HR-LCMS Positive and Negative mode chromatograms (Figure 2a and 2b) of SBRK were compared with the spectra in the NIST database and total 110 botanicals were identified (Supplementary table S1). All identified botanicals were screened for ADME using parameters like HIA, and drug-likeness, 44 compounds were qualified through predefined criteria and are given in table 2.
Targets and Network Analysis of SBAK Against Immunity enhancer and pulmonary protection.
The 44 active compounds SMILES were plotted into SwissTargetPrediction to obtain the potential targets of each compound. 109 compound-related targets were collected and after removing the duplicates total 62 targets were finalized shown in table 2.
Besides, a total of 1341 targets related to immunity enhancers having a score ≥ 5.0 (Supplementary Table S2) and 1158 targets related to pulmonary protection having a score ≥ 5.0 (Supplementary Table S3) were retrieved from the GeneCards database. It also found 20 overlapping gene targets (out of 1340) for immunity enhancer-related targets and 24 overlapping gene targets (out of 1158) for pulmonary protection-related targets (Supplementary Table S4). A separate compound–target network of SBAK against immunity enhancer and pulmonary protection was constructed and visualized by importing bioactive and overlapping targets for immunity enhancer and pulmonary protection into software Cytoscape 3.9.1. SBAK immunity enhancer network contained 29 nodes and 30 edges (Figure 3) and Genistein, Luteolin and Kaempferol were the top 3 ingredient nodes linked to more targets while CA2 (Carbonic Anhydrase 2) is the top crucial target of SBAK for immunity enhancer activity ranked by parameters of degree, betweenness centrality, and closeness centrality (Supplementary Table S5), whereas SBAK pulmonary protection network contained 32 nodes and 35 edges (Figure 4) and Luteolin, Kaempferol, 4-Hydroxycinnamic acid and Genistein were the top ingredient nodes linked to more targets and CA2 (Carbonic Anhydrase 2) and ESR2 (Estrogen Receptor 2) is top crucial targets of SBAK for immunity enhancer activity ranked by parameters of degree, betweenness centrality, and closeness centrality (Supplementary Table S6).
Gene Ontology and KEGG Enrichment Analyses
GO and KEGG enrichment analyses were performed on the above-mentioned 20 targets for immunity enhancing activity and 24 targets for pulmonary protection activity of SBAK (Supplementary Table S7 and S8 respectively). It showed that these potential targets for immunity enhancer activity were mainly located in vesicle (GO:0031982), extracellular space (GO:0005615), vesicle lumen (GO:0031983), cytoplasmic vesicle (GO:0031410), intracellular vesicle (GO:0097708), regulating molecular functions like transition metal ion binding (GO:0046914), identical protein binding (GO:0042802), ion binding (GO:0043167), nitric-oxide synthase regulator activity (GO:0030235), RNA polymerase II-specific DNA-binding transcription (GO:0061629), and participated in biological processes like response to oxidative stress (GO:0006979), response to organo-nitrogen and nitrogen compound (GO:0010243, GO:1901698), response to organic cyclic compound (GO:0014070), cell population proliferation (GO:0008283), response to chemical (GO:0042221), results of KEGG analysis for immunity enhancer activity indicated that 20 overlapping genes of Syrup Bal Ayush Kwatha were significantly enriched in 3 signaling pathways (p < 0.05), of which Endocrine resistance (KEGG:01522), Estrogen signaling pathway (KEGG:04915) were involved as shown in Figure 5a and 5b.
Potential targets for pulmonary protection activity were mainly located in cyclin B1-CDK1 complex (GO:0097125), extracellular space (GO:0005615), vesicle lumen (GO:0031983), extracellular region (GO:0005576), mitochondrion (GO:0005739) regulating molecular functions like transition metal ion binding (GO:0046914), ion binding (GO:0043167), nuclear estrogen receptor activity (GO:0030284), estrogen response element binding (GO:0034056), identical protein binding (GO:0042802) and participated in biological processes like response to oxygen-containing compound and nitrogen compound (GO:1901700, GO:1901698), cellular response to oxygen-containing compound (GO:1901701), response to organonitrogen compound (GO:0010243), response to amyloid-beta (GO:1904645) results of KEGG analysis for pulmonary protection activity indicated that 24 overlapping genes of Syrup Bal Ayush Kwatha were significantly enriched in 3 signaling pathways (p < 0.05) of which Endocrine resistance pathway (KEGG:01522), Estrogen signaling pathway (KEGG:04915) were involved as shown in Figure 6a and 6b.