Active constituents and mechanisms of Respiratory Detox Shot, a traditional Chinese medicine prescription, for COVID-19 control and prevention: Network-molecular docking-LC-MSE analysis

Abstract

Objective: Lung-toxin Dispelling Formula No. 1, referred to as Respiratory Detox Shot (RDS), was developed based on a classical prescription of traditional Chinese medicine (TCM) and the theoretical understanding of herbal properties within TCM. Therapeutic benefits of using RDS for both disease control and prevention, in the effort to contain the coronavirus disease 2019 (COVID-19), have been shown. However, the biochemically active constituents of RDS and their mechanisms of action are still unclear. The goal of the present study is to clarify the material foundation and action mechanism of RDS.

Methods: To conduct an analysis of RDS, an integrative analytical platform was constructed, including target prediction, protein-protein interaction (PPI) network, and cluster analysis; further, the hub genes involved in the disease-related pathways were identified, and the their corresponding compounds were used for in vitro validation of molecular docking predictions. The presence of these validated compounds was also measured in samples of the RDS formula to quantify the abundance of the biochemically active constituents. In our network pharmacological study, a total of 26 bioinformatic programs and databases were used, and six networks, covering the entire Zang-fu viscera, were constructed to comprehensively analyze the intricate connections among the compounds-targets-disease pathways-meridians of RDS.

Results: For all 1071 known chemical constituents of the nine ingredients in RDS, identified from established TCM databases, 157 passed drug-likeness screening and led to 339 predicted targets in the constituent-target network. Forty-two hub genes with core regulatory effects were extracted from the PPI network, and 134 compounds and 29 crucial disease pathways were implicated in the target-constituent-disease network. Twelve disease pathways attributed to the Lung-Large Intestine meridians, with six and five attributed to the Kidney-Urinary Bladder and Stomach-Spleen meridians, respectively. One-hundred and eighteen candidate constituents showed a high binding affinity with SARS-coronavirus-2 3-chymotrypsin-like protease (3CL^pro), as indicated by molecular docking using computational pattern recognition. The in vitro activity of 22 chemical constituents of RDS was validated using the 3CL^pro inhibition assay. Finally, using liquid chromatography mass spectrometry in data-independent analysis mode, the presence of seven out of these 22 constituents was confirmed and validated in an aqueous decoction of RDS, using reference standards in both non-targeted and targeted approaches.

Conclusion: RDS acts primarily in the Lung-Large Intestine, Kidney-Urinary Bladder and Stomach-Spleen meridians, with other Zang-fu viscera strategically covered by all nine ingredients. In the context of TCM meridian theory, the multiple components and targets of RDS contribute to RDS's dual effects of health-strengthening and pathogen-eliminating. This results in general therapeutic effects for early COVID-19 control and prevention.

Keywords: 3C-like protease; COVID-19; Lung-toxin Dispelling Formula No. 1; Meridian tropism; Molecular docking; Network pharmacology.

Fig. 1

The ingredient-property-flavor-meridian tropism network of the RDS prescription. The network shows the interconnection of the nine ingredients in RDS and corresponding information about their TCM properties (hot, cool, warm, cold and neutral), flavors (sour, bitter, sweet, pungent and salty) and meridian tropisms (Liver, Gallbladder, Heart, Small Intestine, Spleen, Stomach, Lung, Large Intestine, Kidney and Urinary Bladder). The color scheme of the nodes and edges corresponds with the five elements and colors in TCM theory: green, wood; red, fire; yellow, earth; white, gold; black, water. Blue: the nine ingredients in RDS. ASA: Armeniacae Semen Amarum (Kuxingren); FSF: Forsythiae Fructus (Lianqiao); GLRR: Glycyrrhizae Radix et Rhizoma (Gancao); GS: Gleditsiae Spina (Zaojiaoci); GSRR: Ginseng Radix et Rhizoma (Renshen); LJF: Lonicerae Japonicae Flos (Jinyinhua); RDS: Respiratory Detox Shot; SPH: Schizonepetae Herba (Jingjie); SR: Scrophulariae Radix (Xuanshen); TCM: traditional Chinese medicine; VN: Nidus Vespae (Fengfang).

Fig. 2

RDS ingredient-constituent-target network. After mapping onto the HINT background network, the RDS network showed interconnections among the 148 predicted targets of RDS, the corresponding 155 constituents, and the nine ingredients. Diamond, RDS prescription; triangles, TCM ingredients in RDS; hexagons, chemical constituents TCM ingredients; arrowhead, predicted targets of RDS. ASA: Armeniacae Semen Amarum (Kuxingren); FSF: Forsythiae Fructus (Lianqiao); GLRR: Glycyrrhizae Radix et Rhizoma (Gancao); GS: Gleditsiae Spina (Zaojiaoci); GSRR: Ginseng Radix et Rhizoma (Renshen); HINT: High-quality INTeractomes; LJF: Lonicerae Japonicae Flos (Jinyinhua); RDS: Respiratory Detox Shot; SPH: Schizonepetae Herba (Jingjie); SR: Scrophulariae Radix (Xuanshen); TCM: traditional Chinese medicine; VN: Nidus Vespae (Fengfang).

Fig. 3

The protein–protein interaction network showing the 148 predicted RDS targets. The size of each node is proportional to the node degree. The color depth of the node is proportional to the regulated strength score (RSS) of the target. The targets with the highest RSS were heat-shock protein 90 kD α (cytosolic) class A member 1 (HSP90AA1), estrogen receptor 1 (ESR1), androgen receptor (AR), peroxisome proliferator-activated receptor γ (PPARG) and nuclear receptor coactivator 2 (NCOA2). Forty-two hub genes were circled in red line (solid and dotted line) and 11 genes with an RSS >30 were circled in red dotted line. RDS: Respiratory Detox Shot.

Fig. 4

Cluster analysis and protein-complex recognition of the RDS target network. Cluster analysis was performed on the RDS target network to reveal its highly interconnected regions. Six clusters were identified, indicating their closely related biological functions. RDS: Respiratory Detox Shot.

Fig. 5

Kyoto Encyclopedia of Genes and Genomes pathway enrichment of the 42 RDS hub genes. Forty-two hub genes were analyzed for functional enrichment analysis using the STRING database, with FDR set at 1 × 10⁻⁶. A total of 39 pathways were identified and were mainly related to biological functions such as signal transduction, transport and catabolism and cell growth and death. Symbol size is proportional to the number of genes included. FDR: false discovery rate; RDS: Respiratory Detox Shot.

Fig. 6

The intricate network includes the disease pathways, their corresponding meridian tropism, RDS hub genes, the corresponding chemical constituents and traditional Chinese medicine ingredients. The circle in the center described the disease pathways and their corresponding meridian tropism. Nearly half of the disease pathways were related to the Lung meridian (white quadrangles), followed by the Stomach (yellow quadrangles) and Kidney meridians (black quadrangles). They all matched the primary therapeutic effects and the underlying Zang-fu viscera theory of the RDS prescription. ASA: Armeniacae Semen Amarum (Kuxingren); FSF: Forsythiae Fructus (Lianqiao); GLRR: Glycyrrhizae Radix et Rhizoma (Gancao); GS: Gleditsiae Spina (Zaojiaoci); GSRR: Ginseng Radix et Rhizoma (Renshen); LJF: Lonicerae Japonicae Flos (Jinyinhua); RDS: Respiratory Detox Shot; SPH: Schizonepetae Herba (Jingjie); SR: Scrophulariae Radix (Xuanshen); VN: Nidus Vespae (Fengfang).

Fig. 7

RDS candidate compounds and representative results of molecular docking with severe acute respiratory syndrome coronavirus 2 3CL^pro (6LU7). Schematic diagrams demonstrated the 3CL^pro binding sites and proximate affinity of candidate compounds in RDS and valganciclovir, an antiviral drug approved by the US Food and Drug Administration. Black dots: carbon atoms; blue dots: nitrogen atoms; red dots: oxygen atoms; green dotted lines: hydrogen bonds; red combs: amino acid residues. 3CL^pro: 3-chymotrypsin-like protease; RDS: Respiratory Detox Shot.

Fig. 8

Representative base ion chromatograms of Respiratory Detox Shot from the ultra high-performance liquid chromatography/quadrupole time-of-flight mass spectrometry analysis. Base ion chromatograms of the positive ions (upper) and the negative ions (lower) were obtained for candidate compounds. The X-axis represents retention time (minutes) of the chromatogram. The Y-axis represents intensity (counts) of peaks in the chromatogram. BPI: base peak intensity; Rt: retention time.