Extraction and In-Silico Evaluation of Bioactive Compounds from Pontederia crassipes as Potential Anti-Tubercular Agent

Figure 1.Pontederia crassipes (water hyacinth) growing in its natural aquatic habitat showing the characteristic vegetative and flowering morphology.

Geography Distribution

Habitat- Water hyacinth is native to Amazon basin of South America but presently it was extensively spread in tropical and subtropical part of world. It is now found countries all over the world in various regions like Asia, Africa, and North America. In India, Water hyacinth is commonly observed in freshwater bodies such as ponds, lakes, rivers and irrigation canals. It develops well in warm climate with high nutrient availability in water. It prefers stagnant and slowmoving water and can rapidly increase in volume if proper condition exist, often form dense mats covering surface of water which affect the aquatic life and water quality. In many places, it is seen as invasive in bodies of water.⁹

Tuberculosis

Tuberculosis (TB) is a chronic and granulomatous infectious disease which is caused by the bacillus called Mycobacterium tuberculosis. This infection mainly affects the lungs called pulmonary tuberculosis, but it can also affect extrapulmonary areas such as lymph nodes, pleura, bones, joints, kidneys, meninges etc¹⁰. Tuberculosis is one of the most important infectious diseases in the world. Tuberculosis exists in two forms: latent and active. In latent tuberculosis infection (LTBI), people stay a bacteria in the body called Mycobacterium tuberculosis, but do not show any symptoms and do not spread the disease. However, bacteria can become inactive for many years in the body and only become active later, when their immune system is weak or fails. On the other hand, active tuberculosis shows clear and clear symptoms and can spread to other people, especially if it is pulmonary¹¹.

The pathological feature of tuberculosis is the formation of a granuloma, an organized aggregate of immune cells that develops to contain and control infection caused by Mycobacterium tuberculosis. Tuberculous granulomas are composed of various cell types, including macrophages, epithelioid cells, multinucleated giant cells, and lymphocytes¹². A characteristic feature of these granulomas is caseous necrosis, in which the central region undergoes tissue destruction and develops a soft, cheese-like appearance¹³. Although granuloma formation plays a protective role by restricting bacterial spread, it also provides a microenvironment that allows M. tuberculosis to persist in a dormant state, contributing to latent infection and the potential for disease reactivation^14.

Tuberculosis is an airborne infectious disease that is primarily transmitted through the inhalation of aerosolized droplets containing Mycobacterium tuberculosis. Transmission occurs when individuals with active pulmonary tuberculosis cough, sneeze, speak, or sing, releasing infectious particles into the air that can remain suspended for prolonged periods and be inhaled by susceptible individuals¹⁵. Exposure to even a small number of bacilli may result in infection and contribute to disease transmission¹⁶. Despite global efforts to control tuberculosis, it remains a major public health problem due to factors such as poverty, malnutrition, overcrowding, and poor living conditions¹⁷. In addition, conditions that impair the immune system, particularly human immunodeficiency virus (HIV) infection, significantly increase the risk of developing active tuberculosis. Individuals with weakened immune responses are less able to contain M. tuberculosis infection, resulting in an increased likelihood of progression from latent infection to active disease ¹⁶.

MATERIALS AND METHODS

Chemicals, Materials and Reagents

Distilled water, Methanol, Ethanol, Hydrochloric acid, Concentrated sulphuric acid, Analytical weighing balance, Soxhlet apparatus / Maceration apparatus, Beakers, Conical flasks, Measuring cylinders, Pipettes and droppers, Glass rods, Funnel and filter paper, Watch glass, Magnetic stirrer.

Collection and authentication of plant material

Fresh flowers of Pontederia crassipes were collected from a pond in Guntur during the month of March. The collected plant material was cleaned to remove adhering impurities and authenticated by a taxonomist from Acharya Nagarjuna University.

Preparation of ethanolic extract of pontederia crassipes flower

Fresh flowers of Pontederia crassipes were collected and washed thoroughly to remove dust and other adhering impurities. The petals were separated manually and weighed accurately. About 150 g of fresh flower petals were taken for extraction. The petals were immersed in 150 mL of ethanol in a clean glass container and kept in a dark place for three days to facilitate extraction of phytoconstituents. After completion of the extraction process, the extract was filtered using filter paper to remove insoluble materials and plant debris. The filtrate obtained was initially shade dried and then subjected to sun drying for further evaporation of the solvent. The partially concentrated extract was later heated gently using a heating mantle to obtain a concentrated solid mass. The dried mass obtained was triturated using a mortar and pestle to produce a fine powder. The prepared ethanolic extract powder was stored in an airtight container and used for preliminary phytochemical screening and in vitro anti-tubercular activity studies.

Figure 2. Freshly collected flowers of Pontederia crassipes used for phytochemical and biological investigations.

Preliminary phytochemical screening of Water Hyacinth

The ethanolic and aqueous extracts of Water Hyacinth were subjected to preliminary phytochemical screening using standard qualitative tests to detect the presence of various secondary metabolites¹⁸. The results of the screening, indicating the presence or absence of each phytoconstituent, are presented in Table below.

Table 1. Preliminary phytochemical screening of ethanolic and aqueous extracts of Pontederia crassipes flowers.

+ = present, − = absent

IN SILICO STUDIES

Selection and Preparation of Target Protein

Molecular docking studies were carried out to investigate the interaction between the phytoconstituents of Pontederia crassipes flower and the selected target protein of Mycobacterium tuberculosis. The three-dimensional crystal structure of the target protein, Enoyl-acyl Carrier Protein Reductase (InhA), was retrieved from the Protein Data Bank (PDB) with PDB ID 4P8N. The downloaded protein structure was carefully examined and prepared for docking studies. Water molecules, co-crystallized ligands, and other unwanted heteroatoms present in the structure were removed to avoid interference during docking. The prepared protein structure was then saved in the appropriate format and utilized as the receptor molecule for further molecular docking analysis. The active binding site of the protein was identified based on the reported ligand-binding residues and structural information available in the Protein Data Bank. The prepared receptor structure served as the target for evaluating the binding potential of the selected phytoconstituents.

Figure 3: Three-dimensional structure of InhA enzyme (PDB ID: 4P8N)

• PDB ID: 4P8N
• PDB DOI: https://doi.org/10.2210/pdb4P8N/pdb

• Classification: Oxidoreductase/ Oxidoreductase Inhibitor
• Organism(s): Mycobacterium tuberculosis H37Rv
• Source: Protein Data Bank (PDB)
• Experimental Method: X-ray diffraction
• Expression System: Escherichia coli BL21(DE3)

• Resolution: 1.79 Å

Ligand Preparation

The phytoconstituents reported from Pontederia crassipes flower were selected for molecular docking studies. The chemical structures of the selected compounds were retrieved from the PubChem database in SDF format. The downloaded structures were converted into the appropriate format and subjected to energy minimization prior to docking. The prepared ligands were then used for docking against the target protein. Selected ligands included rutin, β-sitosterol, orientin, isovitexin, luteolin, myricetin, quercetin, chlorogenic acid, kaempferol, tricin, gossypetin, azaleatin, apigenin, naringenin, chrysoeriol and other phytoconstituents identified from Pontederia crassipes flowers.

Molecular Docking Studies

Molecular docking was performed using pyrxautodock vina docking software to predict the binding orientation and affinity of the selected phytoconstituents within the active site of the target protein. Docking is a computational technique that predicts the most favorable interaction between a ligand and a receptor molecule. The prepared protein was used as the receptor, while the selected phytoconstituents were treated as ligands. During the docking process, multiple binding conformations were generated and evaluated using a scoring function. The binding affinity values were expressed in kcal/mol. A more negative docking score indicates stronger binding between the ligand and the receptor protein. The docking study was performed to identify potential anti-tubercular compounds from Pontederia crassipes flower extract and to understand their molecular interactions with the target protein.

Protein–Ligand Interaction Analysis

Following docking, the best binding pose of each ligand was selected based on the docking score and interaction profile. The docked complexes were analyzed to identify amino acid residues involved in binding.Various molecular interactions such as hydrogen bonding, hydrophobic interactions, van der Waals forces, π-π interactions, and electrostatic interactions were evaluated. The strength and nature of these interactions were considered important factors influencing ligand binding and stability within the active site of the target protein.

Visualization of Docked Complexes

The docked protein–ligand complexes were visualized using molecular visualization software. Both two-dimensional (2D) and three-dimensional (3D) interaction diagrams were generated to provide a detailed understanding of the binding mechanism. The interaction maps were used to identify the key amino acid residues involved in ligand binding and to compare the interaction patterns of different phytoconstituents with the target protein.

ADME Prediction Studies

The pharmacokinetic properties of the selected phytoconstituents were evaluated using the ADMETlab 2.0 web server. ADME analysis was carried out to assess the Absorption, Distribution, Metabolism, and Excretion characteristics of the compounds. Parameters including molecular weight, hydrogen bond donors, hydrogen bond acceptors, lipophilicity (LogP), gastrointestinal absorption, bioavailability score, and Lipinski's Rule of Five were determined. The obtained results were used to evaluate the drug-likeness and pharmacokinetic suitability of the selected phytoconstituents as potential anti-tubercular agents.

IN VITRO STUDIES

In Vitro Anti-Tubercular Activity by Microplate Alamar Blue Assay (MABA)

The anti-tubercular activity of Water Hyacinth (Eichhornia crassipes) flower extract was evaluated using the Microplate Alamar Blue Assay (MABA) against Mycobacterium tuberculosis. MABA is a rapid, sensitive, and cost-effective colorimetric method widely used for the screening of compounds with anti-tubercular activity. The assay is based on the reduction of the blue-colored Alamar Blue reagent (resazurin) to a pink-colored product (resorufin) by metabolically active bacterial cells. The color change serves as an indicator of bacterial growth, viability, and metabolic activity of the microorganism. In the presence of an effective anti-tubercular agent, bacterial growth is inhibited and the blue color is retained, whereas active bacterial growth results in a change from blue to pink.

Procedure

1. 1. Mycobacterium tuberculosis culture grown on Lowenstein–Jensen (LJ) medium was suspended in sterile Middlebrook 7H9 broth supplemented with 0.2% glycerol and 10% OADC (oleate-albumin dextrose-catalase) enrichment. The suspension was diluted and a 1:20 dilution used as the inoculum for the assay.
   2. All experimental procedures were carried out under appropriate biosafety conditions.
   3. Sterile deionized water (200 µL) was added to the outer perimeter wells of a sterile 96-well microplate to minimize evaporation during incubation.
   4. Each test well received 100 µL of Middlebrook 7H9 broth, followed by serial dilutions of the Water Hyacinth flower extract to obtain concentrations of 25, 12.5, 6.25, 3.125, 1.56, and 0.78 µg/ml.
   5. The bacterial inoculum was added to the wells containing the test sample. Rifampicin (3.125 µg/ml) was used as the reference standard.
   6. The microplates were covered and sealed with parafilm and incubated at 37°C for five days.
   7. After incubation, 25 µL of a freshly prepared 1:1 mixture of Alamar Blue reagent and 10% Tween 80 was added to each well, followed by further incubation for 24 hours.
   8. A blue color indicated inhibition of bacterial growth, whereas a pink color indicated bacterial growth.
   9. The Minimum Inhibitory Concentration (MIC) was determined as the lowest concentration that prevented the color change from blue to pink.

RESULT AND DISSCUSSION

Molecular Docking Score (pdb id: 4P8N)

Molecular docking studies were carried out to evaluate the binding affinity of selected phytoconstituents reported from Pontederia crassipes flowers against the selected Mycobacterium tuberculosis target protein. The docking analysis was performed to predict the interaction of each phytoconstituent with the active site of the protein and to identify compounds with potential anti-tubercular activity, A total of 35 phytoconstituents were screened and their binding scores were compared with the standard ligand BTZ043. The docking score and amino acid interactions obtained for each compound are presented in Table 6.1. Compounds with more negative binding scores were considered to possess stronger binding affinity toward the target protein. The results revealed variations in binding affinity among the selected phytoconstituents, indicating differences in their interaction with the target protein. The detailed docking scores and interacting amino acid residues are shown below. Among all the compounds studied, rutin showed the highest binding affinity with a docking score of -11.4 kcal/mol, followed by β-sitosterol (-10.2 kcal/mol), orientin (-9.8 kcal/mol), and isovitexin (-9.7 kcal/mol). The standard drug BTZ043 showed a docking score of -6.7 kcal/mol. Several phytoconstituents(i.e, 18 ligands) showed better binding scores than the standard drug, indicating good interaction with the target protein.

Table 2 - Molecular Docking Results of Selected Phytoconstituents of Pontederia crassipes

Protein–Ligand Interaction Analysis

The docked complexes were further analyzed to understand the interaction pattern of the selected phytoconstituents with the active site of the target protein. The 2D and 3D interaction diagrams were generated to visualize the binding mode and identify the amino acid residues involved in ligand binding.

Figure 4: 3D and 2D interactions of Rutin

Figure 5: 3D and 2D interactions of Beta-sitosterol

Figure 6: 3D and 2D interactions of Orientin

Figure7: 3D and 2D interactions of Isovitexin

Figure 8: 3D and 2D interactions of Luteolin

Figure 9: 3D and 2D interactions of Myrcetin

Figure 10: 3D and 2D interactions of Quercetin

Figure 11: 3D and 2D interactions of Chlorogenic acid

Figure 12: 3D and 2D interactions of Kaempferol

Figure 13: 3D and 2D interactions of Tricin

Figure 14: 3D and 2D interactions of Gossypetin

Figure 15: 3D and 2D interactions of Azaleatin

Figure 16: 3D and 2D interactions of Apigenin

Figure 17: 3D and 2D interactions of Naringenin

Figure 18: 3D and 2D interactions of Chrysoeriol

Figure 19: 3D and 2D interactions of Ferulic acid

Figure 20: 3D and 2D interactions of P-Coumaric acid

Figure 21: 3D and 2D interactions of Syringic acid

Figure 22: 3D and 2D interactions of (Standard) BTZ043

Figure 23: 3D and 2D interactions of Caffeic acid

Figure 24: 3D and 2D interactions of Oleic acid

Figure 25: 3D and 2D interactions of Linolenic acid

Figure 26:3D and 2D interactions of Vanillic acid

Figure 27: 3D and 2D interactions of Gallic acid

Figure 28: 3D and 2D interactions of Protocatechuic acid

Figure 29: 3D and 2D interactions of Stearic acid

Figure 30: 3D and 2D interactions of Salicylic acid

Figure 31: 3D and 2D interactions of Palmitic acid

Figure 32: 3D and 2D interactions of 2-Methyl Resorcinol

Figure 33: 3D and 2D interactions of 4-Methyl Resorcinol

Figure 34: 3D and 2D interactions of Lauric acid

Figure 35: 3D and 2D interactions of Myristic acid

Figure 36:3D and 2D interactions of Catechol

Figure 37: 3D and 2D interactions of Pyrogallol

Figure 38: 3D and 2D interactions of Resorcinol

ADMET Result

ADMET prediction studies were carried out to evaluate the drug-likeness and pharmacokinetic properties of the selected phytoconstituents of Pontederia crassipes. The compounds were analyzed for their absorption, distribution, metabolism, excretion and toxicity characteristics using an online prediction tool.

The ADME profiles of the selected compounds, along with their SMILES notation, chemical structures, and bioavailability radar plots, are presented below. These results provide useful information regarding the suitability of the compounds for further drug development studies.

Table 3: ADMET Properties of Selected Phytoconstituents of Pontederia crassipes

Sr. No	Compound name	SMILES	Structures	Radar images
1	Rutin	C[C@H]1[C@@H] ([C@H]([C@H]([C@@H] (O1)OC[C@@H]2[C@H] ([C@@H]([C@H]([C@@H] (O2)OC3=C(OC4=CC(=CC (=C4C3=O)O)O)C5=CC(=C (C=C5)O)O)O)O)O)O)O)O
2	Beta-sitosterol	CC[C@H](CC[C@@H](C) [C@H]1CC[C@@H]2[C@@] 1(CC[C@H]3[C@H]2CC=C4 [C@@]3(CC[C@@H](C4)O) C)C)C(C)C
3	Orientin	C1=CC(=C(C=C1C2=CC(=O) C3=C(O2)C(=C(C=C3O)O) [C@H]4[C@@H]([C@H] ([C@@H]([C@H](O4)CO)O) O)O)O)O
4	Isovitexin	C1=CC(=CC=C1C2=CC(=O) C3=C(O2)C=C(C(=C3O) [C@H]4[C@@H]([C@H] ([C@@H]([C@H](O4)CO)O)O)O)O)O
5	Luteolin	C1=CC(=C(C=C1C2=CC (=O)C3=C(C=C(C=C3O2) O)O)O)O
7	Quercetin	C1=CC(=C(C=C1C2=C (C(=O)C3=C(C=C(C=C3O2) O)O)O)O)O
8	Chlorogenic acid	C1[C@H]([C@H]([C@@H] (C[C@@]1(C(=O)O)O)OC (=O)/C=C/C2=CC(=C(C=C2) O)O)O)O
9	Kaempferol	C1=CC(=CC=C1C2=C (C(=O)C3=C(C=C(C=C3O2) O)O)O)O
10	Tricin	COC1=CC(=CC(=C1O)OC) C2=CC(=O)C3=C(C=C (C=C3O2)O)O
11	Gossypetin	C1=CC(=C(C=C1C2=C (C(=O)C3=C(O2)C(=C (C=C3O)O)O)O)O)O
12	Azaleatin	COC1=CC(=CC2=C1C(=O) C(=C(O2)C3=CC(=C(C=C3) O)O)O)O
13	Apigenin	C1=CC(=CC=C1C2=CC (=O)C3=C(C=C(C=C3O2) O)O)O
14	Naringenin	C1[C@H](OC2=CC(=CC (=C2C1=O)O)O)C3=CC=C (C=C3)O
15	Chrysoeriol	COC1=C(C=CC(=C1)C2= CC(=O)C3=C(C=C(C=C3O2) O)O)O
16	Ferulic acid	COC1=C(C=CC(=C1)/ C=C/C(=O)O)O
17	P-Coumaric acid	C1=CC(=CC=C1/C= C/C(=O)O)O
18	Syringic acid	COC1=CC(=CC(=C1O) OC)C(=O)O
19	(Standard) BTZ043	C[C@H]1COC2(O1) CCN(CC2)C3=NC(=O) C4=C(S3)C(=CC(=C4)C(F) (F)F)[N+](=O)[O-]

Physiochemical Properties of selected Compounds

The physicochemical properties of the selected phytoconstituents were evaluated to understand their chemical characteristics and suitability for drug development. Parameters such as molecular weight, molecular volume, density, hydrogen bond acceptors, hydrogen bond donors, topological polar surface area, rotatable bonds, ring count, aqueous solubility, and lipophilicity were analyzed. The physicochemical properties of the selected compounds are lipophilic were analyzed. The physiochemical properties of the selected compounds are presented in the table 4

Table 4: Physicochemical Properties of Selected Phytoconstituents of Pontederia crassipes

Drug-likeness properties of selected compounds

The drug-likeness properties of the selected phytoconstituents were evaluated using various prediction models. Parameters such as QED, Lipinski's Rule of Five, Ghose filter, Pfizer rule, GSK rule, Golden Triangle rule, and bioavailability score were analyzed to assess the suitability of the compounds for drug development. The results obtained for the selected compounds are presented in Table 5

Table 5: Drug-Likeness Properties of Selected Phytoconstituents of Pontederia crassipes

Absorption parameter of selected compounds

The absorption properties of the selected phytoconstituents were evaluated to predict their ability to be absorbed and transported in the body. Parameters such as Caco-2 permeability, MDCK permeability, P-glycoprotein (P-gp) inhibition, P-gp substrate status, human intestinal absorption (HIA), and oral bioavailability at different levels (F20%, F30%, and F50%) were analyzed. The results obtained for the selected compounds are presented in Table 6.

Table 6: Absorption Parameters of Selected Phytoconstituents of Pontederia crassipes

Distribution and metabolism parameter of selected molecules

The distribution and metabolism properties of the selected phytoconstituents were evaluated to predict their behavior in the body after absorption. Distribution parameters such as plasma protein binding (PPB), volume of distribution (VD), blood-brain barrier (BBB) permeability, and fraction unbound (Fu) were analyzed. Metabolic properties were assessed based on their interaction with major cytochrome P450 enzymes, including CYP1A2, CYP2C19, CYP2C9, CYP2D6, and CYP3A4, as inhibitors or substrates. The results are presented in Table 7

Table 7: Distribution and Metabolism Parameters of Selected Phytoconstituents of Pontederia crassipes

Excretion and toxicity parameters of selected compounds

The excretion and toxicity properties of the selected phytoconstituents were evaluated to assess their safety and elimination characteristics. Excretion parameters such as plasma clearance (CL), half-life (T½), and other related properties were analyzed. Toxicity assessment included hepatotoxicity (HHT), drug-induced liver injury (DILI), mutagenicity, acute oral toxicity, maximum recommended daily dose (FDA MDD), skin sensitization, carcinogenicity, eye corrosion, eye irritation, and respiratory toxicity. The results obtained for the selected compounds are presented in Table 8

Table 8: Excretion and Toxicity Parameters of Selected Phytoconstituents of Pontederia crassipes