Development and validation of a rapid high-performance thin-layer chromatographic method for quantification of gallic acid, cinnamic acid, piperine, eugenol, and glycyrrhizin in Divya-Swasari-Vati, an ayurvedic medicine for respiratory ailments.

Divya-Swasari-Vati is a calcium containing polyherbal ayurvedic medicine prescribed for the lung-related ailments observed in the current pandemic of Severe Acute Respiratory Syndrome Coronavirus 2 infections. The formulation is a unique quintessential blend of nine herbs cited in Ayurvedic texts for chronic cough and lung infection. Analytical standardization of herbal medicines is the pressing need of the hour to ascertain the quality compliance. This persuaded us to develop a simple, rapid, and selective high-performance thin-layer chromatographic method for Divya-Swasari-Vati quality standardization. The developed method was validated for the quantification of marker components, gallic acid, cinnamic acid, piperine, eugenol and glycyrrhizin, against reference standards in five different batches of Divya-Swasari-Vati. The analytes were identified by visualization at 254 nm, and by matching their retention factor with authentic standards. The developed method was validated as per the guidelines recommended by the International Council for Harmonization for parameters like, linearity, limit of detection, limit of quantification, accuracy, and precision. Therefore, the developed novel high-performance thin-layer chromatographic process could be employed for rapid standardization of Divya-Swasari-Vati and other related herbal formulation, which would aid in quality manufacturing and product development.

Comprehensive and Rapid Quality Evaluation Method for the Ayurvedic Medicine Divya-Swasari-Vati Using Two Analytical Techniques: UPLC/QToF MS and HPLC-DAD.

Divya-Swasari-Vati (DSV) is a calcium-containing herbal medicine formulated for the symptomatic control of respiratory illnesses observed in the current COVID-19 pandemic. DSV is an Ayurvedic medicine used for the treatment of chronic cough and inflammation. The formulation has shown its pharmacological effects against SARS-CoV-2 induced inflammation in the humanized zebrafish model. The present inventive research aimed to establish comprehensive quality parameters of the DSV formulation using validated chromatographic analytical tools. Exhaustive identification of signature marker compounds present in the plant ingredients was carried out using ultra performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC/QToF MS). This was followed by simultaneous estimation of selected marker components using rapid and reliable high-performance liquid chromatography (HPLC) analysis. Eleven marker components, namely gallic acid, protocatechuic acid, methyl gallate, ellagic acid, coumarin, cinnamic acid, glycyrrhizin, eugenol, 6-gingerol, piperine and glabridin, were selected out of seventy-four identified makers for the quantitative analysis in DSV formulation. Validation of the HPLC method was evaluated by its linearity, precision, and accuracy tests as per the International Council of Harmonization (ICH) guidelines. Calibration curves for the eleven marker compounds showed good linear regression (r2 > 0.999). The relative standard deviation (RSD) value of intraday and interday precision tests were within the prescribed limits. The accuracy test results ranged from 92.75% to 100.13%. Thus, the present inclusive approach is first of its kind employing multi-chromatographic platforms for identification and quantification of the marker components in DSV, which could be applied for routine standardization of DSV and other related formulations.

Calcio-Herbal Medicine Divya-Swasari-Vati Ameliorates SARS-CoV-2 Spike Protein-Induced Pathological Features and Inflammation in Humanized Zebrafish Model by Moderating IL-6 and TNF-α Cytokines.

PURPOSE: Severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) infection has grown into a pandemic and without a specific cure, disease management is the need of the hour through symptomatic interventions. Studies with severe acute respiratory syndrome-coronavirus (SARS-CoV) have highlighted the role of herbal medicines either in combination with antiviral drugs or by themselves in curtailing the severity of infection and associated inflammation. Divya-Swasari-Vati is an Indian ayurvedic formulation used in the treatment of chronic cough and lung inflammation, which is one of the first symptoms of SARS-CoV-2 infections. METHODS: In this study, we used a A549 cell xenotransplant in the swim bladder of zebrafish and modeled the SARS-CoV-2 infection by injecting the fish with a recombinant spike protein. The different groups were given normal feed or feed mixed with either dexamethasone (as the control drug) or Divya-Swasari-Vati. The changes in behavioral fever, infiltration of pro-inflammatory cells in the swim bladder, degeneration or presence of necrotic cells in the kidney, and gene expression of pro-inflammatory cytokines were studied to determine the rescue of the diseased phenotype. RESULTS: Challenge with the spike protein caused changes in the swim bladder cytology with infiltrating pro-inflammatory cells, skin hemorrhage, and increase in behavioral fever. This was also accompanied by increased mortality of the disease control fish. Treatment with Divya-Swasari-Vati reversed most of the disease symptoms including damage to the kidney glomerulocytes, and complete reversal of behavioral fever. Dexamethasone, used as a comparator, was only able to partly rescue the behavioral fever phenotype. Divya-Swasari-Vati also suppressed the pro-inflammatory cytokines, IL-6 and TNF-α, levels in a dose-dependent manner, under in vivo and in vitro conditions. CONCLUSION: The study showed that the A549 xenotransplanted zebrafish injected with the recombinant spike protein of SARS-CoV-2 is an efficient model for the disease; and treatment with Divya-Swasari-Vati medicine rescued most of the inflammatory damage caused by the viral spike protein while increasing survival of the experimental fish.