The Southeast Asian Journal of Tropical Biology Vol. 32 No. 3, 2025: 310 - 327 DOI: 10.11598/btb.2025.32.3.2456 ISSN: 0215-6334 | e-ISSN: 1907-770X 310 IN SILICO STUDY OF JATI (Tectona grandis) LEAF CONSTITUENTS AS TRADITIONAL WOUND CARE Gabriella Chandrakirana Krisnamurti1, Anak Agung Sagung Alit Sukmaningsih2 and Cicilia Novi Primiani3,4* 1Biotechnology Program, School of Bioresources and Technology, King Mongkut’s University of Technology Thonburi, Bang Khun Thian 10150, Thailand 2Biology Study Program, Faculty of Mathematics and Natural Sciences, Udayana University, Bali 80361, Indonesia 3Department of Pharmacy, Faculty of Health and Science, Universitas PGRI Madiun 63118, Indonesia 4Department of Biology Education, Faculty of Teacher Training and Education, Universitas PGRI Madiun 63118, Indonesia ARTICLE HIGLIGHTS • T. grandis leaf methanolic extract contains 82 compounds, with 17 being predominant. • Epigallocatechin 3-O cinnamate, epigallocatechin 3-O-p-coumarate, and tectograndinol predicted as the potential bioactive compounds in wound healing • Molecular docking suggests inhibitory activities against NF- κB, MMP-2, and MMP-9, and stimulatory activity against EGFR- 1 that comparable to commercial drugs. Article Information Received : 12 February 2025 Revised : 29 August 2025 Accepted : 22 September 2025 *Corresponding author, e-mail: primiani@unipma.ac.id Research Paper ABSTRACT Wound-healing process involves a physiological cascade to restore skin integrity, which includes inflammatory response, cell proliferation, and tissue reconstruction. Prolonged inflammation in wound-healing process may lead to a chronic wound stage. Proper wound care is needed to prevent wound-caused mortality. Several studies showed the potential of T. grandis leaf in wound-healing process. However, the bioactive compounds and the molecular mechanism of T. grandis leaf remains unknown. This study aimed to identify bioactive compounds and biological activity contained in T. grandis leaf extract as well as analyze its molecular mechanism in wound-healing process by conducting in silico study using NF- κB, MMP-2, MMP-9, and EGFR-1. Bioactive compounds of T. grandis leaf extract were identified with LC-MS. Three potential compounds, epigallocatechin 3-O cinnamate, epigallocatechin 3-O-p-coumarate, and tectograndinol, were selected based on the Pa score screening with the PASS prediction. Drug-likeness and pharmacokinetics properties of the selected compounds were virtually identified by SwissADME and Protox. The interactions of T. grandis bioactive compounds toward NF-κB, MMP-2, MMP-9, and EGFR-1 were compared to those of curcumin, phenytoin, and nitrofurazone as control. Molecular docking to identify the protein- ligand interaction was performed by Autodock Vina integrated in PyRx v.0.8. Among 82 bioactive compounds detected in the LC- MS analysis, epigallocatechin 3-O-cinnamate, epigallocatechin 3-O-p coumarate, and tectograndinol exhibited anti-inflammatory, antioxidant, free radical scavenger, and MMP-9 inhibitor activities. According to Lipinski’s rule of five, bioactive compounds are possible to be administered as medication. Molecular docking showed that bioactive compounds potentially bound to the active sites of NF-κB, MMP-2, and MMP-9, resulting in proteins inhibition. This study suggested that the wound-healing mechanism of T. grandis bioactive compounds were driven by EGFR-1 stimulation indicated by the ability of bioactive compounds to interact with EGFR-1 in similar manner to those of nitrofurazone. We concluded that bioactive compounds of T. grandis leaf extract has significant potential to be used as traditional wound treatment and therapy. These compounds demonstrated wound-healing activity in silico by interacting with the key molecular targets, including NF-κB, MMP-2, MMP-9, and EGFR-1. Keywords: bioactive, EGFR, MMP, Tectona grandis, wound Copyright (c) 2025@author(s). https://doi.org/10.11598/btb.2025.32.3.2456 https://creativecommons.org/licenses/by-nc-nd/4.0/ In silico study of jati leaf constituents as traditional wound care - Krisnamurti et al. 311 INTRODUCTION Skin is the largest barrier of human’s body, which provides protection, homeostasis, signal transduction, and excretion. Wound, defined as skin damage, caused by external or internal diseases is threatening and needs proper cure (Tottoli et al. 2020). The repairing process included hemostasis, inflammation, proliferation, and remodeling that requires coordination and interaction of various cells, extracellular matrix (ECM), pro- inflammatory mediators, and growth factor (Tyavambiza et al. 2022). Though skin has self- recovery ability, various factors delay the wound healing process. Prolonged inflammation might contribute to the development of chronic wounds (Singh et al. 2016). Angiogenesis is a process that promotes the development of chronic wounds which occur immediately after the injury. The mechanism involves matrix metalloproteinases (MMPs) which play major roles in each stage of wound-healing process, such as wound matrix modification, cell migration, angiogenesis, and tissue remodeling. Two types of MMPs, MMP-2 and MMP-9, play a major role in angiogenesis regulation during wound healing via proangiogenic cytokines activation and antiangiogenic peptides generation (Reiss et al. 2010; Caley et al. 2015; Bergant Suhodolčan et al. 2021). Expressions of MMP-2 and MMP-9 are naturally beneficial during the wound-healing process. However, the imbalance level of MMP-2 and MMP-9 may lead to chronic wounds. The abnormal increased level of MMP- 9 has been observed in the chronic wounds (Reiss et al. 2010; Cabral-Pacheco et al. 2020). Elevated MMP-9 expression in the chronic wounds is associated with MMP-2, due to the function of MMP-2 in stimulating MMP-9 activation (Ratzinger et al. 2002). The expressions MMP-2 and MMP-9 are often detected in cardiovascular disease, diabetes mellitus, colorectal cancer tissue, and neurodegenerative disease (Groblewska et al. 2014; Cabral-Pacheco et al. 2020). Acceleration of wound-healing process is also regulated by the re-epithelialization which occurred during the migration of keratinocytes. Epidermal growth factor receptor (EGFR) is a protein that has been involved in the process of re-epithelization, including cell migration and proliferation (Repertinger et al. 2004). Previous findings reported that the increased EGFR expression accelerates wound re-epithelialization on the incisional wound (Nanney et al. 2000). The skin of EGFR wild-type mice (EGFR +/+) exhibited smaller erythema at the wound margin compared to EGFR-null mice (EGFR -/-). Five days after the injury, the wound appearance of EGFR-null mice was visibly similar to that of the EGFR wild-type mice (Repertinger et al. 2004). EGFR also stimulates angiogenesis in murine skin carcinomas (Casanova et al. 2002) which is essential in the early stage of wound healing (Caley et al. 2015; Shanthi Kumari et al. 2020). It is important to achieve effective, safe, less cost treatment that provides long-term relief for wounds (Tottoli et al. 2020). Natural products are the source to develop traditional and synthetic herbal treatment and therapy with several advantages, such as being readily available in nature, less side effects, and efficiency. Some medicinal plants have been implemented for wound care for several decades (Varma & Giri 2013). Tectona grandis, or teak, or jati in Indonesia has been known for its medicinal properties, majorly antioxidant, anti-inflammatory, antibacterial, anti-tumor, and antidiabetic (Varma & Giri 2013; Arief et al. 2014; Asdaq et al. 2022). Previously, T. grandis leaf extract effectively reduced the wound area in the incision and excision wound. It has been reported that the wound area is significantly reduced by treating the wound with 5% and 10% ointment of T. grandis leaf extract (Varma & Giri 2013). The anti-inflammatory, antioxidant, immunomodulatory, and antibacterial potential of T. grandis leaf are associated in the wound-healing process (Mikhal’chik et al. 2006; Dégbé et al. 2018; Comino-Sanz et al. 2021; Han et al. 2023). Post-surgical wounds and diabetic wounds are more likely to drive a significant impact on a patient’s health. Poor healthcare to the patient’s wound may develop into chronic wounds. Without proper treatment and therapy, wounds could contribute to mortality (Akiki et al. 2021). Thus, treatment of wounds is crucial to reduce the risk of chronic wound development and mortality. Previous in vivo studies reported extensive exploration on the potential of T. grandis leaf extract in wound-healing process even though the mechanism is still questionable. This study aimed to provide scientific proof about T. grandis potent biological activities, focusing on wound-healing activity. The study was performed by identifying T. grandis bioactive compounds, also predicted the biological activity and molecular mechanism in wound-healing process using NF-κB, EGFR1, MMP-2, and MMP-9. BIOTROPIA Vol. 32 No. 3, 2025 312 MATERIALS AND METHODS Preparation of Tectona grandis Leaf Extract T. grandis leaf were obtained from Hutan Jati Gunung Wilis, Madiun. The leaves were identified at the Taxonomy Laboratory of the Universitas PGRI Madiun and were assigned the identification number: 0023/Taxo-Plant/Biology/IV/2021. The leaves were prepared by drying and grinding into powder, then sieved using 60 mesh size. The processed leaf powder was weighed in 0.5 to 2 g. Leaf powder was extracted with 95% methanol with sample and methanol ratio of 1 : 5 g/mL (Onivogui et al. 2015). The powder was macerated with the solution and filtered with Whatman grade 1 which was placed a vacuum filter. The filtrate was evaporated with rotary evaporator to separate the methanol from the extract. The extract obtained was prepared for analysis with Liquid Chromatography-Mass Spectrometry (LC-MS). Leaf extract was diluted by methanol homogenization until the concentration reached approximately 100 ppm. Sample was centrifuged at 8,000 rpm for 10 minutes to remove any insoluble fine solid particles that could have remained in the extract, thus preventing potential clogging of the analytical column. Two milliliters of supernatant was collected, then 3 mL of acidified acetonitrile was added to the supernatant. The extract was separated by centrifugation at 8,000 rpm for 30 seconds to obtain clear supernatant containing purified sample. Screening of Bioactive Compounds in Tectona grandis Leaf Methanolic Extract The extract of T. grandis leaf was purified using solid phase extraction (SPE) to remove interfering compounds according to the procedure of Fernand et al. (2009). The sample was loaded to the C18 Sep-Pak cartridge column (1 mL, 100 mg) which had previously been conditioned with 1 mL mixture of acetonitrile : water with ratio of 80 : 20 to activate the stationary phase. Approximately 0.5 mL of the liquid leaf extract was then loaded onto the conditioned column. The column was washed with a series of solutions to selectively elute different compound classes. Specifically, 1 mL of water was added to remove highly polar impurities. Subsequently, 0.5 mL of acetonitrile : water (80 : 20 ratio) was added to elute semi-polar compounds. Finally, 0.5 mL of acetonitrile was used to elute the most non-polar compounds of interest. The final purified solution was filtered using a 0.45 µm cellulose acetate membrane filter and degassed before injection. The sample was analyzed using a Shimadzu LCMS- 8040 Liquid Chromatography-Mass Spectrometry system. The injection volume was 1 µL. Chromatographic separation was performed on a Shimadzu Shim-pack FC-ODS column (2 mm x 150 mm, 3 µm). The mobile phase consisted of a single solvent system in isocratic mode. The mobile phase was methanol : water (90 : 10 ratio), delivered at a constant flow rate of 0.5 mL/minute. The total run time was 80 minutes. The mass spectrometer was operated in positive ion mode. The ion source was Electrospray Ionization (ESI), interface voltage +4.5 kV, desolvation line (DL) temperature of 250 °C, heat block temperature of 400 °C, drying gas flow of 15 L/minute, nebulizing gas flow of 3 L/ minute, and scan mode with full scan (m/z range 100–1000). Bioactivity Prediction of Tectona grandis Bioactive Compounds Seventeen most abundant bioactive compounds of T. grandis leaf extract were identified for their potency, which we selected for further analyses (Table 1). The canonical SMILES of 17 bioactive compounds were obtained from PubChem and loaded to PASS prediction for bioactivity prediction (Filimonov et al. 2014). Each bioactive compound was later identified for the potency as antioxidant, free radical scavenger, MMP expression inhibitor, and anti-inflammatory. Bioactive compounds with Pa score ≥ 0.7 were chosen for further analysis, as they indicate a high similarity to compounds in the database that have been proven for treatment and therapy. Prediction of Drug-likeness and Toxicity of Bioactive Compounds Three bioactive compounds selected for bioactivity predictions were continued for identification and prediction of its drug-likeness and pharmacokinetics (Banerjee et al. 2024). The canonical SMILES of three bioactive compounds were loaded to SwissADME (http://www. swissadme.ch/index.php) and Protox tool (https:// tox.charite.de/protox3/index.php). In silico study of jati leaf constituents as traditional wound care - Krisnamurti et al. 313 Table 1 Dominant bioactive compounds of Tectona grandis leaf extract Retention time Composition (%) Bioactive compound Pubchem ID 23.293 2.48141 Epigallocatechin-3-O-p coumarate CID6474788 23.705 2.17803 Epigallocatechin gallate CID65064 33.505 2.15389 Procyanidin B5 CID124017 22.176 2.13232 6C-glucopyranosylepicatechin CID131752183 33.434 2.09243 Proanthocyanidin A-2 CID124025 33.496 1.98748 Procyanidin B1 CID11250133 11.566 1.84348 Isoobtusilactone A CID6442493 3.042 1.80174 Gallic acid CID370 11.958 1.77578 Tectograndinol C_ID C00022743 (from knapsack) 21.584 1.7546 Epigallocatechin-3-O cinnamate CID21629801 19.211 1.73830 Epiafzelechin-3-O-gallate CID467295 33.498 1.71418 Procyanidin B2 CID124017 10.322 1.70537 Kaempferol CID5280863 33.429 1.58901 Procyanidin A1 CID5089889 11.562 1.43720 Isolinderanolide B SID274339182 12.421 1.43717 Chlorogenic acid CID1794427 11.427 1.42081 Quercetin CID5280343 Molecular Docking Selected bioactive compounds of Tectona grandis leaf extract were further analyzed for the interactions toward proteins involved in wound- healing process. The 3D structures of selected bioactive compounds, i.e., epigallocathecin 3-O- p coumarate (CID6474788) and epigallocatechin 3-O-cinnamate (CID21629801), were downloaded from PubChem database. The 3D structure of tectograndinol was obtained from the canonical SMILES structure provided by the Knapsack family C_ID C00022743 and loaded to OpenBabel to construct its 3D chemical structure, later converted into SDF format. Curcumin (CID969516), nitrofurazone (CID5447130) and phenytoin (CID1775), as a control, were obtained from PubChem. All ligands, including bioactive compounds and control, were prepared using PyRx 0.8 by minimizing the energy and converting into PDB format. The 3D structure of EGFR-1 (3POZ), MMP9 (1L6J), MMP2 (7XGJ), and NF-κB (1KN) were downloaded from PDB (Liu et al. 2021; Al Mousa et al. 2024). Proteins were prepared by removing water molecules and the natural ligands with Discovery Studio 4.1. Ligands were prepared by minimizing the energy and converting to PDB format in PyRx version 0.8. Docking was performed by AutoDock Vina integrated in PyRx 0.8. Bioactive compounds were interacted to EGFR-1 (x = 19.5327, y = 24.7294, z = 15.1872), MMP-9 (x = 39.3102138564, y = 35.2175327806, z = 34.6209), MMP-2 (x = 20.3416, y = -0.3234, z = 20.0629), and NF-κB (x = 38.4173, y = 25.9442, z = 28.1869) in the specific grid box optimized by the software and exhaustiveness 50. Interactions between ligands and proteins were visualized by Discovery Studio 4.1. RESULTS AND DISCUSSION Prediction on Biological Activity of T. grandis Leaf Extract Methanolic extracts of T. grandis leaf contained 82 bioactive compounds based on the LC-MS analysis (Fig. 1). Bioactive compounds with high peak in the LC-MS result as well as longer retention time were categorized as the abundant bioactive compound and predicted as the dominants. However, some bioactive compounds may elute later due to the strong interaction between analyte and the stationary phase, showing the late in retention time (Katajamaa & Oresic 2005). Our study found that the most abundant bioactive compounds were flavonoids group, followed by proanthocyanidin, phenolic acids, diterpenoids, and lignans. Other studies showed that the T. grandis leaf extract contains various bioactive compounds, such as flavonoids, tannins, alkaloids, anthraquinones, anthocyanins, naphthoquinone, and cyanidine (Arief et al. 2014; Murukan & Murugan 2018). Bioactive compounds exhibit various biological activities, BIOTROPIA Vol. 32 No. 3, 2025 314 such as anti-inflammatory, antibacterial, and antioxidants causes the plant to be a traditional agent for treating diabetes, cancer, malaria, and skin diseases (Rajuri et al. 2010; Suryanti et al. 2020). Phenolic contents are the major constituent of T. grandis leaf extract (Budianto et al. 2023). Phenolic and anthocyanins of T. grandis leaf methanol extract exerts strong antioxidant activity (Suryanti et al. 2020). The antioxidant activity of T. grandis allowed the acceleration of the wound- healing process by reducing the oxidative stress and performing anti-inflammatory activity (Ponugoti et al. 2013; Comino-Sanz et al. 2021). Seventeen bioactive compounds were screened for biological activity-related to wound-healing mechanisms, which are on anti-inflammatory activity, free radical scavenger, antioxidant activity, and MMP inhibitory activity (Table 2). According to the screening, bioactive compounds of T. grandis leaf extract mostly exhibited antioxidant and anticarcinogenic activities. Tectograndinol and epigallocatechin- 3-O-p coumarate showed exceptional activities as anti-inflammatory and MMP-9 inhibitor, respectively. Epigallocatechin 3-O-p-coumarate and epigallocatechin-3-O-cinnamate also showed antioxidant potential, as indicated by their high predicted Pa scores for free radical scavenging and antioxidant activities. Furthermore, these bioactive compounds exhibited the highest Pa scores for all biological activities included in the prediction. Therefore, epigallocathecin 3-O-cinnamate, epigallocathecin 3-O-p coumarate, and tectograndinol were selected for further analysis. The anti-inflammatory activity of epigallocathecin 3-O-cinnamate and epigallocathecin 3-O-p coumarate may be performed by flavonoids with two connecting benzene rings (A and B) via oxygen-containing heterocycle (C), also with the A ring of glycosyl mode (Maleki et al. 2019; Han et al. 2023). Tectograndinol which appeared with the highest anti-inflammatory activity, categorized as diterpenoid group. Diterpenoids are the promising class of secondary metabolites with presenting high anti-inflammatory capacity (González et al. 2015). Commercial medicinal plant extracts containing high diterpenoids showed direct anti-inflammatory activity by inhibiting NF-κB, which effectively reduced pain and symptoms of rheumatoid arthritis (González et al. 2015; Lv et al. 2015). Seven diterpenoid glucosides from fruit extracts presents anti-inflammatory activity against LPS- induced NO production in RAW 264.7 (Liu et al. 2023). Previous study revealed the decreased pro- inflammatory mediators, TNF-α and interleukins (IL-1β, IL-6), on the macrophage-stimulated LPS following treatment with T. grandis leaf extract (Han et al. 2023). The complexity of phytochemical constituents from T. grandis also allowed physiological mechanisms. Additionally, bioactive compounds from the extract could modulate the immune response to impede the parasitic effect on antigen presenting cells by increasing the cytokine production (Dégbé et al. 2018). Figure 1 Bioactive compounds of Tectona grandis leaf extract analyzed by liquid chromatography-mass spectrometry (LC-MS) spectra In silico study of jati leaf constituents as traditional wound care - Krisnamurti et al. 315 Table 2 Biological activity prediction of Tectona grandis leaf extract Bioactive compound Free radical scavenger Antioxidant MMP9 expression inhibitor Anticarcinogenic Anti- inflammatory Epigallocatechin 3-O-p-coumarate Pa 0.96 0.87 0.706 0.897 Pi 0.001 0.003 0.006 0.003 Epigallocatechin gallate Pa 0.934 0.814 0.841 Pi 0.001 0.003 0.004 Procyanidin B5 Pa 0.856 0.787 0.848 Pi 0.002 0.004 0.004 (-)-Epicatechin 6-C-glucoside Pa 0.894 0.831 Pi 0.002 0.003 Proanthocyanidin A-2 Pa 0.742 0.704 0.713 Pi 0.003 0.004 0.008 Procyanidin B1 Pa 0.798 0.803 0.757 Pi 0.003 0.003 0.007 Isoobtusilactone A Pa Pi Gallic acid Pa Pi Tectograndinol Pa 0.746 Pi 0.011 Epigallocatechin-3- O-cinnamate Pa 0.953 0.863 0.891 Pi 0.001 0.003 0.003 Epiafzelechin-3-O- gallate Pa 0.933 0.81 0.844 Pi 0.001 0.003 0.004 Procyanidin B2 Pa 0.798 0.803 0.757 Pi 0.003 0.003 0.007 Kaempferol Pa 0.771 0.856 0.738 0.715 Pi 0.003 0.003 0.005 0.008 Procyanidin A1 Pa 0.742 0.704 0.713 Pi 0.003 0.004 0.008 Isolinderanolide B Pa Pi Chlorogenic acid Pa 0.856 0.785 0.846 Pi 0.002 0.004 0.004 Quercetin Pa 0.811 0.872 0.734 0.757 Pi 0.003 0.003 0.005 0.007 Drug-likeness and Pharmacokinetics Prediction of Tectona grandis Bioactive Compounds Prior to drug development, it is necessary to predict the drug-likeness and pharmacokinetics properties of the bioactive compounds. Drug- likeness properties according to Lipinski’s rule of five and pharmacokinetics include absorption, distribution, metabolism, excretion, and toxicity (ADME/T) which contribute to the physiological effect of a compound (Krisnamurti et al. 2021; Banerjee et al. 2024). According to the ADME/T analysis, epigallocatechin 3-O-p coumarate has 1 violation (Table 3). However, the bioavailability score of bioactive compounds was 0.55, which is still in the range of good bioavailability score. It means the bioactive compounds can be easily absorbed by the body (Martin 2005). Compared to others, tectograndinol exhibits the highest gastrointestinal absorption and blood brain barrier permeability. Epigallocatechin 3-O-cinnamate, epigallocatechin 3-O-p- coumarate, and tectograndinol shared similar properties in which lipophilicity and water solubility. The bioactive compounds are more likely lipophilic and moderately soluble in water. BIOTROPIA Vol. 32 No. 3, 2025 316 The log Kp score is important in the analysis of T. grandis bioactive compounds, as wound treatment is mostly used for skin permeability. The log Kp value represents how easy the chemical can penetrate the skin, which indicates the significance of skin absorption. The more negative log Kp value the lower the potency of a bioactive compound to penetrate the skin (Scheler et al. 2014). All three bioactive compounds showed negative scores of skin permeation, while tectograndinol showed the less negative score. Virtual prediction indicates tectograndinol as the potent bioactive compound for external application for wound treatment, such as an ointment. Though bioactive compounds are known for its safety and less adverse effect, it is important to test the toxicity. Toxicity analysis was carried out by predicting LD50 and various toxicity properties in the system. LD50 and class of toxicity represent the amount of a substance that is expected to cause death in 50% of a group of test animals from a single dose. Meanwhile, various toxicity properties specifically predict the types of adverse effects that can emerge in an organism (Oduola et al. 2010). The bioactive compounds tested in this study were categorized in class 4 and class 5. The LD50 were divided into six categories, i.e., class I (LD50 ≤ 5 mg/kg), class II (5 < LD50 ≤ 50 mg/ kg), class III (50 < LD50 ≤ 300 mg/kg), class IV (300 < LD50 ≤ 2,000 mg/kg), class V (2,000 < LD50 ≤ 5,000 mg/kg), and class VI (LD50 > 5,000 mg/kg). The higher the class of LD50 the less harmful the bioactive compound to the body (Banerjee et al. 2024). Our study showed that tectograndinol possessed less toxicity compared to those of epigallocatechin 3-O-p-coumarate and epigallocatechin 3-O-cinnamate. Toxicity prediction showed that epigallocathecin 3-O-cinnamate and epigallocatechin 3-O-p- coumarate had the probability of nephrotoxicity and respiratory toxicity of 0.72 and 0.78, respectively, for both compounds. The nephrotoxicity and respiratory toxicity were probably inactive at the probability of 1 – 0.72 and 1 – 0.78, respectively. Moreover, those compounds probably caused immunotoxicity as the score were the highest at 0.9 and 0.94. The probability of compounds to be inactive was less than 10%. Tectograndinol showed the highest toxicity in cardiotoxicity with score of 0.83, while it may inactive with probability of 1 – 0.83 which is score 0.17 (17%). Overall, bioactive compounds of T. grandis leaf extract showed its safety by not causing hepatotoxicity, neurotoxicity, carcinogenicity, mutagenicity, and cytotoxicity. The bioactive compounds are also safe for the environment, represented by ecotoxicity, so it could be predicted that the bioactive compounds are ecofriendly as if they were developed as a drug (Gan et al. 2024). However, development of epigallocatechin 3-O-cinnamate and epigallocatechin 3-O-p-coumarate as a medication may need further consideration as they might be toxic in nephrons, indicated by the red color in the table. According to ADME/T analysis, tectograndinol is potentially the safest bioactive compound for drug development. Table 3 Drug-likeness and pharmacokinetics properties of Tectona grandis bioactive compounds Epigallocathecin 3-O-cinnamate Epigallocatechin 3-O-p- coumarate Tectograndinol Lipinski Yes; 0 violation Yes; 1 violation Yes; 0 violation Bioavailability score 0.55 0.55 0.55 Lipophilicity (Po/w) 2.45 2.07 3.4 Water solubility (LogS) -5.18 (Moderately soluble) -5.24 (Moderately soluble) -4.24 (Moderately soluble) GI absorption Low Low High BBB permeant No No Yes P-gp substrate No No No CYP1A2 inhibitor No No No CYP2C19 No No No CYP2C9 No No No CYP2D6 No No No CYP3A4 No No No Log Kp (skin permeation) -7.07 cm/s -7.42 cm/s -5.93 cm/s LD50 1000 mg/kg 1000 mg/kg 5000 mg/kg Toxicity 4 4 5 In silico study of jati leaf constituents as traditional wound care - Krisnamurti et al. 317 Epigallocathecin 3-O-cinnamate Epigallocatechin 3-O-p- coumarate Tectograndinol Lipinski Yes; 0 violation Yes; 1 violation Yes; 0 violation Hepatotoxicity 0.68 0.68 0.86 Neurotoxicity 0.85 0.85 0.85 Nephrotoxicity 0.72 0.72 0.74 Respiratory toxicity 0.78 0.78 0.73 Cardiotoxicity 0.87 0.87 0.83 Carcinogenicity 0.52 0.52 0.61 Immunotoxicity 0.90 0.94 0.63 Mutagenicity 0.64 0.64 0.75 Cytotoxicity 0.77 0.77 0.96 BBB-barrier 0.54 0.54 0.61 Ecotoxicity 0.6 0.6 0.54 Clinical toxicity 0.54 0.54 0.68 Nutritional toxicity 0.64 0.64 0.74 Notes: Less active Active Less inactive Inactive Interactions of Tectona grandis Bioactive Compounds to Wound Healing-Related Protein Identification of wound-healing mechanism was performed by in silico study using key wound healing proteins, NF-κB, MMP-9, MMP-2, and EGFR-1. The potency of three selected T. grandis bioactive compounds on wound-healing-related protein were compared to curcumin, nitrofurazone, and phenytoin as control. Curcumin, a polyphenol derived from Curcuma longa, is widely known as potential anti-inflammatory and antioxidant agent. Study demonstrated the activity of curcumin in reducing the healing time on excisional wound in rats, which mostly plays in the proliferative phase of wound- healing process (Patenall et al. 2024). Nitrofurazone is an antibacterial agent that has been acknowledged for its potency in wound care. Study revealed the activity of nitrofurazone in promoting tissue granulation in the wound, thus leads to wound-healing process (Erdur et al. 2008). Phenytoin or diphenylhydantoin was firstly established as convulsive disorders treatments. It has an effect on connective tissue and is potentially used in wound care. Study demonstrated the wound-healing process in rat burn skin wound model following phenytoin administration. Phenytoin aids wound-healing process by developing vascularized and granulation tissue, also synthesizing collagen by re-epithelization (Patenall et al. 2024). The activity of bioactive compounds toward NF-κB and MMPs were compared with phenytoin, while nitrofurazone was used to compare the activity of bioactive compounds toward EGFR- 1. The activity of T. grandis bioactive compounds against NF-κB were shown in Figure 2. NF-κB is an essential transcriptional factor in wound-healing process, which plays major role in cell migration in inflammatory phase and proliferation in proliferative phase. The activation of NF-κB affects the cytokines and growth factor secretion, cell proliferation, also MMPs expression (Yadav et al. 2024). Our study revealed that bioactive compounds from T. grandis leaf extract performed inhibition toward NF-κB in the similar inhibitory sites as phenytoin (Table 4). Phenytoin posed binding with Glu49, Arg50, Glu222, Gln241, and Gly259 of NF-κB (-8 kcal/ mol) in which those amino acid residues indicated as the key amino acid residues of NF-κB inhibition. Epigallocatechin 3-O-cinnamate performed inhibition via Glu222 and Gln241 (-9 kcal/mol), epigallocatechin 3-O-p-coumarate via Arg50 (-9.8 kcal/mol), while tectograndinol via Glu49, Gln241, and Gly259 (-8 kcal/mol). It could be predicted that T. grandis compounds performed wound-healing process in similar manner as the commercial wound-healing medication. BIOTROPIA Vol. 32 No. 3, 2025 318 Figure 2 Interaction of with ligands Notes: A = epigallocathecin 3-O-cinnamate; B = epigallocatechin 3-O-p-coumarate; C = tectograndinol; D = curcumin; E = phenytoin. Bioactive compounds from T. grandis leaf extract potentially formed binding with the similar amino acid residues as phenytoin. Table 4 Interactions of Tectona grandis bioactive compounds toward NF-κB Ligand Name Category Type Binding energy (kcal/mol) NF-κB Epigallocatechin 3-O-cinnamate Glu222, Gly237 Hydrogen Bond Conventional Hydrogen Bond -9Gln241 Hydrogen Bond Pi-Donor Hydrogen Bond Glu225 Electrostatic Pi-Anion Pro275, Lys28 Hydrophobic Pi-Alkyl Epigallocatechin 3-O-p-coumarate Phe239, Glu225, Pro275, Ile224, Arg273 Hydrogen Bond Conventional Hydrogen Bond -9.8 Phe239 Hydrophobic Pi-Pi T-shaped Arg50, Pro275 Hydrophobic Pi-Alkyl Tectograndinol Glu49. Gly259, Gln241 Hydrogen Bond Conventional Hydrogen Bond -7.2Glu49 Hydrogen Bond Carbon Hydrogen Bond Arg236 Hydrophobic Alkyl Phe239 Hydrophobic Pi-Alkyl Curcumin Lys177, Thr256, Gly183 Hydrogen Bond Conventional Hydrogen Bond -8 Lys177, His173 Hydrogen Bond Carbon Hydrogen Bond Thr179 Hydrogen Bond Pi-Donor Hydrogen Bond Thr179 Hydrophobic Pi-Sigma Leu346 Hydrophobic Pi-Alkyl Phenytoin Glu222, Gln241 Hydrogen Bond Conventional Hydrogen Bond -8 Glu49;Arg50, Gly259;Arg260 Hydrophobic Amide-Pi Stacked Arg50 Hydrophobic Pi-Alkyl Asp390 Electrostatic Pi-Anion Tyr393 Hydrophobic Pi-Pi Stacked Pro97 Hydrophobic Pi-Alkyl In silico study of jati leaf constituents as traditional wound care - Krisnamurti et al. 319 The binding sites of T. grandis bioactive compounds toward NF-κB were different from that of curcumin, which suggested that T. grandis bioactive compounds act toward NF-κB differently from curcumin, the common nature-based wound- healing agent. The potency of T. grandis bioactive compounds to inhibit NF-κB also indicated by lower binding energy than those of curcumin and phenytoin. A low binding energy indicates the efficient ability of ligands to bind strongly to their receptors. A receptor-ligand complex with low binding energy is expected to form a strong and stable complex. In contrast, a higher binding energy suggests a weaker binding interaction, which may negatively affect the ligand’s ability to effectively perform its function with the receptor (Hasan et al. 2023). Epigallocatechin 3-O-cinamate, epigallocatechin 3-O-p-coumarate, and tectograndinol formed binding sites toward MMP-2 differently to each other (Fig. 3). In the binding to MMP-2, tectograndinol did not share any identical amino acid residues with the other compounds. However, tectograndinol was an exception to this observation, as it shared two key amino acid residues with the control drug phenytoin, specifically at Pro25 and His98 with binding energy of -6.6 kcal/mol. A study conducted by Takeuchi et al. (2022) revealed that the interaction of an inhibitor with amino acid residues in the catalytic site, including His121, His131, Glu130, Ala88, Phe87, Ala86, Phe5, Tyr74, and Leu82 is crucial for the downregulation of MMP-2 expression. Based on molecular docking study, epigallocathecin 3-O-cinnamate may perform MMP-2 inhibition by binding on MMP-2 catalytic sites Phe87, Ala88, and Tyr74 with strongest binding energy -7.9 kcal/ mol (Hasan et al. 2023). Epigallocatechin 3-O-p- coumarate moderately acts as MMP-2 inhibitor by binding to Phe5 with -7.4 kcal/mol. Our study showed that curcumin possessed the potential activity to bind with MMP-2 indicated by the highest binding energy score (-9.5 kcal/ mol), while it did not interact with MMP-2 catalytic sites. This molecular docking study revealed that T. grandis bioactive compounds are more potent to bind with MMP-2, compared to binding with curcumin and phenytoin, as the bioactive compounds bind with catalytic sites with lower binding energy than phenytoin. It suggested that T. grandis bioactive compounds may inhibit collagen, fibronectin, laminin, and elastin degradation (Wolosowicz et al. 2024). Our study revealed that bioactive compounds of T. grandis leaf extract showed less inhibitory activity on MMP-9 (Fig. 4). The results suggested that the binding mechanism of T. grandis bioactive compounds to MMP-9 is distinct from that of curcumin or phenytoin, as evidenced by the lack of shared amino acid residues at the binding site (Table 5). Figure 3 Interaction of MMP-2 with ligands Notes: A = epigallocathecin 3-O-cinnamate; B = epigallocatechin 3-O-p-coumarate; C = tectograndinol; D = curcumin; E = phenytoin. All compounds posed different binding sites, suggesting the difference of mechanism of action to MMP-2. BIOTROPIA Vol. 32 No. 3, 2025 320 Figure 4 Interaction of MMP-9 with ligands Notes: A = epigallocathecin 3-O-cinnamate; B = epigallocatechin 3-O-p-coumarate; C = tectograndinol; D = curcumin; E = phenytoin. Tectograndinol appeared to have similar binding site as phenytoin. Table 5 Interactions of Tectona grandis bioactive compounds toward MMPs Ligand Name Category Type Binding energy (kcal/mol) MMP-2 Epigallocathecin 3-O-cinnamate Thr91, Tyr74, Gly94 Hydrogen Bond Conventional Hydrogen Bond -7.9Tyr74 Hydrophobic Pi-Pi Stacked Phe87, His85 Hydrophobic Pi-Pi T-shaped Ala88 Hydrophobic Pi-Alkyl Epigallocatechin 3-O-p-coumarate Lys79, Asp102 Hydrogen Bond Conventional Hydrogen Bond -7.4 Trp68 Hydrophobic Pi-Pi T-shaped Phe5;Pro6 Hydrophobic Amide-Pi Stacked Lys8 Hydrophobic Alkyl Gly71, Phe4 Unfavorable Unfavorable Donor-Donor Tectograndinol Phe65, Glu58 Hydrogen Bond Conventional Hydrogen Bond -6.6Pro25, Val93 Hydrophobic Alkyl His98 Hydrophobic Pi-Alkyl Curcumin Arg150, Thr144 Hydrogen Bond Conventional Hydrogen Bond -9.5 Gly136, Val118 Hydrogen Bond Carbon Hydrogen Bond Leu83 Hydrophobic Pi-Sigma Val118 Hydrophobic Pi-Alkyl Phenytoin Arg67 Hydrogen Bond Conventional Hydrogen Bond -6.9 His98 Hydrophobic Pi-Pi T-shaped Pro25, Ile21, Met62 Hydrophobic Pi-Alkyl In silico study of jati leaf constituents as traditional wound care - Krisnamurti et al. 321 Ligand Name Category Type Binding energy (kcal/mol) MMP-9 Epigallocathecin 3-O-cinnamate Asp206, Glu130 Hydrogen Bond Conventional Hydrogen Bond -7.4Thr336 Hydrogen Bond Pi-Donor Hydrogen Bond Thr336, Met338 Hydrophobic Pi-Sigma Epigallocatechin 3-O-p-coumarate Asp182 Hydrogen Bond Conventional Hydrogen Bond -7.9 Asp185 Hydrogen Bond Carbon Hydrogen Bond Asp182 Electrostatic Pi-Anion Arg95 Hydrophobic Alkyl; Pi-Alkyl Tectograndinol Asp390 Hydrogen Bond Conventional Hydrogen Bond -5.9 Phe425 Hydrophobic Pi-Alkyl Curcumin Asn38, Leu39, Arg51, Tyr52 Hydrogen Bond Conventional Hydrogen Bond -7.4Lys184 Hydrogen Bond Carbon Hydrogen Bond Leu39 Hydrophobic Pi-Sigma Arg95 Hydrophobic Pi-Alkyl Phenytoin Tyr393 Hydrogen Bond Conventional Hydrogen Bond; Pi-Donor Hydrogen Bond -6.1Asp390 Electrostatic Pi-Anion Tyr393 Hydrophobic Pi-Pi Stacked Pro97 Hydrophobic Pi-Alkyl However, tectograndinol was an exception, as it shared the same amino acid residue, Asp390, with the phenytoin. Tectograndinol was suggested to inhibit MMP-9 in the inflammation, as the compound also formed binding with Phe425, which is known as the active site of MMP-9 (Elkins et al. 2002). Neither epigallocatechin 3-O-cinnamate nor epigallocatechin 3-O-p- coumarate formed binding interactions with MMP-9 at the same amino acid residues as phenytoin or curcumin, suggesting a different inhibitory mechanism. Bioactive compounds of T. grandis actively inhibit MMP-9, as shown by their lower binding energy in the docking study. Epigallocathecin 3-O-p-coumarate performed the strongest binding to MMP-9 (-7.9 kcal/ mol). Epigallocathecin 3-O-cinnamate binding to MMP-9 was the second strongest (-7.4 kcal/ mol) and tectograndinol binding to MMP-9 was the least strong (-5.9) kcal/mol). The ability of tectograndinol to interact with MMP-9 was relatively lower as compared with control. Molecular docking result showed the optimum binding of T. grandis leaf bioactive compounds toward EGFR-1 (Table 6). T. grandis compounds, epigallocathecin 3-O-cinnamate, epigallocathecin 3-O-p-coumarate, and tectograndinol, were specifically bind to EGFR-1 through Val726, Ala743, and Lys745 (Fig. 5). Moreover, nitrofurazone and curcumin also occupy the same binding pocket as T. grandis bioactive compounds, which indicate the potential of T. grandis bioactive compounds to promote wound-healing process in a similar manner to those of nitrofurazone and curcumin. Based on the binding energy, epigallocathecin 3-O-cinnamate was the easiest to bind with EGFR-1 (-9.1 kcal/mol). Epigallocathecin 3-O-p-coumarate performed a moderate strength with -8.9 kcal/mol binding affinity. The least binding energy was on tectograndinol with -7.4 kcal/mol. Binding of T. grandis leaf bioactive compounds to the active sites of EGFR-1 may stimulate the activation of EGFR-1 for cell migration and proliferation, leading to re-epithelialization and dermal maturation (Repertinger et al. 2004; Shin et al. 2022). Based on the virtual prediction, T. grandis bioactive compounds have as much potential as nitrofurazone in accelerating the wound- healing process. Treatment with nitrofurazone increased the re-epithelialization process and decreased the number of inflammatory cells. The bactericidal effect of nitrofurazone contributed to the acceleration of the wound-healing process (Karapolat et al. 2020). Interestingly, a previous study reported the antibacterial activity of anthraquinones in T. grandis extract. The methanolic and aqueous extract of T. BIOTROPIA Vol. 32 No. 3, 2025 322 grandis has also demonstrated the inhibitory effect on Gram-positive and Gram-negative bacteria (Irinmwinuwa et al. 2023). Treatment of wounds with T. grandis leaf extract may have more potency than that of nitrofurazone. The process of wound healing requires various types of cells and proteins. MMP is one of the proteins regulated during wound-healing process that is activated by NF-κB and upregulated during inflammation stage which mostly driven by reactive oxygen species (ROS) (Caley et al. 2015; Kandhwal et al. 2022; Yadav et al. 2024). The balance level of ROS may stimulate the signaling pathway of NF- κB activation and MMPs upregulation, which play major role during wound-healing process. However, ROS could also stimulate the ECM turnover, causing tissue destruction and interference with the repair process (Hingorani et al. 2019). Besides, the prolonged inflammation phase would also delay the wound-healing process. Treatment with T. grandis leaf extract with exhibit antioxidant and free radical scavenger activity may balance the ROS level during wound-healing process, leading to the acceleration of wound-healing process (Sun et al. 2021). A molecular docking study revealed the potential of bioactive compounds from T. grandis in treating wounds, showing a similar mechanism to the known drugs phenytoin and nitrofurazone. In contrast, the wound-healing mechanism of T. grandis leaf compounds may differ slightly from that of curcumin, a natural product. This is supported by the finding that curcumin and the T. grandis compounds bind to different sites on the key wound-healing proteins. Furthermore, the study revealed that T. grandis leaf bioactive compounds bind to the active sites of NF-κB, MMP-2, MMP-9, and EGFR-1 with high efficiency. Based on virtual prediction, our study suggested the mechanism of T. grandis leaf extract in wound-healing process via NF-κB, MMP-2, MMP-9, and EGFR-1 (Fig. 6), however, further investigation through in vivo and in vitro studies is required for definitive confirmation. Table 6 Interaction of Tectona grandis bioactive compounds toward EGFR-1 Ligand Name Category Type Binding energy (kcal/mol) EGFR-1 Epigallocathecin 3-O-cinnamate Arg841, Asn842 Hydrogen Bond Conventional Hydrogen Bond -9.1Leu718 Hydrophobic Pi-Sigma Val726, Ala743, Lys745 Hydrophobic Alkyl Lys745, Leu788, Val726 Hydrophobic Pi-Alkyl Epigallocatechin 3-O-p-coumarate Lys745, Arg841 Hydrogen Bond Conventional Hydrogen Bond -8.8 Cys797 Other Pi-Sulfur Val726 Hydrophobic Alkyl Val726, Leu718, Ala743, Lys745 Hydrophobic Pi-Alkyl Tectograndinol Lys745, Thr854, Ala743 Hydrogen Bond Conventional Hydrogen Bond -7.3 Val726, Ala743, Lys745, Leu844, Leu718, Cys797 Hydrophobic Alkyl Curcumin Met793, Leu788 Hydrogen Bond Conventional Hydrogen Bond -8.4Gln791 Hydrogen Bond Carbon Hydrogen Bond Leu718 Hydrophobic Pi-Sigma Leu844, Val726 Hydrophobic Pi-Alkyl Nitrofurazone Thr854, Asp855, Phe856 Hydrogen Bond Conventional Hydrogen Bond -6.2Phe856 Hydrogen Bond Pi-Donor Hydrogen Bond Val726, Ala743, Lys745 Hydrophobic Pi-Alkyl In silico study of jati leaf constituents as traditional wound care - Krisnamurti et al. 323 Figure 5 Interaction of EGFR-1 with ligands Notes: A = epigallocathecin 3-O-cinnamate; B = epigallocatechin 3-O-p-coumarate; C = tectograndinol; D = curcumin; E = nitrofurazone. All bioactive compounds posed similar binding sites toward EGFR-1. Figure 6 Prediction of the molecular mechanism of T. grandis bioactive compounds in promoting wound healing via EGFR-1 and NF-κB signaling, leading to regulated MMP-2/MMP-9 expression During wound-healing process, NF-κB naturally activated as innate immune response in all cells as a response to infection or injury. It is essential for migration of phagocytic and inflammatory cells to the tissue. The binding of EGF and EGFR initiates the cell proliferation and migration by PI3K factors activation, JAK/STAT, PLC and PKC, also Ras/Raf signaling via EGF/EGFR pathway. The mechanism initiates upregulation of NF-κB signalling which stimulates prolonged inflammation (Bodnar 2013; Tomas et al. 2014; Yang et al. 2022). The upregulated NF-κB may lead to impaired wound-healing process due to the role of NF-κB as precursor of inflammation. In addition, NF-κB activation also facilitate in upregulated MMP-9 expression, leading to prolonged inflammation and development of chronic wound (Ambrozova et al. 2017; Yadav et al. 2024). Moreover, MMP-2 and MMP-9 are jointly involved in the hemostasis process of wound-healing mechanism (Kandhwal et al. 2022). Expression of MMP-2 during inflammation triggers the MMP-9 in promoting cell migration and re-epithelialization (Caley et al. BIOTROPIA Vol. 32 No. 3, 2025 324 2015; Lee & Kim 2022). However, upregulated MMP-2 and MMP-9 is associated with chronic wounds. In the vascular cells, overexpressed MMP-2 and MMP-9 promote ECM degradation (Ayuk et al. 2016; Hingorani et al. 2019). Thus, maintaining the expression of MMP-2 and MMP- 9 is necessary to accelerate wound-healing process (Chiang et al. 2023). The bioactive compounds from T. grandis leaves form a strong binding with the receptor, as suggested by the presence of hydrogen bonds. These bonds play a pivotal role in stabilizing the receptor-ligand complex and increasing the ligand’s efficiency in performing its action (Krisnamurti et al. 2021). Wound care using T. grandis leaves extract could balance each wound healing phase, leading to acceleration of the healing process. The mechanism predicted from this study was that the bioactive compounds of T. grandis leaf act as MMP- 2 inhibitor and MMP-9 inhibitor. It prevents the upregulation of MMP-2 and MMP-9 expression during inflammation phase in the wound-healing process, leading to the failure of chronic wound development. The controlled MMP-2 and MMP- 9 expressions initiate the migration of fibroblast to the wound site via ECM remodeling (Hingorani et al. 2019). Moreover, bioactive compounds from T. grandis leaf have the potential to stimulate EGFR- 1, as shown by molecular docking studies. This protein is identified for its pivotal role in wound- healing process by regulating cell proliferation, survival, and differentiation (Chakraborty et al. 2014; Sun et al. 2021). A previous study showed that mice treated with EGFR-1 exhibited a reduced incision wound width compared to the untreated group. Additionally, abundant hair growth was observed in the EGFR-treated mice without incision wounds, showing the importance of EGFR stimulation during the wound healing process (Repertinger et al. 2004). The binding of ligands to EGFR-1 may stimulate cell proliferation and migration via the PI3K, JAK/STAT, PLC, and PKC pathways, all of which aid the wound healing process (Tomas et al. 2014; Yang et al. 2022). Our study suggested that the bioactive compounds of T. grandis leaf downregulate NF-κB expression, leading to anti-inflammatory activity. The mechanism also contributes in lowering expression of MMP-2 and MMP-9. The regulated expression of MMP-2 and MMP-9 would allow cell migration during the wound-healing process (Lee & Kim 2022). Treatment and therapy of wounds with T. grandis leaf could be an alternative treatment with high safety and effectiveness. However, the biological activity of T. grandis bioactive compounds in wound treatment should be further studied. CONCLUSION Epigallocatechin 3-O-cinnamate, epigallocatechin 3-O-p-coumarate, and tectograndinol were identified as the most abundant bioactive compounds from a total of 82 bioactive compounds contained in T. grandis methanolic extract. The bioactive compounds was predicted to have biological activities related to wound-healing process as antioxidant, free radical scavenger, anti- inflammatory, and MMP-9 expression inhibitor. In silico analysis of drug-likeness and ADME/T showed that T. grandis bioactive compounds possessed favorable properties and were predicted to be highly safe. This study suggested that bioactive compounds hold potential for developing new medication with reduced side effects. Further study by molecular docking demonstrated the wound- healing process via NF-κB, MMP-2, MMP-9, and EGFR-1 binding, which showed similar results as that of commonly prescribed wound- healing pharmaceuticals. This study predicted the molecular mechanism of wound-healing process using the bioactive compounds of T. grandis by performing inhibition of NF-κB, MMP-2, MMP- 9 and stimulating EGFR-1. Further in vivo or in vitro studies are required to evaluate the activity of T. grandis bioactive compounds in wound treatment. REFERENCES Akiki RK, Anand RS, Borrelli M, Sarkar IN, Liu PY, Chen ES. 2021. Predicting open wound mortality in the ICU using machine learning. J Emerg Crit Care Med 5:13. DOI: 10.21037/jeccm-20-154 Al Mousa AA, Abouelela ME, Mansour A, Nasr M, Ali YH, Al Ghamidi NS, …, Hassane AM. 2024. 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