms like fever, cough and breathing di culties (Vincent et al, 2020). In more severe cases, they can cause pneumonia, severe acute respiratory syndrome, kidney failure and even death (Pitchiah et al, 2020) WHO initiated the "Solidarity Trial" in many countries to compare the medicines' effectiveness, such as Remdesivir, Lopinavir/ Ritonavir with interferon beta and Hydroxycholroquine against the Coronavirus infection (WHO-Guidelines 2019). Because of the side effects, antiviral therapy cannot be continued in the long run and relief from ailments is only symptomatic (Aminu Saleh Ahmad & Ruchi Sharma 2020). There is a need to look out for solutions present in other health care systems like Ayurveda, Siddha, Herbal medicine etc.
The concept of epidemics is very well de ned and established in Ayurveda and Siddha. As per the siddha literature the contamination and vitiation of environmental factors such as-water, air, environment and season is responsible for the epidemics along with the disequilibrium of the doshas in the hosts that makes them more receptive to the disease (Jain et al, 2019) In this study, Kabasura Kudineer is suggested as an intervention to in uence the immune response in a way which is bene cial for the host and stop the viral replication. In case of Siddha medicine, many ingredients are immune-modulators and have the capacity to inhibit the virus by enhancing and restoring immunity. Kabasura Kudineer, an o cial Siddha formulation described in Siddha manuscript is used for phlegmatic fevers and is a dependable siddha prescription with u-like symptom. (Kiran et al, 2020).
The Siddha & Ayurveda medicines are not usually tested in labs and there is lack of in vitro studies on these medicines. Kabasura Kudineer has shown great potential in RCTs and is required to explore its antiviral e cacy and mechanism of action. Kabasura Kudineer is typically used as a concoction. To increase the ease of consumption, it was converted to a tablet form by Sri Sri Tattva

Virus cells:
The SARS-CoV-2 viral isolate was obtained by BEI resources managed by ATCC. Isolate USA-WA1/2020 was isolated from an oropharyngeal swab from a patient with a respiratory illness who had recently returned from travel to the affected region of China and developed clinical disease  in January 2020 in Washington, USA.
(Catalogue-BEI Resources) 2.4 Test Material preparation: A stock solution of the Kabasura Kudineer tablet powder was dissolved in DMSO and was made to 100X concentration. The Kabasura Kudineer was manufactured by Sriveda Sattva Pvt Ltd, Bangalore (Sri Sri Tattva). The stock solution was serially diluted at 1/20 with Phosphate Buffer saline (PBS) to obtain 5X solution, used from the assay. 100µl of the stock solution was added in all the wells. In positive cell control well, 100µl of PBS was added while for positive test control 100µl of remdesivir solution was added.
2.5 Plaque reduction assay: The assay plate was coated with approximately 30,000 Vero E6 cells in a solution of 200 µL of DMEM containing 10 % FBS per well. A 96 well plate was used for the assay. The plates were incubated overnight (12-18 h) at 37° C. Three plates were used for controls as following: a) positive control (virus infected cells treated with remdesivir), b) virus only control (Vero E6 cells infected with virus and no test material), c) cell only control (Vero E6 cells without the infection or test materials). After overnight incubation, the excess of cell culture media was removed and 100µl of test material diluted to required concentrations was added. The following concentrations were used for Kabasura Kudineer: 0.5 mg/mL, 0.25 mg/mL, 0.12 mg/mL, 0.06 mg/mL and 0.03 mg/mL.
The test was performed in duplicates to nullify any error. After loading of the test material, the plates were incubated in 5% CO2 incubator for an hour. After 1h of pre-incubation with the test material, the test material was removed and 30µL/well of a virus mix (prepared in infection medium) was added. The virus mix contained virus at multiplicity of Infection, (MOI) of 0.01. After virus was added, the plates were again incubated at 37° C in CO2 incubator (5%) for 1 h with shaking at every 15 minutes. After 1 h, the medium containing the test material and the virus were removed from the wells. Thereafter, 200 µL of DMEM: Carboxymethylcellouse (CMC) mixture again containing the test material at desired concentration was added to each well of the 96-well plates. The infected cell lines were incubated at the 37 o C, 5% CO2 for 3 days. After 3 days of incubation the CMC overlay was removed gently with a pipette and the cells were washed twice with PBS buffer. The cells were xed with 200 µL of 4% formaldehyde to each well and incubated for 30 minutes. Formaldehyde was removed and 100 µL of 0.05% (w/v) crystal violet in 20 % methanol was added to each well and incubated for 20-30 minutes. After 30 minutes the excess crystal violet was removed with distilled water and plaques were visualized. The number of plaques were counted to determine the PFU/mL, the log reduction and percentage viral load reduction in the presence of test material. The IC-50 of the test material was determined using GraphPad Prism software (Version 9.0.1) FNDR's research and handling of SARS-CoV-2 has been endorsed by its Institutional Biosafety Committee. All SARS-CoV-2 studies were performed with approved standard operating procedures and conform to the safety requirements recommended by the Department of Biotechnology, Government of India.

Results
Plaque Assay Data

Test Material
The test material (NF1) Kabasura Kudineer was found to exhibit good antiviral activity against SARS-CoV-2. The highest antiviral activity was 81.5% at a concentration of 0.5 mg/ml ( Table 1). The IC-50 was calculated by considering the top value of 100 and baseline value of 0 and was found to be 0.2 mg/mL (Fig. 2)

Control
The IC-50 value of positive control (Remdesivir) was calculated by considering the top and baseline value 100 and 0, respectively ( Table 1). The reported IC-50 was 1.3 µM.
The test and negative control also ensured the e cacy of viral isolates, cell lines used and the test procedure. By providing respective controls, it was ensured that the antiviral effect in test material arm was caused only due to the antiviral e cacy of Kabasaura Kudineer and not due to any toxicity.

Discussion
Herbal medicines have been used as therapeutic agents throughout the human civilization. Their use was challenged largely during 19th and 20th century by the advent of semi-synthetic and synthetic drugs. In recent times, though, the consumption of plant-based medicines has increased manifold because of increasing reports of adverse side effects and bacterial resistance to antibiotics (Mohammed, 2012; Capodice and Chubak 2021).
This research was aimed at investigating the antiviral properties of Kabasura Kudineer tablets in an in vitro experiment.
In vitro means that it is done outside of a living organism and usually involves isolated tissues, organs or cells. Natarajan et al, 2020). The Kabasura Kudineer increases the immunity and could act as immunomodulator, it can reinstate the respiratory health (Ramya et al, 2021). There was reduction in viral load of SARS-CoV-2 at the end of treatment (10 days) and time taken to convert patient for symptomatic to asymptomatic based on clinical symptoms in 10 days (Srivastava et al, 2021). Ministry of AYUSH has launched a massive nationwide campaign to distribute its proven poly herbal Siddha drug Kabasura Kudineer to strengthen its position in ght againt COVID 19 pandemic (Jabaris SLS and Venkataraman K 2020)

Conclusion
This study supports the antiviral e cacy of Kabasura Kudineer by demonstrating an 81.5% reduction in the SARS-nCOV-2 viral load, which is in line with the symptomatic relief provided by Kabasura Kudineer in COVID patients in clinical studies.
The results of our study support a wider usage of Kabasura Kudineer in clinical settings as a treatment for COVID-19.

Funding
We would like to thank Cooper Family Foundation, Australia for providing us the funding for this in vitro study Plaque reduction assay of infected Vero E6 Cells