The Role of In-vitro Cytotoxicity Assays to Confirm Non-toxicity Subsequent to the Detoxification Procedure of Mercuric Sulphide and Arsenic Trisulphide, Two Integral Metal Components Used in the Formulation of a Novel Herbo-mineral Antiviral Drug Targeted for COVID-19, ZingiVir-H

herbo-mineral drugs are unique Ayurvedic metallic preparations known in the Indian subcontinent for several centuries and have several benefits that have been instrumental in their widespread use in treatment of different disorders by traditional medicinal practitioners. These include better stability, lower dosage, ease of storability and sustained availability. In the herbo-metallic preparations, the particles used have a diameter of about 10–15 nm. Bhasma is now been considered Ayurvedic nanomedicine and provides opportunity for drug designing employing modern concept of nanomedicine. In order to combat the harmful effects produced as a result of single drug or formulations containing the metal, Ayurveda emphasizes a process, namely Shodhana (purification). It is stated in the various treatises that processing of the mineral drugs mentioned in Rasashastra (Indian Iatrochemistry) with classically prescribed traditional purification procedures consisting of medicinal herb juices/decoctions can help reduce poisonous effects and also potentiate their therapeutic value. Recently Pankajakasthuri formulated ZingiVir-H, a herbo-mineral Ayurvedic medicine targeted for managing various viral fevers, Bronchitis and Acute Respiratory tract infections. This medicine was formulated using five potent herbs and two purified heavy metals (Mercuric sulphide - HgS and Arsenic trisulphide - As2S3). The toxicity associated with metallic constituents used for preparing the medicine was removed by subjecting to various detoxifying processes as described in the century-old classical literatures of Ayurveda. However, one criticism levelled at Ayurvedic metal-derived drugs is a lack of empirical evidence for their claimed non-toxicity after detoxification. Given the widespread misconception, we intend to use MTT assay and ROS production to assess the cellular toxicity of ZingiVir-H and its two heavy metal ingredients (raw and at various stages of detoxification) in the L6 rat myoblast cell line. Purified HgS and As2S3 had significantly lower cellular toxicity in the MTT assay when compared to their raw forms. In the MTT assay, ZingiVir-H recorded non-toxicity when compared to that of raw heavy metals. Alternatively, purified HgS and As2S3 also recorded a significant reduction in ROS production when compared to that of raw HgS and As2S3. Finally, ZingiVir-H and the drug without its metal constituents recorded only 4.5±0.33% and 1.1±0.23% of ROS. These results established that the detoxification of HgS and As2S3 leads to a significant reduction of toxicity as well as the ROS production. Further, the quantitative analysis by X-ray diffractometer evidently revealed that the percentage of arsenic, mercury and sulphur was significantly reduced after the detoxification/purification of HgS and As2S3. By abiding the scientifically validated traditional purification techniques for poisonous medicinal plants and metals, it is possible to ameliorate their toxic effects and enhance their therapeutic efficacy. In addition to this, we conducted an oral toxicity study of ZingiVir-H in wistar rats. Treatment with ZingiVir-H daily for a continuous 28 days in experimental animals did not show any signs of toxicity. Histopathological examination of vital organs revealed normal architecture, suggesting no morphological alterations after ZingiVir-H treatment. These results demonstrated that ZingiVir-H did not possess the potential to induce any toxicity in the animals. Finally, the safety of ZingiVir-H tablets for the use of humans at a maximum dose of 1500 mg/ kg/day is scientifically established. Then we conducted a pilot clinical study to demonstrate the preliminary evaluation of the efficacy and safety of ZingiVir-H in hospitalized adults diagnosed with viral infection. Here in this study ZingiVir-H recorded a significant effect in managing viral infection. The Liver Function Test (LFT) and Renal Function Test (RFT) parameters before and after treatments were within the recommended clinical range, which established the hepato-renal safety of the ZingiVir-H in the study subjects. Thus, this study eventually proved that ZingiVir-H is highly effective and safe in managing viral fever in humans. Based on our findings, we hope that this research paper will attract widespread interest in order to confirm the toxicity threshold of herbomineral drugs and purified heavy metals according to Ayurvedic principles via cytotoxicity testing. Unambiguously, we can conclude that ZingiVir-H is nontoxic for human administration using scientific investigations such as in-vitro cytotoxicity assays, in-vivo animal toxicity experiments, and a pilot clinical study.