INTRODUCTION

Medicinal plants continue to play a central role in the search for biologically active compounds for therapeutic use and drug development. Despite advances in synthetic chemistry and high-throughput screening technologies, a significant proportion of contemporary pharmaceuticals are either directly obtained from plant sources or designed based on the structural framework of plant-derived secondary metabolites. Nearly 80% of people worldwide rely, to some degree, on traditional medicine for basic healthcare, according to the World Health Organization [1]. This is especially true in low- and middle-income nations where plant-based therapies are still both economically and culturally relevant. This continued reliance, alongside the increasing challenges of antimicrobial resistance, chronic inflammatory diseases, cancer prevalence, and emerging viral infections, has strengthened the need to identify and validate new phytochemicals with therapeutic relevance.

One plant that has attracted attention in ethnomedicine is Bursera simaruba (L.) Sarg., commonly referred to as Gumbo Limbo. It is widely used across Central America, the Caribbean, and some tropical regions of Africa. As a member of the Burseraceae family, it belongs to a group of resin-producing plants known for their richness in terpenoids, flavonoids, and phenolic constituents. Traditionally, B. simaruba has been utilized in the treatment of a wide range of conditions, including inflammation, fever, gastrointestinal disturbances, infections, wounds, and various skin disorders. Ethnobotanical reports highlight the use of different plant parts such as the bark, leaves, and resin, often prepared as decoctions or topical applications [2]. These longstanding uses point toward the presence of active phytochemicals that merit detailed scientific investigation.

Studies exploring the phytochemical profile of B. simaruba and related species have identified several important classes of compounds, including triterpenoids, lignans, flavonoids, tannins, and essential oils. Many of these metabolites have demonstrated notable antioxidant, antimicrobial, anti-inflammatory, and cytotoxic activities. In particular, terpenoids derived from members of the Burseraceae family have shown potential in modulating key biological pathways, such as NF-κB signaling and apoptosis, which are relevant in inflammation and cancer. Despite these promising findings, there is still a need for more comprehensive in vitro studies, especially those that integrate molecular target prediction, to better understand their mechanisms of action.

At the same time, nanotechnology has introduced new possibilities in biomedical research, particularly in enhancing the delivery and effectiveness of bioactive compounds. The use of plant extracts in the green synthesis of nanoparticles has emerged as a widely accepted approach due to its sustainability and eco-friendly nature compared to traditional methods. In this process, bioactive constituents found in plants, including polyphenols, flavonoids, and various sugars, play a dual role by facilitating the reduction of metal ions and stabilizing the nanoparticles formed. Compared to traditional chemical methods, this strategy tends to produce nanoparticles with better biocompatibility and lower toxicity [3]. Notably, nanoparticles such as silver, zinc oxide, and gold synthesized through plant-mediated processes have demonstrated improved antimicrobial, antioxidant, anticancer, and anti-inflammatory effects, often due to synergistic interactions between the metal core and the phytochemical coating.

To fully understand and optimize these nanoparticles, proper characterization is essential. A range of analytical techniques is typically employed, alongside a range of characterization techniques such as UV–Visible spectroscopy, Fourier-transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM), and dynamic light scattering (DLS). These methods help confirm nanoparticle formation and provide detailed information about size, structure, surface properties, and stability. Thorough physicochemical characterization not only ensures consistency and reproducibility but also supports accurate evaluation of biological activity, ultimately enhancing the potential for real-world biomedical applications [4].

Beyond experimental validation, computational approaches such as molecular docking have revolutionized early-stage drug discovery by enabling prediction of ligand–protein interactions at the molecular level. Molecular docking provides insights into binding affinity, interaction energy, hydrogen bonding patterns, and active site compatibility between phytochemicals and therapeutic targets. This approach accelerates medicinal target prediction, reduces experimental cost, and guides rational drug development strategies. Recent studies have successfully integrated phytochemical screening with docking simulations to identify potential inhibitors of key targets involved in inflammation (COX-2), cancer (EGFR, Bcl-2), microbial infections (DNA gyrase), and metabolic disorders (α-amylase, DPP-4) [5].

Importantly, combining in vitro bioassays with molecular docking enhances the robustness of pharmacological investigations. While in vitro assays provide empirical evidence of biological activity, such as antioxidant scavenging capacity, antimicrobial inhibition zones, cytotoxicity profiles, or enzyme inhibition, molecular docking offers mechanistic explanations at the atomic level. This integrative strategy bridges the gap between ethnomedicine and evidence-based pharmacology.

Despite the documented ethnomedicinal use of Bursera simaruba, there remains limited comprehensive research integrating green synthesis of nanoparticles, detailed physicochemical characterization, in vitro biological evaluation, and molecular docking-based medicinal target prediction. Most existing studies focus on isolated pharmacological aspects without correlating phytochemical composition to specific molecular targets. Furthermore, given the global urgency to discover novel antimicrobial and anti-inflammatory agents in the era of rising resistance and chronic disease prevalence, systematic evaluation of traditionally valued plants such as Gumbo Limbo is both timely and scientifically justified.

Therefore, this study is designed to investigate the in vitro biological potential of Bursera simaruba, employ green synthesis approaches for nanoparticle fabrication using its extract, characterize the synthesized nanomaterials using advanced analytical techniques, and utilize molecular docking tools to predict plausible medicinal targets. By integrating phytochemistry, nanotechnology, experimental pharmacology, and computational modelling, this research seeks to provide a multidimensional understanding of the therapeutic potential of Gumbo Limbo and contribute to the expanding field of plant-based nanomedicine and rational drug discovery.

MATERIALS AND METHODS

Plant Identification Authentication

Fresh leaves of Bursera simaruba (commonly known as Gumbo limbo) were harvested on March 10, 2025, from Ado-Ekiti in Ekiti State, Nigeria. Botanical identification and authentication were carried out at the herbarium unit of the Department of Plant Science, Faculty of Science, Ekiti State University, Ado-Ekiti. Voucher specimens were prepared and deposited with assigned accession numbers for reference. The plant was further verified by Mr. Omotayo, the Chief Technologist at the facility. Subsequent procedures involving sample preparation, biochemical and antimicrobial evaluation, nanoparticle characterization, and molecular docking for therapeutic target prediction were carefully designed to preserve the integrity and bioactivity of the aqueous leaf extract.

Preparation and Extraction of Plant Material

Fresh leaves of Bursera simaruba were carefully harvested and rinsed thoroughly under running tap water to remove adhering dirt, debris, and potential microbial contaminants. The washed leaves were then spread out and allowed to dry in the shade for several days. This approach was deliberately chosen to preserve heat- and light-sensitive phytochemicals, particularly flavonoids and polyphenols, which are prone to degradation when exposed to direct sunlight. Once completely dried, the leaves were ground into a fine powder using an appropriate milling device.

To enable effective extraction of bioactive components, a weighed quantity (20 g) of the powdered material was submerged in 100 mL of distilled water and permitted to stand overnight with sporadic stirring. Whatman filter paper was then used to filter the mixture, and the resulting clear filtrate was gathered and kept for use in green synthesis, phytochemical analysis, and antioxidant assessment.

Phytochemical Screening (Fresh Extract)

The aqueous extract was subjected to qualitative phytochemical investigations to identify major secondary metabolites, including flavonoids, alkaloids, saponins, phenolics, and steroidal compounds. These analyses were performed using established standard procedures as outlined by Ogbuagu et al. [6].

Determination of DPPH Free Radical Scavenging Activity

Using a slightly modified procedure outlined by Airaodion et al. [7], the DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging experiment was used to evaluate the extract’s antioxidant activity. This process involved transferring 1.0 mL of the extract into different test tubes at concentrations of 20, 40, and 80 mg/mL. Each tube received an equivalent volume (1.0 mL) of 0.1 mM methanolic DPPH solution. To ensure that the extract and the DPPH radicals had enough time to interact, the reaction mixtures were thoroughly mixed and allowed to sit at room temperature in the dark for half an hour.

A spectrophotometer was used to assess each sample’s absorbance at 516 nm after incubation. The extract’s capacity to scavenge radicals was demonstrated by a drop in absorbance when compared to the control. The extract was substituted with distilled water for the control. Next, the conventional formula was used to determine the % inhibition of DPPH radicals:

% DPPH radical scavenging ability    =

Determination of Ferric Reducing Antioxidant Power

A slightly modified version of the technique described by Airaodion et al. [8] was used to evaluate the extract’s ferric-reducing ability. This technique is predicated on the idea that when excess ferricyanide is present, antioxidants can convert ferric ions (Fe³⁺) to ferrous ions (Fe²⁺). Briefly, 1.0 mL of 200 mM sodium phosphate buffer (pH 6.6) and 1.0 mL of 1% potassium ferricyanide solution were combined with different quantities of the methanolic extract (10–50 μg/mL). To enable the process, the resultant mixtures were incubated for 20 minutes at 50 °C. Following incubation, the solutions were cooled before 1.0 mL of 10% trichloroacetic acid (TCA) was added. After that, the samples were centrifuged for ten minutes at 2000 rpm to produce a clear supernatant.

After that, 1.0 mL of the supernatant was moved and mixed with 0.25 mL of a 0.1% ferric chloride solution and 1.0 mL of distilled water. The control sample had all of the ingredients except potassium ferricyanide, and the blank was distilled water. A spectrophotometer was used to test each sample’s absorbance at 700 nm. Higher ferric-reducing antioxidant activity in this experiment is indicated by lower absorbance readings. The following formula was then used to get the % ferric-reducing antioxidant power (FRAP):

FRAP (%) = x 100

Determination of Total Phenolic Content

According to Airaodion and Onabanjo [9], the Folin-Ciocalteu method was used to measure the total phenolic content. This process involved reacting 0.2 mL of the extract with 2.0 mL of a 7.5% sodium carbonate solution and 1.5 mL of 10% Folin-Ciocalteu reagent. To enable colour development, the mixture was incubated for 40 minutes at 45°C. Next, absorbance was measured at 700 nm.

Milligrams of gallic acid equivalents per gram of dry extract (mg GAE/g) were used to express the phenolic content. A gallic acid calibration curve represented by the following equation was used to accomplish quantification:

Y = 0.005x + 0.464 (R² = 0.961).

Determination of Total Flavonoid Content

A colorimetric technique outlined by Esonu et al. [10] was used to determine the total flavonoid content. First, 0.2 mL of the extract and 0.3 mL of 5% sodium nitrite were combined. 0.6 mL of 10% aluminium chloride was added after five minutes. After six minutes, 2.1 mL of distilled water and 2.0 mL of 1 M sodium hydroxide were added to the mixture. After fully mixing the solution, absorbance was measured at 510 nm in comparison to a reagent blank.

Gallic acid equivalents (mg GAE/g of dry extract) were used to express the flavonoid content. Under the same circumstances, a standard gallic acid solution (0.5 mg/mL in methanol) was utilised for calibration. Every analysis was carried out twice. The standard curve equation used was:
Y = 0.005x + 0.464 (R² = 0.961).

Estimation of Vitamin C

The determination of ascorbic acid was carried out following the method outlined by Airaodion et al. [11]. Briefly, 0.5 mL and 1.0 mL portions of the filtered sample were transferred into separate volumetric flasks. Bromine water was then carefully added dropwise until a stable coloration persisted, indicating that ascorbic acid had been fully oxidized to dehydroascorbic acid. Any excess bromine present was subsequently neutralized with a few drops of 10% thiourea solution, yielding a clear mixture.

Following this, 0.5 mL of 2,4-dinitrophenylhydrazine (DNPH) reagent (prepared as 2% in 9N H₂SO₄) was introduced and mixed thoroughly. The final volume of the reaction mixture was adjusted to 2 mL using 10% acetic acid. Standard solutions of ascorbic acid (2 mg/mL), ranging from 0.2 to 1.0 mL, were processed under identical conditions. Absorbance readings were taken at 520 nm, and the vitamin C concentration was determined using a calibration curve generated through linear regression. The results were expressed in milligrams per gram (mg/g) of the sample.

Green Synthesis of Nanoparticles

Nanoparticles were synthesized using the aqueous extract of Bursera simaruba in accordance with the procedure described by Adewumi et al. [12].

Antimicrobial Analysis

The antimicrobial properties of the extract were assessed against selected pathogenic microorganisms using standard laboratory techniques. The agar well diffusion method was applied for bacterial isolates, while antifungal activity was evaluated using the mycelial growth inhibition assay, as previously described [13]. The test organisms included Escherichia coli, Streptococcus mutans, Salmonella typhi, Staphylococcus aureus, Klebsiella pneumoniae, Macrophomina phaseolina, and Alternaria alternata.

RESULTS

The proximate composition of Bursera simaruba leaf revealed that carbohydrates constituted the highest proportion (33.65 ± 0.07%), followed by crude protein (17.11 ± 0.01%), ash (17.07 ± 0.10%), crude fat (15.08 ± 0.11%), crude fibre (10.82 ± 0.10%), and moisture content (6.27 ± 0.05%) as presented in Table 1. Antinutrient analysis showed relatively low oxalate (1.61 ± 0.02 mg/100 g) and tannin (4.14 ± 0.01 mg/100 g) levels, moderate phytate content (13.14 ± 0.06 mg/100 g), and a trypsin inhibitor value of 42.00 ± 1.14% (Table 2).

Mineral profiling indicated potassium (5.25 ± 0.01 mg/100 g) as the most abundant macroelement, followed by calcium (3.91 ± 0.01 mg/100 g), sodium (2.85 ± 0.01 mg/100 g), and magnesium (1.99 ± 0.01 mg/100 g). Trace elements detected included iron (2.19 ± 0.00 ppm), copper (0.49 ± 0.01 ppm), manganese (0.43 ± 0.00 ppm), chromium (0.11 ± 0.00 ppm), and lead (0.10 ± 0.00 ppm) (Table 3).

Qualitative phytochemical screening showed strong presence of saponins (++++) and mild presence of phenolics (+) and alkaloids (+), while anthraquinones, terpenoids, Keller-Killani, and Liebermann’s tests were negative (Table 4). Quantitative antioxidant constituents revealed high flavonoid content (82.86 ± 0.02 mg AAE/g), phenolic content (57.00 ± 0.03 mg GAE/g), and vitamin C (1.97 ± 0.02 mg/g) (Table 5), with calibration curves illustrated in Figures 2, 3, and 4. In vitro antioxidant assays demonstrated nitric oxide scavenging activity as the highest (75.00 ± 0.02%), followed by FRAP (66.01 ± 0.01%), DPPH (57.00 ± 0.02%), and hydrogen peroxide scavenging (40.00 ± 0.03%) (Table 6).

Antibacterial evaluation showed that the crude extract produced inhibition zones ranging from 2.00–4.00 mm, while silver (AgNP), zinc (ZnNP), and copper (CuNP) nanoparticles exhibited enhanced activity. CuNP showed the highest inhibition against Streptococcus mutans (8.50 mm) and Salmonella typhi (8.00 mm), while AgNP recorded 8.00 mm against both Staphylococcus aureus and Klebsiella pneumoniae (Table 7).

Antifungal activity demonstrated moderate inhibition by the crude extract (60.37% against Microphomina phaseolina and 56.32% against Alternaria alternata), while nanoparticle formulations showed higher mycelial growth inhibition, particularly AgNP (72.63%) and ZnNP (71.58%) against M. phaseolina (Table 8). Nanoparticle synthesis and characterization were confirmed by UV–Visible spectroscopy (Figure 1) and FTIR analyses of the extract and synthesized nanoparticles (Figures 5–8).

Ligand identification of major phytochemicals is presented in Table 9, while predicted ADME properties are summarized in Table 10, showing generally high gastrointestinal absorption for most compounds. Molecular docking analysis against DPP4 revealed binding affinities ranging from −4.3 to −6.8 kcal/mol, with 4-dimethylamino-3,5-dinitrobenzoic acid (−6.8 kcal/mol), benzenepropanoic acid derivative (−6.4 kcal/mol), and squalene (−6.2 kcal/mol) showing the strongest interactions (Table 11). Predicted molecular targets of selected phytochemicals are presented in Table 12.

Target gene network analyses, including transcription factor enrichment (Figure 9a), protein–protein interaction expansion (Figure 9b), kinase enrichment analysis (Figure 9c), and expression-to-kinase network (Figure 9d), further illustrated the molecular interaction landscape. Detailed 2D binding interactions of selected ligands with DPP4 are shown in Figures 10a–10e.

Discussion

Bursera simaruba (commonly known as gumbo limbo) has long been recognized in traditional medicine for treating inflammatory and metabolic conditions. Despite its widespread use, there is still a gap in comprehensive scientific studies that combine phytochemical characterization, eco-friendly nanoparticle synthesis, antimicrobial assessment, and computational prediction of molecular targets. In this study, we examined the proximate and phytochemical constituents of Bursera simaruba leaves, assessed their antioxidant and antimicrobial activities, characterized metallic nanoparticles synthesized through green methods, and explored potential therapeutic targets using molecular docking and in silico approaches.

The proximate composition showed that carbohydrates (33.65%) constituted the highest fraction of the leaf, followed by crude protein (17.11%), ash (17.07%), crude fat (15.08%), crude fibre (10.82%), and low moisture content (6.27%). The relatively low moisture level enhances shelf stability and reduces susceptibility to microbial spoilage, supporting its suitability for pharmaceutical preparation. The high ash value reflects a considerable mineral reservoir, corroborating the mineral profile observed in this study.

Comparable studies on medicinal plants have reported similar macronutrient distributions. For instance, nutritional profiling of tropical medicinal leaves demonstrated carbohydrate ranges of 30–45% and protein values between 12–20%, supporting the classification of B. simaruba as nutritionally valuable [14]. The relatively high crude fat content observed in this study is consistent with the detection of key fatty acids, including oleic acid, linoleic acid, and palmitic acid. These fatty acids are well recognized for their roles in modulating inflammatory responses and supporting metabolic regulation [15].

The levels of oxalate (1.61 mg/100 g), phytate (13.14 mg/100 g), and tannin (4.14 mg/100 g) were found to be low to moderate, remaining within acceptable dietary thresholds. Although phytates and tannins may reduce mineral bioavailability, they also exert antioxidant and antimicrobial effects through metal chelation and free radical scavenging. Recent evidence indicates that moderate phytate intake is associated with protective cardiovascular and anticancer effects due to its antioxidant activity [16].

The trypsin inhibitor activity (42%) suggests the presence of protease inhibitory compounds. While high levels may reduce protein digestibility, plant-derived protease inhibitors have been increasingly recognized for their anticancer and anti-inflammatory potential [17]. Therefore, within a pharmacological context, these compounds may contribute positively to bioactivity.

The mineral profile demonstrated the presence of potassium, calcium, magnesium, sodium, iron, copper, manganese, and chromium, with minimal lead contamination. Potassium dominance supports electrolyte balance and cardiovascular regulation, while calcium and magnesium contribute to neuromuscular function and metabolic homeostasis.

Trace elements such as iron and copper play central roles in antioxidant enzyme systems, including catalase and superoxide dismutase. The detection of chromium, known to influence insulin sensitivity, further supports the plant’s potential relevance in metabolic disorders. Similar mineral compositions have been reported in other medicinal plants used for glycemic control [18].

Qualitative screening revealed abundant saponins and detectable phenols and alkaloids. Quantitatively, flavonoid content (82.86 mg AAE/g) and phenolic content (57.00 mg GAE/g) were high, accompanied by measurable vitamin C (1.97 mg/g). The strong antioxidant performance observed in DPPH (57%), nitric oxide (75%), FRAP (66%), and H₂O₂ (40%) assays confirms the extract’s robust redox-modulating capacity.

Phenolics and flavonoids are widely reported as major contributors to antioxidant activity in medicinal plants [19,20]. The strong nitric oxide scavenging effect observed in this study is particularly relevant, as excessive NO production is implicated in inflammatory and metabolic diseases. Comparable antioxidant capacities have been reported for other Bursera species, which were attributed to high polyphenolic content [21].

The successful synthesis of AgNPs, ZnONPs, and CuNPs was confirmed by characteristic UV-Visible surface plasmon resonance peaks and FTIR-identified functional groups (–OH, –C=O, –NH). These findings demonstrate that phenolics and flavonoids served as reducing and capping agents.

Green synthesis using plant extracts is widely recognized for producing stable, biocompatible nanoparticles with enhanced antimicrobial properties [12]. The FTIR peaks observed in this study align with previously reported functional groups involved in metal ion reduction during phytogenic nanoparticle synthesis.

The crude extract exhibited mild antibacterial activity, whereas nanoparticle formulations significantly enhanced inhibition zones. AgNPs showed strong activity against Staphylococcus aureus and Klebsiella pneumoniae, while CuNPs demonstrated superior inhibition against Streptococcus mutans and Salmonella typhi. Similarly, ZnNPs and AgNPs showed enhanced antifungal activity against Microphomina phaseolina and Alternaria alternata. These findings are consistent with reports that silver and copper nanoparticles disrupt microbial membranes, generate reactive oxygen species, and interfere with intracellular proteins [22]. The enhanced antimicrobial efficacy compared to crude extract suggests synergistic interactions between metal ions and phytochemicals.

The ADME analysis demonstrated favourable pharmacokinetic profiles for several phytochemicals, including high gastrointestinal absorption and acceptable bioavailability scores (0.55–0.85). Compounds such as 1,2-benzenediol and benzaldehyde derivatives were predicted to be highly soluble and BBB-permeant, suggesting central nervous system accessibility. Lipophilic compounds such as squalene and fatty acids exhibited moderate solubility but acceptable permeability, consistent with the Lipinski rule of five for drug-likeness [23].

Docking analysis against DPP4 revealed strong binding affinities for 4-dimethylamino-3,5-dinitrobenzoic acid (−6.8 kcal/mol), benzenepropanoic acid derivative (−6.4 kcal/mol), and squalene (−6.2 kcal/mol). These values are within ranges reported for natural DPP4 inhibitors.Fatty acids such as oleic acid and palmitic acid demonstrated stable hydrophobic interactions within the DPP4 active site, consistent with previous reports indicating that long-chain fatty acids can modulate enzymatic targets via hydrophobic binding [22].

Target prediction analysis revealed interactions with CA5B, HSD17B1/2, SQLE, FABP3, LPAR4, and LSS, implicating pathways involved in glucose metabolism, steroid regulation, lipid biosynthesis, and signal transduction. Network analyses (TFEA, KEA, PPI expansion) further indicated multi-target activity, consistent with polypharmacology principles in phytomedicine [24].

Conclusion

Bursera simaruba demonstrates significant antioxidant, antimicrobial, and nanoparticle-mediated bioactivities. Molecular docking and target prediction support its potential antidiabetic mechanism through DPP4 inhibition and metabolic pathway modulation. These findings provide mechanistic insight into its traditional medicinal use and justify further pharmacological and clinical investigations.

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