In Vitro and In Silico Characterization of Crocatin A from Red Betel Leaves: Targeting DNA Gyrase B and DNA Ligase of Enterococcus faecalis with ADMET-Based Druglikeness Analysis

In Vitro and In Silico Characterization of Crocatin A from Red Betel Leaves: Targeting DNA Gyrase B and DNA Ligase of Enterococcus faecalis with ADMET-Based Druglikeness Analysis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article In Vitro and In Silico Characterization of Crocatin A from Red Betel Leaves: Targeting DNA Gyrase B and DNA Ligase of Enterococcus faecalis with ADMET-Based Druglikeness Analysis Devi Meliani, Trisna Yuliana, Dikdik Kurnia This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7015807/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 08 Mar, 2026 Read the published version in BMC Chemistry → Version 1 posted 10 You are reading this latest preprint version Abstract Dental caries tooth tissue disease that can cause complications. The gram-positive bacteria that play a role in the process of infection are Enterococcus faecalis . Red betel leaves ( Piper crocatum Ruiz and Pav.) contained active substances in their phytochemicals. However, there is no additional information on the antibacterial properties of P. crocatum or the molecular mechanisms that affect DNA Gyrase B and DNA Ligase of E. faecalis ATCC 29212. This study aimed to screen and test compounds from P. crocatum for their ability to inhibit E. faecalis and predict the mechanism of inhibition of certain proteins using a molecular docking approach. Isolation of Crocatin A from P. crocatum was carried out by column chromatography and then characterized via infrared (IR), nuclear magnetic resonance (NMR), and mass spectroscopy, then compound was tested using Kirby Bauer and microdilution methods. The active compound and derivatives were predicted to act against DNA gyrase B and DNA ligase from E. faecalis and ADMET properties by in silico . The study showed that Crocatin A has been isolated from P. crocatum . It exhibited antibacterial properties against E. faecalis (MIC 1250 µg/mL) as well as in silico against DNA Gyrase B (-6.34 kcal/mol) and DNA Ligase (-5.77 kcal/mol) enzymes. Therefore, it can be concluded that Crocatin A present in Red Betel leaves has moderate activity in inhibiting E. faecalis by in vitro and potential to inhibit DNA synthesis in E. faecalis by in silico. Crocatin A Piper crocatum Ruiz & Pav Enterococcus faecalis DNA gyrase DNA ligase Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction In 2018, oral health problems, particularly dental caries, affected almost half of the world's population of 3.58 billion people. According to the World Health Organization (WHO), in 2019, 60–90% of school-aged children and almost 100% of adults had dental issues( 1 ). Dental caries are the primary cause of root canal infections. Caries is a periodontal disease caused by damage to the hard tissues of the teeth (enamel and dentin layers), which can extend to the nerves of the teeth due to the fermentation of carbohydrates, particularly sucrose, by pathogenic microorganisms in the mouth( 2 ). E. faecalis plays a major role in root canal infections are Enterococcus faecalis ( 3 ). E. faecalis is a Gram-positive cocci microorganism that can survive facultatively without oxygen (facultative anaerobes). In addition, these microorganisms can survive in the root canal for a long period of time without being supplied with nutrients( 4 ). Many attempts have been made to treat oral infections by using synthetic antibacterial agents. The use of the tetracycline class of antibiotics should be limited to the root canals because of the risk of discoloration of the teeth( 5 ). The use of various antibiotics can also induce bacterial resistance. Therefore, to overcome the problems of drug and bacterial resistance, a solution is needed to identify active compounds that can be used as new drugs that are effective and safe. Sources of bioactive compounds can be found in plants, one of which is Red betel leaf( 6 ). Red betel leaf (P. crocatum) is known to have good pharmacological effects, such as antifungal( 7 ), antibacterial ( 8 )antiinflammatory, antioxidant, antidiabetic, antitumour, anticancer, hepatoprotective, immunomodulatory, antihyperglycaemic, and analgesic ( 9 ),( 10 ),( 11 ),( 12 ). The compounds in this plant include flavonoids, phenolics, terpenoids, steroids, tannins, and essential oils, all of which are known to have various bioactivities( 13 ). Bacterial growth can be inhibited by several mechanisms, such as the inhibiton of DNA replication, which inhibits protein synthesis and microbial growth( 14 ). Computational chemistry plays an important role in drug discovery and development. Computational chemistry also facilitates the development of new drugs from traditional medicinal plants( 15 ). In the in silico molecular docking study, the inhibition of bacterial DNA replication was carried out by inhibiting one of the enzymes involved, such as DNA gyrase B, which mediates the introduction of negative supercoils, and DNA ligase, which acts as a splicer of fragments formed during the replication process or when the DNA repair process takes place; thus, inhibition of DNA gyrase B and DNA ligase enzymes can be used as potential target( 16 ). Interaction modelling of compounds as drug candidates and proteins as receptor targets can be used to search for new drugs that are more effective and efficient( 17 ). Therefore, the aim of this study was to identify the antibacterial components of P. crocatum against E. faecalis . We predicted the molecular interactions that inhibit key enzymes in bacterial DNA synthesis, namely, DNA gyrase B and DNA ligase. Material and methods Materials Red Betel ( Piper cocatum Ruiz and Pav) is a plant species that is native to Bandung, West Java, Indonesia. Plant specimens were obtained from the Systematics and Molecular Laboratory located at the Department of Biology, Faculty of Mathematics and Natural Sciences, Padjadjaran University, Sumedang, Indonesia. Organic solvents, namely, methanol, n -hexane, and ethyl acetate, were used for extraction, separation, and purification. Column chromatography was performed using Silica G 60 (Merck, Darmstadt, Germany) and ODS RP-18, whereas thin-layer chromatography (TLC) was performed using Silica G 60 F 254 plates and ODS RP-18 F 254 S (Merck, Darmstadt, Germany). Compound spots on the TLC plate were visualized under ultraviolet (UV) light at 254 nm and 365 nm by spraying with 10% H 2 SO 4 in Ethanol. In vitro antibacterial tests were performed using Enterococcus faecalis ATCC 29212, Mueller-Hinton broth, and Mueller-Hinton agar as media, methanol as a negative control, and chlorhexidine as a positive control. The 3D structures of DNA Gyrase B and DNA Ligase used in this study were obtained from the Protein Data Bank (PDB) ID: 4K4O,1TA8. Crocatin A, a compound isolated from Piper crocatum , was obtained from ChemDraw3D. The antibacterial positive control, chlorhexidine (CID 9552079), and the native ligand for DNA Gyrase B (CID 66560858), a native ligand for DNA Ligase (CID 14181), were used. All data were obtained from PubMed ( https://www.ncbi.nlm.nih.gov/pccompound ). Instruments The structure of the active compound was determined by infrared (IR) spectroscopy with an FTIR Spectrum-100 spectrometer (Buckinghamshire, England), and HR-ESI-MS spectra were acquired using a quadrupole time-of-flight mass spectrometer (Xevo G2-XS QTOF, Waters Corp.) and, Nuclear Magnetic Resonance (NMR) ( 1 H-NMR, 13 C-NMR, and135º DEPT) with a Bruker Avance 700 MHz (Bruker, Germany). The TLC plate was visualized using a UV lamp detector with λmax wavelengths of 254 and 365 nm. For the antibacterial activity test, 96-well microplates (NEST Biotechnology, Wuxi, China), micropipettes (Winlab, Grogol, Jakarta), microtubes (Chemikalie, Pandan Loop, Singapore), an incubator (Memmert, Schwabach, Germany), paper discs (Grainger, Origin, USA), and a Biochrom microplate reader (Biochrom, Ltd., Cambridge, UK) were used. Isolation Compound from Extract of P. crocatum A sample of 1.6 kg of fresh P. crocatum leaf was extracted with methanol (20 L) by the maceration method for 3×24 h. The filtrate from the maceration process was evaporated using a rotary evaporator at 40°C, and 100 g of the methanol extract was obtained. For the bioactivity evaluation of antibacterial activity, extracts were prepared at a series of concentrations according to assay protocols( 18 ). The methanol extract (10 g), which showed the best activity against E. faecalis ATCC 29212, was purified by gradient chromatography using Silica G 60 (0.063-0.200 mm) and a combination of n -hexane-ethyl acetate-methanol solvents for elution. Fractions F.1–10 were obtained and visualized under UV light at 254 and 356 nm by spraying with 10% H 2 SO 4 in EtOH, followed by heating. Next, each fraction from F1-F10 was tested for antibacterial activity using the agar disk Kirby-Bauer method. Among the fractions, fraction 7 (493.7 mg) exhibited the largest inhibition zone (8 mm). The F7 subfraction (493.7 mg) was purified by column chromatography on an ODS RP-18 column and eluted with H 2 O-MeOH at a gradient of 10% (v/v) to yield compound 1 (11.1 mg). Structure Determination of Isolated Compound Comprehensive spectroscopic analysis data were used to determine the structure of the isolated compound, including ultraviolet (UV), mass spectrometry (MS), 1D and 2D-NMR ( 1 H-NMR, 13 C-NMR, DEPT 135°, HMQC, 1 H- 1 H COSY, HMBC), and infrared (IR) spectra. Assessment of the Extracts and Isolated Compound of P. crocatum Leaf Activity Against E. faecalis The Kirby-Bauer disk diffusion method was used to evaluate P. crocatum extracts against E. faecalis ATCC 29212. The diameter of the growth area around the paper disk was measured to determine the zone of each sample( 19 ). The assay determines the resistance or sensitivity of E. faecalis ATCC 29212 strains to extracts guided by CLSI protocols (CLSI, 2012)( 20 ). Methanol was used to dilute all samples, except for the water fraction, and the positive control (chlorhexidine) was diluted in water. Stock solutions were prepared for each extract and 75 mg of each extract was diluted in methanol, except for the water fraction, to obtain a 5% stock solution. Next, 2, 2.5, 5, 10, 20, and 30% concentrations of all the samples, together with 2% chlorhexidine, were prepared for the assay. Then, 20 µL of each sample was impregnated onto a 6 mm paper disk and placed on the agar surface. Repeated tests were required to obtain better results. 1 ose of bacteria was grown in 5 mL of broth media to prepare the bacteria. The solution was incubated at 37°C for 24 h. A microplate reader was used to measure the optical density of the solution at 620 nm after the incubation. The solution was diluted to reach 0.5 McFarland standard or approximately 180 CFU/mL in broth media (100 µL). The resulting culture was swabbed onto the surface of agar medium and a paper disk was prepared. The samples were incubated at 37°C for 24 h. Repeated tests were required to obtain better results. The MIC and MBC of the compound against E. faecalis ATCC 29212 were determined using the microdilution method in a 96-well microplate( 21 ). Bacterial cells were pre-cultured under aerobic conditions in Mueller-Hinton broth at 37°C. In the presence of several concentrations of a compound inserial two-fold dilutions, bacterial cells were incubated at 37°C without shaking for 48 h in the same broth on a microplate, as shown in the procedure used at the Clinical and Laboratory Standards Institute. The optical density of the solution was measured at 620 nm wavelength using a microplate reader. The MICs of the cells were defined as the lowest concentrations where, visually the bacterial cells were not observed by the OD value, as reported previously, and given by duplicate assessments. The compound was dissolved in methanol and used as a negative control, while chlorhexidine was used as a positive control. Each concentration of the compound solution in the microplate well was spread onto the surface of the agar and incubated at 37°C for 24 h. After incubation, the amount of liquid in each well of the microplate was measured at 620 nm using a microplate reader. MIC was determined by comparing the absorption value of the sample well (compound plus bacteria) with that of the blank well (bacteria). Furthermore, the liquid from the wells was spread on Mueller-Hinton agar and incubated for 24 h to evaluate the MBC, the lowest concentration of the sample required to kill the bacteria. Ramachandran plot for enzyme validation Ramachandran plot assessed the enzyme’s structural accuracy in this study. Each three-dimensional structure DNA Gyrase B (PDB ID : 4K4O) and Lygase (PDB ID : 1TA8) were input as .pdb format on the PROCHECK SAVES v6.1 by UCLA web server ( https://saves.mbi.ucla.edu/ ). In silico Characterization of Crocatin A AutoDock 4.0, in the open-source software PyRx 0.8 was used for ligand-protein docking and virtual screening of antibacterial activity. Crocatin A and chlorhexidine were used for binding to DNA gyrase B and DNA ligase as protein targets, and the ligands were free for blind docking. 4-[(3aR,6aR)-2,3,3a,4,6,6a-hexahydro-1H-pyrrolo[2,3-c]pyrrol-5-yl]-6-fluoro-N-methyl-2-(2 methylpyrimidin-5-yl)oxy-9H-pyrimido[4,5-b]indol-8-amine, the native ligand for the DNA Gyrase B enzyme, and [(2 R ,3 S ,4 R ,5 R )-5-(3-carbamoylpyridin-1-ium-1-yl)-3,4-dihydroxyoxolan-2-yl]methyl dihydrogen phosphate, the native ligand for the DNA ligase enzyme, was being re-docked to the enzyme. The conformation was selected based on binding energy: the one with the lowest binding energy score that has a value of root-mean-square deviation less than 2.0 Å was selected( 22 ) , ( 23 ). ADME/Tox prediction and drug-likeness analysis ADME (absorption, distribution, metabolism, and excretion) predictions were analyzed on the pkCSM web server (biosig.lab.uq.edu.au/pcksm/), while drug-likeness predictions were completed from the SwissADME web server ( http://www.swissadme.ch/ ). Results and Discussion Oral infections caused by pathogenic bacteria such as E. faecalis can cause various oral health problems, including dental root canal infections. The use of several antibiotics and antiseptics to prevent and cure such infections over a long period can cause side effects, with the emergence of bacterial resistance( 24 ). Therefore, new antibacterial agents that are more effective and efficient are needed. One alternative is to utilize the potential of medicinal plants such as the red betel plant (P. crocatum) ( 25 ). Ethnobotanical and ethnopharmacological data show that the herbal red betel (P. cocatum) has various biological activities, including antitumor, antibacterial, antioxidant, antifungal, anti-inflammatory, anticancer, and antidiabetic( 26 ),( 27 ). Red Betel (P. crocatum ) can be used as an alternative to obtain herbal antibacterial compounds against pathogenic bacteria in dental root canals, namely E. faecalis ( 28 ). Structural Determination of Compound The isolation of compounds from the methanol extract of red betel leaves yielded compound 1 with the characteristics of white crystals that can dissolve in chloroform. The pure compound isolated from red betel leaves had an Rf value of 0.72 as thin layer chromatography (TLC) Silica G 60 F 254 eluted with eluent n -hexane: ethyl acetate (7:3). While on 2-dimensional TLC, compound 1 had an R f value of 0.62 with a combination of n -hexane: ethyl acetate and n-hexane: acetone (7:3) as the eluent. Compound 1 shows in the FTIR spectrum of 1 shows CH S p 3 absorption at 2947 cm − 1 , C = O acetyl at 1742 cm − 1 , C = O unsaturated carbon at 1704 cm − 1 , C = C aromatic at 1590 and 1511 cm − 1 , C-O-CH 3 stretching at 1235 cm − 1 and CH 2 -O stretching at 1123 cm − 1 . The structure of compound 1 was determined by NMR spectroscopy using a Bruker Avance 700 MHz spectrometer. 13 C-NMR, DEPT, and HMQC spectra of compound 1 showed 25 carbon signals for seven methyl carbons, two methylene carbons, seven methine carbons, and nine quaternary carbons. Important signals including aromatic carbon appeared at \(\:\delta\:\) C 152.6, 136.8, 152.6, 133.5, 105.9 and 105.9; olefinic carbon at \(\:\delta\:\text{C}\) 152.2 and 126.2; allyl group at \(\:\delta\:\) C 34.5, 133.9 and 118.1; methoxy-substituted aromatic group at \(\:\delta\:\) C 54.9, 59.6, and 54.9; methoxy-aliphatic carbon at \(\:\delta\:\) C 54.6 and 54. A methyl carbon was observed at \(\:\delta\:\) C 16.1, and another methyl group attached to the carbonyl was found at \(\:\delta\:\) C 19.4. Two quaternary carbons were observed at \(\:\delta\:\) C 94.8 and 48.3, while a carbinol carbon appeared at \(\:\delta\:\) C 78.2. The 1 H NMR spectrum showed five methoxy signals [ \(\:\delta\:\) H 3.79 (3H, s), 3.79 (3H, s), 3.71 (3H, s), 3.71 (3H, s), and 3.24 (3H, s)] and one methyl signal [ \(\:\delta\:\) H 1.31 (3H, d)]. Signals corresponding to allyl groups appeared at \(\:\delta\:\) H 5.90 (1H, m), 5.27 (1H, d, J = 12.18 Hz), 5.20 (1H, d, J = 19.07 Hz), 2.53 (1H, m), and 2.43 (2H, m). An aromatic proton was observed at \(\:\delta\:\) H 6.34 (2H, s). An olefinic proton appeared at \(\:\delta\:\) H 6.45 (1H, s), possibly indicative of an enone system, and a benzylic methine was observed at \(\:\delta\:\) H 3.34 (3H, s). The additional signals at \(\:\delta\:\) H 5.32 (1H, s) and 2.43 (2H, m) correspond to the carbinol and methine protons, respectively. HMBC revealed a correlation between C-5 ( \(\:\delta\:\) C 136.8), C-3 ( \(\:\delta\:\) C 152.6), and C-4 ( \(\:\delta\:\) C 152.6) methoxy protons as benzene substituents ( 29 ) , ( 30 ), a correlation between protons of C-2' ( \(\:\delta\:\) C 78.2) and C-4' ( \(\:\delta\:\) C 192.5) ketones, a correlation between protons of C-2' ( \(\:\delta\:\) C 78.2) and acetyl, a correlation between protons of C-9' ( \(\:\delta\:\) C 118.1) allyl and C-7' ( \(\:\delta\:\) C 34.5), and a correlation between C-9 ( \(\:\delta\:\) C 16.1) methyl protons and C-8 ( \(\:\delta\:\) C 47.6). Based on analysis of the NMR spectral data, compound 1 was identified as a compound from the neolignan group, crocatin A (Table 1 ), with the chemical formula C 25 H 32 O 8 and a molecular mass value 460.52 m/z . The structure of compound 1 (Fig. 1). Table 1 Comparison compound 1 and crocatin A with literature Crocatin A ( 31 ) Compound 1 (700 MHz, CD 3 OD) Posisi \(\:\delta\:\) H \(\:\delta\:\text{C}\) \(\:\delta\:\) H \(\:\delta\:\text{C}\) 1 133 133.5 2 6.26 (3H, s) 105.9 6.34 (2H, s) 105.9 3 152.8 152.6 4 137.1 136.8 5 152.8 152.6 6 6.26 (3H, s) 105.9 6.34 (2H, s) 105.9 7 3.33 (1H, d) 59.6 3.34 (3H, s) 3.71 (3H, s) 59.5 8 2.30 (1H, m) 48.8 2.43 (2H, m) 47.6 9 1.28 (3H, d) 17.2 1.31 (3H, d,7.07 Hz) 16.1 1' 48 48.3 2' 5.35 (1H, s) 78.7 5.32 (1H, s) 78.2 3' 94.7 94.8 4' 191.5 192.5 5' 152.6 152.2 6' 6.17 (1H, s) 124.5 6.45 (1H, s) 126.2 7' 2.46 (1H, m) 2.34 (1H, m) 35.1 2.43 (2H, m) 2.53 (1H, m) 34.5 8' 5.83 (1H, m) 133.7 5.90 (1H, m) 133.9 9' 5.23 (1H, d) 5.20 (1H, s) 119.4 5.20 (1H, d, 19.04 Hz) 5.27 (1H, d, 12.18 Hz) 118.1 COO 169.4 169.5 3-OCH 3 3.78 (3H, s) 55.8 3.79 (3H, s) 54.9 4-OCH 3 3.77 (3H, s) 60.7 3.71 (3H, s) 59.6 5-OCH 3 3.78 (3H, s) 55.8 3.79 (3H, s) 54.9 3՛-OCH 3 3.68 (3H, s) 55.4 3.71 (3H, s) 54.6 5՛-OCH 3 3.29 (3H, s) 55 3.24 (3H, s) 54 COOCH 3 2.25 (3H, s) 21 2.25 (3H, s) 19.4 Determination of the antibacterial activity of compound 1 The process of identifying potential compounds as antibacterial agents against oral pathogens of red betel ( P. crocatum) was guided by bioactivity testing. The extract of the isolated compound and bioactivity tests against E. faecalis were performed. Isolation and structural elucidation showed that the compound was a secondary metabolite of the lignan group, crocatin A. The results of the inhibition zone against E. faecalis showed that crocatin A could inhibit E. faecalis with inhibition zone values of 7, 7.2, 7.8 mm at concentrations of 1, 2, and 5%, respectively. Based on this value, crocatin A inhibited E. faecalis , had the highest value at a concentration of 5% in the moderate category. The MIC test against E. faecalis showed that Crocatin A inhibited the weak range at a concentration of 1250 µg/ml. In contrast, the MBC value of Crocatin A against E. faecalis showed that the bactericidal potential of Crocatin A weak, with an MBC of 5000 µg/mL (Fig. 2). This indicates that crocatin A can inhibit E. faecalis within the assay range, and it is likely that the concentration of crocatin A must be increased to kill E. faecalis . Therefore, crocatin A shows weak activity against E. faecalis . Antibacterial data for crocatin A are presented in Table 2 . The antibacterial activity of compound 1 against E. faecalis was evaluated using the disc diffusion test. Based on the reference inhibition zone values, compound 1 (crocatin A) showed moderate antibacterial activity against the strain at concentrations of 1, 2, and 5%, which were classified using the antibacterial in the Kirby-Bauer test, and has several categories for the strength of the antibacterial effect: no inhibition zone; inhibition zone diameter less than 5 mm was considered weak; inhibition zone diameter 5–10 mm was considered moderate; and inhibition zone diameter more than 10–20 mm was considered strong( 32 ),( 33 ). This indicated the potential of crocatin A, which was successfully isolated from red betel leaves. Based on reference, the MIC value is strong if MIC < 100 µg/mL, moderate if 100 625 µg/mL( 34 ). Table 2 Inhibition zone, MIC, and MBC of crocatin A against E. faecalis. Compound Inhibition zone (mm) MIC (µg/mL) MBC (µg/mL) 1% 2% 5% Compound 1 7 7.3 7.8 1250 5000 Chlorhexidine 2% (+) 20 - - Methanol (-) - - - Ramachandran plot The Ramachandran plot is a tool used to evaluate protein structure by displaying the distribution of the backbone dihedral angles φ (phi) and ψ (psi) of amino acid residues. Favorable rotations fall within the allowed regions, particularly the most favored (red) and additionally allowed (yellow) areas, which indicate sterically stable conformations. Typically, φ around − 60° and ψ around − 40° correspond to α-helices, while φ around − 120° and ψ around 120° indicate β-sheets. A high percentage of residues in these regions reflects a structurally valid protein model suitable for further analyses such as molecular docking or simulation( 35 ). Based on the results of the Ramachandran plot, most of the residues (black dots) of both enzymes are located in the highly allowed region (red) and allowed region (yellow) of the Ramachandran diagram. This indicates that the Gyrase B protein structure model has a stable conformation and good stereochemistry. Thus, the Gyrase B and Ligase structures have good stereochemical validity, with over 90% of residues located in the allowed regions. Therefore, this structure can be used with high confidence in docking simulations and further ligand–protein interaction analyses (Fig. 3). In Silico test (Molecular docking) Molecular interactions between crocatin A as a ligand and a protein as a receptor were predicted through molecular docking to determine the mechanism of action of crocatin A as an antibacterial agent. A scoring function from the docking results was required to examine the quality of the docking results. Physico chemical information of geometric complementarity was used to achieve more accurate results, such as binding affinity and intermolecular interactions between protein and ligand, including hydrogen bonds and hydrophobic interaction( 36 ),( 37 ). In addition to molecular docking studies involving Crocatin A ( 1 ) across various enzyme targets, docking was also performed on four structurally related derivatives that differ by a single functional group and have been previously isolated from natural sources. This approach aimed to assess how specific structural modifications influence binding affinity toward the target enzymes. By comparing binding energies and interaction profiles, the impact of functional group variations on inhibitory activity can be evaluated. The selected derivatives examined in this study, as shown in Fig. 4, include Crocatin B ( 2 ), Pipcroside A ( 3 ), Pipcroside B ( 4 ), and Pipcroside C ( 5 ). In this study, in silico analyses were used to predict the molecular docking of the inhibitory mechanism of crocatin A against key enzymes involved in bacterial growth. The key enzymes targeted in the in silico antibacterial assay were DNA gyrase B and DNA ligase. The strength of the ligand-protein interaction was determined by analyzing the binding affinity (∆G), inhibition constant (Ki)( 38 ), and intermolecular interaction values between Crocatin A, its derivatives, and positive controls for DNA gyrase B and DNA lygase. The results of the analysis of binding affinity values and inhibition constants of crocatin A and its derivatives for the two receptors are shown in Tables 3 and 4 . Table 3 Analysis of binding affinity values and inhibition constants of Crocatin A and its derivatives against DNA gyrase B enzyme. No. Compound Binding affinity (Kcal/mol) Inhibition constant/Ki 1 Compound 1 -6.34 22.43 µM 2 Crocatin B -7.38 3.88 µM 3 Pipcroside A -6.27 14.45 µM 4 Pipcroside B -6.02 15.25 µM 5 Pipcroside C -5.68 68.60 µM 6 Chlorhexidin -8.64 460.64 nM 7 4-[(3aR,6aR)-2,3,3a,4,6,6a-hexahydro-1H-pyrrolo[2,3-c] pyrrol-5-yl]-6-fluoro-N-methyl-2-(2-methylpyrimidin-5-yl) oxy-9H-pyrimido[4,5-b] indol-8-amine -9.20 180.49 nM Table 4 Analysis of binding affinity values and inhibition constants of Crocatin A and its derivatives against DNA ligase enzyme. No. Compound Binding affinity (Kcal/mol) Inhibition constant/Ki 1 Compound 1 -5.77 58.78 µM 2 Crocatin B -5.84 52.21 µM 3 Pipcroside A -7.18 5.49 µM 4 Pipcroside B -5.07 191.36 µM 5 Pipcroside C -4.91 250.59 µM 6 Chlorhexidin -10.37 25.08 nM 7 [(2 R ,3 S ,4 R ,5 R )-5-(3-carbamoylpyridin-1-ium-1-yl)-3,4-dihydroxyoxolan-2-yl] methyl dihydrogen phosphate -7.94 1.51 µM Based on several bacterial inhibition mechanisms, including inhibition of DNA protein synthesis, several key enzymes involved in this mechanism were selected, including DNA Gyrase B and DNA Ligase enzymes. This in silico study was conducted by rigid docking on the lattice box region of the native ligand of each receptor, that is the isolated compound was tethered to the native ligand tethering region, so that the potential of the compound to interact with the receptor could be determined and compared with that of the native ligand as an inhibitor. The native ligands of DNA Gyrase B were docked at positions 11.036 (X), -5.747 (Y), and 14.749 (Z). In addition to the native ligand, chlorhexidine was used as the positive control in the in vitro antibacterial assay. The binding affinity of the native ligand and chlorhexidine was compared with that of Crocatin A. DNA Gyrase B enzyme was obtained from the RCSB Protein Data Bank with enzyme code 1K4O, which has an RMSD value of 1.10 Å. Based on these results, crocatin A ( 1 ) showed binding affinity that was not significantly different from that of Crocatin B ( 2 ) and the native ligand and chlorhexidine. A greater binding affinity reduces the Ki value, and a small amount of the drug is required to achieve antibacterial activity. This value is important to indicate the ability of the compound to inhibit bacteria, as the inhibition constant correlates with the IC 50 value, regardless of competitive or non-competitive kinetics( 39 ). The test ligand crocatin A interacted more with the same amino acid residues as the amino acid residues that interacted with chlorhexidine, namely ASN A:48, SER A:49, THR A:168, SER A:122, ILE A:74, GLY A:79, and LYS A:138. The crocatin A ligand binds to the same five amino acid residues as chlorhexidine through van der Waals interactions. Van der Waals interactions are the most common interactions among all the ligands, and play a major role in the formation of hydrogen bonds and Coulomb interactions( 40 ). This made the binding affinity value of the test ligand close to that of chlorhexidine. The native ligand binds to only the same four amino acid residues as chlorhexidine. These types of interactions are illustrated in Fig. 5. The native ligands in the DNA ligase were docked at positions 6.062 (X), 32.743 (Y), and 26.151 (Z). Similar to DNA Gyrase B, chlorhexidine was used as a positive control in the in vitro antibacterial test in addition to the native ligand. The DNA ligase enzyme was obtained from the RCSB Protein Data Bank with the enzyme code 1TA8, which had an RMSD value of 1.80 Å. Molecular docking using Autodock 4.0, with 100 trials, showed four clusters of native ligand (-5.77 Kcal/mol) 4 times, Crocatin A (-10.37 Kcal/mol) 9 times, and chlorhexidine (-7.94 Kcal/mol) 15 time. Based on these results, crocatin A showed binding affinity values that were not significantly different from those of their derivatives and the native ligand; however, chlorhexidine had the best affinity value. The test ligand crocatin A interacted more with the same amino acid residues as those of the native ligand, namely ASP A:39, ASP A:43, TYR A:30, TYR A:29, ARG A:68, PRO A:65, and TYR A:25. Crocatin A binds to the same three amino acid residues as its native ligand via van der Waals interactions. This makes the binding affinity of the test ligand almost equal to that of the native ligand. Chlorhexidine binds only to the same two amino acid residues as in the native protein. All the types of interactions are illustrated in Fig. 6. ADMET and drug-likeness analysis Natural products contribute to nearly 50% of existing drugs, driving interest in their bioactive compounds for novel drug discovery due to their structural diversity and favorable pharmacokinetic properties. While experimental ADMET evaluation is costly and time-consuming, web-based computational tools, including AI models and drug-likeness scoring systems, offer efficient and reliable alternatives for predicting a compound’s drug potential. The aqueous solubility values of the tested compounds ranged from − 5 to 0, indicating poor solubility across all compounds. Despite this, Crocatin A ( 1 ) and ( 2 ) demonstrated high intestinal absorption rates (> 80%), as shown in Fig. 7. This suggests that, while solubility is limited, these compounds may still exhibit favorable oral bioavailability if supported by suitable formulation strategies. In terms of distribution, logVDss values were used to evaluate tissue distribution. All compounds showed logVDss values ≤ -0.15 (Table 5 ), indicating low distribution volume and a tendency to remain within the plasma compartment. This pharmacokinetic profile may allow lower doses to achieve therapeutic plasma concentrations, potentially reducing systemic side effects. The metabolic predictions indicated that none of the compounds inhibited CYP1A2, CYP2C19, CYP2C9, or CYP2D6 enzymes. However, both Crocatin A ( 1 ) and Crocatin B ( 2 ) were predicted to inhibit CYP3A4. This suggests a potential for drug–drug interactions when co-administered with other compounds metabolized by CYP3A4, warranting further evaluation in future pharmacokinetic studies. Total clearance values were used to estimate the elimination rate of the compounds. Pipcroside C ( 5 ) exhibited the highest predicted clearance (log 1.091 mL/min/kg), followed by Pipcroside B ( 4 ) and Pipcroside A ( 3 ), indicating relatively rapid elimination. In contrast, Crocatin B ( 2 ) showed the lowest clearance (log 0.393), suggesting a longer half-life and possible risk of accumulation upon repeated dosing. Crocatin A ( 1 ) presented a moderate clearance value (log 0.567), implying a balanced excretion profile. Toxicity assessments based on LD 50 values revealed relatively low acute toxicity across all compounds, with predicted LD50 values ranging from 2,541 to 3,977 mg/kg. Crocatin A showed the lowest LD 50 value, indicating slightly higher toxicity compared to its derivatives, though still within an acceptable safety margin. Additionally, none of the compounds exhibited potential for skin sensitization, suggesting good dermal safety profiles. Table 5 ADMET prediction of Crocatin A and its derivative compounds Properties Parameters Predicted Value Crocatin A ( 1 ) Crocatin B ( 2 ) Pipcroside A ( 3 ) Pipcroside B ( 4 ) Pipcroside C ( 5 ) Adsorption Water solubility (log mol/L) -4.36 -3.762 -3.21 -3.22 -3.36 Intestinal Absorption (% absorbed) 100 100 59.039 59.581 46.407 Skin Permeability -2.75 -2.787 -2.735 -2.735 -2.735 Distribution Volume Distribution (VDss, log L/kg) 0.022 0.099 -0.077 -0.031 0.13 BBB Permeability (log BBB) -1.131 -0.328 -1.683 -1.633 -1.72 CNS Permeability (log PS) -2.87 -2.868 -4.392 -4.374 -4.755 Metabolism Inhibitor of: CYP1A2 No No No No No CYP2C19 No No No No No CYP2C9 No No No No No CYP2D6 No No No No No CYP3A4 Yes Yes No No No Excretion Total Clearance (log ml/min/kg) 0.567 0.393 0.966 0.977 1.091 Toxicity Lethal Dose 50% (mg/kg) 2.541 2.979 3.598 3.498 3.977 Skin sensitisation No No No No No The drug-likeness profiles of the compounds were assessed using established predictive rules, including those proposed by Lipinski, Ghose, Veber, Egan, and Muegge, to evaluate their suitability as oral drug candidates. Crocatin A ( 1 ) and Crocatin B ( 2 ) showed the most favorable profiles, fully complying with all five rule sets without any violations. Both compounds also demonstrated a relatively high bioavailability score of 0.55, indicating good potential for oral absorption. Their predicted log P values (across multiple models) fall within acceptable ranges, suggesting adequate lipophilicity to support membrane permeability and absorption. In contrast, Pipcroside A ( 3 ), Pipcroside B ( 4 ), and Pipcroside C ( 5 ) violated multiple criteria. All three compounds exceeded the molecular weight and hydrogen bond acceptor limits set by Lipinski’s rule and failed additional thresholds in the Ghose, Veber, Egan, and Muegge filters, particularly due to high topological polar surface area (TPSA) values. Furthermore, their bioavailability scores were lower (0.17), implying limited oral bioavailability (Table 6 ). Overall, Crocatin A and Crocatin B stand out as the most promising drug-like candidates based on in silico evaluations, supporting their potential for further development as orally active agents. Table 6 Physicochemical properties of Crocatin A and its derivative compounds Physicochemical properties Compounds Crocatin A ( 1 ) Crocatin B ( 2 ) Pipcroside A ( 3 ) Pipcroside B ( 4 ) Pipcroside C ( 5 ) Chemical Formula C25H32O8 C23H30O7 C28H38O12 C28H38O12 C28H38O12 Molecular mass (≤ 500 g/mol) 460.52 418.48 566.59 566.24 566.59 Hydrogen bond acceptor (≤ 10) 8 7 12 12 12 Hydrogen bond donor (≤ 5) 0 1 5 5 5 Molar refractivity (130 ≥ MR index ≥ 40) 121.25 111.51 139.16 139.16 139.46 Lipophilicity Log P o/w (iLOGP) 3.67 2.84 1.37 3.01 2.67 Log P o/w (XLOGP3) 3.62 3.04 0.91 0.91 0.15 Log P o/w (WLOGP) 3.44 2.87 0.04 0.04 -0.17 Log P o/w (MLOGP) 1.12 0.75 -1.67 -1.67 -1.67 Log P o/w (SILICOS-IT) 4.20 3.66 1.11 1.11 1.11 Druglikeness Lipinski Yes; 0 violation MLOGP ≤ 4.15 Yes; 0 violation MLOGP ≤ 4.15 No; 2 violations: MW > 500, NorO > 10 No; 2 violations: MW > 500, NorO > 10 No; 2 violations: MW > 500, NorO > 10 Ghose Yes Yes No; 3 violations: MW > 480, MR > 130, #atoms > 7 No; 3 violations: MW > 480, MR > 130, #atoms > 7 No; 3 violations: MW > 480, MR > 130, #atoms > 7 Veber Yes Yes No; 1 violation: TPSA > 140 No; 1 violation: TPSA > 140 No; 1 violation: TPSA > 140 Egan Yes Yes No; 1 violation: TPSA > 131.6 No; 1 violation: TPSA > 131.6 No; 1 violation: TPSA > 131.6 Muegge Yes Yes No; 2 violations: TPSA > 150, H-acc > 10 No; 2 violations: TPSA > 150, H-acc > 10 No; 2 violations: TPSA > 150, H-acc > 10 Bioavailability Score 0.55 0.55 0.17 0.17 0.17 Conclusion Crocatin A isolated from the methanol extract of Red Betel leaves, showed antibacterial properties against E. faecalis . The inhibitory potential of crocatin A against E. faecalis in the inhibition zone wass categorized as moderate, and MIC and MBC showed weak results. In a molecular docking study, crocatin A showed binding affinity to DNA gyrase B and DNA Ligase enzyme is -6.34 and − 5.77 Kcal/mol. The results of drug likeness analysis also show promising results as oral medications with several parameter considerations. Based on in vitro and in silico results, it can be concluded that Crocatin A and its derivatives have good potential as antibacterial agents. Abbreviations E. faecalis Enterococcus faecalis P. crocatum Piper crocatum MIC Minimum Inhibition Concentration MBC Minimum Bactericidal Concentration ADMET Adsorption, Distribution, Metabolism, Excretion, Toxicity LD 50 Lethal Dose 50% Ki Inhibition Konstanta NMR Nuclear Magnetic Resonance DEPT Distortionless Enhancement by Polarization Transfer HMQC Heteronuclear Multiple Quantum Coherence HMBC Heteronuclear Multiple Bond Correlation IR Infra-Red UV Ultraviolet Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare no competing interests. Funding No funding. Availability of data and materials No datasets were generated or analysed during the current study. Author contributions Devi Meliani: writing—review and editing, writing—original draft and reviewing process handling. Trisna Yuliana: writing—review and editing, writing—original draft. 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2","display":"","copyAsset":false,"role":"figure","size":27233,"visible":true,"origin":"","legend":"\u003cp\u003eInhibition zone of crocatin A against \u003cem\u003eE. faecalis.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Fig.2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7015807/v1/c21a66a8c8cc68e8a3715280.jpg"},{"id":88277084,"identity":"bb95d8c5-2e57-4a49-bb79-5839c588cb80","added_by":"auto","created_at":"2025-08-04 18:30:25","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":187123,"visible":true,"origin":"","legend":"\u003cp\u003eRamachandran plot of DNA Gyrase B and Lygase\u003c/p\u003e","description":"","filename":"Fig.3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7015807/v1/b9ec886e00b7a5ff40fb1a69.jpg"},{"id":88276342,"identity":"418d5961-4935-4de4-a889-c4c4115ae359","added_by":"auto","created_at":"2025-08-04 18:22:25","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":391316,"visible":true,"origin":"","legend":"\u003cp\u003eThe structure of Crocatin A and its derivatives (the difference is marked by pink highlights).\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-7015807/v1/3fc3c0b41b4c85648ea414c2.png"},{"id":88277086,"identity":"819b0750-554c-46e9-bbda-97d4dd0093b1","added_by":"auto","created_at":"2025-08-04 18:30:25","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2392333,"visible":true,"origin":"","legend":"\u003cp\u003eMolecular docking of crocatin A (a), crocatin B (b), pipcroside A (c), pipcroside B (d), pipcroside C (e), chlorhexidine (f), and native ligand (g) against DNA gyrase B.\u003c/p\u003e","description":"","filename":"Fig.5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7015807/v1/ce986c0b3eb2abe65006a8cb.jpg"},{"id":88276346,"identity":"3f48e661-b67f-4405-a42b-c33db476c917","added_by":"auto","created_at":"2025-08-04 18:22:25","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":5943005,"visible":true,"origin":"","legend":"\u003cp\u003eMolecular docking of crocatin A (a), crocatin B (b), pipcroside A (c), pipcroside B (d), pipcroside C (e), chlorhexidine (f), and native ligand (g) against DNA ligase.\u003c/p\u003e","description":"","filename":"Fig.6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7015807/v1/d8e82bf07c285394752b9a45.jpg"},{"id":88277087,"identity":"98b42550-2996-42bf-a7b6-f4174404f547","added_by":"auto","created_at":"2025-08-04 18:30:25","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":86359,"visible":true,"origin":"","legend":"\u003cp\u003eSwissADME bioavailability radar of different bioactive drug-likeness molecules\u003c/p\u003e","description":"","filename":"Fig.7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7015807/v1/08ab04584bb3899586c5f316.jpg"},{"id":104250782,"identity":"019026ae-dd71-4955-ae4a-3260de41bb06","added_by":"auto","created_at":"2026-03-09 16:08:27","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":10327622,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7015807/v1/46ee2c3b-53e9-4cf8-b2cc-51144d0081d9.pdf"},{"id":88277613,"identity":"3ce06f5b-5883-4cfe-9605-37786e878b2b","added_by":"auto","created_at":"2025-08-04 18:38:25","extension":"docx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":1700068,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterialBMCChemistryResearchpaper.docx","url":"https://assets-eu.researchsquare.com/files/rs-7015807/v1/305f00dd3cae9574f729b51c.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"In Vitro and In Silico Characterization of Crocatin A from Red Betel Leaves: Targeting DNA Gyrase B and DNA Ligase of Enterococcus faecalis with ADMET-Based Druglikeness Analysis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIn 2018, oral health problems, particularly dental caries, affected almost half of the world's population of 3.58\u0026nbsp;billion people. According to the World Health Organization (WHO), in 2019, 60\u0026ndash;90% of school-aged children and almost 100% of adults had dental issues(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Dental caries are the primary cause of root canal infections. Caries is a periodontal disease caused by damage to the hard tissues of the teeth (enamel and dentin layers), which can extend to the nerves of the teeth due to the fermentation of carbohydrates, particularly sucrose, by pathogenic microorganisms in the mouth(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). \u003cem\u003eE. faecalis\u003c/em\u003e plays a major role in root canal infections are \u003cem\u003eEnterococcus faecalis\u003c/em\u003e(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). \u003cem\u003eE. faecalis\u003c/em\u003e is a Gram-positive cocci microorganism that can survive facultatively without oxygen (facultative anaerobes). In addition, these microorganisms can survive in the root canal for a long period of time without being supplied with nutrients(\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eMany attempts have been made to treat oral infections by using synthetic antibacterial agents. The use of the tetracycline class of antibiotics should be limited to the root canals because of the risk of discoloration of the teeth(\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). The use of various antibiotics can also induce bacterial resistance. Therefore, to overcome the problems of drug and bacterial resistance, a solution is needed to identify active compounds that can be used as new drugs that are effective and safe. Sources of bioactive compounds can be found in plants, one of which is Red betel leaf(\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Red betel leaf \u003cem\u003e(P. crocatum)\u003c/em\u003e is known to have good pharmacological effects, such as antifungal(\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e), antibacterial (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e)antiinflammatory, antioxidant, antidiabetic, antitumour, anticancer, hepatoprotective, immunomodulatory, antihyperglycaemic, and analgesic (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e),(\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e),(\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e),(\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). The compounds in this plant include flavonoids, phenolics, terpenoids, steroids, tannins, and essential oils, all of which are known to have various bioactivities(\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eBacterial growth can be inhibited by several mechanisms, such as the inhibiton of DNA replication, which inhibits protein synthesis and microbial growth(\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Computational chemistry plays an important role in drug discovery and development. Computational chemistry also facilitates the development of new drugs from traditional medicinal plants(\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). In the \u003cem\u003ein silico\u003c/em\u003e molecular docking study, the inhibition of bacterial DNA replication was carried out by inhibiting one of the enzymes involved, such as DNA gyrase B, which mediates the introduction of negative supercoils, and DNA ligase, which acts as a splicer of fragments formed during the replication process or when the DNA repair process takes place; thus, inhibition of DNA gyrase B and DNA ligase enzymes can be used as potential target(\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). Interaction modelling of compounds as drug candidates and proteins as receptor targets can be used to search for new drugs that are more effective and efficient(\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Therefore, the aim of this study was to identify the antibacterial components of \u003cem\u003eP. crocatum\u003c/em\u003e against \u003cem\u003eE. faecalis\u003c/em\u003e. We predicted the molecular interactions that inhibit key enzymes in bacterial DNA synthesis, namely, DNA gyrase B and DNA ligase.\u003c/p\u003e"},{"header":"Material and methods","content":"\u003cp\u003e\u003cb\u003eMaterials\u003c/b\u003e\u003c/p\u003e\u003cp\u003eRed Betel (\u003cem\u003ePiper cocatum\u003c/em\u003e Ruiz and Pav) is a plant species that is native to Bandung, West Java, Indonesia. Plant specimens were obtained from the Systematics and Molecular Laboratory located at the Department of Biology, Faculty of Mathematics and Natural Sciences, Padjadjaran University, Sumedang, Indonesia. Organic solvents, namely, methanol, \u003cem\u003en\u003c/em\u003e-hexane, and ethyl acetate, were used for extraction, separation, and purification. Column chromatography was performed using Silica G 60 (Merck, Darmstadt, Germany) and ODS RP-18, whereas thin-layer chromatography (TLC) was performed using Silica G 60 F\u003csub\u003e254\u003c/sub\u003e plates and ODS RP-18 F\u003csub\u003e254\u003c/sub\u003e S (Merck, Darmstadt, Germany). Compound spots on the TLC plate were visualized under ultraviolet (UV) light at 254 nm and 365 nm by spraying with 10% H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e in Ethanol.\u003c/p\u003e\u003cp\u003eIn vitro antibacterial tests were performed using \u003cem\u003eEnterococcus faecalis\u003c/em\u003e ATCC 29212, Mueller-Hinton broth, and Mueller-Hinton agar as media, methanol as a negative control, and chlorhexidine as a positive control.\u003c/p\u003e\u003cp\u003eThe 3D structures of DNA Gyrase B and DNA Ligase used in this study were obtained from the Protein Data Bank (PDB) ID: 4K4O,1TA8. Crocatin A, a compound isolated from \u003cem\u003ePiper crocatum\u003c/em\u003e, was obtained from ChemDraw3D. The antibacterial positive control, chlorhexidine (CID 9552079), and the native ligand for DNA Gyrase B (CID 66560858), a native ligand for DNA Ligase (CID 14181), were used. All data were obtained from PubMed (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/pccompound\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/pccompound\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003eInstruments\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe structure of the active compound was determined by infrared (IR) spectroscopy with an FTIR Spectrum-100 spectrometer (Buckinghamshire, England), and HR-ESI-MS spectra were acquired using a quadrupole time-of-flight mass spectrometer (Xevo G2-XS QTOF, Waters Corp.) and, Nuclear Magnetic Resonance (NMR) (\u003csup\u003e1\u003c/sup\u003eH-NMR, \u003csup\u003e13\u003c/sup\u003eC-NMR, and135\u0026ordm; DEPT) with a Bruker Avance 700 MHz (Bruker, Germany). The TLC plate was visualized using a UV lamp detector with λmax wavelengths of 254 and 365 nm. For the antibacterial activity test, 96-well microplates (NEST Biotechnology, Wuxi, China), micropipettes (Winlab, Grogol, Jakarta), microtubes (Chemikalie, Pandan Loop, Singapore), an incubator (Memmert, Schwabach, Germany), paper discs (Grainger, Origin, USA), and a Biochrom microplate reader (Biochrom, Ltd., Cambridge, UK) were used.\u003c/p\u003e\u003cp\u003e\u003cb\u003eIsolation Compound from Extract of\u003c/b\u003e \u003cb\u003eP. crocatum\u003c/b\u003e\u003c/p\u003e\u003cp\u003eA sample of 1.6 kg of fresh \u003cem\u003eP. crocatum\u003c/em\u003e leaf was extracted with methanol (20 L) by the maceration method for 3\u0026times;24 h. The filtrate from the maceration process was evaporated using a rotary evaporator at 40\u0026deg;C, and 100 g of the methanol extract was obtained. For the bioactivity evaluation of antibacterial activity, extracts were prepared at a series of concentrations according to assay protocols(\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe methanol extract (10 g), which showed the best activity against \u003cem\u003eE. faecalis\u003c/em\u003e ATCC 29212, was purified by gradient chromatography using Silica G 60 (0.063-0.200 mm) and a combination of \u003cem\u003en\u003c/em\u003e-hexane-ethyl acetate-methanol solvents for elution. Fractions F.1\u0026ndash;10 were obtained and visualized under UV light at 254 and 356 nm by spraying with 10% H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e in EtOH, followed by heating. Next, each fraction from F1-F10 was tested for antibacterial activity using the agar disk Kirby-Bauer method. Among the fractions, fraction 7 (493.7 mg) exhibited the largest inhibition zone (8 mm). The F7 subfraction (493.7 mg) was purified by column chromatography on an ODS RP-18 column and eluted with H\u003csub\u003e2\u003c/sub\u003eO-MeOH at a gradient of 10% (v/v) to yield compound \u003cb\u003e1\u003c/b\u003e (11.1 mg).\u003c/p\u003e\u003cp\u003e\u003cb\u003eStructure Determination of Isolated Compound\u003c/b\u003e\u003c/p\u003e\u003cp\u003eComprehensive spectroscopic analysis data were used to determine the structure of the isolated compound, including ultraviolet (UV), mass spectrometry (MS), 1D and 2D-NMR (\u003csup\u003e1\u003c/sup\u003eH-NMR, \u003csup\u003e13\u003c/sup\u003eC-NMR, DEPT 135\u0026deg;, HMQC, \u003csup\u003e1\u003c/sup\u003eH-\u003csup\u003e1\u003c/sup\u003eH COSY, HMBC), and infrared (IR) spectra.\u003c/p\u003e\u003cp\u003e\u003cb\u003eAssessment of the Extracts and Isolated Compound of\u003c/b\u003e \u003cb\u003eP. crocatum\u003c/b\u003e \u003cb\u003eLeaf Activity Against\u003c/b\u003e \u003cb\u003eE. faecalis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe Kirby-Bauer disk diffusion method was used to evaluate \u003cem\u003eP. crocatum\u003c/em\u003e extracts against \u003cem\u003eE. faecalis\u003c/em\u003e ATCC 29212. The diameter of the growth area around the paper disk was measured to determine the zone of each sample(\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). The assay determines the resistance or sensitivity of \u003cem\u003eE. faecalis\u003c/em\u003e ATCC 29212 strains to extracts guided by CLSI protocols (CLSI, 2012)(\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). Methanol was used to dilute all samples, except for the water fraction, and the positive control (chlorhexidine) was diluted in water. Stock solutions were prepared for each extract and 75 mg of each extract was diluted in methanol, except for the water fraction, to obtain a 5% stock solution.\u003c/p\u003e\u003cp\u003eNext, 2, 2.5, 5, 10, 20, and 30% concentrations of all the samples, together with 2% chlorhexidine, were prepared for the assay. Then, 20 \u0026micro;L of each sample was impregnated onto a 6 mm paper disk and placed on the agar surface. Repeated tests were required to obtain better results. 1 ose of bacteria was grown in 5 mL of broth media to prepare the bacteria. The solution was incubated at 37\u0026deg;C for 24 h. A microplate reader was used to measure the optical density of the solution at 620 nm after the incubation. The solution was diluted to reach 0.5 McFarland standard or approximately 180 CFU/mL in broth media (100 \u0026micro;L). The resulting culture was swabbed onto the surface of agar medium and a paper disk was prepared. The samples were incubated at 37\u0026deg;C for 24 h. Repeated tests were required to obtain better results.\u003c/p\u003e\u003cp\u003eThe MIC and MBC of the compound against \u003cem\u003eE. faecalis\u003c/em\u003e ATCC 29212 were determined using the microdilution method in a 96-well microplate(\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Bacterial cells were pre-cultured under aerobic conditions in Mueller-Hinton broth at 37\u0026deg;C. In the presence of several concentrations of a compound inserial two-fold dilutions, bacterial cells were incubated at 37\u0026deg;C without shaking for 48 h in the same broth on a microplate, as shown in the procedure used at the Clinical and Laboratory Standards Institute. The optical density of the solution was measured at 620 nm wavelength using a microplate reader. The MICs of the cells were defined as the lowest concentrations where, visually the bacterial cells were not observed by the OD value, as reported previously, and given by duplicate assessments. The compound was dissolved in methanol and used as a negative control, while chlorhexidine was used as a positive control. Each concentration of the compound solution in the microplate well was spread onto the surface of the agar and incubated at 37\u0026deg;C for 24 h. After incubation, the amount of liquid in each well of the microplate was measured at 620 nm using a microplate reader. MIC was determined by comparing the absorption value of the sample well (compound plus bacteria) with that of the blank well (bacteria). Furthermore, the liquid from the wells was spread on Mueller-Hinton agar and incubated for 24 h to evaluate the MBC, the lowest concentration of the sample required to kill the bacteria.\u003c/p\u003e\u003cp\u003e\u003cb\u003eRamachandran plot for enzyme validation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eRamachandran plot assessed the enzyme\u0026rsquo;s structural accuracy in this study. Each three-dimensional structure DNA Gyrase B (PDB ID : 4K4O) and Lygase (PDB ID : 1TA8) were input as .pdb format on the PROCHECK SAVES v6.1 by UCLA web server (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://saves.mbi.ucla.edu/\u003c/span\u003e\u003cspan address=\"https://saves.mbi.ucla.edu/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003eIn silico\u003c/b\u003e \u003cb\u003eCharacterization of Crocatin A\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAutoDock 4.0, in the open-source software PyRx 0.8 was used for ligand-protein docking and virtual screening of antibacterial activity. Crocatin A and chlorhexidine were used for binding to DNA gyrase B and DNA ligase as protein targets, and the ligands were free for blind docking. 4-[(3aR,6aR)-2,3,3a,4,6,6a-hexahydro-1H-pyrrolo[2,3-c]pyrrol-5-yl]-6-fluoro-N-methyl-2-(2 methylpyrimidin-5-yl)oxy-9H-pyrimido[4,5-b]indol-8-amine, the native ligand for the DNA Gyrase B enzyme, and [(2\u003cem\u003eR\u003c/em\u003e,3\u003cem\u003eS\u003c/em\u003e,4\u003cem\u003eR\u003c/em\u003e,5\u003cem\u003eR\u003c/em\u003e)-5-(3-carbamoylpyridin-1-ium-1-yl)-3,4-dihydroxyoxolan-2-yl]methyl dihydrogen phosphate, the native ligand for the DNA ligase enzyme, was being re-docked to the enzyme. The conformation was selected based on binding energy: the one with the lowest binding energy score that has a value of root-mean-square deviation less than 2.0 \u0026Aring; was selected(\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e)\u003csup\u003e,\u003c/sup\u003e(\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cb\u003eADME/Tox prediction and drug-likeness analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eADME (absorption, distribution, metabolism, and excretion) predictions were analyzed on the pkCSM web server (biosig.lab.uq.edu.au/pcksm/), while drug-likeness predictions were completed from the SwissADME web server (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.swissadme.ch/\u003c/span\u003e\u003cspan address=\"http://www.swissadme.ch/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003eOral infections caused by pathogenic bacteria such as \u003cem\u003eE. faecalis\u003c/em\u003e can cause various oral health problems, including dental root canal infections. The use of several antibiotics and antiseptics to prevent and cure such infections over a long period can cause side effects, with the emergence of bacterial resistance(\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e). Therefore, new antibacterial agents that are more effective and efficient are needed. One alternative is to utilize the potential of medicinal plants such as the red betel plant \u003cem\u003e(P. crocatum)\u003c/em\u003e(\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e). Ethnobotanical and ethnopharmacological data show that the herbal red betel \u003cem\u003e(P. cocatum)\u003c/em\u003e has various biological activities, including antitumor, antibacterial, antioxidant, antifungal, anti-inflammatory, anticancer, and antidiabetic(\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e),(\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e). Red Betel \u003cem\u003e(P. crocatum\u003c/em\u003e) can be used as an alternative to obtain herbal antibacterial compounds against pathogenic bacteria in dental root canals, namely \u003cem\u003eE. faecalis\u003c/em\u003e(\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStructural Determination of Compound\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe isolation of compounds from the methanol extract of red betel leaves yielded compound \u003cstrong\u003e1\u003c/strong\u003e with the characteristics of white crystals that can dissolve in chloroform. The pure compound isolated from red betel leaves had an Rf value of 0.72 as thin layer chromatography (TLC) Silica G 60 F\u003csub\u003e254\u003c/sub\u003e eluted with eluent \u003cem\u003en\u003c/em\u003e-hexane: ethyl acetate (7:3). While on 2-dimensional TLC, compound \u003cstrong\u003e1\u003c/strong\u003e had an R\u003cem\u003ef\u003c/em\u003e value of 0.62 with a combination of \u003cem\u003en\u003c/em\u003e-hexane: ethyl acetate and n-hexane: acetone (7:3) as the eluent.\u003c/p\u003e\n\u003cp\u003eCompound \u003cstrong\u003e1\u003c/strong\u003e shows in the FTIR spectrum of 1 shows CH \u003cem\u003eS\u003c/em\u003ep\u003csup\u003e3\u003c/sup\u003e absorption at 2947 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, C\u0026thinsp;=\u0026thinsp;O acetyl at 1742 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, C\u0026thinsp;=\u0026thinsp;O unsaturated carbon at 1704 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, C\u0026thinsp;=\u0026thinsp;C aromatic at 1590 and 1511 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, C-O-CH\u003csub\u003e3\u003c/sub\u003e stretching at 1235 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and CH\u003csub\u003e2\u003c/sub\u003e-O stretching at 1123 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe structure of compound \u003cstrong\u003e1\u003c/strong\u003e was determined by NMR spectroscopy using a Bruker Avance 700 MHz spectrometer. \u003csup\u003e13\u003c/sup\u003eC-NMR, DEPT, and HMQC spectra of compound \u003cstrong\u003e1\u003c/strong\u003e showed 25 carbon signals for seven methyl carbons, two methylene carbons, seven methine carbons, and nine quaternary carbons. Important signals including aromatic carbon appeared at \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\delta\\:\\)\u003c/span\u003e\u003c/span\u003eC 152.6, 136.8, 152.6, 133.5, 105.9 and 105.9; olefinic carbon at \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\delta\\:\\text{C}\\)\u003c/span\u003e\u003c/span\u003e 152.2 and 126.2; allyl group at \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\delta\\:\\)\u003c/span\u003e\u003c/span\u003eC 34.5, 133.9 and 118.1; methoxy-substituted aromatic group at \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\delta\\:\\)\u003c/span\u003e\u003c/span\u003eC 54.9, 59.6, and 54.9; methoxy-aliphatic carbon at \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\delta\\:\\)\u003c/span\u003e\u003c/span\u003eC 54.6 and 54. A methyl carbon was observed at \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\delta\\:\\)\u003c/span\u003e\u003c/span\u003eC 16.1, and another methyl group attached to the carbonyl was found at \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\delta\\:\\)\u003c/span\u003e\u003c/span\u003eC 19.4. Two quaternary carbons were observed at \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\delta\\:\\)\u003c/span\u003e\u003c/span\u003eC 94.8 and 48.3, while a carbinol carbon appeared at \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\delta\\:\\)\u003c/span\u003e\u003c/span\u003eC 78.2.\u003c/p\u003e\n\u003cp\u003eThe \u003csup\u003e1\u003c/sup\u003eH NMR spectrum showed five methoxy signals [\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\delta\\:\\)\u003c/span\u003e\u003c/span\u003eH 3.79 (3H, s), 3.79 (3H, s), 3.71 (3H, s), 3.71 (3H, s), and 3.24 (3H, s)] and one methyl signal [\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\delta\\:\\)\u003c/span\u003e\u003c/span\u003eH 1.31 (3H, d)]. Signals corresponding to allyl groups appeared at \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\delta\\:\\)\u003c/span\u003e\u003c/span\u003eH 5.90 (1H, m), 5.27 (1H, d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;12.18 Hz), 5.20 (1H, d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;19.07 Hz), 2.53 (1H, m), and 2.43 (2H, m). An aromatic proton was observed at \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\delta\\:\\)\u003c/span\u003e\u003c/span\u003eH 6.34 (2H, s). An olefinic proton appeared at \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\delta\\:\\)\u003c/span\u003e\u003c/span\u003eH 6.45 (1H, s), possibly indicative of an enone system, and a benzylic methine was observed at \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\delta\\:\\)\u003c/span\u003e\u003c/span\u003eH 3.34 (3H, s). The additional signals at \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\delta\\:\\)\u003c/span\u003e\u003c/span\u003eH 5.32 (1H, s) and 2.43 (2H, m) correspond to the carbinol and methine protons, respectively. HMBC revealed a correlation between C-5 (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\delta\\:\\)\u003c/span\u003e\u003c/span\u003eC 136.8), C-3 (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\delta\\:\\)\u003c/span\u003e\u003c/span\u003eC 152.6), and C-4 (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\delta\\:\\)\u003c/span\u003e\u003c/span\u003eC 152.6) methoxy protons as benzene substituents (\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e)\u003csup\u003e,\u003c/sup\u003e(\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e), a correlation between protons of C-2\u0026apos; (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\delta\\:\\)\u003c/span\u003e\u003c/span\u003eC 78.2) and C-4\u0026apos; (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\delta\\:\\)\u003c/span\u003e\u003c/span\u003eC 192.5) ketones, a correlation between protons of C-2\u0026apos; (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\delta\\:\\)\u003c/span\u003e\u003c/span\u003eC 78.2) and acetyl, a correlation between protons of C-9\u0026apos; (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\delta\\:\\)\u003c/span\u003e\u003c/span\u003eC 118.1) allyl and C-7\u0026apos; (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\delta\\:\\)\u003c/span\u003e\u003c/span\u003eC 34.5), and a correlation between C-9 (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\delta\\:\\)\u003c/span\u003e\u003c/span\u003eC 16.1) methyl protons and C-8 (\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\delta\\:\\)\u003c/span\u003e\u003c/span\u003eC 47.6).\u003c/p\u003e\n\u003cp\u003eBased on analysis of the NMR spectral data, compound \u003cstrong\u003e1\u003c/strong\u003e was identified as a compound from the neolignan group, crocatin A (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e), with the chemical formula C\u003csub\u003e25\u003c/sub\u003eH\u003csub\u003e32\u003c/sub\u003eO\u003csub\u003e8\u003c/sub\u003e and a molecular mass value 460.52 \u003cem\u003em/z\u003c/em\u003e. The structure of compound\u0026nbsp;\u003cstrong\u003e1\u003c/strong\u003e (Fig. 1).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eComparison compound \u003cstrong\u003e1\u003c/strong\u003e and crocatin A with literature\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003eCrocatin A (\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eCompound 1 (700 MHz, CD\u003csub\u003e3\u003c/sub\u003eOD)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePosisi\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\delta\\:\\)\u003c/span\u003e\u003c/span\u003eH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\delta\\:\\text{C}\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\delta\\:\\)\u003c/span\u003e\u003c/span\u003eH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\delta\\:\\text{C}\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e133\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e133.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.26 (3H, s)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e105.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.34 (2H, s)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e105.9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e152.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e152.6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e137.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e136.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e152.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e152.6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.26 (3H, s)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e105.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.34 (2H, s)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e105.9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.33 (1H, d)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e59.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.34 (3H, s) 3.71 (3H, s)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e59.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.30 (1H, m)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e48.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.43 (2H, m)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e47.6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.28 (3H, d)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.31 (3H, d,7.07 Hz)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e48.3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.35 (1H, s)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e78.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.32 (1H, s)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e78.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e94.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e94.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e191.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e192.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e152.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e152.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.17 (1H, s)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e124.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.45 (1H, s)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e126.2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.46 (1H, m) 2.34 (1H, m)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e35.1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.43 (2H, m) 2.53 (1H, m)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e34.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.83 (1H, m)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e133.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.90 (1H, m)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e133.9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.23 (1H, d) 5.20 (1H, s)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e119.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.20 (1H, d, 19.04 Hz) 5.27 (1H, d, 12.18 Hz)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e118.1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCOO\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e169.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e169.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3-OCH\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.78 (3H, s)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e55.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.79 (3H, s)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e54.9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4-OCH\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.77 (3H, s)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e60.7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.71 (3H, s)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e59.6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5-OCH\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.78 (3H, s)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e55.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.79 (3H, s)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e54.9\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3՛-OCH\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.68 (3H, s)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e55.4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.71 (3H, s)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e54.6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5՛-OCH\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.29 (3H, s)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.24 (3H, s)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e54\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCOOCH\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.25 (3H, s)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.25 (3H, s)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e19.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003eDetermination of the antibacterial activity of compound 1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe process of identifying potential compounds as antibacterial agents against oral pathogens of red betel (\u003cem\u003eP. crocatum)\u003c/em\u003e was guided by bioactivity testing. The extract of the isolated compound and bioactivity tests against \u003cem\u003eE. faecalis\u003c/em\u003e were performed. Isolation and structural elucidation showed that the compound was a secondary metabolite of the lignan group, crocatin A. The results of the inhibition zone against \u003cem\u003eE. faecalis\u003c/em\u003e showed that crocatin A could inhibit \u003cem\u003eE. faecalis\u003c/em\u003e with inhibition zone values of 7, 7.2, 7.8 mm at concentrations of 1, 2, and 5%, respectively. Based on this value, crocatin A inhibited \u003cem\u003eE. faecalis\u003c/em\u003e, had the highest value at a concentration of 5% in the moderate category. The MIC test against \u003cem\u003eE. faecalis\u003c/em\u003e showed that Crocatin A inhibited the weak range at a concentration of 1250 \u0026micro;g/ml. In contrast, the MBC value of Crocatin A against \u003cem\u003eE. faecalis\u003c/em\u003e showed that the bactericidal potential of Crocatin A weak, with an MBC of 5000 \u0026micro;g/mL (Fig. 2). This indicates that crocatin A can inhibit \u003cem\u003eE. faecalis\u003c/em\u003e within the assay range, and it is likely that the concentration of crocatin A must be increased to kill \u003cem\u003eE. faecalis\u003c/em\u003e. Therefore, crocatin A shows weak activity against \u003cem\u003eE. faecalis\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eAntibacterial data for crocatin A are presented in Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e. The antibacterial activity of compound \u003cstrong\u003e1\u003c/strong\u003e against \u003cem\u003eE. faecalis\u003c/em\u003e was evaluated using the disc diffusion test. Based on the reference inhibition zone values, compound \u003cstrong\u003e1\u003c/strong\u003e (crocatin A) showed moderate antibacterial activity against the strain at concentrations of 1, 2, and 5%, which were classified using the antibacterial in the Kirby-Bauer test, and has several categories for the strength of the antibacterial effect: no inhibition zone; inhibition zone diameter less than 5 mm was considered weak; inhibition zone diameter 5\u0026ndash;10 mm was considered moderate; and inhibition zone diameter more than 10\u0026ndash;20 mm was considered strong(\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e),(\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e). This indicated the potential of crocatin A, which was successfully isolated from red betel leaves. Based on reference, the MIC value is strong if MIC\u0026thinsp;\u0026lt;\u0026thinsp;100 \u0026micro;g/mL, moderate if 100\u0026thinsp;\u0026lt;\u0026thinsp;MIC\u0026thinsp;\u0026le;\u0026thinsp;625 \u0026micro;g/mL, and weak if MIC\u0026thinsp;\u0026gt;\u0026thinsp;625 \u0026micro;g/mL(\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e).\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eInhibition zone, MIC, and MBC of crocatin A against \u003cem\u003eE. faecalis.\u003c/em\u003e\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eCompound\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003eInhibition zone (mm)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eMIC (\u0026micro;g/mL)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eMBC (\u0026micro;g/mL)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e1%\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e2%\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e5%\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCompound \u003cstrong\u003e1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1250\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5000\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eChlorhexidine 2% (+)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMethanol (-)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e\u003cstrong\u003eRamachandran plot\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Ramachandran plot is a tool used to evaluate protein structure by displaying the distribution of the backbone dihedral angles \u0026phi; (phi) and \u0026psi; (psi) of amino acid residues. Favorable rotations fall within the allowed regions, particularly the most favored (red) and additionally allowed (yellow) areas, which indicate sterically stable conformations. Typically, \u0026phi; around \u0026minus;\u0026thinsp;60\u0026deg; and \u0026psi; around \u0026minus;\u0026thinsp;40\u0026deg; correspond to \u0026alpha;-helices, while \u0026phi; around \u0026minus;\u0026thinsp;120\u0026deg; and \u0026psi; around 120\u0026deg; indicate \u0026beta;-sheets. A high percentage of residues in these regions reflects a structurally valid protein model suitable for further analyses such as molecular docking or simulation(\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eBased on the results of the Ramachandran plot, most of the residues (black dots) of both enzymes are located in the highly allowed region (red) and allowed region (yellow) of the Ramachandran diagram. This indicates that the Gyrase B protein structure model has a stable conformation and good stereochemistry. Thus, the Gyrase B and Ligase structures have good stereochemical validity, with over 90% of residues located in the allowed regions. Therefore, this structure can be used with high confidence in docking simulations and further ligand\u0026ndash;protein interaction analyses (Fig. 3).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn Silico\u003c/strong\u003e \u003cstrong\u003etest (Molecular docking)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMolecular interactions between crocatin A as a ligand and a protein as a receptor were predicted through molecular docking to determine the mechanism of action of crocatin A as an antibacterial agent. A scoring function from the docking results was required to examine the quality of the docking results. Physico chemical information of geometric complementarity was used to achieve more accurate results, such as binding affinity and intermolecular interactions between protein and ligand, including hydrogen bonds and hydrophobic interaction(\u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e),(\u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eIn addition to molecular docking studies involving Crocatin A (\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e) across various enzyme targets, docking was also performed on four structurally related derivatives that differ by a single functional group and have been previously isolated from natural sources. This approach aimed to assess how specific structural modifications influence binding affinity toward the target enzymes. By comparing binding energies and interaction profiles, the impact of functional group variations on inhibitory activity can be evaluated. The selected derivatives examined in this study, as shown in Fig.\u0026nbsp;4, include Crocatin B (\u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e), Pipcroside A (\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e), Pipcroside B (\u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e), and Pipcroside C (\u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eIn this study, in silico analyses were used to predict the molecular docking of the inhibitory mechanism of crocatin A against key enzymes involved in bacterial growth. The key enzymes targeted in the in silico antibacterial assay were DNA gyrase B and DNA ligase. The strength of the ligand-protein interaction was determined by analyzing the binding affinity (∆G), inhibition constant (Ki)(\u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e), and intermolecular interaction values between Crocatin A, its derivatives, and positive controls for DNA gyrase B and DNA lygase. The results of the analysis of binding affinity values and inhibition constants of crocatin A and its derivatives for the two receptors are shown in Tables \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e and\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab3\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eAnalysis of binding affinity values and inhibition constants of Crocatin A and its derivatives against DNA gyrase B enzyme.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNo.\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCompound\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eBinding affinity (Kcal/mol)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eInhibition constant/Ki\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCompound \u003cstrong\u003e1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-6.34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e22.43 \u0026micro;M\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCrocatin B\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-7.38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.88 \u0026micro;M\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePipcroside A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-6.27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14.45 \u0026micro;M\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePipcroside B\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-6.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e15.25 \u0026micro;M\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePipcroside C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-5.68\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e68.60 \u0026micro;M\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eChlorhexidin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-8.64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e460.64 nM\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4-[(3aR,6aR)-2,3,3a,4,6,6a-hexahydro-1H-pyrrolo[2,3-c] pyrrol-5-yl]-6-fluoro-N-methyl-2-(2-methylpyrimidin-5-yl) oxy-9H-pyrimido[4,5-b] indol-8-amine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-9.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e180.49 nM\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003cdiv align=\"left\" class=\"colspec\"\u003e\u003cbr\u003e\u003c/div\u003e\n \u003cdiv align=\"left\" class=\"colspec\"\u003e\u003cbr\u003e\u003c/div\u003e\n \u003ctable id=\"Tab4\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eAnalysis of binding affinity values and inhibition constants of Crocatin A and its derivatives against DNA ligase enzyme.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eNo.\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCompound\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eBinding affinity (Kcal/mol)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eInhibition constant/Ki\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCompound \u003cstrong\u003e1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-5.77\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e58.78 \u0026micro;M\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCrocatin B\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-5.84\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e52.21 \u0026micro;M\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePipcroside A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-7.18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.49 \u0026micro;M\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePipcroside B\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-5.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e191.36 \u0026micro;M\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePipcroside C\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-4.91\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e250.59 \u0026micro;M\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eChlorhexidin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-10.37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25.08 nM\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e[(2\u003cem\u003eR\u003c/em\u003e,3\u003cem\u003eS\u003c/em\u003e,4\u003cem\u003eR\u003c/em\u003e,5\u003cem\u003eR\u003c/em\u003e)-5-(3-carbamoylpyridin-1-ium-1-yl)-3,4-dihydroxyoxolan-2-yl] methyl dihydrogen phosphate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-7.94\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.51 \u0026micro;M\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eBased on several bacterial inhibition mechanisms, including inhibition of DNA protein synthesis, several key enzymes involved in this mechanism were selected, including DNA Gyrase B and DNA Ligase enzymes. This in silico study was conducted by rigid docking on the lattice box region of the native ligand of each receptor, that is the isolated compound was tethered to the native ligand tethering region, so that the potential of the compound to interact with the receptor could be determined and compared with that of the native ligand as an inhibitor. The native ligands of DNA Gyrase B were docked at positions 11.036 (X), -5.747 (Y), and 14.749 (Z). In addition to the native ligand, chlorhexidine was used as the positive control in the in vitro antibacterial assay. The binding affinity of the native ligand and chlorhexidine was compared with that of Crocatin A. DNA Gyrase B enzyme was obtained from the RCSB Protein Data Bank with enzyme code 1K4O, which has an RMSD value of 1.10 \u0026Aring;.\u003c/p\u003e\n\u003cp\u003eBased on these results, crocatin A (\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e) showed binding affinity that was not significantly different from that of Crocatin B (\u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e) and the native ligand and chlorhexidine. A greater binding affinity reduces the Ki value, and a small amount of the drug is required to achieve antibacterial activity. This value is important to indicate the ability of the compound to inhibit bacteria, as the inhibition constant correlates with the IC\u003csub\u003e50\u003c/sub\u003e value, regardless of competitive or non-competitive kinetics(\u003cspan class=\"CitationRef\"\u003e39\u003c/span\u003e). The test ligand crocatin A interacted more with the same amino acid residues as the amino acid residues that interacted with chlorhexidine, namely ASN A:48, SER A:49, THR A:168, SER A:122, ILE A:74, GLY A:79, and LYS A:138.\u003c/p\u003e\n\u003cp\u003eThe crocatin A ligand binds to the same five amino acid residues as chlorhexidine through van der Waals interactions. Van der Waals interactions are the most common interactions among all the ligands, and play a major role in the formation of hydrogen bonds and Coulomb interactions(\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e). This made the binding affinity value of the test ligand close to that of chlorhexidine. The native ligand binds to only the same four amino acid residues as chlorhexidine. These types of interactions are illustrated in Fig. 5.\u003c/p\u003e\n\u003cp\u003eThe native ligands in the DNA ligase were docked at positions 6.062 (X), 32.743 (Y), and 26.151 (Z). Similar to DNA Gyrase B, chlorhexidine was used as a positive control in the in vitro antibacterial test in addition to the native ligand. The DNA ligase enzyme was obtained from the RCSB Protein Data Bank with the enzyme code 1TA8, which had an RMSD value of 1.80 \u0026Aring;. Molecular docking using Autodock 4.0, with 100 trials, showed four clusters of native ligand (-5.77 Kcal/mol) 4 times, Crocatin A (-10.37 Kcal/mol) 9 times, and chlorhexidine (-7.94 Kcal/mol) 15 time.\u003c/p\u003e\n\u003cp\u003eBased on these results, crocatin A showed binding affinity values that were not significantly different from those of their derivatives and the native ligand; however, chlorhexidine had the best affinity value. The test ligand crocatin A interacted more with the same amino acid residues as those of the native ligand, namely ASP A:39, ASP A:43, TYR A:30, TYR A:29, ARG A:68, PRO A:65, and TYR A:25.\u003c/p\u003e\n\u003cp\u003eCrocatin A binds to the same three amino acid residues as its native ligand via van der Waals interactions. This makes the binding affinity of the test ligand almost equal to that of the native ligand. Chlorhexidine binds only to the same two amino acid residues as in the native protein. All the types of interactions are illustrated in Fig. 6.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eADMET and drug-likeness analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNatural products contribute to nearly 50% of existing drugs, driving interest in their bioactive compounds for novel drug discovery due to their structural diversity and favorable pharmacokinetic properties. While experimental ADMET evaluation is costly and time-consuming, web-based computational tools, including AI models and drug-likeness scoring systems, offer efficient and reliable alternatives for predicting a compound\u0026rsquo;s drug potential.\u003c/p\u003e\n\u003cp\u003eThe aqueous solubility values of the tested compounds ranged from \u0026minus;\u0026thinsp;5 to 0, indicating poor solubility across all compounds. Despite this, Crocatin A (\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e) and (\u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e) demonstrated high intestinal absorption rates (\u0026gt;\u0026thinsp;80%), as shown in Fig. 7. This suggests that, while solubility is limited, these compounds may still exhibit favorable oral bioavailability if supported by suitable formulation strategies. In terms of distribution, logVDss values were used to evaluate tissue distribution. All compounds showed logVDss values \u0026le; -0.15 (Table \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e), indicating low distribution volume and a tendency to remain within the plasma compartment. This pharmacokinetic profile may allow lower doses to achieve therapeutic plasma concentrations, potentially reducing systemic side effects. The metabolic predictions indicated that none of the compounds inhibited CYP1A2, CYP2C19, CYP2C9, or CYP2D6 enzymes. However, both Crocatin A (\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e) and Crocatin B (\u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e) were predicted to inhibit CYP3A4. This suggests a potential for drug\u0026ndash;drug interactions when co-administered with other compounds metabolized by CYP3A4, warranting further evaluation in future pharmacokinetic studies. Total clearance values were used to estimate the elimination rate of the compounds. Pipcroside C (\u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e) exhibited the highest predicted clearance (log 1.091 mL/min/kg), followed by Pipcroside B (\u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e) and Pipcroside A (\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e), indicating relatively rapid elimination. In contrast, Crocatin B (\u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e) showed the lowest clearance (log 0.393), suggesting a longer half-life and possible risk of accumulation upon repeated dosing. Crocatin A (\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e) presented a moderate clearance value (log 0.567), implying a balanced excretion profile. Toxicity assessments based on LD\u003csub\u003e50\u003c/sub\u003e values revealed relatively low acute toxicity across all compounds, with predicted LD50 values ranging from 2,541 to 3,977 mg/kg. Crocatin A showed the lowest LD\u003csub\u003e50\u003c/sub\u003e value, indicating slightly higher toxicity compared to its derivatives, though still within an acceptable safety margin. Additionally, none of the compounds exhibited potential for skin sensitization, suggesting good dermal safety profiles.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab5\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eADMET prediction of Crocatin A and its derivative compounds\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eProperties\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\" rowspan=\"2\"\u003e\n \u003cp\u003eParameters\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"5\"\u003e\n \u003cp\u003ePredicted Value\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCrocatin A (\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCrocatin B (\u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePipcroside A (\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePipcroside B\u003c/p\u003e\n \u003cp\u003e(\u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePipcroside C (\u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003eAdsorption\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eWater solubility (log mol/L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-4.36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-3.762\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-3.21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-3.22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-3.36\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eIntestinal Absorption (% absorbed)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e59.039\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e59.581\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e46.407\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eSkin Permeability\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-2.75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-2.787\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-2.735\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-2.735\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-2.735\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"3\"\u003e\n \u003cp\u003eDistribution\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eVolume Distribution (VDss, log L/kg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.022\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.099\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-0.077\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-0.031\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.13\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eBBB Permeability (log BBB)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-1.131\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-0.328\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-1.683\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-1.633\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-1.72\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eCNS Permeability (log PS)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-2.87\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-2.868\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-4.392\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-4.374\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-4.755\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"5\"\u003e\n \u003cp\u003eMetabolism\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" rowspan=\"5\"\u003e\n \u003cp\u003eInhibitor of:\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCYP1A2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCYP2C19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCYP2C9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCYP2D6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCYP3A4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eExcretion\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eTotal Clearance (log ml/min/kg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.567\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.393\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.966\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.977\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.091\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eToxicity\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eLethal Dose 50% (mg/kg)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.541\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.979\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.598\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.498\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.977\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eSkin sensitisation\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eThe drug-likeness profiles of the compounds were assessed using established predictive rules, including those proposed by Lipinski, Ghose, Veber, Egan, and Muegge, to evaluate their suitability as oral drug candidates. Crocatin A (\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e) and Crocatin B (\u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e) showed the most favorable profiles, fully complying with all five rule sets without any violations. Both compounds also demonstrated a relatively high bioavailability score of 0.55, indicating good potential for oral absorption. Their predicted log P values (across multiple models) fall within acceptable ranges, suggesting adequate lipophilicity to support membrane permeability and absorption. In contrast, Pipcroside A (\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e), Pipcroside B (\u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e), and Pipcroside C (\u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e) violated multiple criteria. All three compounds exceeded the molecular weight and hydrogen bond acceptor limits set by Lipinski\u0026rsquo;s rule and failed additional thresholds in the Ghose, Veber, Egan, and Muegge filters, particularly due to high topological polar surface area (TPSA) values. Furthermore, their bioavailability scores were lower (0.17), implying limited oral bioavailability (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e). Overall, Crocatin A and Crocatin B stand out as the most promising drug-like candidates based on in silico evaluations, supporting their potential for further development as orally active agents.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab6\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003ePhysicochemical properties of Crocatin A and its derivative compounds\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003ePhysicochemical properties\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"5\"\u003e\n \u003cp\u003eCompounds\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCrocatin A (\u003cspan class=\"CitationRef\"\u003e1\u003c/span\u003e)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCrocatin B (\u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePipcroside A (\u003cspan class=\"CitationRef\"\u003e3\u003c/span\u003e)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePipcroside B\u003c/p\u003e\n \u003cp\u003e(\u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003ePipcroside C (\u003cspan class=\"CitationRef\"\u003e5\u003c/span\u003e)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eChemical Formula\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC25H32O8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC23H30O7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC28H38O12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC28H38O12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eC28H38O12\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMolecular mass (\u0026le;\u0026thinsp;500 g/mol)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e460.52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e418.48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e566.59\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e566.24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e566.59\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHydrogen bond acceptor (\u0026le;\u0026thinsp;10)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eHydrogen bond donor (\u0026le;\u0026thinsp;5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMolar refractivity (130\u0026thinsp;\u0026ge;\u0026thinsp;MR index\u0026thinsp;\u0026ge;\u0026thinsp;40)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e121.25\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e111.51\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e139.16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e139.16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e139.46\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"6\"\u003e\n \u003cp\u003eLipophilicity\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLog P\u003csub\u003eo/w\u003c/sub\u003e (iLOGP)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.84\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.67\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLog P\u003csub\u003eo/w\u003c/sub\u003e (XLOGP3)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.62\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.91\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.91\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.15\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLog P\u003csub\u003eo/w\u003c/sub\u003e (WLOGP)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.44\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.87\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-0.17\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLog P\u003csub\u003eo/w\u003c/sub\u003e (MLOGP)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-1.67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-1.67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-1.67\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLog P\u003csub\u003eo/w\u003c/sub\u003e (SILICOS-IT)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.66\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.11\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"6\"\u003e\n \u003cp\u003eDruglikeness\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLipinski\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eYes; 0 violation\u003c/p\u003e\n \u003cp\u003eMLOGP\u0026thinsp;\u0026le;\u0026thinsp;4.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eYes; 0 violation\u003c/p\u003e\n \u003cp\u003eMLOGP\u0026thinsp;\u0026le;\u0026thinsp;4.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo; 2 violations: MW\u0026thinsp;\u0026gt;\u0026thinsp;500, NorO\u0026thinsp;\u0026gt;\u0026thinsp;10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo; 2 violations: MW\u0026thinsp;\u0026gt;\u0026thinsp;500, NorO\u0026thinsp;\u0026gt;\u0026thinsp;10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo; 2 violations: MW\u0026thinsp;\u0026gt;\u0026thinsp;500, NorO\u0026thinsp;\u0026gt;\u0026thinsp;10\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGhose\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo; 3 violations: MW\u0026thinsp;\u0026gt;\u0026thinsp;480, MR\u0026thinsp;\u0026gt;\u0026thinsp;130, #atoms\u0026thinsp;\u0026gt;\u0026thinsp;7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo; 3 violations: MW\u0026thinsp;\u0026gt;\u0026thinsp;480, MR\u0026thinsp;\u0026gt;\u0026thinsp;130, #atoms\u0026thinsp;\u0026gt;\u0026thinsp;7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo; 3 violations: MW\u0026thinsp;\u0026gt;\u0026thinsp;480, MR\u0026thinsp;\u0026gt;\u0026thinsp;130, #atoms\u0026thinsp;\u0026gt;\u0026thinsp;7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eVeber\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo; 1 violation: TPSA\u0026thinsp;\u0026gt;\u0026thinsp;140\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo; 1 violation: TPSA\u0026thinsp;\u0026gt;\u0026thinsp;140\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo; 1 violation: TPSA\u0026thinsp;\u0026gt;\u0026thinsp;140\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eEgan\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo; 1 violation: TPSA\u0026thinsp;\u0026gt;\u0026thinsp;131.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo; 1 violation: TPSA\u0026thinsp;\u0026gt;\u0026thinsp;131.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo; 1 violation: TPSA\u0026thinsp;\u0026gt;\u0026thinsp;131.6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMuegge\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eYes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo; 2 violations: TPSA\u0026thinsp;\u0026gt;\u0026thinsp;150, H-acc\u0026thinsp;\u0026gt;\u0026thinsp;10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo; 2 violations: TPSA\u0026thinsp;\u0026gt;\u0026thinsp;150, H-acc\u0026thinsp;\u0026gt;\u0026thinsp;10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNo; 2 violations: TPSA\u0026thinsp;\u0026gt;\u0026thinsp;150, H-acc\u0026thinsp;\u0026gt;\u0026thinsp;10\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBioavailability Score\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.17\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eCrocatin A isolated from the methanol extract of Red Betel leaves, showed antibacterial properties against \u003cem\u003eE. faecalis\u003c/em\u003e. The inhibitory potential of crocatin A against \u003cem\u003eE. faecalis\u003c/em\u003e in the inhibition zone wass categorized as moderate, and MIC and MBC showed weak results. In a molecular docking study, crocatin A showed binding affinity to DNA gyrase B and DNA Ligase enzyme is -6.34 and \u0026minus;\u0026thinsp;5.77 Kcal/mol. The results of drug likeness analysis also show promising results as oral medications with several parameter considerations. Based on in vitro and in silico results, it can be concluded that Crocatin A and its derivatives have good potential as antibacterial agents.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e\u003cem\u003eE. faecalis\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Enterococcus faecalis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eP. crocatum\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Piper crocatum\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eMIC\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Minimum Inhibition Concentration\u003c/p\u003e\n\u003cp\u003eMBC\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Minimum Bactericidal Concentration\u003c/p\u003e\n\u003cp\u003eADMET\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Adsorption, Distribution, Metabolism, Excretion, Toxicity\u003c/p\u003e\n\u003cp\u003eLD\u003csub\u003e50\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/sub\u003eLethal Dose 50%\u003c/p\u003e\n\u003cp\u003eKi\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Inhibition Konstanta\u003c/p\u003e\n\u003cp\u003eNMR\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Nuclear Magnetic Resonance\u003c/p\u003e\n\u003cp\u003eDEPT\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Distortionless Enhancement by Polarization Transfer\u003c/p\u003e\n\u003cp\u003eHMQC\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Heteronuclear Multiple Quantum Coherence\u003c/p\u003e\n\u003cp\u003eHMBC\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Heteronuclear Multiple Bond Correlation\u003c/p\u003e\n\u003cp\u003eIR\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Infra-Red\u003c/p\u003e\n\u003cp\u003eUV \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Ultraviolet\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo funding.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo datasets were generated or analysed during the current study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDevi Meliani: writing—review and editing, writing—original draft and reviewing process handling. Trisna Yuliana: writing—review and editing, writing—original draft. Dikdik Kurnia: writing—review and editing, writing—original draft.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors are grateful for Universitas Padjadjaran for all research facilities, .\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eShamsudin NF, Ahmed QU, Mahmood S, Shah SAA, Khatib A, Mukhtar S et al. Antibacterial Effects of Flavonoids and Their Structure-Activity Relationship Study: A Comparative Interpretation. Molecules. 2022;27(4).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eYadav KPSA. A Review of Dental Caries. Asian J Biomed Pharm Sci. 2016;73\u0026ndash;80.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePitts NB, Zero DT, Marsh PD, Ekstrand K, Weintraub JA, Ramos-Gomez F et al. Dental caries. 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Indones J Pharm Sci Technol. 2020;7(2):64.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-chemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ccjo","sideBox":"Learn more about [BMC Chemistry](https://bmcchem.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/ccjo/default.aspx","title":"BMC Chemistry","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Crocatin A, Piper crocatum Ruiz \u0026 Pav, Enterococcus faecalis, DNA gyrase, DNA ligase","lastPublishedDoi":"10.21203/rs.3.rs-7015807/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7015807/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eDental caries tooth tissue disease that can cause complications. The gram-positive bacteria that play a role in the process of infection are \u003cem\u003eEnterococcus faecalis\u003c/em\u003e. Red betel leaves (\u003cem\u003ePiper crocatum Ruiz\u003c/em\u003e and Pav.) contained active substances in their phytochemicals. However, there is no additional information on the antibacterial properties of \u003cem\u003eP. crocatum\u003c/em\u003e or the molecular mechanisms that affect DNA Gyrase B and DNA Ligase of \u003cem\u003eE. faecalis\u003c/em\u003e ATCC 29212. This study aimed to screen and test compounds from \u003cem\u003eP. crocatum\u003c/em\u003e for their ability to inhibit \u003cem\u003eE. faecalis\u003c/em\u003e and predict the mechanism of inhibition of certain proteins using a molecular docking approach. Isolation of Crocatin A from \u003cem\u003eP. crocatum\u003c/em\u003e was carried out by column chromatography and then characterized via infrared (IR), nuclear magnetic resonance (NMR), and mass spectroscopy, then compound was tested using Kirby Bauer and microdilution methods. The active compound and derivatives were predicted to act against DNA gyrase B and DNA ligase from \u003cem\u003eE. faecalis\u003c/em\u003e and ADMET properties by \u003cem\u003ein silico\u003c/em\u003e. The study showed that Crocatin A has been isolated from \u003cem\u003eP. crocatum\u003c/em\u003e. It exhibited antibacterial properties against \u003cem\u003eE. faecalis\u003c/em\u003e (MIC 1250 \u0026micro;g/mL) as well as in silico against DNA Gyrase B (-6.34 kcal/mol) and DNA Ligase (-5.77 kcal/mol) enzymes. Therefore, it can be concluded that Crocatin A present in Red Betel leaves has moderate activity in inhibiting \u003cem\u003eE. faecalis\u003c/em\u003e by in vitro and potential to inhibit DNA synthesis in \u003cem\u003eE. faecalis\u003c/em\u003e by in silico.\u003c/p\u003e","manuscriptTitle":"In Vitro and In Silico Characterization of Crocatin A from Red Betel Leaves: Targeting DNA Gyrase B and DNA Ligase of Enterococcus faecalis with ADMET-Based Druglikeness Analysis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-04 18:22:20","doi":"10.21203/rs.3.rs-7015807/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-09-04T08:25:56+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-25T13:28:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"178120303744118467677435637198667832214","date":"2025-07-31T11:40:43+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-31T09:10:32+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"230929500208172122349376704508285587898","date":"2025-07-30T05:51:43+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-30T05:26:59+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-21T14:31:57+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-07-21T11:00:21+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-21T10:43:27+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Chemistry","date":"2025-07-21T10:13:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-chemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ccjo","sideBox":"Learn more about [BMC Chemistry](https://bmcchem.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/ccjo/default.aspx","title":"BMC Chemistry","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c19e0fb8-df03-405a-9dd7-38840cde4d3a","owner":[],"postedDate":"August 4th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-03-09T16:05:06+00:00","versionOfRecord":{"articleIdentity":"rs-7015807","link":"https://doi.org/10.1186/s13065-026-01747-8","journal":{"identity":"bmc-chemistry","isVorOnly":false,"title":"BMC Chemistry"},"publishedOn":"2026-03-08 15:59:37","publishedOnDateReadable":"March 8th, 2026"},"versionCreatedAt":"2025-08-04 18:22:20","video":"","vorDoi":"10.1186/s13065-026-01747-8","vorDoiUrl":"https://doi.org/10.1186/s13065-026-01747-8","workflowStages":[]},"version":"v1","identity":"rs-7015807","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7015807","identity":"rs-7015807","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}