In Silico Antibacterial Activity of Polyacetilene Derivatives Against Mycobacterium tuberculosis and In-Vitro Antioxidant Properties from Ethanol Extraction of Blackjack ( Bidens pilosa L.)

was used as standard, and the absorbance was measured at 426 nm. Antioxidant activity was evaluated using DPPH scavenging 517 nm and Frap Assay 596 nm. The molecular docking results showed that the compounds (5-(2-Phenylethynyl)-2-b-glucosylmethyl-thiophene, Glucopyranosyloxy-3-hydroxy-6(E)-tetradecane-8,10,12-triyne and Phenylhexa-1,3,5-triyn-1-yl acetate were most potentially anti-tuberculosis on the target protein InhA with the binding energy produced <-6.55 Kcal/mol and the lowest inhibitory cost of 1.838×10 -6 . The compound has amino acids similar to natural ligands and standard compounds of isoniazid. The given ADME-T predictions showed good pharmacokinetic properties and safe levels of toxicity (5-(2-Phenylethynyl)-2- b-glucosylmethyl-thiophene, Glucopyranosyloxy-3-hydroxy-6(E)- tetradecane-8,10,12-triyne in hepatotoxic and LD 50 . The highest antioxidant properties in blackjack are produced by FRAP assay 722.905±7.985 mMTE/gr. This proves that blackjack has antioxidant activity and is required for bioassay-guided isolation testing against Mycobacterium tuberculosis .


Introduction
Tuberculosis is one of the diseases that can be transmitted through the air and is caused by the Mycobacterium tuberculosis bacteria (MTB).Tuberculosis cases are one of the leading causes of death worldwide [1].Indonesia has the second-highest in terms of tuberculosis infections, following India.This is evident from the incidence rate in 2022, which reached 969 thousand people and increased 17% compared to 2020 [1].This case shows the challenges associated with tuberculosis treatment, such as drug resistance and adverse side effects, which contribute to patient noncompliance during the course of therapy.
Treatment of tuberculosis is a comprehensive and ongoing treatment.It takes at least six months with a combination of at least four kinds of anti-tuberculosis drugs (OAT) [2].Resistance to OAT occurs due to mutations in the Mycobacterium tuberculosis InhA gene, so this resistance is the driver for developing antituberculosis drugs [3,4].
One of the resistances that gives rise to mutations in the OAT target coding gene is the inhA gene for isoniazid [5].Active isoniazid binds to the inhA-NADH complex.This complex will deactivate enoyl reductase and inhibit mycolic acid biosynthesis in the cell wall of Mycobacterium tuberculosis.The alternation of amino acids at the NADH bond site of inhA results in isoniazid resistance by preventing mycolic acid biosynthesis.The substitution of Ser94Ala results in a decrease in the affinity of inhA bonds to NADH; thus, inhibition of mycolic acid synthesis occurs.InhA promoter mutations are more common at positions -24(G-T), -16(A-G), or -8(T-G/A) and -15(CT) [6,7].These promoter mutations produce inhA overexpression and trigger isoniazid resistance.This spurred the development of antituberculosis drugs.One way to do this is to use active compounds from plants that can potentially be used as anti-tuberculosis agents [8].
The development of natural ingredients has the potential to support tuberculosis therapy blackjack (Bidens pilosa L.), which became one of the innovations in its development.Blackjack are wild plants that, ethnobotanically in Jombang, West Java, are used as vegetables, whereas in China, it is processed and utilized as herbal tea [9,10].Blackjack plants have bioactive compounds such as phenolics, flavonoids, polyacetylene, chalcones, okanine glycosides, and terpenoids [11].In their activity, blackjack plants have been proven to be antimalarial, anti-inflammatory, antihypertensive, colorectal cancer, and antibacterial in modulating immunity [12,13,14,15].
Polyacetylene compounds are potential candidates for producing antibacterial activity against Grampositive and Gram-negative bacteria [16]; however, further development of anti-tuberculosis activity has yet to be carried out.This research screens polyacetylene derivatives in blackjack, tested through molecular docking as an initial step in developing new drug ingredients.Additionally, the antioxidant properties of the ethanol extract of blackjack are examined as supporting data to assess the provided antioxidant characteristics.Molecular docking generates an energy affinity output that describes the bond strength resulting from the interaction between ligands and receptors.

Experimental
The research method used was in silico experimental research of polyacetylene derivative compounds from the blackjack plant.This included applying the Lipinski Rule of Five, conducting molecular docking studies, and predicting the pharmacokinetics and toxicity of the active compounds to assess their potential as anti-tuberculosis agents.Additionally, the antioxidant properties of the ethanol extract of the blackjack plant were evaluated using UV-Vis spectrophotometry.

Tools
The software used included ChemDraw version 16.0, Chem 3D version 16.0, AutoDockTools version 1.5.6, and Discovery Studio 2019.The hardware employed was the Asus TUF Gaming F15 featuring an Intel (R) Core (TM) i5-10300H CPU @2.50 GHz and 8 GB RAM.UV-Vis spectrophotometry (Shimadzu, Japan) was utilized as the required instrument for testing antioxidant properties.

Preparation of Protein and Native Ligand
Protein preparation involved isolating proteins in their active form, specifically those binding to native ligands, utilizing AutoDockTools.Eliminating the water component during protein preparation was essential to prevent interference with the ligand process.Then, polar hydrogen and Kollman charges were added, and the results were saved in .pdbformat.

Validation of Molecular Docking Method
The molecular docking method was validated using natural ligands with target proteins that have undergone preparation using the AutoDockTools program.The grid box arrangement was used to determine the ligandbound space to be in-docking, obtained grid box dimensions (X: 40, 50; Z: 36) with spacing amstrong (0.375 Å) and natural ligand coordinates (x: -3.047; y: 32.422; z: 14.205).The docking result obtained the banding energy value and Root Mean Square Deviation (RMSD).The docking validation method is said to be valid if the RMSD value is ≤ 2 Å [25].The docking simulation was conducted using AutoDock4 software, which was configured with AutoDockTools and executed via the Command Prompt.The docking parameters were set using the "Genetic Algorithm (GA)" parameter, the number of GA runs was generated 100 times the ligandreceptor interaction, and the parameter settings were set by default.The output of the docking simulation was stored based on the Lamarckin GA settings, and the docking simulation was run using the command prompt.

Molecular Docking Analysis
All test ligands were docked using the same methodology as in the validation process, maintaining identical grid box dimensions and positioning for each receptor.The docking procedure, performed using AutoDockTools, yielded two key parameters: binding affinity (ΔG; kcal/mol) and ligand-receptor interactions, which were derived from interactions with similar amino acids [26].

Preparation of Ethanol Extraction
Blackjack was found in the Banyumas area, Central Java, and had been proven to be authenticated.The dry powder of blackjack plants was extracted using 70% ethanol (1:10) by stirring and allowed to stand for 24 hours, and the macerated sample was filtered.The residue was remacerated with the same solvent.The filtrate obtained was evaporated using a rotary vacuum evaporator to obtain a thick extract.

Determination of Total Phenolic and Total Flavonoid Content
The ethanol extract of blackjack was utilized to determine total phenolic and total flavonoid levels [21].To measure the total phenolic content (TPC), 1 mL of the extract or standard solution was combined with 5 mL of Folin-Ciocalteu reagent at room temperature for 8 minutes.Subsequently, 4 mL of 1% NaOH was added, and the mixture was left to stand for 40 minutes, followed by measuring the absorption at a wavelength of 741 nm.The total phenolic content is expressed in mgGAE/gram.
To measure total flavonoid content (TFC), 0.5 mL of the extract solution or standard was mixed with 1.5 mL of ethanol, 0.1 mL of 10% AlCl3, 0.1 mL of CH3COONa, and 2.8 mL of distilled water.The mixture was allowed to stand for 15 minutes at room temperature, and the wavelength was measured at an absorption of 426 nm.Total flavonoid content was reported in mgQE/gram.

Determination of Antioxidant Activity
Antioxidant analysis was carried out using DPPH scavenging and FRAP Assay following the method [27].DPPH analysis was performed by taking 0.5 mL of extract, or standard solution reacted with 5 mL of DPPH (25 μg/mL ethanol), allowed to sit in a dark place for 30 minutes, and measured at a wavelength of 517 nm.DPPH scavenging was reported in mMTE/gr.The FRAP assay was conducted by mixing 0.21 mL of the extract solution or standard with 3.99 mL of FRAP reagent (300 mM acetate buffer, 10 mM TPTZ in HCl, and 20 mM FeCl3 (ratio 10:1:1 pH 3.6)).The mixture was then allowed to stand in a dark place for 40 minutes before being measured at a wavelength of 596 nm.The results were reported in mMTE/gr.

Analysis Result
Data analysis encompassed several parameters, including bond energy and inhibition constant (Ki).Visualization tools, such as Discovery Studio, were utilized to observe interactions between ligands and amino acid residues.The formula for calculating the inhibition constant (IC) is described in Equation (1).
Where ΔG is the binding energy (kcal/mol), R is the gas constant (1.986 kcal/mol), and T is the temperature (2981.5 K).Data analysis of the total phenolic and flavonoid content, as well as antioxidant activity measured by DPPH scavenging and FRAP assay, was conducted by performing three repetitions with standard deviation (±SD) and calculated using the assay Equation (2).

Validation of Molecular Docking Method
The target protein utilized in the study was the InhA GDP, obtained from the rcsb.orgwebsite with a PDB ID: 1ENY.This protein complex included one natural ligand: Nicotinamide-Adenin-Dinucleotide.InhA protein is crucial for the survival of Mycobacterium, particularly in the synthesis of mycolic acid [23].Method validation is performed by separating the protein structure from the natural ligand (Figure 1).
The method validation results were carried out by looking at the RMSD parameters.RMSD is the deviation distance between the binding position of natural ligands and proteins after docking against the actual position of natural ligand bonds.Docking validation is carried out by determining the grid box, which is the area where interactions between natural ligands and proteins occur [28].The results of the visualization of natural proteins and ligands in RMSD and bond energy can be seen in Table 2.
Based on the validation results, it produces an RMSD value of 1.47 Å, which means that the molecular docking method used is validated and reliable for docking other test ligands [29].The parameter used to assess docking results is the binding energy, which evaluates a drug's capacity to bind to receptors.A lower ∆G value indicates a stronger interaction between receptors and ligands, signifying stronger attachment.This is reflected in the ∆G value, where a smaller value indicates stronger binding affinity [30].The inhibition constant (Ki) indicates the concentration required of ligands in inhibiting the target protein.A lower Ki value indicates a stronger complex formed by the ligand and the protein.This is attributed to the rise in torsional energy within the complex, contributing to the stability of the enzymeligand complexes [31].The results obtained from isoniazid standard compounds demonstrate the smallest bond energy and inhibition constant among other standard compounds, specifically -6.53 kcal/mol and 2.045×10 -4 μM (Table 3).
The similarity of amino acid residues to natural ligands (%) 0 22.22 44.44 0 The similarity of amino acid residues to standard compounds (%) 80 12.50 80 The bond energy value of isoniazid plays a significant role in its interaction with proteins, particularly through intramolecular interactions such as hydrogen bonds and van der Waals forces.The amino acid residue produced by isoniazid is Ile21, Ile95, Phe41, Val65, and Asp64 (Figure 2).Hydrogen bonds are typically stronger than van der Waals bonds, as they can form even when the distance between the ligand and the receptor is relatively far.
Residues produced by selected active compounds of blackjack plants against target proteins are known to be hydrophilic, and most amino acid residues are hydrophilic.Hydrophobic amino acids have properties that are difficult to dissolve in water.On the other hand, amino acids are hydrophilic, with ionized side chains easily soluble in water [33].The similarity of polyacetylene-derived amino acid residues between natural ligands and the standard isoniazid indicates a similarity in the type of interaction and binding to the target protein.Pratama et al. [34] stated that the higher similarity of amino acid residues indicates a high probability that the active compound's ligand will interact similarly with natural ligands.
The enzymatic metabolism of the drug indicates the biotransformation of a drug in the body; it is essential to consider drug metabolism.The superclass of cytochrome P450 enzymes plays a vital role in drug metabolism.The CYP families responsible for drug metabolism include 1A2, 2C9, 2C19, 2D6, and 3A4 (Table 6).The compounds 5-(2-Phenylethynyl)-2-b-glucosylmethyl-thiophene and Phenylhexa-1,3,5-triyn-1-yl acetate have lower total clearance values than the standard compounds of 0.275 and 0.658 (Table 6).Drug toxicology is one of important areas in preclinical investigation, as toxicity is a major factor leading to reduced drugs at the discovery and development stage [38].Therefore, reliable prediction of compound toxicity in silico plays a crucial role in minimizing costs and saving time throughout the drug discovery and development process [39,40].The risk of toxicity for compounds 5-(2-phenylethynl)-2-bglucosylmethyl-thiophene, β-D-glucopyranosyloxy-3hydroxy-6(E)-tetradecen-8,10,12-triyne, and Phenylhexa-1,3,5-triyn-1-yl acetate is shown in Table 6.Three toxicity descriptors, namely AMES and Hepatotoxic LD50, become the main estimated component.The AMES test is a test to detect the mutagenicity of a compound that can suppress genetic damage and mutations [41].According to the findings, 5-(2-Phenylethynl)-2-bglucosylmethyl-thiophene exhibits mutagenic properties, while the other compounds do not demonstrate mutagenic properties (Table 6).
Hepatotoxicity is a major factor leading to reduced drugs.Unfortunately, a quarter of marketed drugs were recalled due to their adverse effects on the liver.The phenylhexa-1,3,5-triyn-1-yl acetate compound can provide hepatotoxic effects, while the other two compounds do not exhibit hepatotoxic effects (Table 6).Determination of acute toxicity is another important task in the goal of drug discovery and development.Acute toxicity expressed in median lethal dose (LD50); the number of doses of the test compound that killed 50% of treated animals within a given time [42].LD50 of compounds 5-(2-Phenylethynyl)-2-b-glucosylmethylthiophene and β -D-Glucopyranosyloxy-3-hydroxy-6(E)-tetradecen-8,10,12-triyne indicate low acute toxicity, as they are higher than those of standard compounds, measured at 2.569 and 2.321 mol/kg, respectively, in rat models (Table 6).

Determination of Antioxidant Properties
The ethanol extracts of blackjack demonstrated a total phenolic content of 74.754±1.145mgGAE/g and a total flavonoid content of 22.589±0.089mgQE/g.Additionally, the antioxidant activity assessed by DPPH and FRAP assays yielded levels of 601.756±2.587mMTE/g and 722.905±7.985mMTE/g, respectively (Table 7).
Plant-derived natural antioxidants play a crucial role in reducing or mitigating the detrimental effects of oxidative stress.The DPPH assay is a handy method for assessing the antioxidant capacity of plants, among other assays.Flavonoids and phenols, which are antioxidant chemicals with hydrogen-donating groups, lead to reducing the methanolic DPPH solution by forming nonradical compounds [43].In addition to their antioxidant capabilities, flavonoids and other phenolics also possess other biological activities, including antibacterial, antiviral, and anticancer effects [44].The ability of these substances to bind proteins and scavenge free radicals is typically responsible for their biological and pharmacological effects [45].Antioxidants are crucial compounds that can shield the body from damage caused by oxidative stress created by free radicals.Plant polyphenols function as reducing agents and antioxidants due to the hydrogen-donating characteristic of their hydroxyl groups [46].

Figure 1 .
Figure 1.Separation of proteins and natural ligands

Figure 2 .
Figure 2. Interaction of amino acid residues and hydrogen bonds of isoniazid

Table 2 .
RMSD and free binding energy of natural ligands

Table 3 .
Simulation results of docking of standard compounds and polyacetylene derivatives of blackjack against MTB protein

Table 4 .
Similarity of amino acid residuals

Table 5 .
Lipinski's Rule of Five

Table 6 .
Prediction of ADME-T properties of selected compounds

Table 7 .
Determination of antioxidant properties