Bangladesh Journal of Pharmacology Research Article Diabetic wound healing activity of Myrmecodia pendens ethyl acetate fraction BJP Introduction Diabetic wound is among the most serious complica- tions of diabetes, with high morbidity rates. Although approximately 60–80% of the diabetic wound can heal, 10–15% remain chronic, and 5–24% of cases result in amputation within 6 to 18 months (McDermott et al., 2023). Neuropathic ulcer often requires more than 20 weeks to heal, while neuroischemic wound takes even longer and carry a higher risk of amputation (Boulton and Whitehouse, 2000; Alexiadou and Doupis, 2012). Beyond being a significant health burden, diabetic wound also imposes substantial financial pressure due to prolonged medical care, advanced wound therapies, and surgical intervention such as amputation in severe case. Unlike normal wound healing, diabetic wound takes longer time to heal (Spampinato et al., 2020; Burgess et al., 2021). This is due to its inability to progress to the proliferative phase and the persistence of chronic inflammation. This wound is characterized by unique conditions such as high moisture level, eleva- ted blood sugar, hypercoagulability, and weakened immune response. These factors increase the risk of infection, hinder nutrient supply to the wound tissue, and slow the healing process (Dasari et al., 2021). Therefore, managing diabetic wound requires a com- plex therapeutic approach. One promising solution is to use multiple compounds present in natural products, which work synergistically to support the wound healing process. Natural pro- ducts that exhibit anti-inflammatory activity, suppress or inhibit microbial growth, and modulate cytokines and growth factors are potential therapeutic leads for Abstract This study investigates the diabetic wound healing potential of M. pendens using an in vivo diabetic animal model and molecular docking targeting MMP, EGF, and FGF pathways. Ethyl acetate hydrogel formulations (0.05%, 0.10%, and 0.15%) were tested over 14 days, with wound closure measured using ImageJ. The fraction contained 91.5 mg QE/g of flavonoids. Docking analysis showed strong binding affinities of quercetin, cholesta-22,24-dien-5- ol, and procyanidin B1 to MMP, EGFR, and FGFR (−8.4 to −10.2 kcal/mol). By day 14, the negative control group showed 30-40% wound closure, while tetrachlorodecaoxide reached 80% (p<0.01). The 0.05% hydrogel achieved 85% closure (p<0.05), and the 0.10% hydrogel showed complete healing (100%, p<0.001). The 0.15% hydrogel was less effective than 0.10%, with significant differences between days 7 and 14 (p<0.05). These findings suggest that M. pendens hydrogel may aid diabetic wound healing through molecular path- way modulation. Article Info Received: 6 March 2025 Accepted: 21 May 2025 Available Online: 23 May 2025 DOI: 10.3329/bjp.v20i2.80330 Cite this article: Falya Y, Umar AK, Suharyani I, Anugra CY. Diabetic wound healing activity of Myrmecodia pendens ethyl acetate fraction. Bangladesh J Pharmacol. 2025; 20: 62-71. Diabetic wound healing activity of Myrmecodia pendens ethyl acetate fraction Yuniarti Falya1, Abd Kakhar Umar2, Ine Suharyani3, and Cindra Yulianto Anugra3 1Faculty of Pharmacy, Universitas Islam Kalimantan Muhammad Arsyad Al Banjari, Banjarmasin, South Borneo, Indonesia; 2Medical Informatics Laboratory, ETFLIN, Palu City 94225, Indonesia; 3Faculty of Pharmacy, Universitas Muhammadiyah Ahmad Dahlan Cirebon, Cirebon 45153, Indonesia. This work is licensed under a Creative Commons Attribution 4.0 License. You are free to copy, distribute and perform the work. You must attribute the work in the manner specified by the author or licensor. A Journal of the Bangladesh Pharmacological Society (BDPS) Bangladesh J Pharmacol 2025; 20: 62-71 Journal homepage: www.bdpsjournal.org; www.banglajol.info Abstracted/indexed in Academic Search Complete, Agroforestry Abstracts, Asia Journals Online, Bangladesh Journals Online, Biological Abstracts, BIOSIS Previews, CAB Abstracts, Current Abstracts, Directory of Open Access Journals, EMBASE/Excerpta Medica, Global Health, Google Scholar, HINARI (WHO), International Pharmaceutical Abstracts, Open J-gate, Science Citation Index Expanded, SCOPUS and Social Sciences Citation Index ISSN: 1991-0088 treating diabetic wound. There are several plants that have potential diabetic wound healing activity, including Crocus sativus (saff- ron) (Soheilifar et al., 2024); Musa paradisiaca (Cheng et al., 2020); Sphenocentrum jollyanum pierre (Adeleke et al., 2022)., and Myrmecodia pendens (Najah et al., 2024). Myrmecodia pendens has antioxidant, antimicrobial, and immunomodulatory properties (Sudiono et al., 2015; Widyawati et al., 2020; Dirgantara et al., 2022; Daulay et al., 2024; Lisnanti et al., 2024). Its diabetic wound- healing effect has not yet been examined. In addition, this study aimed to explore its ability to modulate key molecular pathways such as matrix metalloproteinases (MMP), epidermal growth factor receptor (EGFR), and fibroblast growth factor receptor (FGFR), supported by phytochemical, in silico, and in vivo evaluations. The ethyl acetate fraction of M pendens extract is known to contain flavonoids that have been shown to positive- ly regulate MMP-2, MMP-8, MMP-9, MMP-13, and the Ras/Raf/MEK/ERK, PI3K/Akt, and nitric oxide (NO) signaling pathways (Chanu et al., 2023). Compounds such as procyanidin B1 dimer (3.2 mg/g dry sample) and rosmarinic acid (20.7 mg/g dry sample) exhibit significant free radical scavenging activity, with IC50 values of 27.6 µg/mL and 35.8 µg/mL, respectively (Engida et al., 2015). Additionally, six phenolic and terpenoid compounds have been identified in M. pendens, some of which show antibacterial activity against various pathogenic bacteria, including dibenzo- p-dioxin-2,8-dicarboxylic acid exhibits an inhibition zone of 8.6 mm against E. faecalis, while stigmast-4-ene- 3-one and pomolic acid show inhibition zones of 9.0 mm and 10.2 mm against S. mutans, respectively. Phloroglucinol sesquiterpene demonstrates the largest inhibition zone of 12.3 mm against P. gingivalis. Other studies have reported that the ethyl acetate fraction of M. pendens exhibits antibacterial activity against S. mutans biofilms at a concentration of 50 µg/mL, with a Minimum Biofilm Eradication Concentration (MBEC) of 40% for a one-minute induction period (Gartika et al., 2018). A combination of hexane-ethyl acetate fractions shows the best antibacterial activity, with a Minimum Inhibitory Concentration (MIC) of 0.05 mg/mL and a Minimum Bactericidal Concentration (MBC) of 12.5 mg/mL (Kuswandani et al., 2019). This study explores the potential of ethyl acetate fraction-based hydrogel of M pendens for diabetic wound healing, both in silico and in vivo. Materials and Methods The materials used include M pendens plants sourced from Borneo, West Kalimantan, filter paper (What- man® Grade 1), aquadest (Brataco®), 70% ethanol (Sigma-Aldrich®), n-hexane (Merck®), chloroform (Merck®), sodium hydroxide (Sigma-Aldrich®), 0.1% ferric chloride/FeCl3 (Merck®), sulfuric acid (Sigma- Aldrich®), hydrochloric acid (Merck®), aluminum chlo- ride (Sigma-Aldrich®), methanol (Merck®), Dragen- dorff's, Carbopol (Carbopol 940), hydroxypropyl methylcellulose/HPMC (Ashland® Benecel™), glycerin (Brataco®), methylparaben (Brataco®), propylparaben (Brataco®), triethanolamine (Merck®), and phosphate- buffered saline. Plant collection, identification, and simplification This plant is an epiphytic species that grows attached to various types of trees. When collected, the plants were harvested randomly from several tree species that were not identified at the time. The plants were sourced from the Nanga Pinoh region of West Kalimantan, Indonesia, and subsequently identified as M. pendens by Prof. Ria Yulia Gloria, at Biology Laboratory of the State Islamic Institute of Syekh Nurjati Cirebon, with notification number 30/LN.08/LB.1.1/PP.009/12/2023. The part of the plant collected was the caudex (the swollen, hollow stem), which was separated and brought to the pharma- cognosy laboratory for further processing. The outer surface of the caudex was initially cleaned of dirt and fibrous roots. It was then split and cut into large sections, which were sun-dried to remove ants. Once all sections were free from ants, they were sliced into thin, flat pieces approximately 1 cm thick and washed to remove soil, debris, and ant eggs. Sections with inconsistent tissue characteristics (e.g., at the junctions of the caudex with the stem or roots) were discarded. The cleaned slices were then oven-dried at 40°C for one week. The dried pieces were subsequently referred to and labeled as simplicia. Extraction and fractionation The dried simplicia was blended until it became a coarse powder and dried in an oven for a day (40°C). The coarse powder (350 g) was then moistened with 70% ethanol until all parts were submerged. This maceration process was carried out for 3 days at room temperature (25°C) with regular manual shaking (twice a day). The solution was then filtered to obtain a dilute extract solution. The residue was, then, remacerated following the same procedure as before. This process was repeated three times. The filtrates from these processes were combined, and the solvent was then evaporated using a rotary evaporator at 40°C, 200 mBar, and 150 rpm until a thick extract consistency was achieved. The extract was subsequently fractionated using ethyl acetate and water in a 1:1 ratio. The two phases were then separated, and the ethyl acetate phase was further dried using a rotary evaporator until a thick fraction was obtained. Bangladesh J Pharmacol 2025; 20: 62-71 63 Simplician and extract standardization M. pendens simplicia was standardized to ensure its quality and consistency by assessing key parameters as described in the Indonesian Herbal Pharmacopoeia. The analyzed parameters included organoleptic properties, water-soluble content, ethanol-soluble content, mois- ture content, total ash content, acid-insoluble ash con- tent, and percentage of drying loss. Phytochemical screening The crude methanolic extract and fractions of M pendens were analyzed for phytochemical constituents, inclu- ding alkaloids, flavonoids, steroids, tannins, saponins, terpenoids, and phenolics. Phytochemical reagents were added to the extract and fraction solutions, and the qualitative results were recorded as the presence and the absence of each phytochemical. Total flavonoid content Total flavonoid content of the ethyl acetate extract was determined using the aluminum chloride colorimetric method and using standard solutions (4, 5, 6, 7, and 9 ppm). For the analysis, 1 mL extract solution in 80% methanol (1 mg/mL) was mixed with 0.5 mL 95% ethanol (v/v), 0.2 mL aluminum chloride solution (10%), 0.2 mL 1 M potassium acetate, and 1 mL distilled water to a total volume of 2.5 mL. The mixture was well mixed and incubated at room temperature for 30 min. Total flavonoids were measured using a UV-Vis spec- trophotometer at wavelengths 520 nm vs. a reagent blank containing water instead of the sample. Quercetin was used as the standard for the quantification of total flavonoids. Results were expressed as milligrams of quercetin equivalent per gram of dry weight extract (mg QE/g). Total content of flavonoid was calculated as follows: Total flavonoid content = QE × V/m Where QE is the quercetin equivalence (mg/mL) or concentration of quercetin solution established from the calibration curve; V is the volume of extract (mL), and m is the weight (g) of the dry extract (Sulastri et al., 2018) FTIR analysis Approximately 2 mg of each sample fraction was weighed and thoroughly mixed with 100 mg of dry potassium bromide powder. The mixture is then homo- genized to ensure an even distribution of the sample in the potassium bromide and pressed into a thin, trans- parent disc using a high-pressure hydraulic press (approximately 10 tons of pressure). The potassium bromide disc was then placed in the FT-IR spectro- meter, where scanning was performed in the infrared range of 4000–400 cm⁻¹. Before analyzing the sample, a background spectrum (air) was recorded to eliminate atmospheric interference (Ol'ha and Hovorun, 2021). Computational study Ligand preparation A total of 70 compounds from 3 literature sources were collected (6-8), and structures with the same CID were manually removed. The 2D structures were then con- verted to 3D using Open Babel in PDB format. Similar structures and molecules with identical descriptors were also removed using the --unique command in Open Babel. The structures were protonated at pH 7.4, and charges were added using the MMFF94 force field. Before saving, the energy of the compounds was mini- mized, and the MMFF94 force field was applied. All PDB files were converted to PDBQT format using the repair_ligand4.py script. The entire process was perfor- med simultaneously using custom Python code. Receptor preparation The structures of EGFR, FGFR, and MMP were obtain- ed from the RCSB with IDs MMP [1CIZ], EGFR [2GS6], FGFR [4QQ5]. Water molecules, ions, and non-ligand compounds were removed using Discovery Studio. The position of the native ligand was set as grid coor- dinates, with the grid size adjusted to match the native ligand's structure. The native ligand was then separated and saved as a PDB file. The native ligand was prepar- ed following the same process as the test ligands until it was in PDBQT format. The cleaned protein structure was saved as a PDB file. The protein structure was subsequently input into AutoDock Tools, where Koll- man charges were applied, polar hydrogens were added, and the structure was saved in PDBQT format. Finally, the initial native ligand structure and the pose after docking were superimposed, and the RMSD value was calculated as a validation of the docking method. Molecular docking Molecular docking was performed using DockFlin (Version 2.0, ETFLIN, Indonesia). All ligands and pro- teins were loaded into their respective panels. The grid files, containing the coordinates and size, were input according to the sequence of the proteins in the panel. After setting the output folder, the docking process was initiated using the Vina scoring function. Diabetic wound activity Animal preparation Forty male Wistar rats (aged 8-10 weeks, weighing 180- 220 g) were used in the study and were adapted to cage conditions for two weeks. Subsequently, the rats were given a special diabetes diet (diabetic rat feed) for another two weeks. Before the diet, the rats were induced with alloxan 110 mg/kg via intraperitoneal injection. At the end of the diet period, which lasted for 7 days post-alloxan induction, fasting blood glucose levels were measured to confirm diabetes, blood 64 Bangladesh J Pharmacol 2025; 20: 62-71 glucose levels of the rats were measured using a digital glucometer (Autocheck®), and those with hypergly- cemia (140-220 mg/dL) were included in the study. Wound creation and treatment Wound creation began with shaving the dorsal fur using an electric shaver, followed by anesthesia with ketamine administered intraperitoneally at the wound site. A full-thickness wound was induced using a 4 mm biopsy punch, and the initial wound size was recorded using a camera positioned 30 cm away from the wound. Daily treatment was administered for 14 days, with wound size assessed on day 0, 3, 5, 7, 9, and 14. The rats were divided into five treatment groups (6 rats per group): a negative control group (hydrogel base), a positive control group (tetrachlorodecaoxide hydrogel), and three experimental groups treated with varying concentrations of the ethyl acetate fraction of M pendens (0.05%, 0.10%, and 0.15%) in a hydrogel base. The hydrogel base was prepared by dissolving 0.5% hydro- xypropyl methylcellulose in water. Wound healing was assessed by calculating the percentage of wound coverage, determined by comparing the initial wound area with the area at each observation point. Statistical analysis Statistical analysis was performed to determine the significance of differences between the treatment groups in the in vivo study. One-way ANOVA followed by post hoc testing using the Tukey method was appli- ed using RStudio (version 2024.09.1). Results Extract standardization and phytochemicals M. pendens ethyl acetate extract contained various bio- active compound groups, including alkaloids, flavo- noids, terpenoids, tannins, and saponins (data not shown). The total flavonoid content in the M. pendens extract was found as 48.5 mg QE/g (4.9 ± 0.9% QE), while in the ethyl acetate, the value significantly incre- ased to 91.5 mg QE/g (91.5 ± 0.014% QE). The peaks at 3341 cm-1 and 1564 cm-1 show a significant absorbance, indicating that the ethyl acetate fraction of M pendens contained compounds with numerous hydroxyl or phenolic groups (carboxyl or phenolic; Figure 1). This is further supported by 937 cm-1 and 503 cm-1 peaks, which are characteristic signs of C-O coupled with O-H and cis-HCOOH. Extract hydrogel formulation Figure 2 presents the organoleptic analysis results of four hydrogel formulas. In terms of aroma, all formulas exhibited a stable characteristic scent throughout the storage period. However, the formulas containing the ethyl acetate fraction of M pendens (0.05%, 0.10%, and 0.15%) showed a slight increase in aroma intensity com- pared to the base formula A (Figure 2A). Regarding color, each formula displayed distinct differences. The base formula was white, while formulas containing the ethyl acetate fraction of M. pendens showed a gradient of yellow, deepening as the extract concentration increased. The 0.05% formula had a clear yellow color, the 0.10% formula was dark yellow, and the 0.15% formula appeared yellow-brown. In terms of texture, all hydrogel formulas exhibited a smooth consistency and were easy to apply to the skin. The base formula, without any extract, had a light, non- sticky texture and provided a soft sensation upon appli- cation, which can be attributed to hydroxypropyl methylcellulose gel that was lightweight and non- sticky. In contrast, the formulas containing the ethyl acetate fraction of M. pendens (0.05%, 0.10%, and 0.15%) showed a thicker and slightly stickier texture, with % T ra n s m it ta n c e 4000 3000 2000 1000 100 90 80 70 60 50 Figure 1: FT-IR spectrum ethyl acetate fraction of M. pendens cm-1 Bangladesh J Pharmacol 2025; 20: 62-71 65 viscosity increasing as the extract concentration rose. Molecular docking Method validation was carried out through the redoc- king process of the native ligand into the binding site of each receptor to calculate the Root Mean Square Devia- tion (RMSD) value and evaluate the accuracy of the method used. The redocking results shown in Figure 3 A-C indicate RMSD values of 0.0251 Å for MMP, 1.6272 Å for EGFR, and 1.1429 Å for FGFR. The low RMSD values (≤2.0 Å) for all three complexes suggest that the docking method successfully replicated the position and orientation of the native ligand within the binding site with a high degree of accuracy. Thus, these results confirm that the docking method used is valid and reliable for evaluating ligand-receptor interactions in this study. Based on the findings, natural flavonoid compounds such as quercetin, kaempferol, and apigenin found in the ethyl acetate fraction of M pendens exhibit strong interaction to MMP1. Figure 3 D-F shows selected potential compounds that demonstrate the best binding conformations with the receptors. The highest binding energies were quercetin for MMPs receptor with a binding energy of -9.6 kcal/mol, cholesta-22,24-dien-5- ol, 4,4-dimethyl-(22E)-4,4-dimethylcholesta-22,24-dien-6 -ol for EGFR receptor with a binding energy of -9.1 kcal/mol, and procyanidin B1 for FGFRs receptor with a binding energy of -9.3 kcal/mol. Figure 3D illustrates the molecular interactions of compounds from the ethyl acetate fraction of M. pendens with the MMP receptor, showing several important molecular mechanisms. Hydrophobic interactions between the aromatic group of quercetin and hydrophobic residues such as Leu218 Conventional hydrogen bond Pi-Carbon Pi-Pi stocked Pi-Pi T-shaped Alkyl Pi-Sigma Interactions A C B D F E Figure 3: Native ligand (green) and native ligand redocked (blue) comparisons of MMP (A), EGFR (B), and FGFR (C). Binding of quercetin in the active site of MMP (D), binding of cholesta-22,24-dien-5-ol, 4,4-dimethyl-(22E)-4,4-dimethylcholesta-22,24-dien-6- ol in the active site of EGFR (E), binding of procyanidin B1 in the active site of FGFRs (F) Figure 2: Hydrogel formulations - base (A); formula 0.05% (B); formula 0.10% (C); formula 0.15% (D) A B C D 66 Bangladesh J Pharmacol 2025; 20: 62-71 and Leu164 strengthen the binding of the compound to the active site. Figure 3E shows the interaction of cholesta-22,24-dien-5 -ol, 4,4-dimethyl-(22E)-4,4-dimethylcholesta-22,24-dien- 6-ol from the ethyl acetate fraction of M. pendens with the EGFR receptor. The alkyl chain from the steroid compound interacts with hydrophobic residues such as Leu768, Leu694, Leu820, and Val702 through hydro- phobic interactions. Figure 3F shows the interaction of procyanidin B1 from the ethyl acetate fraction of M. pendens with FGFRs, involving the formation of hydro- gen bonds with residues such as Ala558, Glu565, Glu480, Gly484, and Phe483. This FGF interacted with the external domain of the receptor, which consists of subunits containing important residues like Glu565 and Glu480. Diabetic wound healing The morphological observation of wound healing at various time points for 14 days revealed significant differences between the negative control, positive control, and treatment groups with hydrogel based on the ethyl acetate fraction of M pendens (Figure 4). In the negative control group, wound healing was the slowest, with wound closure not reaching 50% by day 14. In the treatment group with 0.05% ethyl acetate hydrogel, wound contraction started to be visible by day 14, although scabbing was uneven and the wound was not fully healed, yet still better than the negative control. In the 0.10% concentration group, the wound was still relatively large on day 3, with a small amount of new tissue at the wound edges. By days 5 to 7, scabbing began to form, and by day 9, the wound showed shrinkage with slower epithelialization compared to the 0.15% concentration group. By day 14, the wound was only partially closed, with more noticeable scarring. In contrast, at the 0.15% concentration, the wound area began to shrink by day 3 with more uniform and rapid new tissue formation. Scabbing formed more quickly by days 5 to 7, and the wound shrank more significantly compared to the 0.05% concentration. By day 9, the wound was almost completely closed, and by day 14, the wound was fully closed with minimal scarring, indicating fast healing and effective remodeling. Hair growth was observed on the newly formed skin in the 0.15% fraction group, showing good skin function recovery. Data analysis in Figure 4C showed that the negative control group experienced suboptimal wound healing, with a low percentage of wound closure (around 30- 40%) by day 14. Meanwhile, the positive control (tetra- chlorodecaoxide) showed significant improvement (p<0.01) from day 5, with wound closure reaching 80% by day 14. Hydrogel with 0.05% ethyl acetate fraction of M. pendens showed significant improvement compared to the negative control (p<0.05), although still slower than the higher concentrations, with 85% wound clo- sure by day 14. The 0.10% concentration showed the best results, with the fastest and most significant wound closure at all time points (p<0.001), reaching 100% closure by day 14, compared to the positive con- trol and other groups. Although the 0.15% concentra- tion was effective, its speed was slightly slower compared to the 0.10%, with significant differences observed from days 7 to 14 (p<0.05). % W o u n d c o v e ra g e C Figure 4: A) Picture of real wound with diameter by scale (left) and automatic wound marking (right, arrow head) by ImageJ soft- ware. B) Morphology of wound healing in diabetic rats following treatment with hydrogel based on the ethyl acetate fraction of M pendens at differences concentrations (0.05%, 0.10%, and 0.15%), compared to negative and positive controls. C) Wound closure of rats observed during the treatment. Note: (*, p<0.05) indicates a significant difference compared to the 0.15% group. (#, p<0.05) indicates a significant difference compared to the positive control group. (@, p<0.05) indicates a significant difference compared to the 0.10% group. ($, p<0.05) indicates a significant difference compared to the 0.05% group 100 75 50 25 0 Day 0 Day 3 Day 5 Day 7 Day 9 Day 14 Base Positive control Hydrogel containing fraction (0.05%) 0.10% 0.15% Day 3 Day 5 Day 7 Day 9 Day 14 A B Bangladesh J Pharmacol 2025; 20: 62-71 67 Discussion The phytochemical profile of M. pendens confirms the presence of several key secondary metabolites, inclu- ding alkaloids, flavonoids, terpenoids, tannins, and saponins. The high concentration of flavonoids in the ethyl acetate fraction, recorded at 91.5 ± 0.0% QE, indi- cates that this fraction is particularly bioactive. This value is notably higher than that reported in a study, where documented 63.3 ± 1.8 mg QE/g in ethanolic extracts containing compounds like kaempferol, quer- cetin, and apigenin (Engida et al., 2015). Similarly, lower levels of rosmarinic acid and procyanidin B1 were observed in the ethyl acetate fraction, and even less in supercritical extracts (Larit et al., 2019). FTIR analysis findings show that compounds such as rosmarinic acid, procyanidin B1, gallic acid, caffeic acid, ferulic acid, p-coumaric acid, quercetin, rutin, kaempferol, luteolin, apigenin, palmitic acid, oleic acid, cholesta-22,24-dien-5-ol, and 4,4-dime- thyl are likely responsible for the observed peaks (Kanimozhi and Prasad, 2015). On the other hand, the peak at 1393 cm-1 is a distinctive feature indicating the presence of compounds with an amide group (Lv et al., 2018; Ellerbrock and Gerke, 2021). Flavonoid compounds with an amide group identified in M. pendens include cinnamic acid and methanamide, which have shown anti-neurodegene- rative effects (Hofmann et al., 2022), as well as strong anti-inflammatory, antibacterial, and antifungal proper- ties (Wibawa et al., 2020; Chan et al., 2021). Increased extract concentration was also associated with changes in physical characteristics of the formula- tion, such as stronger aroma and deeper color intensity. These sensory changes are likely due to the higher levels of terpenoids and phenolic compounds, which are known to produce distinctive scents and visual pigmentation (Gutierrez-del-Rio et al., 2021; Masyita et al., 2022). Flavonoids, in particular, contribute yellow hues, while tannins impart brown tones (Manzoor et al., 2021; Mallick et al., 2024). Both compound groups increase in concentration as the extract content rises, affecting the final appearance of the formulation. The observed increase in viscosity with higher extract con- centrations is consistent with reports that flavonoids and tannins can interact with hydrogel matrices, such as hydroxypropyl methylcellulose, enhancing gel strength and formulation stability (Micale et al., 2020; Ferreira et al., 2024; Studzińska-Sroka et al., 2024; Valero et al., 2024). This not only improves texture but also helps retain the active ingredients longer on the skin, potentially enhancing therapeutic effect (Juncan et al., 2021; Salvioni et al., 2021; Studzińska-Sroka et al., 2024) Molecular docking studies provide mechanistic insight into how the ethyl acetate fraction supports wound healing. Quercetin, a dominant flavonoid in the extract, interacts with MMPs by forming hydrogen bonds with key residues (e.g., Glu202 and His201), inhibiting enzy- matic activity at the catalytic site (Liu et al., 2021; Li et al., 2024). Inhibition of MMP-1, -2, and -9 reduces ECM degradation, a process that is often excessive in diabetic wounds and contributes to poor healing (Varghese et al., 2021; Liang et al., 2023). Quercetin also disrupts signaling pathways such as NF-κB and MAPK, down- regulating MMP gene expression. Moreover, it pro- motes the expression of TIMPs (tissue inhibitors of metalloproteinases), helping restore protease balance and supporting ECM stabilization (Fu et al., 2022; Kamal et al., 2024). Hydrogen bonds occur between the hydroxyl group of the steroid compound and amino acid residues such as Thr830, which contains a hydro- xyl group on its side chain, and alkyl interactions with amino acid residues like Leu768, Lys704, Cys773, Leu694, Leu820, Ala719, Val702, and Lys721 at the active site of EGFR, which play a key role in modula- ting EGFR activation and related signaling pathways (Panigrahi, 2008). Other compounds, including steroid derivatives like cholesta-22,24-dien-5-ol, demonstrate interaction with EGFR through hydrogen bonding with key residues such as Leu768 and Lys721. These interactions facilitate conformational changes necessary for EGFR activation and subsequent signaling through the MAPK and PI3K/Akt pathways. This activation promotes keratino- cyte proliferation and migration, which are critical for wound closure (Xu et al., 2023). Additionally, interac- tions with FGFR via compounds like procyanidin B1 suggest activation of angiogenic pathways. Binding with residues such as Lys704 and His553 initiates recep- tor dimerization and tyrosine phosphorylation, trigger- ing downstream signaling cascades involved in fibro- blast migration and neovascularization. The presence of glycosaminoglycan-binding residues also enhances FGFR activation, contributing to tissue regeneration (Shanmugam et al., 2022). The in vivo results suggest that the wound healing efficacy of the ethyl acetate fraction of M. pendens is positively correlated with concentration. The gradual enhancement of healing with increasing doses indicates that the bioactive constituents actively promote key regenerative processes, such as fibroblast proliferation, extracellular matrix remodeling, and neovascularize- tion (Addis et al., 2020). Although minor variations in healing speed were observed between concentrations, the highest concentration (0.15%) ultimately achieved the most complete wound closure, implying that higher doses maintain sufficient biological activity to over- come potential delays in the early phases of repair. This trend supports the notion that, within the tested range, increasing concentration does not induce cytotoxic effects severe enough to impair healing. Instead, the data emphasizes the potential for dose escalation to 68 Bangladesh J Pharmacol 2025; 20: 62-71 B achieve maximal therapeutic outcomes, highlighting the importance of careful dose optimization when deve- loping phytochemical-based wound healing therapies. Beyond enhancing wound closure rates, treatment with the ethyl acetate fraction markedly improved the quality of wound remodeling. The absence of scab formation throughout the healing process, coupled with the lack of visible scar tissue, suggests that the extract modulated the repair process toward a regenerative rather than a fibrotic pathway (Mamun et al., 2024). This phenomenon may reflect a favorable influence on keratinocyte proliferation and migration, as well as modulation of myofibroblast activity, minimizing exce- ssive extracellular matrix deposition (Dong et al., 2025). These in vivo outcomes are consistent with molecular docking findings, which showed that major bioactive compounds in the fraction, such as quercetin and steroid derivatives, interact with key targets like MMPs, EGFR, and FGFR to regulate ECM stability, promote keratinocyte activation, and stimulate angiogenesis. Moreover, the observed regrowth of hair follicles at the wound site indicates successful restoration of dermal architecture and adnexal structures, implying activation of resident stem cell populations and full reestablish- ment of skin function. Collectively, these findings posi- tion the ethyl acetate fraction of M. pendens as a promi- sing candidate not only for accelerating wound closure but also for promoting functional tissue regeneration, a hallmark objective in the development of next-genera- tion wound therapeutics. This study was limited to in silico, in vitro, and short- term in vivo evaluations, which may not fully reflect the long-term safety and efficacy of M. pendens hydrogel in clinical settings. The in vivo tests were conducted on a rat animal model, and the findings may not be directly applicable to human physiology. Furthermore, the investigation focused solely on selected molecular path- ways (MMP, EGFR, FGFR), while other important mechanisms involved in diabetic wound healing such as inflammatory cytokines and angiogenesis factors were not assessed. Conclusion The ethyl acetate fraction of M. pendens demonstrates therapeutic potential for diabetic wound healing by inhibiting MMPs and activating EGFR and FGFR pathways. Its rich phytochemical content and in vivo efficacy support further development into advanced pharmaceutical formulations. Financial Support This research is funded through the Hibah Penelitian Dosen Pemula Kemendikbud (Ministry of Education and Culture) No. 106/E5/PG.02.00.PL/2024. Ethical Issue The preparation process and animal wound healing tests were approved by the Ethics Committee (KEPK), Faculty of Pharmacy, Universitas Muhammadiyah Ahmad Dahlan Cirebon, under approval number 003/VIII/2024/9110/KEPK/ STFMC. Animal handling in this study followed the AECD guidelines. 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