**3. Discussion**

The medications used for TB were beneficial in the past but, at present, the insufficiency of these medications has raised the need for the identification and discovery of novel therapeutics. *A. aspera* is a widely known medicinal plant with various anti-bacterial activities. The plant is famous for its contents of alkaloids, saponins, carbohydrates, glycosides, flavonoids, tannins, and triterpenoids [11,12]. *C. gigantea* is also a traditional medicinal herb, used to cure common ailments, such as fevers, cough, cold, asthma etc. [20]. The root bark is an expectorant, febrifuge, anthelmintic, depurative, and laxative. Asthma, bronchitis, and dyspepsia are treated with the powdered root. Paralysis, arthralgia, swellings, and intermittent fevers can all be treated with the leaves. The flowers have a range of properties, being bitter, digestive, astringent, stomachic, anthelmintic, and tonic [22] and make a well-known homoeopathic remedy [22]. *C. procera* is a shrub with purgative, anthelmintic, anticoagulant, palliative (for respiratory and blood pressure disorders), antipyretic, analgesic and neuromuscular blocking properties [24]. The family members of this plant are high in cardiac glycosides [25,26].

The aerial and root parts of *A. aspera* and the flowers of *C. gigantea* and *C. procera* are widely grown, and are commonly used herbal medications [33]. The plants are distinguished through the different appearances of their flowers [34]. The plant parts were used to prepare the extracts in hexane, ethyl acetate, ethanol, methanol and water. The phytochemical analysis showed the presence of flavonoids, alkaloids, phenol, anthraquinone, terpenoids, tannins, steroids, saponins, carbohydrates, glycosides in the extracts [35]. Many flavonoids are key components of medicinal plants and are employed in the regulation of inflammation and cancer prevention, due to their ubiquity in the human diet [40]. The highest total flavonoid content in *A. aspera* was found in the root ethyl acetate fraction (103.76 <sup>±</sup> 14.8 mg RE g−<sup>1</sup> ) and the aerial ethanol fraction (97.61 <sup>±</sup> 10.65 mg RE g−<sup>1</sup> ). The highest TFC was found in the ethyl acetate fraction of *C. gigantea* ash (184.28 <sup>±</sup> 11.64 mg RE g−<sup>1</sup> ) (Figure 1). The total polyphenolic content of the plant aerial and root parts was analyzed in water, methanol, ethyl acetate, ethanol and hexane fractions. Polyphenols are important components of plants as they defend plants from reactive oxygen species [39]. The highest TPC was found in the ethyl acetate fraction i.e., 15.1 <sup>±</sup> 0.36 mgGAE g−<sup>1</sup> and 18.27 <sup>±</sup> 0.56 mgGAE g−<sup>1</sup> in the aerial and root parts of *A. aspera*, respectively (Figure 2). The flavonoid and phenolic content of the extracts was higher in the ash of *C. gigantea*, indicating heating the extracts caused solubilization of these organic compounds. *M. tuberculosis* H37Rv ATCC 27294, *M. fortuitum* ATCC 6841, *M. abscessus* ATCC 19977 and *M. chelonae* ATCC 35752 were used to determine the minimum inhibitory concentrations of the extracts against the mycobacterial strains. *A. aspera*'s aerial ethyl acetate fraction (3) and *C. gigantea*'s flower ash ethyl acetate fraction (23) was found to be active against the *M. tuberculosis* H37Rv ATCC 27294 strain with an MIC value of 64 mg/L. The non-tuberculous bacteria showed the maximum growth against all extracted compounds (Table S1). Anti-tuberculosis drugs, such as isoniazid, rifampicin, streptomycin, and ethambutol, with MIC values of 0.03 mg/L, 0.3 mg/L, 1 mg/L, and 1 mg/L, respectively, were used as positive control [41,42].

As the ethyl acetate fraction of *A. aspera* and *C. gigantea* plants were the most effective fractions in containing the flavonoid, phenolic content and bacteriostatic effects against *M. tuberculosis* H37Rv, it was further analyzed by GC-MS for the detection of various phytoconstituents (Figure 3). The presence of the compounds, as confirmed in the GC-MS analysis, are listed in Tables S3 and S4.

The mycobacterial proteins were categorized into virulence, detoxification, adaptation (VDA) categories, and the proteins having known structural information. The VDA category is a vast family of proteins that participates in maintaining mycobacterial metabolism and, therefore, targeting these proteins would be an asset in targeting mycobacterial cells [43]. After the evaluation of the complete mycobacterial database, 238 proteins were found to belong to the VDA category and 135 proteins had available structural information in the RSCB-PDB databank. In Table 3, a counteractive study on these proteins led to the determination of 10 proteins, which were *Rv1477*, *Rv1495*, *Rv1566*, *Rv2801c*, *Rv2010*, *Rv2623, Rv0554*, *Rv1636*, *Rv2549c*, and *Rv2757c* [44]. *Rv0554* is an integral part of the menaquinone regulatory operon in Mycobacterium. Menaquinone is a crucial factor in the mycobacterial electron transport chain. There are many genes that were identified, which participate in the menaquinone biosynthesis pathway and the operon is present between *Rv0534c* and *Rv0558*. A gene known as *yfbB* in *E. coli* is known as *menH* and *Rv0554* in *M. tuberculosis* and was predicted to encode an enzyme with a similar function to *menH*. Structurally, it was categorized in the alpha beta hydrolase fold of *E. coli* and is the only protein which is located nearest to the *M. tuberculosis* menaquinone synthesis pathway [45]. *Rv1477* is a mycobacterial resuscitation promoting factor interacting protein (*RipA*) that participates in the cleavage of peptide cross linkages between peptidoglycans similar to other cleavage enzymes, such as *OwlT* and *Spr* in *B. subtilis* and *E. coli*, respectively. *Rv1477* was found to colocalize at bacterial septa with resuscitation promoting factor B (*RpfB*). This protein is important for mycobacterial growth as the depletion strains of this protein in *M. smegmatis* showed abnormal phenotype and decreased growth pattern and, therefore, this protein is a

wonderful candidate for drug target strategies [46]. *Rv1495* is designated as *mazEF*, which is a subcomponent of type II toxin antitoxin system in mycobacteria. Out of 7 homologs of *mazF* which are identified in *M. tuberculosis*, four homologs comprise endoribonuclease activity. *MazF* acts as a toxin and it recognizes and cleaves to the intracellular RNA sequence in a ribosome-independent manner. As it is a sequence specific ribonuclease, it inhibits translation with a lesser degree than other non-specific toxins. Previous studies have also reported extracellular death factor functioning in mediated quorum sensing [47]. *Rv1566c* (*RipD*) is a predicted peptidoglycan specific peptidase of NlpC/p60 family. The unusual peptide linkages of *Mycobacterium* i.e., L-D and D-D and other iso-peptide linkages makes the peptide resilient to cleavage by mycobacterial peptidases. However, during the cell division process, the mycobacterial cells produce specific peptidases that weaken these linkages and help in generation of daughter cells. *Rv1566c* is such a specific peptidase. The known specific peptidases in mycobacterium are *RipA* and *RipB*, which cleave the peptide stem between D-glutamic acid and meso-Dap residue. *Rv1566c* containing peptidoglycan specific peptidase domain has 52% and 51% similarity with the *RipB* and *RipA* proteins, respectively. The *Rv1566c* is the first example of a peptidase domain, which binds to the peptidoglycan in a non-catalytic manner, and this feature is specific to mycobacteria only [48]. *Rv1636* is a universal stress protein (USP) in *M. tuberculosis*. In *M. tuberculosis* it is classified into class I USP, based on the presence of only a single conserved domain of USP which is of similar size of *UspA*. Rv1636 amino acid sequence contains the GXXG-9X-G-S/T conserved domain. The exact role of *Rv1636* in stress condition is yet to be detected but it was predicted that this protein might exclusively be expressed in hypoxia and other stress conditions. One interesting feature of *Rv1636* is its cAMP binding activity. The significant fraction of intracellular cAMP bound to *Rv1636* regulates the signaling associated with the cAMP molecule [49]. *Rv2010* of *M. tuberculosis* codes for *VapC*-15 toxin. These proteins belong to the PIN domain family proteins and contain ribonuclease activity. *VapC* toxin is deleterious to cells, but its effect gets neutralized by *VapB* antitoxin. This protein works in a similar manner to T4 RNase and Mja FEN-1 endonuclease. *VapBC* complex is a significant toxin–antitoxin system and an important participator in mycobacterial metabolism [50]. *Rv2549c* is designated as *VapC20* of *M. tuberculosis* and along *VapB* it forms the *VapBC* toxin–antitoxin complex. *VapC20* interacts with its cognate partner *VapB20* to form a stable complex. Both proteins in their individual states are present in dimer states, which form stable homo-tetramers or homo-octamers upon interaction. *Rv2623* is a universal stress protein of *M. tuberculosis* that helps mycobacteria in different stress conditions. This protein is also important for mycobacterial growth and persistence. *Rv2623* protein is a highly induced protein of mycobacteria in response to stress conditions, such as hypoxia and nitrosative stress, which the bacteria face in infected host cells. Apart from its role as a USP, this protein also has the ability to bind ATP. *Rv2623* also interacts with *Rv1747*, which is an ABC transporter protein and helps in exporting lipo-oligosaccharides to negatively regulate mycobacterial growth [52]. *Rv2757c* of *M. tuberculosis* codes for *VapC*-21 toxin. These proteins belong to the PIN domain family of proteins and have ribonuclease activity. *VapC* toxin is deleterious to cells, but its effects become neutralized by *VapB* antitoxin. *VapC21* is similar in function to the other known *VapC* proteins of *M. tuberculosis* [53]. *Rv2801c* encodes for *MazF*-mt1 protein of *M. tuberculosis*. *MazF* is a known component of *MazEF* toxin–antitoxin system in many prokaryotic cells. *MazEF* is part of the TA system that forms persister cells of *M. tuberculosis*. *M. tuberculosis* has ten such *MazEF* proteins from numbers 1 to 10 and all *MazF* proteins are RNases. *MazF*-mt1 specifically cleaves mRNA. *MazF* family members play important roles in antibiotic and immune tolerance mechanisms [54].

In silico characterization was performed to determine the secondary structure, polarity, instability index and localization of all the selected 10 proteins. *Rv1477*, *Rv1566c*, *Rv2010*, and *Rv2623* were found to be unstable proteins, based on their instability indices, which were based on protein sequence information. *Rv2010* and *Rv2623* were categorized into polar proteins and, therefore, these proteins might be more vulnerable towards surrounding nature (Figure 4a) [56]. Rv0554 was also found to be a non-essential gene for mycobacterial

growth, but it is listed as an important virulence factor that codes for a peroxidase. Most of the proteins participated in mycobacterial metabolism (Figure 4b) [57]. Rv1495 was found to be a lipid anchored protein, and it is a probable toxin (MazF4). Rv1636, Rv2010, Rv2549c, Rv2623, Rv2757c are cytoplasmic proteins and, therefore, components of the secretory system of mycobacteria (Figure 4c) [58]. The secondary structure analysis showed that most of the regions of the proteins were comprised of an alpha helical pattern, which confirmed the stable structural state of the proteins (Figure 4d) [59].

The phylogenetic analysis was performed by Mega11 program and showed close proximity of Rv1636 of *M. tuberculosis* H37Rv and *Mycobacterium bovis* Rv1662, where both code for USP TB15.3. *M. tuberculosis* H37Rv Rv1636, *M. marinum* 2440, *Mycobacterium bovis* 1662, *M. leprae* 1390, *M. smegmatis* mc2155 3811 showed significant identity percentages among their USP domain (Figure 5) [60]. The protein interaction was determined by STRING server and properties like betweenness, closeness, radiality, stress and degree were used as the parameters for the interactive analysis. The analysis configured three proteins Rv1636, Rv2623 and Rv1566 in betweenness, closeness, radiality, and degree category, whereas Rv1636 was the only protein which was highlighted in the stress parameter (Figure 6) [61,62].

Molecular docking studies were executed to determine the highly interactive compound for their binding capacity with the proteins. The structure of the compound is mentioned in (Figure S3). *β*-amyrin (PubChem CID: 225689) was found to have higher binding free energies against *Rv1636*, *Rv1566*, *Rv2549c*, and *Rv1495* proteins (Figure 7). *β*-amyrin strongly integrated with most of the proteins and the interaction involved the pi-alkyl bonds, and hydrogen bonds (Table S7, Figure 8). The ADMET properties confirmed that shortlisted and highly interactive compounds can be a putative drug candidate, as they passed all the qualifying parameters (Tables 5 and 6) [70–72]. Most of the proteins showed high and significant binding affinity with *β*-amyrin, and, thus, it was selected for further analysis [77].

*Rv1636* protein was found to be the top candidate in all the examinations (interaction, docking, biological process etc.), and, therefore, this protein was further analyzed for its stability with *β*-amyrin by molecular dynamic simulation. The RMSD plot showed that the protein and its complex were stable in the initial period till 45 ns, but started to experience a little destabilizing after 45 ns (Figure 9a). This destabilization might be due to the change in the protein structure, as in the instability index *Rv1636* was found to be an unstable protein. The RMSF plot showed instability in most of the residues, whereas the complex showed lesser fluctuation as compared to the protein alone (Figure 9b) [73–75]. The SASA result suggested that the binding of *β*-amyrin to the protein stabilizes the complex (Figure 9d) and this stability was further confirmed by Rg plot (Figure 9c) and FEL, which also confirmed the compactness and folding of the protein in complex form with *β*-amyrin (Figure 9e,f) [76].
