Indian Herb-Derived Phytoconstituent-Based Antiviral, Antimicrobial and Antifungal Formulation: An Oral Rinse Candidate for Oral Hygiene and the Potential Prevention of COVID-19 Outbreaks

Abstract

Outbreaks of emerging infectious diseases continue to challenge human health. Novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has triggered a global coronavirus pandemic, known as COVID-19. Multiple variants of SARS-CoV-2 virus are circulating, thus raising questions with respect to the effectiveness of different lines of treatment, such as vaccines and antiviral drugs. To find the appropriate prevention/treatment, 21 plant-based ingredients (Glycyrrhizin, Withanone, Aloe-emodin, Rhein, Emodin, Chrysophanol, Physcion, Kaempferol, Progallin A, Gallic acid, Naringin, Quercetin, Luteolin, and Apigenin) having antiviral, antibacterial and antifungal properties were identified. We pseudo-typed SARS-CoV-2 on a lentiviral vector plasmid and tested the impact of five different herbal formulations in mammalian HEK293T cells. Viral inactivation assay showed that the natural extracts in a herb-derived phytoconstituent-based formulation, BITS-003, comprising Bacopa monnieri, Glycyerrhiza glabra, Asparagus racemosus-wild, and Nigella sativa had strong virucidal properties, inactivating enveloped viruses from 2log10 (or 99%) to >4log10 (or 99.99%). Moreover, bacterial and yeast cells treated with BITS-003 displayed reduced growth. Topical use of the formulation as a mouthwash/gargle could be effective in reducing symptoms of respiratory viral infections, with the potential to decrease the viral load in the buccal/oral cavity. This may inhibit the coronavirus spreading to the lungs of infected persons and at the same time may reduce the risk of viral transmission to other susceptible persons through micro-droplets originating from the oral cavity of the infected person.

Keywords: COVID-19; SARS-CoV-2; antiviral agents; coronavirus; gargle; horizontal transmission; mouthwash; natural herb; phytoconstituents; shedding.

Figure 1

The phytoconstituents of 14 out of 17 selected herbal ingredients used in the preparation of oral herbal mouthwash. (A) Total Polyphenols, (B) Total Flavonoids (C) Total Saponins. MT-1: Oryza Sativa; MT-2: Phaseolus Munga L; MT-3: Asparagus racemosus; MT-4: Solanum tuberosum; MT-5: Glycyrrhiza glabra; MT-6: Withania somnifera; MT-7: Zingiber officinale; MT-8: Bacopa monnieri; MT-9: Rheum palmatum; MT-10: Rosmarinus officinalis; MT-11: Capsicum annuum; MT-12: Colocasia antiquorum Schott; MT-13: Trigonella foenum graceum and MT-14: Nigella Sativa. (D) Phytoconstituents of herb-based formulation BITS-003. Y-axis represents concentration of the phytoconstituent in microgram/ml. Note: Extracts of Ananas Comosus, Morus nigra and Utica dioica could not be tested due to either background color of the extract or interference with reactants and subsequent precipitation.

Figure 2

Representation of maximum non-cytotoxic concentration of BIT-003 natural extract formulation in HEK293TN cells. (a) Raw data analysis of the relative light units (RLU). (b) Cell viability analysis.

Figure 3

Viral inactivation assay using natural extracts (flow cytometry analysis): Fluorescence Activated Cell Sorting (FACS) analysis of cells transduced using lentiviruses treated with natural extracts at 1:30 dilution for 1 hour. Green Fluorescent Protein (GFP)–positive cells (M4-2) are an indication of biologically active viral particles. A decrease in GFP-positive cells is a direct indication of lentivirus inactivation. A validated chemical (remdesivir) was used as a positive control for viral inactivation.

Figure 4

Viral inactivation assay (graphical representation). Bar graph represents quantification of GFP cells at indicated conditions. Virus infectivity observed is 30.16% in 1X PBS-treated samples, whereas natural extract–treated samples (1:30 dilution for 10 min treatment) showed between 0.00% and 0.03% infectivity. Final strength of the formulation in the cell culture medium is at 1:240 dilution.

Figure 5

Microscope imaging at 10× magnification (Scale bar: 100 µ) and FACS analysis of cells transduced using lentiviruses treated with BITS-003 at 1:30 dilution for 10 minutes. Decrease in GFP-positive cells is a direct indication of lentivirus inactivation.

Figure 6

(a) The synergistic antibacterial activity of the herb-based formulation. Well C: control; Well 1: Asparagus rasemosus at 1:40 dilution; Well 2: Asparagus rasemosus at 1:80 dilution; Well 3: Glycerrhiza glabra at 1:40 dilution; Well 4: Glycerrhiza glabra at 1:80 dilution; Well 5: Bacopa monnieri at 1:40 dilution; Well 6: Bacopa monnieri at 1:80 dilution; Well 7: Rosmarinus officinalis at 1:40 dilution; Well 8: Rosmarinus officinalis at 1:80 dilution; Well 9: Nigella sativa at 1:40 dilution; Well 10: Nigella sativa at 1:80 dilution; Well 11: herbal composition BITS-003 at 1:80 dilution. No bacterial growth was seen at 1:80 dilution in bacterial cells treated with the herb-based formulation. (b) The antifungal activity of the herb-based formulation BITS-003 against Saccharomyces cerevisiae. The herb-based formulation displayed potent antifungal activity at 1:20, 1:40 and 1:80 dilutions. Control: Saccharomyces cerevisiae grown in the absence of herb-based formulation; 1:20 MW: Saccharomyces cerevisiae grown in the presence of 1:20 diluted herb-based formulation; 1:40 MW: Saccharomyces cerevisiae grown in the presence of 1:40 diluted herb-based formulation; 1:80 MW: Saccharomyces cerevisiae grown in the presence of 1:80 diluted herb-based formulation. Complete inhibition of growth was seen for yeast cells treated with the herb-based formulation.