https://orcid.org/0009-0004-5911-5416
Figures
Abstract
Natural resources are vital for identifying novel treatments for noncommunicable diseases and multidrug-resistant pathogens. Lagerstroemia speciosa (L. speciosa) Linn. is traditionally used to manage conditions like diabetes, cancer, and oxidative stress-related diseases. Current studies frequently lack comprehensive bioactivity assessments coupled with molecular simulation analyses of L. speciosa leaf extracts. We aimed to assess the phytochemical profiling, bioactivity, in silico pharmacological properties, and molecular interactions of L. speciosa leaf extracts prepared using organic solvents. Methanol (5.1) and ethanol (4.3) extracts yielded the highest concentrations of total phenolics, flavonoids, and proanthocyanidins due to their high polarity indices. Antioxidant activities were robustly evaluated using DPPH, ABTS, superoxide, and nitric oxide assays, with the ethanol extract demonstrating significant free radical scavenging (IC50 = 75.53 µg/mL for DPPH). The methanol extract exhibited broad-spectrum antibacterial activity against multidrug-resistant strains, including Escherichia coli and Staphylococcus aureus, alongside moderate antifungal activity against Candida albicans and Saccharomyces cerevisiae. The brine shrimp lethality assay revealed moderate cytotoxicity (LC50 = 601.8 µg/mL), suggesting potential anticancer properties likely mediated by bioactive compounds such as ellagic acid and gallic acid. Methanol and ethanol extracts of L. speciosa significantly inhibited α-amylase and α-glucosidase compared to standard acarbose, indicating substantial antidiabetic potential by delaying carbohydrate digestion and reducing postprandial glucose levels. Complementary molecular docking and ADME pharmacokinetic studies provided in silico support for the observed antioxidant, antimicrobial, antidiabetic, and chemopreventive activities. The compelling evidence collectively suggests that L. speciosa extracts are a promising source of bioactive compounds.
Citation: Haque MS, Ahmad R, Sarkar BC, Sana M, Saha HR, Sana NK (2025) Integrating bioactivity and molecular simulations to explore the pharmacological landscape of Lagerstroemia speciosa leaf extract. PLoS One 20(12): e0339566. https://doi.org/10.1371/journal.pone.0339566
Editor: Jorddy Neves Cruz, Universidade Federal do Para, BRAZIL
Received: December 2, 2024; Accepted: November 25, 2025; Published: December 29, 2025
Copyright: © 2025 Haque et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript and its Supporting Information files.
Funding: The author(s) received no specific funding for this work.
Competing interests: The authors have declared that no competing interests exist.
Abbreviations: L. speciosa, Lagerstroemia speciosa; GPx, Glutathione Peroxidase; TPC, Total Phenolic Content; GAE, Gallic Acid Equivalents; TFC, Total Flavonoid Content; CE, Catechin Equivalents; QE, Quercetin Equivalent; CFU, Colony Forming Units; SDA, Sabouraud Dextrose Agar; RNS, Reactive Nitrogen Species; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; SRB, Sulforhodamine B; FCR, Folin Ciocalteu reagent; NBT, Nitro Blue Tetrazolium; DPPH, 2,2-Diphenyl-1-picrylhydrazyl; ABTS, 2,2′-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid); DNSA 3, 5-Dinitrosalicylic Acid; C50, Half Maximal Inhibitory Concentration; BSLA, Brine Shrimp Lethality Assay; IZ, Inhibition Zone; MIC, Minimum Inhibitory Concentration; MBC, Minimum Bactericidal Concentration; MFC, Minimum Fungicidal Concentration; LC50, Lethal Concentration for 50% of the population; R2, Coefficient of Determination; NO, Nitric Oxide; SOD, Superoxide Dismutase; PBS, Phosphate Buffered Saline; DMSO, Dimethyl Sulfoxide; ANOVA, Analysis of Variance; SD, Standard Deviation; PPAR-γ, Peroxisome proliferator-activated receptor gamma; Pa, Probable activity; Pi, Probable inactivity; PASS, Prediction of Activity Spectra for Substances; LogP, Lipophilicity; SMILES, Simplified Molecular Input Line Entry System; ADME, Absorption, Distribution, Metabolism and Excretion; TPSA, Topological Polar Surface Area; AC, Attracting Cavities; AdmetSAR, Absorption, Distribution, Metabolism, Excretion, and Toxicity Structure-Activity Relationship.
Introduction
Noncommunicable diseases are increasingly becoming major global health concerns, with over two billion people affected by conditions linked to oxidative stress [1–3]. Oxidative stress can be mitigated by bioactive compounds with antioxidant properties present in the body [4]. These compounds enhance the production of endogenous antioxidants, including SOD (superoxide dismutase), GPx (glutathione peroxidase), and catalase. These enzymes are essential for protecting the vascular endothelium against free radical-induced damage [5]. Antioxidants are necessary compounds that play a vital role in maintaining human health [6]. The plant kingdom represents a rich source of potential antioxidants for combating oxidative stress-related diseases [7]. Antimicrobial resistance is approaching critical levels, with global deaths projected to reach ten million by the 2030s [8,9]. This evolving health crisis emphasizes the urgent need for novel therapeutic agents derived from natural sources [10,11]. Natural products are inherently more complex, functioning as multitarget molecules. This complexity often leads to better therapeutic outcomes and fewer side effects compared to synthetic molecules. Synthetic molecules typically target a single receptor site [12,13]. Reactive oxygen species (ROS) are continuously generated in human cells. These include superoxide radicals, hydroxyl radicals, singlet oxygen, hydrogen peroxide, and nitric oxide [14]. These reactive entities damage cellular components such as membrane lipids, proteins, enzymes, and DNA [15]. As a result, natural products have gained significant scientific interest for their antioxidant and chemo-preventive properties [16].
L. speciosa Linn. is commonly known as ‘Banaba’ in the Tagalog dialect of the Philippines [17]. In Bangladesh, it is locally referred to as ‘Jarul’. This tropical flowering tree belongs to the family Lythraceae. It is a medium-to-large deciduous tree distinguished by its rounded crown. The species is indigenous to Southeast Asia, with its native range encompassing Bangladesh, India, Indonesia, the Philippines, and Malaysia [18]. The plant has been widely used in traditional medicine across these regions to treat various ailments [19]. Banaba has been used as a traditional diabetes remedy in the Philippines since ancient times [20]. In Malaysian traditional medicine, the bark of L. speciosa has been used as a treatment for dysentery and diarrhea [21]. The study on Banaba indicated that pure corosolic acid significantly reduced blood glucose levels in humans [22,23]. It exhibits diverse pharmacological effects, including anti-inflammatory, analgesic, hepatoprotective, nephroprotective, anti-gout and alpha-amylase inhibitory properties [24–27]. These findings highlight its potential as a versatile therapeutic agent.
Previous studies have demonstrated the antioxidant capabilities and α-glucosidase inhibitory activity of its leaf extracts [28]. However, comprehensive investigations using diverse assays and exploring multiple mechanistic pathways are still lacking. The effects of L. speciosa on other metabolically critical enzymes, such as α-amylase, lipase, and tyrosinase, have not been systematically investigated [28]. This limitation hinders the comprehensive utilization of its therapeutic potential. Toxicological evaluations have largely been confined to acute toxicity assessments [29,30]. Chronic toxicity, genotoxicity, and organ-specific toxicity profiles remain underexplored. Moreover, safety parameters for various extraction methods and their associated bioactive compounds are to be clearly defined yet [19]. Current literature notably lacks robust structure-activity relationship studies and comprehensive molecular simulations. These relationships are essential to elucidate the connection between phytochemicals and their biological functions. Although numerous bioactive compounds have been identified, their synergistic interactions are not fully understood yet. Addressing these critical gaps is essential to unlock the full medicinal potential of L. speciosa.
This study systematically evaluates the bioactive potential of L. speciosa leaf extracts obtained through sequential solvent extraction using methanol, ethanol, acetone, and n-hexane. A multiparametric assessment of antioxidant, antimicrobial, cytotoxic and enzymatic inhibition properties was conducted to determine the extracts’ safety profile and therapeutic index for drug development potential. We further employed comprehensive phytochemical profiling, pharmacokinetics, and molecular docking to quantify and characterize the bioactive compounds present in L. speciosa leaf extracts. Our research represents a major advancement in the field of phytochemistry by establishing structure-activity connections that bridge the gap between classical ethnopharmacology and contemporary drug discovery.
Materials and methods
Chemicals and reagents
All chemicals used were of analytical grade. Folin-Ciocalteu reagent (FCR), 2,2-diphenyl-1-picryl-hydrazyl (DPPH), ABTS [2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt], 3,5-dinitrosalicylic acid (DNSA), Dimethyl sulfoxide (DMSO), Griess reagent (1% sulfanilamide, 2% H3PO4, and 0.1% naphthyl ethylenediamine dihydrochloride), PBS, NaCl, methanol, and catechin (CA) were purchased from Sigma Aldrich, MO, USA. Other reagents for the extraction, such as n-hexane, acetone, ethanol, peptone, agar, nystatin, α-glucosidase, α-amylase, vanillin, and ampicillin, were purchased from Wako Pure Chemical Industries Ltd., Osaka, Japan, while sodium nitroprusside, HCl, sodium acetate, and gallic acid were obtained from Tokyo Chemical Industry Co., Ltd., Tokyo, Japan. Aluminum chloride (AlCl3), sodium carbonate, Nitro blue tetrazolium (NBT), sodium carbonate, trypan blue dye, and other remaining analytical-grade chemicals were purchased from Merck Chemical Company, Darmstadt, Germany.
Plant material
The leaves of L. speciosa used in the present study were sourced from the local gardens of Rajshahi district (24° 22’ 23.911’‘ N and 88° 36’ 17.538 E), Bangladesh, and were harvested in July. Taxonomic authentication of plants was done by Professor Dr. A.H.M. Mahbubur Rahman of the Department of Botany, University of Rajshahi, Bangladesh. A voucher specimen (Accession No. RHB-10) has also been deposited in the University of Rajshahi Herbarium (RUH) according to herbarium codes. Sample collection and preservation were done based on the institutional protocols and policies and national rules governing plant tissue collection.
Preparation of extracts
The collected leaves were washed with distilled water and air-dried at room temperature in a shaded area for two weeks. The samples were then dried, and the dried leaves were ground into a fine powder using a mechanical grinder and finally placed in a tight container. Around 500 g of dried leaf powder was placed in a clean, flat-bottomed glass container and immersed in 1000 mL of methanol for 15 days at room temperature. The resulting extract was filtered through Whatman No. 1 filter paper and concentrated using a rotary evaporator under reduced pressure at 40°C. We evaporated the total filtrate to dryness in a vacuum at 40°C to yield a brownish-red methanolic extract (350 g). The last extract was transferred to a sealed glass vessel for performing the experiment. We also kept aside the 50 g crude methanolic extract of L. speciosa leaves for further experiments. In a similar manner, we distilled other solvents- ethanol, acetone, and n-hexane for extraction from the crude L. speciosa leaves for the experiments in this study.
Total phenolic content
The total phenolic content (TPC) of the L. speciosa leaf extracts was estimated using the Folin Ciocalteu reagent (FCR) assay [31]. When 5 mL of FCR was mixed with 1 mL of methanol containing gallic acid, the mixture was then incubated with L. speciosa for 5 mins at room temperature. After that, an equal volume of 1 mL of 7.5% sodium carbonate (Na2CO3) was also added into the mixture. To the above mixture, we added distilled water up to 10 mL and stirred it well. The optical density at 765 nm was measured in a UV-visible spectrophotometer (Shimadzu, Japan) after incubation at room temperature for 60 minutes, with methanol serving as the blank. Similarly, we followed the same protocol to measure the total phenolic content for the extracts obtained using other solvents. Total phenolics of the extract were determined using gallic acid as a standard reference. The values were expressed as milligrams of gallic acid equivalent (GAE) per gram of the dry extract. A calibration curve for gallic acid was constructed using the linear regression equation y = 0.004797*x + 0.04165, demonstrating a strong correlation with a coefficient of determination (R2) of 0.9648 (S1 Fig.).
Total flavonoid content
The total flavonoid content (TFC) of L. speciosa leaf extracts was measured using a colorimetric assay by the aluminum chloride (AlCl3) method [32]. A mixture was prepared by adding 1 mL of L. speciosa leaf extract, 5 mL of distilled water, and 0.3 mL of 5% NaNO2 in a test tube and allowed to stand for 5 minutes. Following the incubation period, we added 0.6 mL of 10% aluminum chloride, let it stand for 5 minutes, and then added 2 mL of 1M NaOH. At a wavelength of 510 nm, the absorbance of the reaction mixture was measured. A calibration curve for catechin was established using the linear regression equation y = 0.002137*x + 0.03542, which exhibited a strong linear relationship with a coefficient of determination (R2) of 0.9917. The flavonoid content was quantified based on the catechin standard and expressed as milligrams of catechin equivalent (CE) per gram of dry extract (S2 Fig.).
Total flavonol content
Determination of the total flavonol content was followed by the method described Kumaran et al. [33] with minor changes. Briefly, 2.0 mL of L. speciosa leaf extracts at a concentration of 0.1 mg/mL were reacted with 2.0 mL of aluminum chloride at a concentration of 2% w/v in ethanol and 3.0 mL of a sodium acetate solution of 50 g/L. The reaction mixture was then maintained at 20°C for 2.5 hours, after which absorbance was recorded on a UV-visible spectrophotometer (Shimadzu Corporation, Japan) at 440 nm. A calibration curve for quercetin was generated using the linear regression equation y = 0.002490*x + 0.04230, demonstrating a strong linear relationship with a coefficient of determination (R2) of 0.9935. The flavonol content was quantified using quercetin as a reference standard and expressed as milligrams of quercetin equivalent (QE) per gram of dry extract (S3 Fig.).
Total proanthocyanidin content
Total proanthocyanidin content was determined after quantitating using the vanillin-HCl method according to Sun et al. [34] with slight modifications. The reaction mixture for the extract solution consisting of 1.5 mL of extract solution (0.1 mg/mL of methanol) diluted to 3 mL with vanillin reagent (4% w/v in methanol) was first diluted with 1.5 mL of concentrated hydrochloric acid (37% v/v). The chromophores were allowed to develop in the reaction mixture at room temperature for 15 minutes. Absorbance at 500 nm was read on a UV visible spectrophotometer (Shimadzu, Japan). The proanthocyanidin content was determined and expressed as milligrams of catechin equivalents (CE) per gram of dry extract. A standard calibration curve, generated from a series of catechin concentrations, was established using the linear regression equation y = 0.002128*x + 0.01226, demonstrating a robust linear relationship with a coefficient of determination (R2) of 0.9949 (S4 Fig).
DPPH free radical scavenging assay
The L. speciosa extracts were employed for their DPPH (2,2-diphenyl-1-picryl-hydrazyl) radical scavenging activity following the methodology of Brand-Williams et al. [35]. We prepared concentrations of 0.5 mg/mL by dissolving 5 mg of L. speciosa leaf extracts- methanol, ethanol, acetone, and n-hexane in 10 mL of their respective solvents. Ascorbic acid was used as a positive control. The reaction mixtures were vortexed and incubated in the dark for 30 minutes. Various concentrations of the extracts (20, 40, 80, 160, 320, and 500 μg/mL) were used to monitor the transition of oxidized DPPH (initially violet) to reduced DPPH (final product, yellow). Absorbance readings were recorded at 517 nm. The percentage of scavenging activity was calculated using the following formula:
The percentage of scavenging activities was plotted against the corresponding concentrations to determine the IC50 value using a nonlinear regression model with four parameters.
ABTS free radical scavenging assay
The antioxidant activity of the L. speciosa leaf extracts was determined using the ABTS radical cation decolorization assay [36]. The ABTS•+ radical cation was prepared by combining 7 mM ABTS with 2.45 mM potassium persulfate in a glass tube. The mixture was left to stand at room temperature in the dark for 12–16 hours to allow the accumulation of the ABTS•+ radical cation. ABTS•+ exhibits a stable blue-green fluorescence, which diminishes upon reaction with hydrogen-donating antioxidants. For the assay, 1 mL of L. speciosa extracts at concentrations of 20, 40, 80, 100, and 150 μg/mL was mixed with 3 mL of ABTS•+ solution. The absorbance of the reaction mixture was measured at 734 nm to monitor the reduction in color intensity. Ascorbic acid was used as a positive control. A dose-response nonlinear regression curve was generated by plotting the percentage of scavenging activity against the extract concentrations to determine the IC50 value.
Superoxide radical scavenging assay
The superoxide radical scavenging activity of L. speciosa was determined by the riboflavin-light-NBT (nitro blue tetrazolium) assay [37]. This assay measures the ability of superoxide dismutase (SOD) to neutralize superoxide radicals generated during the reaction of riboflavin with nitro blue tetrazolium (NBT) under light, leading to formazan formation [38]. The reaction mixture contained 50 mM phosphate buffer (pH 7.6), 20 μg riboflavin, 12 mM EDTA, and 0.1 mg NBT in a total volume of 3 mL. The reaction was initiated by exposing the mixture to a fluorescent lamp for 90 seconds, both with and without the addition of L. speciosa extracts at concentrations of 20, 40, 80, 160, and 300 μg/mL. The reduction in formazan formation was quantified by measuring the absorbance of the solution at 590 nm. Ascorbic acid was used as a positive control. The percentage of superoxide scavenging activity was calculated as follows:
where A0 is the absorbance of the distilled water and A1 is an absorbance in the presence of the extract. A dose-response curve was generated using nonlinear regression, plotting scavenging activity percentages relative to extract concentrations. The IC50 value was derived from this curve.
Nitric oxide free radical scavenging assay
The nitric oxide (NO) free radical of L. speciosa leaf extracts was determined by the modified Sreejayan et al. method [39]. Under anoxic conditions, sodium nitroprusside generates nitric oxide (NO), which reacts with oxygen to form nitrite ions. These ions are detected using the Griess reagent, where lower NO production indicates stronger scavenging activity by the extracts. 2 μL of sodium nitroprusside standard solution (5 mM) was aliquoted into tubes containing phosphate-buffered saline (PBS). L. speciosa leaf extracts (methanol, ethanol, acetone, and n-hexane) were added at concentrations of 20, 40, 80, 160, and 300 μg/mL. The mixture was incubated for 2 hours at room temperature (25°C). After incubation, the Griess reagent (1% sulfanilamide, 2% phosphoric acid, and 0.1% naphthyl ethylenediamine dihydrochloride) was added to the reaction mixture. This facilitated the formation of a chromophore through nitrite-sulfanilamide diazotization. The optical density of each sample was measured at 546 nm. Ascorbic acid was used as a positive control. The nitric oxide scavenging activity was calculated using the following formula:
where A0 is the absorbance of the control and A1 is the absorbance of the sample extract. A nonlinear regression dose-response curve was plotted and IC50 value was determined from this curve.
Brine shrimp lethality assay
L. speciosa leaf extract cytotoxicity was determined using the brine shrimp lethality assay [40]. Brine shrimp eggs were hatched in artificial seawater (3.8% NaCl solution) in a divided hatching aquarium tank (pH 7). A light source was positioned near the eggs to attract the hatched nauplii. After 48 hours of incubation, brine shrimp nauplii (Artemia salina) were collected for the experiment. To ensure the reliability of the test results, both experimental and control groups were established. 2 mg of L. speciosa methanol leaf extract was dissolved in 100 µL of dimethyl sulfoxide (DMSO) to prepare a stock solution of 20 mg/mL. The extract was diluted in artificial seawater to achieve concentrations of 25, 50, 100, 200, 400, and 800 μg/mL. Twenty nauplii were placed in each experimental bottle containing the prepared concentrations. The nauplii were incubated at room temperature for 24 hours. After incubation, the surviving nauplii were counted using a hand lens, and the percentage of mortality at each concentration was calculated. The LC50 value was determined by plotting the post-treatment concentration against mortality and performing a probit nonlinear regression analysis.
Antibacterial activity
An antimicrobial activity of L. speciosa methanol extract was carried out using the agar disk diffusion method [41]. A stock solution was prepared by dissolving 0.1 g of the methanol extract in 100 mL of methanol, resulting in a final concentration of 100 mg/mL. This stock solution was further diluted to 1 mg/mL to serve as the working solution. Specimens were pre-moistened by applying 20 µL of each dilution, with 5 µL carefully applied to both sides of the disks. The disks were allowed to dry between applications to ensure homogeneous impregnation. Methanol and water-soaked disks were used as negative controls. Bacterial strains were used in the tests: Escherichia coli (ATCC 25922), Salmonella enterica (ATCC 14028), Bacillus subtilis (ATCC 6633), Listeria monocytogenes (ATCC 19115), and Staphylococcus aureus (ATCC 25923). These strains were obtained from the Institute of Nutrition and Food Science, University of Dhaka, Bangladesh. The strains were cultured on nutrient agar and stored at 4°C. Before the experiment, nutrient agar plates were inoculated with a bacterial suspension at a concentration of approximately 1 × 108 CFU/mL. Small (6 mm in diameter) wells were made aseptically in the agar using a sterile cork borer, and disks were placed on the bacterial lawn. The plates were incubated at 37°C for 24 hours, and the antibacterial activity was determined from the inhibition zone (IZ) around the disks. To further characterize the antibacterial activity, the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined. Antibacterial activity was measured in millimeters (mm), with ampicillin disks serving as positive controls for all tested bacterial strains.
Antifungal activity
The antifungal effectiveness of L. speciosa methanol leaf extract was tested using a well-diffusion technique [42]. The fungal strains selected for the study included Aspergillus niger (ATCC 16404), Saccharomyces cerevisiae (ATCC 9763), Candida albicans (ATCC 10231), and Cladosporium sp. (ATCC 16022). These strains were preserved on appropriate agar media to support their growth. Sabouraud dextrose agar (SDA) plates were prepared and streaked with a fungal suspension at a concentration of approximately 1 × 106 colony-forming units per milliliter (CFU/mL). Wells (6 mm in diameter) were made using a sterile cork borer in the agar. A stock solution of the methanol extract of L. speciosa was prepared at 100 mg/mL and diluted to a working concentration of 1 mg/mL. Each well was loaded with the extract solution, and the plates were incubated at 28°C for 48 hours. After incubation, the antifungal activity was assessed by measuring the diameter of the inhibition zones around the wells using a digital caliper. We also quantitatively assessed antifungal activity by determining both the minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) values. Nystatin disks were included in each plate as a positive control.
α-amylase inhibitory assay
Determination of α-amylase inhibitory activity of L. speciosa leaf extracts was carried out using the DNSA modified method [43]. Stock solutions with concentrations of 50, 100, 200, 400, and 500 µg/mL were prepared from the extracts using 10% DMSO. These solutions were mixed with a buffer solution consisting of Na2HPO4/NaH2PO4 (0.02 M) and NaCl (0.006 M) at pH 6.9. For the assay, 200 μL of α-amylase solution (2 units/mL) was combined with 200 μL of the extract and incubated at 30°C for 10 minutes. Subsequently, 200 μL of 1% starch solution was added, and the mixture was incubated for an additional 3 minutes. The reaction was terminated by adding 200 μL of DNSA reagent, followed by heating the solution to 85–90°C for 10 minutes. After cooling, the solution was diluted to 5 mL with distilled water, and the absorbance was measured at 540 nm. Acarbose was used as a positive control. The α-amylase inhibition (%) was calculated as:
where A0 is the absorbance of the control and A1 is the absorbance of the sample extracts. The IC50 value was determined by plotting inhibition percentages against concentrations using nonlinear regression analysis with four variables.
α-glucosidase inhibition assay
The inhibitory effect of L. speciosa leaf extracts on α-glucosidase was determined the following protocol of Kazeem et al. [44]. A substrate solution of p-NPG (p-nitrophenyl glucopyranoside) was prepared in 20 mM phosphate buffer (pH 6.9). For the assay, 100 μL of the extract was preincubated with 50 μL of the extract at varying concentrations for 10 minutes. Subsequently, 50 μL of p-NPG substrate was added to initiate the reaction. Next, 20 μL of phosphate buffer (pH 6.9) was introduced, and the mixture was incubated at 37°C for 20 minutes. The reaction was terminated by adding 2 mL of 0.1 M Na2CO3, which facilitated the release of yellow p-nitrophenol. The absorbance of the released p-nitrophenol was quantified spectrophotometrically at 405 nm. The α-glucosidase inhibitory activity was calculated as follows:
where A0 is the absorbance of the control and A1 is the absorbance of the sample extracts. The IC50 value was determined by plotting inhibition percentages against concentrations.
In silico pharmacological activity prediction
The biological activity profiles of major phytochemicals identified in L. speciosa were predicted using the Prediction of Activity Spectra for Substances (PASS) online platform (http://www.way2drug.com/PassOnline/). The analysis encompassed six principal compounds: corosolic acid, ellagic acid, quercetin, gallic acid, γ-sitosterol, and lagerstroemin. The PASS algorithm was employed to predict probable activity (Pa) and probable inactivity (Pi) for multiple therapeutic targets of interest, including anticancer, anti-inflammatory, antiseptic, free radical scavenging, lipid peroxidase inhibition, insulin promotion, and antioxidant properties.
In silico prediction of pharmacokinetic parameters using SwissADME
ADME (Absorption, Distribution, Metabolism and Excretion) and drug-like characteristics of eight major bioactive compounds identified in L. speciosa were predicted using the SwissADME web-based platform (http://www.swissadme.ch/). The selected compounds- corosolic acid, ellagic acid, quercetin, γ-sitosterol, gallic acid, ursolic acid, phytol, and campesterol were chosen based on their documented abundance in L. speciosa leaf extracts from previous phytochemical studies. Drug-likeness assessment was performed according to Lipinski’s rule of five criteria, which evaluate molecular weight, lipophilicity, hydrogen bond donors, hydrogen bond acceptors and topological polar surface area (TPSA). This analysis provided valuable predictions concerning oral bioavailability and therapeutic viability of the target phytochemicals.
In silico prediction of toxicological properties using AdmetSAR
The toxicological properties of major bioactive compounds identified in L. speciosa leaf extracts were evaluated using the AdmetSAR (Absorption, Distribution, Metabolism, Excretion, and Toxicity Structure-Activity Relationship) online prediction platform (https://lmmd.ecust.edu.cn/admetsar1/predict/). Six predominant compounds were selected for analysis: corosolic acid, ellagic acid, quercetin, γ-sitosterol, gallic acid, and phytol based on their reported abundance in L. speciosa extracts. Comprehensive toxicity profiling states multiple parameters, including AMES toxicity, carcinogenicity potential, honeybee toxicity, acute oral toxicity, fish toxicity, rat acute toxicity, and biodegradation properties.
In silico molecular docking analysis
Preparation of ligands.
Major bioactive compounds commonly found in L. speciosa leaf extracts were selected for ligand preparation. The SMILES (Simplified Molecular Input Line Entry System) strings of Corosolic acid, Ellagic acid, Gallic acid, Quercetin, Lagerstroemin, and γ-Sitosterol were retrieved from the PubChem database and used as input for web-based molecular modeling using SwissDock.
Preparation of receptors/proteins.
The three-dimensional crystallographic structures of selected target proteins were downloaded from the RCSB Protein Data Bank (PDB) in PDB format [45]. The chosen targets included α-glucosidase (PDB ID: 3WY1), DNA gyrase B (PDB ID: 6KZV), β-lactamase (PDB ID: 1XPB), peroxiredoxin 6 (PDB ID: 5B6M), and peroxisome proliferator-activated receptor gamma (PPAR-γ; PDB ID: 2P4Y). These proteins were selected based on their relevance to antidiabetic, antimicrobial, and antioxidant pharmacological activities. The prepared receptor structures were docked with selected phytocompounds using SwissDock to predict ligand-protein interactions and assess pharmacological potential.
Molecular docking with attracting cavities using SwissDock.
In silico molecular docking was carried out to predict ligand-receptor interactions and identify pharmacologically relevant binding cavities. The docking studies were performed using the SwissDock web server (http://www.swissdock.ch) with the “attracting cavities” docking mode to enable high-precision predictions. Both receptor and ligand structures were submitted to SwissDock for analysis. The resulting docked complexes were further visualized using PyMOL for structural analysis. Key parameters, including the Attracting Cavities (AC) score, binding free energy (SwissParam score), and root-mean-square deviation (RMSD), were analyzed to assess docking accuracy and complex stability.
Ethical clearance
The entire experimental design was carried out under the comprehensive ethical supervision of the Experimental Animal Committee at the Institute of Biological Sciences, University of Rajshahi. The study design was formally approved by the committee under approval number 225/322-IAMEBBC/IBSc.
Statistical analysis
Data are presented as mean ± standard deviation (SD) for statistical assessments in triplicate. IC50 and LC50 values were calculated from the GraphPad Prism 10.4.1 version (Boston, MA, USA). Molecular docking results were visualized and analyzed using PyMOL version 3.1.1 (Schrodinger, LLC). Ordinary one-way and two-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison tests were performed with significance set at *p < 0.05.
Results
Total phenolic and flavonoid content of L. speciosa leaf extracts
L. speciosa leaf extracts revealed a significant amount of phenolic and flavonoid phytochemicals. Methanol extracts exhibited the highest TPC at 48.7 ± 0.3 mg GAE/g, followed closely by ethanol extracts at 46.2 ± 0.2 mg GAE/g. Acetone and n-hexane extracts yielded significantly lower TPC values of 31.5 ± 0.4 mg GAE/g and 11.8 ± 0.3 mg GAE/g, respectively, highlighting the superior efficacy of polar solvents for phenolic extraction (Fig 1A). Ethanol extracts showed the highest TFC at 9.2 ± 0.6 mg CE/g, followed by methanol at 8.1 ± 0.7 mg CE/g, n-hexane at 7.5 ± 0.8 mg CE/g, and acetone at 5.6 ± 0.5 mg CE/g. We observed the lowest extraction efficiency in the acetone extract of L. speciosa leaves (Fig 1B).
(A) TPC was determined using the Folin-Ciocalteu assay and expressed as milligrams of gallic acid equivalent (GAE) per gram of dry extract. Methanol and ethanol extracts exhibited significantly higher TPC values compared to acetone and n-hexane. (B) TFC was quantified using a standard colorimetric method and expressed as milligrams of catechin equivalent (CE) per gram of dry extract. Ethanol and methanol extracts showed the highest TFC, followed by n-hexane and acetone. Data are presented as mean ± SD from three independent experiments. Statistical significance was determined using one-way ANOVA followed by Tukey’s post-hoc test. Significant differences between solvents are indicated by asterisks (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001) and “ns” denoting not significant.
Total flavonol and proanthocyanidin content of L. speciosa leaf extracts
The leaf extracts of L. speciosa contained substantial amounts of flavonols and proanthocyanidins. Ethanol extracts exhibited the highest flavonol content (8.4 ± 0.7 mg QE/g), followed by methanol (7.8 ± 0.6 mg QE/g), n-hexane (5.2 ± 0.5 mg QE/g), and acetone extracts demonstrated the lowest content (4.1 ± 0.4 mg QE/g).These results emphasize the solvent-dependent extraction efficiency for flavonol (Fig 2A). Quantitative analysis revealed substantial levels of total proanthocyanidins in L. speciosa leaf extracts. Among the tested solvents, ethanol demonstrated the highest proanthocyanidin content (34.8 ± 1.2 mg CE/g), indicating its superior ability to extract these bioactive compounds. Methanol extracts also showed a relatively high proanthocyanidin concentration (29.4 ± 0.8 mg CE/g), suggesting that both ethanol and methanol are effective solvents for polyphenolic extraction. In contrast, n-hexane (19.2 ± 0.5 mg CE/g) and acetone (14.8 ± 0.4 mg CE/g) exhibited lower proanthocyanidin levels, likely due to differences in solvent polarity and their ability to solubilize these compounds. These findings highlight the role of solvent selection in optimizing the extraction of proanthocyanidins from L. speciosa leaves, with ethanol emerging as the most efficient solvent in this study (Fig 2B).
(A) Total flavonol content was determined using aluminum chloride colorimetric method and expressed as milligrams of quercetin equivalent (QE) per gram of dry extract. Methanol and ethanol extracts exhibited significantly higher flavonol content than acetone and n-hexane. (B) Total proanthocyanidin content was quantified using the vanillin-HCl assay and expressed as milligrams of catechin equivalent (CE) per gram of dry extract. Ethanol extracts showed the highest proanthocyanidin content, followed by methanol, n-hexane, and acetone. Data are denoted as mean ± standard error (n = 3). Statistical significance was determined using one-way ANOVA followed by Tukey’s post-hoc test. Significant differences between solvents are indicated by asterisks (**p < 0.01, ***p < 0.001, ****p < 0.0001) and “ns” denoting not significant.
DPPH and ABST free radical scavenging assay of L. speciosa leaf extracts
The DPPH radical scavenging activity was evaluated using L. speciosa extracts at concentrations ranging from 0 to 500 µg/mL. The ethanol extract demonstrated the highest scavenging activity in the DPPH assay, with inhibition ranging from 15.31 ± 0.64% at 20 µg/mL to 85.4 ± 0.46% at 500 µg/mL compared with methanol, acetone, and n-hexane extracts. The IC50 values for the extracts were determined to be 90.39 µg/mL for methanol, 75.53 µg/mL for ethanol, 78.92 µg/mL for acetone, and 241.59 µg/mL for n-hexane. The standard antioxidant, ascorbic acid, demonstrated the highest potency (68.16 µg/mL) among all extracts of L. speciosa (Fig 3A). In the ABTS assay, the antioxidant potential was assessed using L. speciosa extracts at concentrations ranging from 0 to 150 µg/mL. The methanol extract exhibited significant concentration-dependent scavenging activity, reaching 83.45 ± 0.22% inhibition at 150 µg/mL, while the n-hexane extract showed 62.75 ± 0.21% inhibition at the same concentration. Ascorbic acid, the positive control, achieved the highest scavenging activity at 93.13 ± 0.68% (Fig 3B). The methanol extract of L. speciosa showed an IC50 value of 64.19 µg/mL, followed by the ethanol (70.99 µg/mL), acetone (85.26 µg/mL), and n-hexane (98.78 µg/mL) extracts. Ascorbic acid had a stronger activity with an IC50 of 44.41 µg/mL. These results indicate that the methanol and ethanol extracts possess substantial free radical scavenging activity, although slightly lower potency than ascorbic acid.
(A) DPPH radical scavenging activity was evaluated at concentrations ranging from 0 to 500 µg/mL for L. speciosa leaf extracts and compared to ascorbic acid (positive control). Scavenging activity increased in a concentration-dependent manner. Ascorbic acid showed the highest inhibition, followed by ethanol, methanol, acetone, and n-hexane. (B) ABTS radical scavenging activity was assessed using the ABTS decolorization assay at concentrations ranging from 0 to 150 µg/mL. Ascorbic acid demonstrated the highest scavenging potential, followed by methanol, ethanol, acetone, and n-hexane. Data were analyzed using an inhibitor vs. dose-response four-variable parameter model to determine IC50 values. Results represent the mean ± standard deviation from three independent experiments (n = 3).
Superoxide and nitric oxide free radical scavenging assay of L. speciosa leaf extracts
In the superoxide assay, the scavenging potential was assessed at concentrations ranging from 0 to 300 µg/mL. The methanol extract demonstrated the highest scavenging activity, ranging from 18.02 ± 0.51% at 20 µg/mL to 81.21 ± 0.62% at 300 µg/mL. The ethanol extract followed, while the acetone and n-hexane extracts exhibited moderate to low activity. The standard antioxidant, ascorbic acid, achieved 92.73 ± 0.35% inhibition at 300 µg/mL. The half-maximal inhibitory concentration (IC50) values for superoxide anion radical scavenging demonstrated varying efficacies among the extracts. The methanol extract exhibited an IC50 value of 83.58 µg/mL, while the ethanol extract required 104.85 µg/mL to achieve comparable inhibition. The acetone and n-hexane extracts displayed relatively lower antioxidant potencies with IC50 values of 202.17 µg/mL and 138.65 µg/mL, respectively. The reference antioxidant, ascorbic acid, demonstrated superior scavenging capacity with an IC50 of 54.84 µg/mL (Fig 4A). In the nitric oxide assay, the ethanol extract showed the highest inhibition at 79.94 ± 0.34% at 300 µg/mL, followed by the methanol extract at 72.45 ± 0.14%. The nitric oxide radical scavenging potential of the extracts was expressed as IC50 values, demonstrated varying degrees of efficacy across different extraction solvents. The ethanol extract exhibited superior scavenging activity (89.09 μg/mL), followed by methanol (120.45 μg/mL), acetone (149.18 μg/mL), and n-hexane extracts (216.33 μg/mL). While all extracts demonstrated concentration-dependent inhibition, the reference standard ascorbic acid displayed significantly higher potency (58.58 μg/mL). These findings indicate that medium-polarity solvents more effectively extracted compounds with nitric oxide scavenging capabilities (Fig 4B).
(A) Superoxide radical scavenging activity of L. speciosa leaf extracts and ascorbic acid was measured using the riboflavin-light-nitro blue tetrazolium (NBT) reduction method at concentrations ranging from 0 to 300 µg/mL. Methanol extract exhibited the highest activity among the extracts (B) Nitric oxide (NO) radical scavenging activity of the extracts and ascorbic acid was assessed using the Griess reagent method at concentrations ranging from 0 to 300 µg/mL. Ethanol extract showed the highest activity among the extracts. Ascorbic acid demonstrated the highest scavenging activity compared to all solvent extracts. Data were analyzed using an inhibitor vs. dose-response four-variable parameter model to determine IC50 values. Results are expressed as mean ± SD of triplicate measurements from three biologically independent experiments.
Brine shrimp lethality assay of L. speciosa leaf extracts
In the brine shrimp lethality assay (BSLA), concentrations ranging from 0 to 800 µg/mL were employed to assess the cytotoxic effects of L. speciosa. The assay revealed a concentration-dependent increase in the mortality of Artemia salina nauplii, with mortality rates rising from 5% at 25 µg/mL to 60% at 800 µg/mL. Nonlinear regression analysis demonstrated a strong correlation between extract concentration and mortality, as indicated by a high coefficient of determination (R2 = 0.9836). The LC50 value, representing the concentration required to induce 50% mortality in the nauplii population, was determined to be about 601.8 µg/mL. The high R2 value highlights a clear dose-response relationship, suggesting that the cytotoxic activity is likely attributable to bioactive compounds present in the extract. These findings indicate that the methanol extract of L. speciosa exhibits moderate cytotoxic effects (Fig 5).
Cytotoxicity of L. speciosa methanol leaf extract was evaluated using the brine shrimp lethality assay (BSLA). Extracts, prepared in dimethyl sulfoxide (DMSO), were tested at 0 - 800 µg/mL. The concentration-response relationship was analyzed using nonlinear regression (coefficient of determination, R2 = 0.9836), yielding an LC50 value of 601.8 μg/mL. According to Meyer’s toxicity index, the extract demonstrates moderate cytotoxic potential (LC50 between 500 - 1000 μg/mL). Data represent the mean ± standard deviation from three independent experiments (n = 3).
Antibacterial activity of L. speciosa leaf extracts
The antibacterial activity of methanol-derived L. speciosa leaf extract was evaluated against various bacterial species, as summarized in Table 1. The methanol extract (1 mg/disk) demonstrated inhibitory effects, with mean inhibition zones of 17 ± 0.34 mm for Escherichia coli, 14 ± 0.97 mm for Salmonella enterica, 15 ± 0.40 mm for Bacillus subtilis, 16 ± 0.80 mm for Listeria monocytogenes, and 15 ± 0.10 mm for Staphylococcus aureus. In comparison, the standard antibiotic ampicillin (20 µg/disk) exhibited significantly larger inhibition zones: 25 ± 1.50 mm for Escherichia coli, 26 ± 0.46 mm for Salmonella enterica, 27 ± 0.40 mm for Bacillus subtilis, 29 ± 0.83 mm for Listeria monocytogenes, and 28 ± 1.20 mm for Staphylococcus aureus. MIC values ranged from 250 to 500 µg/mL, while MBC values ranged from 500 to 1000 µg/mL. Specifically, the MBC/MIC ratios were as follows: E. coli (MIC: 250 µg/mL, MBC: 500 µg/mL, ratio = 2), S. enterica (MIC: 500 µg/mL, MBC: 1000 µg/mL, ratio = 2), B. subtilis (MIC: 250 µg/mL, MBC: 500 µg/mL, ratio = 2), L. monocytogenes (MIC: 250 µg/mL, MBC: 500 µg/mL, ratio = 2), and S. aureus (MIC: 250 µg/mL, MBC: 500 µg/mL, ratio = 2). All MBC/MIC ratios were ≤ 2, indicating that the methanol extract exhibited bactericidal activity against the tested strains. Methanol extract of L. speciosa displayed substantial antibacterial activity. Their efficacy was considerably lower than standard antibiotics (ampicillin) against all tested bacterial strains. The highest antibacterial activity of the extract was observed against Escherichia coli, while the lowest activity was recorded against Salmonella enterica. All antimicrobial activity differences between the methanol extract and ampicillin control were statistically significant (paired t-test, p < 0.0001).
Antifungal activity of L. speciosa leaf extracts
The antifungal activity of L. speciosa methanol leaf extracts was evaluated against several fungal species using the agar disk diffusion method, as summarized in Table 2. At a concentration of 1 mg/disk, the methanol extract exhibited inhibitory effects against all tested fungal species, with mean inhibition zones of 12 ± 0.11 mm for Aspergillus niger, 13 ± 0.88 mm for Saccharomyces cerevisiae, 12 ± 0.92 mm for Candida albicans, and 14 ± 0.35 mm for Cladosporium sp. In comparison, the standard antifungal agent nystatin (30 µg/disk) demonstrated significantly larger inhibition zones: 22 ± 0.32 mm for Aspergillus niger, 21 ± 0.19 mm for Saccharomyces cerevisiae, 23 ± 0.58 mm for Candida albicans, and 20 ± 0.94 mm for Cladosporium sp. The MIC values for the methanol extract ranged from 300 to 600 µg/mL, while MFC values ranged from 600 to 1200 µg/mL. Specifically, the MFC/MIC ratios were: A. niger (MIC: 600 µg/mL, MFC: 1200 µg/mL, ratio = 2), S. cerevisiae (MIC: 300 µg/mL, MFC: 600 µg/mL, ratio = 2), C. albicans (MIC: 400 µg/mL, MFC: 800 µg/mL, ratio = 2), and Cladosporium sp. (MIC: 300 µg/mL, MFC: 600 µg/mL, ratio = 2). All MFC/MIC ratios were ≤ 2, indicating fungicidal activity of the extract against the tested strains. The methanol extract of L. speciosa displayed significant antifungal activity. Their efficacy was markedly lower than standard antibiotics (nystatin) across all tested fungal strains. Statistical analysis using paired t-tests revealed that all extracts were significant compared to the reference standard (**p < 0.005).
Enzyme inhibition assays of L. speciosa leaf extracts
The enzyme inhibitory properties of L. speciosa leaf extracts were assessed against α-amylase and α-glucosidase. The inhibitory effects of methanol, ethanol, acetone, and n-hexane extracts were evaluated at concentrations ranging from 0 to 500 µg/mL using nonlinear regression analysis. Acarbose was used as the reference standard. All extracts exhibited concentration-dependent inhibition in both enzyme assays. In the α-amylase assay, the ethanol achieved the highest inhibition (61.0 ± 0.6%) at 500 µg/mL, followed by methanol (56.0 ± 0.9%), acetone (45.7 ± 0.5%), and n-hexane (41.0 ± 0.3%). In comparison, acarbose demonstrated superior inhibition at 78.9 ± 0.5%. The IC50 values further supported these findings, with ethanol showing the lowest IC50 (384.3 µg/mL), followed by methanol (442.8 µg/mL), acetone (593.9 µg/mL), and n-hexane (712.7 µg/mL). Acarbose demonstrated the most potent activity with an IC50 of 265.2 µg/mL (Fig 6A). In the α-glucosidase assay, methanol extract yielded approximately 74.3 ± 1.0% inhibition at 500 µg/mL, increasing from 15.7 ± 0.9% at 50 µg/mL. Ethanol extract achieved 61.2 ± 1.4%, followed by acetone (51.4 ± 0.8%) and n-hexane (41.3 ± 1.2%) at 500 µg/mL. Acarbose demonstrated the highest inhibition, peaking at 85.8 ± 0.8% at 500 µg/mL, surpassing the extracts of L. speciosa leaves. The IC50 value of acarbose was estimated to be approximately 159.76 μg/mL. The IC50 value for the methanol extract was significantly lower (240.11 µg/mL) compared to ethanol (306.61 µg/mL), acetone (460.04 µg/mL), and n-hexane (645.39 µg/mL) (Fig 6B). These findings suggest that ethanol and methanol are the most suitable solvents for L. speciosa extraction due to their higher yield of bioactive compounds.
(A) α-amylase and (B) α-glucosidase inhibitory activities of extracts obtained with methanol, ethanol, acetone, and n-hexane. Acarbose served as positive control. Enzyme inhibition was evaluated across a concentration range of 0 - 500 μg/mL. Among the tested extracts, the ethanol extract exhibited the most potent α-amylase inhibition (IC50 = 240.1 μg/mL), while the methanol extract showed the highest α-glucosidase inhibitory activity (IC50 = 442.8 μg/mL). Dose response curves were fitted using a four-parameter variable slope model (inhibitor vs. response) to determine IC50 values. Statistical significance was robust across all experiments, with p < 0.0001.
In silico analysis of pharmacological potential
The PASS algorithm predicted probable activity (Pa) and probable inactivity (Pi) scores for a range of pharmacological targets, with Pa > 0.7 indicating robust activity. This computational analysis revealed diverse bioactivities among the evaluated compounds (Table 3). Among the compounds analyzed, corosolic acid emerged as a particularly promising candidate, demonstrating exceptional potential as an insulin promoter (Pa = 0.924). Enzymatic inhibitory properties were observed for ellagic acid, particularly as a ubiquinol-cytochrome-c reductase inhibitor (Pa = 0.907) and a peroxidase inhibitor (Pa = 0.861). Quercetin showed exceptional activity as a peroxidase inhibitor (Pa = 0.962) coupled with significant MAP kinase stimulation (Pa = 0.933). The analysis further revealed gallic acid’s dual functionality as a glutathione thiolesterase inhibitor (Pa = 0.944) and antiseptic agent (Pa = 0.910). γ-Sitosterol was predicted to be active in lipid regulation and chemoprevention, with Pa values up to 0.960. Moreover, the prediction revealed that lagerstroemin possesses potent histidine kinase inhibitory activity (Pa = 0.924) and antineoplastic potential (Pa = 0.893).
In silico assessment of pharmacokinetic properties
The pharmacokinetic profiles and drug-likeness parameters for key bioactive compounds from L. speciosa are detailed in Table 4. Drug-likeness was assessed based on Lipinski’s Rule of Five which predicts favorable oral bioavailability. Ellagic acid, quercetin, and gallic acid fully complied with Lipinski’s rule of five, demonstrating favorable molecular weight, lipophilicity (LogP), hydrogen bond donor and acceptor counts, and topological polar surface area. These compounds also showed high gastrointestinal (GI) absorption potential and comparable bioavailability scores ranging from 0.55 to 0.56. In contrast, corosolic acid, γ-sitosterol, ursolic acid, phytol, and campesterol each violated one Lipinski’s rule of five primarily due to lipophilicity (LogP). Despite this, corosolic acid retained high GI absorption and a moderate bioavailability score of 0.56. Conversely, γ-sitosterol, ursolic acid, phytol, and campesterol were predicted to have low GI absorption and varied bioavailability scores. The number of rotatable bonds among compounds ranged from 0 (ellagic acid) to 13 (phytol), reflecting varying degrees of molecular flexibility.
In silico assessment of toxicological properties
A comprehensive toxicological assessment was conducted on six major bioactive compounds extracted from L. speciosa leaves: corosolic acid, ellagic acid, quercetin, γ-sitosterol, gallic acid, and phytol. The predicted toxicological parameters are summarized in Table 5. All compounds demonstrated favorable safety profiles, with predictions indicating non-mutagenic properties (Non AMES toxic) and no carcinogenic potential. The evaluated compounds exhibited high toxicity to honeybees (LD50 < 100 µg/bee), indicating potential ecological risks for pollinator species. Gallic acid and phytol were predicted as readily biodegradable, while the other compounds were classified as not readily biodegradable. Regarding acute oral toxicity, ellagic acid and quercetin were categorized as Category II (50 mg/kg < LD50 < 500 mg/kg), whereas corosolic acid, gallic acid, and phytol were assigned to Category III (500 mg/kg < LD50 < 5000 mg/kg). γ-Sitosterol was classified under Category I (LD50 ≤ 50 mg/kg). The phytochemicals uniformly induced fathead minnow toxicity, with LC50 predictions consistently below 0.5 mmol/L. Predicted acute toxicity in rats ranged from 1.6146 for phytol to 3.0200 for quercetin.
In silico analysis of molecular docking interactions
Docking simulations revealed the interactions between major phytocompounds of L. speciosa and selected target proteins related to antidiabetic, antimicrobial, and antioxidant functions (Table 6). Six abundant compounds from L. speciosa: corosolic acid, ellagic acid, quercetin, γ-sitosterol, gallic acid, and lagerstroemin demonstrated potential pharmacological properties (Fig 7). The analysis generated key parameters including the Attracting Cavities (AC) score, binding free energy (SwissParam score), and root-mean-square deviation (RMSD) to assess docking accuracy and stability. Molecular docking predicted optimal binding conformations by identifying favorable interaction sites and visualizing them using PyMOL (Fig 8). Among the ligands, lagerstroemin exhibited the strongest binding affinity toward α-glucosidase (SwissParam score: −8.9076). It was observed to occupy the enzyme’s catalytic region, indicating a well-defined interaction. Similar to lagerstroemin, corosolic acid demonstrated a robust interaction with α-glucosidase (AC score: 32.249751; SwissParam score: −6.9352). γ-Sitosterol showed effective binding to PPAR-γ, aligning stably within its ligand-binding domain (SwissParam score: −7.4781). Docking successfully at the β-lactamase active site, gallic acid validated the enzyme’s microbial activity (SwissParam score: −6.0175). Through hydrogen bonding and hydrophobic contacts, ellagic acid established an interaction with DNA gyrase B. A favorable binding orientation for quercetin was observed within the active site of peroxiredoxin 6. The root-mean-square deviation (RMSD) values of the docked complexes ranged from 13.6882 (gallic acid) to 41.1486 (lagerstroemin).
The compounds depicted are Corosolic acid, Ellagic acid, Quercetin, γ-Sitosterol, Gallic acid, and Lagerstroemin. These compounds were selected based on their abundance and potential pharmacological relevance.
Docking simulations were conducted with SwissDock and analyzed using PyMOL to visualize binding interactions. (A) Corosolic acid docked to α-glucosidase (PDB ID: 3WY1); (B) Ellagic acid docked to DNA gyrase B (PDB ID: 6KZV); (C) Gallic acid docked to β-lactamase (PDB ID: 1XPB); (D) Quercetin docked to Peroxiredoxin 6 (PDB ID: 5B6M); (E) Lagerstroemin docked to α-glucosidase (PDB ID: 3WY1); and (F) γ-Sitosterol docked to PPAR-γ (PDB ID: 2P4Y). The images display the predicted binding modes and interaction sites (magenta) of each compound (blue) with its respective target protein (green), highlighting the interacting amino acids of the receptors.
Discussion
This study encompasses a detailed evaluation of the phytochemical composition, antioxidant activity, and bioactivity of L. speciosa leaf extracts. The findings highlight their potential for biomedical applications. Methanol, ethanol, acetone, and n-hexane were used as solvents to extract bioactive compounds from L. speciosa leaves. Methanol and ethanol extracts yielded the highest concentrations of total phenolics, flavonoids, and proanthocyanidins content [46,47]. This is attributed to their higher polarity indices compared to acetone and n-hexane. Phenolic and flavonoid compounds are more effectively extracted by polar solvents like methanol and ethanol. Methanol (5.1) has the highest polarity index and showed the best extraction efficiency. Ethanol (4.3) is safer and more eco-friendly, also performed well. In contrast, acetone (5.1) and n-hexane (0.1) showed lower extraction efficiencies due to their reduced capacity to solubilize hydrophilic compounds [48]. The presence of these bioactive compounds aligns with previous research indicating their role in managing oxidative stress. They are also linked to mitigating chronic diseases such as cardiovascular disorders, neurodegenerative conditions, and cancer [49–51]. Antioxidants play a crucial role in neutralizing free radicals, reducing disease risk, and preventing cellular damage [52]. The antioxidant activity of L. speciosa extracts was evaluated using IC50 values, representing the concentration needed to inhibit 50% of free radical activity. Methanol and ethanol extracts exhibited significant antioxidant activity, with IC50 values ranging from 64.19 to 241.59 µg/mL. These values are comparable to reference standard (ascorbic acid) and they had substantial antioxidant potential. They are consistent with previous reports on L. speciosa and other phenolic- and flavonoid-rich medicinal plants [53]. The biological relevance of these IC50 values is significant for therapeutic applications. However, translating these in vitro results to practical therapeutic doses requires further pharmacokinetic and bioavailability studies. The extracts’ potential to combat oxidative stress-related diseases warrants further exploration through in vivo models and clinical trials.
The antioxidant potential of L. speciosa extracts was evaluated using DPPH, ABTS, superoxide, and nitric oxide radical scavenging assays. Ethanol extracts demonstrated the highest DPPH scavenging activity (85.4 ± 0.46%) at a concentration of 500 µg/mL. Methanol derived extract showed unparalleled superoxide radical scavenging activity, with a concentration of 300 µg/mL achieving a remarkable inhibition (81.2 ± 0.6%). This suggests that the extracts contain rich flavonoid and phenolic compounds, which act as effective electron donors and scavengers of superoxide radicals. Superoxide radicals are linked to oxidative stress-related diseases and inflammation [54]. They are known to damage macromolecules, lipids, proteins, and DNA, thereby accelerating aging and disease progression. The observed scavenging activities emphasize L. speciosa’s potential as a natural antioxidant source. This potential can help mitigate oxidative stress and improve health [55]. We observed promising nitric oxide (NO) scavenging activity in L. speciosa extract. Ethanol extracts showed the highest inhibition (79.94 ± 0.34%) at a concentration of 300 µg/mL. NO serves as a biological messenger and vasodilator. However, excessive production can generate reactive nitrogen species (RNS) like peroxynitrite, which are toxic to tissues [56]. The ability of L. speciosa extracts to scavenge NO radicals indicates potential protective benefits. These benefits could extend to chronic inflammatory diseases and certain cancers associated with NO overproduction [57,58]. The brine shrimp lethality assay (BSLA) revealed moderate cytotoxicity in the methanol extract of L. speciosa (LC50 = 601.8 µg/mL). Based on the criteria established by Mayer et al. (1982), LC50 values below 1000 μg/mL in cytotoxicity screening are indicative of bioactivity, while values below 250 μg/mL denote high toxicity. The cytotoxicity of our experimental plant extracts (LC50 = 601.8 µg/mL) was significantly lower compared to that of Vincristine sulfate (LC50 = 0.91 μg/mL), as reported by Ullah et al. [59]. The LC50 of L. speciosa suggests the presence of bioactive compounds with anticancer potential. Compounds such as ellagic acid and gallic acid, previously isolated from Lagerstroemia species, have shown significant cytotoxicity in similar assays [60]. The moderate cytotoxicity indicates that L. speciosa may selectively target damaged cells over normal cells. Future studies are required to confirm selective cytotoxicity. These studies should use well-characterized human cancer cell lines, such as HeLa (cervical cancer) and HepG2 (hepatocellular carcinoma), alongside validated assays, including MTT, SRB, and Calcein assays [61]. These assays are widely used to evaluate cell viability and cytotoxicity. They provide reliable data on the selective toxicity of L. speciosa extracts toward cancer cells compared to normal cells. These approaches will aid in confirming the therapeutic efficacy and safety dose of the extracts. Additionally, apoptosis and cell cycle analysis using flow cytometry, combined with in vivo preclinical cancer models, are necessary to validate these findings. These steps will help elucidate the mechanisms of action and therapeutic applicability of L. speciosa extracts.
The antimicrobial activity of L. speciosa extracts is attributed to phenolic compounds, known for their inhibitory effects on microbial growth. Phenolic compounds in L. speciosa likely exert their antimicrobial effects through mechanisms such as disrupting microbial cell walls, inhibiting key enzymes, and chelating essential metal ions [62]. Specifically, Phenolic compounds containing a C3 side chain demonstrate antimicrobial properties, partly through their ability to decrease oxidative stress levels [63]. Moreover, they inhibit critical enzymes involved in microbial metabolic pathways, limiting nutrient acquisition and cellular functions necessary for survival [64]. The inhibition zones indicate moderate antimicrobial activity against bacterial and fungal strains (i.e., Escherichia coli, Staphylococcus aureus, Candida albicans, and Saccharomyces cerevisiae). These strains were chosen for their clinical significance as common pathogens. Escherichia coli and Staphylococcus aureus are key bacterial species associated with gastrointestinal and skin infections, respectively [65]. Candida albicans and Saccharomyces cerevisiae are well-documented fungal pathogens associated with opportunistic infections [66]. While the activity was lower than conventional antibiotics like ampicillin and antifungals like nystatin. The results suggest potential for complementary use in antimicrobial strategies, particularly in addressing antibiotic resistance. These results coincide with the previous studies that phenolic chemicals enhance their antibacterial activities [67]. Further studies could fully unlock its potential for clinical and commercial applications. Major steps like refining extraction methods, testing synergistic effects, and conducting in vivo studies are essential to evaluate efficacy and safety. L. speciosa extracts exhibited significant inhibition of α-amylase and α-glucosidase, key enzymes in carbohydrate metabolism. The methanol extract showed the highest α-glucosidase inhibitory activity, with an IC50 of 240.11 µg/mL, comparable to other natural inhibitors like Camellia sinensis and Moringa oleifera [68,69]. This dual inhibition of α-amylase and α-glucosidase suggests potential antidiabetic effects by delaying carbohydrate digestion and reducing postprandial glucose spikes. The inhibitory mechanism may involve multiple interactions between the bioactive compounds and enzyme structures. Polyphenols from L. speciosa may form hydrogen bonds with polar residues at the active sites of α-amylase and α-glucosidase, blocking substrates from binding [70]. Flavonoids, particularly those with hydroxyl groups at specific positions, can potentially form complexes with metal ions essential for enzyme function, such as the calcium required for α-amylase activity. Tannins, with their multiple phenolic hydroxyl groups, may induce conformational changes in the enzymes through hydrophobic interactions with aromatic amino acid residues surrounding the active site. Additionally, these phytochemicals might act as competitive inhibitors by mimicking the structure of natural substrates, or as non-competitive inhibitors by binding to allosteric sites that alter enzyme conformation and catalytic efficiency. The synergistic effect of multiple compounds present in the extract could also enhance inhibitory potency through simultaneous interactions at different binding sites, collectively modifying enzyme kinetics and substrate accessibility.
Our in silico analysis uncovered considerable pharmacological potential for L. speciosa phytochemicals against a diverse range of biological targets. PASS analysis indicated diverse bioactivities with high probability scores (Pa), specifically potential insulin-promoting, enzymatic inhibitory, and chemopreventive effects [71]. The strong affinities of corosolic acid and lagerstroemin for α-glucosidase support their potential as antidiabetic agents. In a similar fashion, quercetin displayed promising antioxidant activity, while ellagic acid notably exhibited antimicrobial activity [72]. These effects were supported by high docking scores and stable interactions with peroxiredoxin 6 and DNA gyrase B. Pharmacokinetic profiling indicated that ellagic acid, quercetin, and gallic acid satisfied Lipinski’s Rule of Five, having high gastrointestinal (GI) absorption and moderate bioavailability [73]. Despite minor deviations from these rules, corosolic acid maintained favorable oral absorption profiles. Toxicological predictions indicated that all compounds were non-carcinogenic and non-mutagenic. However, predicted toxicity to honeybees and aquatic species raised potential environmental concerns. The binding capabilities of these compounds with their respective target proteins were further validated through molecular docking. The RMSD values indicated varying conformational stabilities where gallic acid formed the most stable complex. These computational insights highlight the therapeutic potential of L. speciosa constituents and provide a strong basis for future experimental validation [74]. The findings of this study elucidate the antioxidant, antimicrobial, cytotoxic, and enzymatic inhibitory properties of L. speciosa leaf extracts, supporting their traditional medicinal uses. Further research should identify and characterize specific bioactive compounds (e.g., quercetin, kaempferol) and evaluate their synergistic effects. In vivo studies are crucial to assess pharmacokinetics, bioavailability, and safety, paving the way for therapeutic applications. The findings emphasize the potential of L. speciosa as a source of plant-based medicinal compounds. These compounds could address oxidative stress, metabolic imbalances, and infections. This aligns with the growing interest in natural products for managing global health challenges.
Conclusions
This study highlights the therapeutic potential of L. speciosa leaf extracts, particularly methanol and ethanol extracts. These extracts are notably rich in key bioactive compounds, including corosolic acid, ellagic acid, gallic acid, quercetin, lagerstroemin, γ-sitosterol, campesterol, and phytol. Our findings reveal significant antioxidant activities through DPPH, ABTS, superoxide, and nitric oxide scavenging assays. Furthermore, the disk diffusion method confirmed their antimicrobial efficacy, demonstrating comparability to standard antibiotics. The brine shrimp toxicity assay also validated their cytotoxic potential, suggesting a promising role in anticancer therapeutics. Beyond these, the extracts exhibited potent α-amylase and α-glucosidase inhibitory activities indicating considerable antidiabetic potential. In silico ADME and molecular docking analysis provided compelling support for the diverse pharmacological activities observed with L. speciosa’s bioactive compounds. The L. speciosa leaf extract holds significant promise as a sustainable and effective natural resource for tackling pressing global health challenges.
Supporting information
S1 Fig. Standard curve of Gallic acid for determining total phenolic compounds.
https://doi.org/10.1371/journal.pone.0339566.s001
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S2 Fig. Catechin (CA) standard curve for determining total flavonoid content.
https://doi.org/10.1371/journal.pone.0339566.s002
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S3 Fig. Quercetin’s standard curve (QU) determination of total flavonol content.
https://doi.org/10.1371/journal.pone.0339566.s003
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S4 Fig. Standard curve of Catechin for the determination of proanthocyanidin.
https://doi.org/10.1371/journal.pone.0339566.s004
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Acknowledgments
The authors cordially acknowledge the Department of Biochemistry and Molecular Biology at the University of Rajshahi, Bangladesh to carry out this study. The authors also extend to appreciate the Laboratory of Biochemistry, University of Hyogo, Japan and the Ibn Sina Specialized Diagnostic Center and Hospital, Dhaka, Bangladesh for supporting the experimental assistance.
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