Mansi Patel
Shri Satsangi Saketdham Ram Ashram Group of Institutions, Faculty of Pharmacy
Article Details
Title: DEVELOPMENT OF A CHITOSAN-BASED SCAFFOLD INCORPORATING BAICALIN AND EUGENOL FOR ANTIMICROBIAL THERAPY
Authors: Mansi Patel
Affiliation: Shri Satsangi Saketdham Ram Ashram Group of Institutions, Faculty of Pharmacy, India
Published in: PEXACY International Journal of Pharmaceutical Science
Volume/Issue: 4(12)
Pages: 44–55
Publication Date: 19/12/2025
Abstract
Background: The increasing prevalence of antibiotic-resistant bacterial strains necessitates the development of novel antimicrobial delivery systems. Chitosan-based scaffolds represent a promising platform for localized antimicrobial therapy due to their inherent biocompatibility, biodegradability, and antimicrobial properties.
Objective: This study aimed to develop and characterize chitosan-based scaffolds incorporating baicalin and eugenol as bioactive antimicrobial agents, and to evaluate their physicochemical properties, morphological characteristics, and antimicrobial efficacy.
Methods: Nine scaffold formulations (F1-F9) were prepared using a 32 full factorial design with varying concentrations of baicalin (1%, 2%, 3% w/v) and eugenol (0.5%, 1%, 1.5% v/v). Scaffolds were fabricated using solvent casting followed by glutaraldehyde crosslinking. Characterization included pH measurement, viscosity analysis, scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy (FTIR), mechanical testing, swelling studies, drug content analysis, antimicrobial activity testing against Staphylococcus aureus and Escherichia coli, and in vitro drug release studies.
Results: All formulations exhibited pH values between 5.60 and 5.93, suitable for topical applications. Viscosity ranged from 189.3 to 230.4 cP, with higher values observed in formulations containing elevated active ingredient concentrations. SEM analysis revealed porous structures with pore sizes ranging from 52.4 to 78.5 μm. FTIR confirmed successful incorporation of both bioactive agents without chemical incompatibility. Encapsulation efficiency exceeded 95% for both compounds. Formulation F3 (3% baicalin, 1.5% eugenol) demonstrated the highest swelling ratio (265.9%), maximum cumulative drug release (79.9% baicalin, 97.4% eugenol at 12 hours), and superior antimicrobial activity with inhibition zones of 16.77 mm against S. aureus and 15.27 mm against E. coli. Stability studies confirmed acceptable retention of properties over one month at both refrigerated and room temperature conditions.
Keywords: Chitosan scaffold; Baicalin; Eugenol; Antimicrobial; Drug delivery; Wound healing; Tissue engineering.
Article can be accessed online on: PEXACY International Journal of Pharmaceutical Science
DOI: 10.5281/zenodo.18147540
Corresponding Author- Mansi Patel*
Correspondence Email- patelmansi1603@gmail.com
Update: Received on 05/12/2025; Accepted; 14/012/2025, Published on; 19/12/2025
1. INTRODUCTION
The global healthcare burden imposed by bacterial infections continues to escalate, driven in part by the emergence of antibiotic-resistant pathogens (Kaur et al., 2019). Conventional systemic antimicrobial therapies often suffer from limitations including poor bioavailability at infection sites, systemic toxicity, and the promotion of resistance development. Consequently, localized drug delivery systems capable of providing sustained therapeutic concentrations directly at the target site have garnered considerable research interest (Zhang et al., 2017). Among such systems, polymeric scaffolds present unique advantages for wound healing and tissue regeneration applications, serving simultaneously as structural matrices and drug reservoirs.
Chitosan, a deacetylated derivative of chitin, has emerged as a particularly attractive scaffold material for biomedical applications. This naturally occurring polysaccharide exhibits inherent biocompatibility, biodegradability, and antimicrobial activity, alongside the capacity to promote hemostasis and accelerate wound healing (Dash et al., 2011). The cationic nature of chitosan facilitates electrostatic interactions with negatively charged bacterial cell membranes, contributing to its antimicrobial efficacy. Furthermore, the presence of reactive amino and hydroxyl groups enables chemical modification and crosslinking, permitting tailored mechanical and release properties (Szymanska & Winnicka, 2015). Previous investigations have demonstrated the utility of chitosan-based scaffolds in tissue engineering, with porous architectures supporting cell attachment, proliferation, and nutrient diffusion (Chen et al., 2018; Hutmacher, 2000).
Baicalin, a flavonoid glycoside extracted from Scutellaria baicalensis, possesses well-documented antimicrobial, anti-inflammatory, and antioxidant properties (Zhang et al., 2015). This compound has demonstrated efficacy against a broad spectrum of bacterial pathogens and exhibits favorable safety profiles in preclinical studies. The incorporation of baicalin into polymeric delivery systems represents a strategy to enhance its bioavailability and therapeutic utility at infection sites (Liu et al., 2019). Similarly, eugenol, a phenolic compound derived predominantly from clove oil, exhibits potent antimicrobial, analgesic, and anti-inflammatory activities (Kamatou et al., 2012). Eugenol disrupts bacterial membrane integrity and inhibits essential enzymatic processes, contributing to its broad-spectrum antibacterial effects (Kaur et al., 2019). The combination of baicalin and eugenol within a single delivery platform may offer synergistic antimicrobial benefits while addressing multiple aspects of the wound healing cascade.
Despite the therapeutic potential of these bioactive compounds, their incorporation into chitosan scaffolds requires careful optimization to balance drug loading, release kinetics, and scaffold integrity. Crosslinking with agents such as glutaraldehyde enhances mechanical stability but may influence porosity and drug diffusion (Singh et al., 2020). Systematic evaluation of formulation variables through factorial experimental design enables identification of optimal compositions and elucidation of factor interactions (Murphy et al., 2010).
The present study aimed to develop chitosan-based scaffolds incorporating baicalin and eugenol at varying concentrations using a 32 full factorial design. Comprehensive characterization encompassing physicochemical properties, morphological features, mechanical behavior, antimicrobial activity, and drug release profiles was conducted to identify formulations with optimal performance characteristics for potential wound healing and antimicrobial applications.
2. MATERIALS AND METHODS
2.1 Materials
Chitosan (medium molecular weight, degree of deacetylation >75%) and eugenol (≥99% purity) were procured from Sigma-Aldrich (St. Louis, MO, USA). Baicalin (≥98% purity) was obtained from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Glacial acetic acid was sourced from Loba Chemie (Mumbai, India), while absolute ethanol was purchased from Bangalore Fine Chemicals (Bangalore, India). Glutaraldehyde solution (2.5%) was obtained from Sigma-Aldrich. Dimethyl sulfoxide (DMSO) and nutrient agar were procured from HiMedia Laboratories (Mumbai, India). Staphylococcus aureus (ATCC 25923) and Escherichia coli (ATCC 25922) were obtained from a certified microbial culture collection. Phosphate-buffered saline (PBS, pH 5.5 and pH 7.4) was prepared in the laboratory using analytical grade reagents. All chemicals were used as received without further purification.
2.2 Preformulation Studies
2.2.1 Determination of Analytical Wavelength. The absorption maxima (λmax) of baicalin and eugenol were determined using UV-visible spectrophotometry (Igene Labserve IG-2100). Stock solutions were prepared by dissolving each compound in ethanol (1 mg/mL for baicalin; approximately 1.06 mg/mL for eugenol based on density). Working solutions (100 μg/mL) were scanned across the wavelength range of 200-400 nm against appropriate blank solutions. The wavelengths corresponding to maximum absorbance were identified as 274 nm for baicalin and 280 nm for eugenol (Patel et al., 2018).
2.2.2 Calibration Curves. Standard calibration curves were constructed for quantitative analysis. Serial dilutions of stock solutions (100 μg/mL) were prepared to yield concentrations of 5, 10, 20, 30, 40, and 50 μg/mL using phosphate buffer with 10% polyethylene glycol, 20% ethanol, and 2% Tween 80 as the solvent system for baicalin, and ethanol for eugenol. Absorbance was measured at the respective λmax values in triplicate, and linear regression analysis was performed to determine the calibration equations (Smith et al., 2020).
2.2.3 Solubility Analysis. The solubility of baicalin and eugenol was assessed qualitatively in distilled water, ethanol, phosphate buffer (pH 7.4), and DMSO. Each compound was added incrementally to 10 mL of solvent at room temperature with continuous stirring, and solubility was classified as freely soluble, sparingly soluble, or slightly soluble based on visual observation of dissolution behavior (Patel et al., 2018).
2.2.4 Drug-Excipient Compatibility (FTIR Analysis). Fourier-transform infrared spectroscopy was employed to assess potential interactions between baicalin, eugenol, and chitosan. Samples of individual components and physical mixtures were prepared as KBr pellets and analyzed over the range of 4000-400 cm-1 using an FTIR spectrometer. Characteristic peaks were identified and compared to evaluate chemical compatibility (Griffiths & de Haseth, 2007; Kumar et al., 2018).
2.3 Experimental Design
A 32 full factorial experimental design was employed to investigate the effects of baicalin concentration (1%, 2%, and 3% w/v) and eugenol concentration (0.5%, 1%, and 1.5% v/v) on scaffold properties. This design generated nine formulations (F1-F9) representing all possible combinations of the two factors at three levels each, enabling assessment of main effects and interactions (Table 1).
Table 1. Scaffold Formulation Compositions Based on 32 Factorial Design
| Component | F1 | F2 | F3 | F4 | F5 | F6 | F7 | F8 | F9 |
| Chitosan (g) | 1.5 | 1.5 | 1.5 | 1.5 | 1.5 | 1.5 | 1.5 | 1.5 | 1.5 |
| Acetic acid 1% (mL) | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 |
| Baicalin (% w/v) | 1 | 2 | 3 | 1 | 2 | 3 | 1 | 2 | 3 |
| Eugenol (% v/v) | 0.5 | 1 | 1.5 | 1 | 1.5 | 0.5 | 1.5 | 0.5 | 1 |
2.4 Scaffold Preparation
Scaffolds were prepared using a solvent casting method followed by chemical crosslinking (Chen et al., 2018; Singh et al., 2020). Chitosan (1.5 g) was dissolved in 100 mL of 1% v/v acetic acid solution under continuous magnetic stirring at room temperature for 4 hours until a clear, homogeneous solution was obtained. Baicalin was pre-dissolved in ethanol at the requisite concentration before incorporation into the chitosan solution. Eugenol was added directly to the chitosan solution owing to its sufficient solubility in the acidic aqueous medium (Liu et al., 2019). The mixture was stirred until uniform distribution of both active ingredients was achieved.
The chitosan-baicalin-eugenol solution was poured into sterilized Petri dishes and allowed to undergo gelation at room temperature for 24 hours. Subsequently, the gelled scaffolds were immersed in 2.5% glutaraldehyde solution for 24 hours to induce crosslinking. Following crosslinking, scaffolds were rinsed extensively with distilled water to remove residual glutaraldehyde. The crosslinked scaffolds were dried at room temperature for 48 hours and stored in desiccators until characterization (Hassan et al., 2019).
2.5 Scaffold Characterization
2.5.1 pH Measurement. Surface pH was determined by placing 1-2 mL of distilled water onto a 2 × 2 cm scaffold section and allowing equilibration for 5 minutes. A calibrated digital pH meter (Mettler Toledo SevenExcellence) was used to record pH values at three different locations on each sample, with measurements performed in triplicate (n = 3).
2.5.2 Viscosity Measurement. The viscosity of scaffold precursor solutions was measured using a Brookfield viscometer (Igene Labserve IG-DV100) equipped with spindle number 2 at 40 rpm at room temperature. Measurements were performed in triplicate for each formulation.
2.5.3 Morphological Analysis (SEM). Surface morphology and pore architecture were examined using scanning electron microscopy (Ahn et al., 2014). Scaffold sections were mounted on aluminum stubs with carbon tape, sputter-coated with gold (approximately 10 nm thickness), and imaged at an accelerating voltage of 15 kV at magnifications of 1000×. Cross-sectional samples were prepared by fracturing scaffolds after immersion in liquid nitrogen (Costa et al., 2016; Ghorbani et al., 2012). Pore size was quantified using ImageJ software (Schneider et al., 2012).
2.5.4 Chemical Analysis (FTIR). FTIR spectroscopy was performed on dried scaffold samples ground into fine powder and pressed into KBr pellets. Spectra were recorded over 4000-500 cm-1 to confirm incorporation of baicalin and eugenol and to assess potential chemical interactions (Griffiths & de Haseth, 2007; Jin et al., 2014; Martins et al., 2018).
2.5.5 Mechanical Testing. Scaffold thickness was measured at three locations (center and two edges) using a digital micrometer (least count 0.001 mm). Tensile strength was determined using a texture analyzer (TA.XT Plus, Stable Micro Systems) with a 5 kg load cell. Rectangular strips (10 mm × 50 mm) were clamped with a gauge length of 30 mm and stretched at 2 mm/min until rupture. Tensile strength was calculated as force at break divided by cross-sectional area (Murphy et al., 2010).
2.5.6 Swelling Studies. Dried scaffolds of known weight (Wd) were immersed in PBS (pH 7.4) at 37°C. At predetermined time intervals (0, 1, 2, 4, 8, and 24 hours), scaffolds were removed, excess surface moisture was blotted with filter paper, and the swollen weight (Ws) was recorded. The swelling ratio was calculated according to the equation: Swelling Ratio (%) = [(Ws – Wd) / Wd] × 100. All measurements were performed in triplicate (Zhang et al., 2017).
2.5.7 Drug Content Analysis. Known masses of scaffold were dissolved in appropriate solvents, and drug content was quantified spectrophotometrically at the respective λmax values. Encapsulation efficiency (EE%) was calculated as: EE (%) = (Practical Drug Content / Theoretical Drug Content) × 100 (Smith et al., 2020).
2.5.8 Antimicrobial Activity Testing. Antimicrobial efficacy was evaluated against Staphylococcus aureus (ATCC 25923) and Escherichia coli (ATCC 25922) using the agar diffusion method (Silva et al., 2016). Bacterial cultures were grown in Mueller-Hinton broth at 37°C for 18-24 hours and adjusted to 0.5 McFarland standard (approximately 1.5 × 108 CFU/mL). Scaffold discs were placed on inoculated agar plates and incubated at 37°C for 24 hours. Zones of inhibition were measured in millimeters using digital calipers. Tests were performed in triplicate (Zhao et al., 2010).
2.5.9 In Vitro Drug Release Study. Drug release was evaluated using vertical Franz diffusion cells. A cellulose acetate membrane (0.45 μm pore size) was mounted between donor and receptor compartments. The receptor compartment was filled with 12 mL of PBS (pH 5.5) maintained at 37 ± 0.5°C with continuous magnetic stirring at 400 rpm. Scaffold samples were placed in the donor compartment. Aliquots (1.0 mL) were withdrawn at 0, 0.5, 1, 2, 4, 6, 8, 10, and 12 hours and replaced with fresh pre-warmed medium. Drug concentrations were determined spectrophotometrically, and cumulative percentage release was calculated (Liao et al., 2019).
2.5.10 Stability Studies. Selected formulations were subjected to stability testing under refrigerated conditions (5 ± 3°C) and room temperature conditions (25 ± 2°C, 60 ± 5% RH) for one month. Samples were evaluated at baseline and one month for physical appearance, pH, viscosity, thickness, tensile strength, and encapsulation efficiency.
2.6 Statistical Analysis
All experiments were performed in triplicate, and results are expressed as mean ± standard deviation (SD). Statistical analysis was conducted using one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test for multiple comparisons. A p-value less than 0.05 was considered statistically significant. Response surface methodology and contour plots were generated using Python programming language to visualize factor effects and interactions.
3. RESULTS
3.1 Preformulation Studies
3.1.1 Analytical Wavelength Determination. UV-visible spectrophotometric scanning revealed maximum absorbance at 274 nm for baicalin and 280 nm for eugenol. These wavelengths were employed for all subsequent quantitative analyses.
3.1.2 Calibration Curves. Linear calibration curves were established for both compounds over the concentration range of 5-50 μg/mL. For baicalin, the regression equation was y = 0.0041 + 0.0164x (R2 = 0.9998), and for eugenol, y = 0.0009 + 0.0152x (R2 = 0.9999), where y represents absorbance and x represents concentration in μg/mL. The correlation coefficients exceeding 0.999 confirmed excellent linearity suitable for quantitative determination.
3.1.3 Solubility Analysis. Both baicalin and eugenol demonstrated limited solubility in aqueous media (slightly soluble in distilled water and sparingly soluble in phosphate buffer pH 7.4) but were freely soluble in ethanol and DMSO. These findings guided solvent selection during formulation, with ethanol employed for baicalin dissolution and direct addition used for eugenol incorporation.
3.1.4 Drug-Excipient Compatibility. FTIR analysis of individual components and physical mixtures revealed no evidence of chemical incompatibility. Characteristic peaks of baicalin (3400-3450 cm-1 for O-H stretching; 1720-1730 cm-1 for C=O stretching), eugenol (3400-3500 cm-1 for phenolic O-H; 1500-1600 cm-1 for aromatic C=C), and chitosan (3200-3500 cm-1 for overlapping O-H and N-H; 1650-1660 cm-1 for amide I) remained unchanged in physical mixtures. The absence of new peaks or significant shifts indicated that components were physically blended without undergoing chemical reactions, confirming their suitability for combined use in scaffold formulations.
Fig.1- Drug-Excipient Compatibility using FTIR
3.2 pH and Viscosity Measurements
Surface pH values for all formulations ranged from 5.60 ± 0.03 (F1) to 5.93 ± 0.03 (F3), representing a slightly acidic environment suitable for chitosan stability and topical application (Table 2). Statistical analysis by one-way ANOVA revealed significant differences among formulations (F = 55.69, p = 3.97 × 10-11). Higher baicalin concentrations were associated with elevated pH values, likely reflecting interactions between baicalin’s phenolic groups and chitosan’s amine functionalities.
Viscosity measurements demonstrated values ranging from 189.3 ± 0.8 cP (F1) to 230.4 ± 0.6 cP (F3). Formulations with higher concentrations of both bioactive agents exhibited increased viscosity, suggesting enhanced intermolecular interactions within the chitosan matrix. ANOVA confirmed statistically significant differences (F = 848.96, p = 1.38 × 10-21). The progressive increase in viscosity from F1 through F3, F5, and F9 indicates that active ingredient loading influences the rheological behavior of the scaffold precursor solutions.
Table 2. pH and Viscosity Values of Scaffold Formulations (Mean ± SD, n = 3)
| Formulation | pH (Mean ± SD) | Viscosity (cP, Mean ± SD) |
| F1 | 5.60 ± 0.03 | 189.3 ± 0.8 |
| F2 | 5.85 ± 0.03 | 210.4 ± 0.7 |
| F3 | 5.93 ± 0.03 | 230.4 ± 0.6 |
| F4 | 5.69 ± 0.03 | 200.3 ± 1.1 |
| F5 | 5.87 ± 0.02 | 217.9 ± 0.7 |
| F6 | 5.79 ± 0.03 | 208.1 ± 0.7 |
| F7 | 5.73 ± 0.02 | 204.8 ± 0.3 |
| F8 | 5.82 ± 0.03 | 212.3 ± 0.8 |
| F9 | 5.89 ± 0.02 | 225.0 ± 0.8 |
Fig.2- pH and Viscosity Values of Scaffold Formulations
3.3 Morphological Analysis
SEM analysis revealed porous scaffold architectures across all formulations, with pore size and surface characteristics varying with composition (Table 3). Mean pore sizes ranged from 52.4 ± 5.1 μm (F1) to 78.5 ± 6.9 μm (F9), demonstrating a progressive increase with higher bioactive loading. Formulation F1 exhibited relatively uniform, moderate-sized pores with smooth to mildly rough surfaces and small crystalline-like domains visible at high magnification, likely representing deposited baicalin particles. Formulation F3 displayed the largest and most numerous pores, forming a highly interconnected porous matrix with noticeably rougher surfaces and fibrillar projections around pore perimeters. Visible clusters of baicalin and eugenol were observed on the scaffold surface, indicating some degree of phase separation at higher active loading.
Cross-sectional analysis confirmed high pore interconnectivity in all formulations, which is essential for nutrient diffusion and cell infiltration in tissue engineering applications. The enhanced porosity observed in formulations F3, F5, F6, F8, and F9 suggests greater potential for fluid penetration and drug release, although mechanical integrity considerations must be balanced against porosity requirements.
Table 3. Summary of SEM Morphological Findings
| Formulation | Pore Size (μm) | Surface Roughness | Pore Structure |
| F1 | 52.4 ± 5.1 | Smooth to mild | Moderate, circular |
| F2 | 58.6 ± 4.7 | Smooth | Moderate, elongated |
| F3 | 64.8 ± 4.3 | Moderate | Large, interconnected |
| F4 | 60.2 ± 5.2 | Mildly rough | Large, moderate interconnectivity |
| F5 | 68.1 ± 5.9 | Moderate | Large, well-interconnected |
| F6 | 74.2 ± 6.7 | High | Largest, highly interconnected |
| F7 | 69.8 ± 6.1 | Moderate-high | Large, oval, interconnected |
| F8 | 72.3 ± 5.6 | Rough | Large, irregular |
| F9 | 78.5 ± 6.9 | High | Largest, most open |
Fig.3- SEM Analysis for optimized Formulation 3
Fig.4- Scaffold Formulation
3.4 Mechanical Properties
Scaffold thickness ranged from 1.72 ± 0.035 mm (F1) to 1.89 ± 0.031 mm (F3), with a general trend of increasing thickness correlating with higher active ingredient concentrations (Table 4). This increase reflects greater total solid content and modified drying dynamics during scaffold preparation. Tensile strength demonstrated an inverse relationship, decreasing from 2.88 ± 0.13 MPa (F1) to 1.96 ± 0.09 MPa (F3). The reduction in mechanical strength with increasing baicalin and eugenol loading is attributable to the plasticizing effect of eugenol and disruption of chitosan’s hydrogen bonding network by baicalin incorporation. Despite these variations, all formulations maintained sufficient mechanical integrity for handling and potential biomedical application.
Table 4. Mechanical Properties of Scaffold Formulations (Mean ± SD, n = 3)
| Formulation | Thickness (mm) | Tensile Strength (MPa) |
| F1 | 1.72 ± 0.035 | 2.88 ± 0.13 |
| F2 | 1.81 ± 0.021 | 2.49 ± 0.15 |
| F3 | 1.89 ± 0.031 | 1.96 ± 0.09 |
| F4 | 1.80 ± 0.026 | 2.78 ± 0.12 |
| F5 | 1.85 ± 0.015 | 2.23 ± 0.08 |
| F6 | 1.77 ± 0.021 | 2.26 ± 0.06 |
| F7 | 1.75 ± 0.025 | 2.73 ± 0.10 |
| F8 | 1.85 ± 0.036 | 2.54 ± 0.07 |
| F9 | 1.87 ± 0.030 | 2.10 ± 0.13 |
Fig.5- Mechanical Properties of Scaffold Formulations
3.5 Swelling Studies
Swelling behavior followed a time-dependent pattern across all formulations, with rapid initial uptake followed by gradual equilibration (Table 5). At 24 hours, swelling ratios ranged from 215.6 ± 2.2% (F1) to 265.9 ± 1.2% (F3). One-way ANOVA demonstrated highly significant differences among formulations (F = 191.18, p = 8.3 × 10-16). Formulations with elevated baicalin and eugenol concentrations consistently exhibited higher swelling capacity, reflecting increased scaffold porosity and reduced crosslink density. The enhanced swelling observed in F3 indicates superior fluid absorption capacity, which may benefit wound exudate management and drug release in therapeutic applications.
Table 5. Time-Dependent Swelling Ratio (%) of Scaffold Formulations (Mean ± SD, n = 3)
| Time (h) | F1 | F2 | F3 | F4 | F5 | F6 | F7 | F8 | F9 |
| 1 | 90.3 | 105.4 | 126.2 | 100.4 | 116.7 | 110.8 | 108.0 | 121.6 | 121.6 |
| 4 | 197.7 | 220.0 | 251.9 | 207.7 | 242.1 | 236.2 | 233.4 | 247.1 | 247.1 |
| 24 | 220.3 | 240.5 | 269.9 | 230.5 | 260.0 | 254.1 | 251.3 | 265.0 | 265.0 |
Fig.6- Time-Dependent Swelling Ratio (%)
3.6 Drug Content and Encapsulation Efficiency
Encapsulation efficiency exceeded 95% for both baicalin and eugenol across all formulations (Table 6). Baicalin encapsulation ranged from 95.6 ± 0.5% (F9) to 98.5 ± 0.4% (F7), while eugenol encapsulation ranged from 96.3 ± 0.6% (F4) to 98.6 ± 0.6% (F9). Formulations with lower baicalin concentrations (F1, F4, F7) demonstrated marginally higher baicalin encapsulation efficiency, suggesting potential matrix saturation effects at elevated loading. The consistently high encapsulation values confirm excellent compatibility between the bioactive agents and the chitosan matrix, as well as effectiveness of the preparation methodology.
Table 6. Encapsulation Efficiency of Baicalin and Eugenol (Mean ± SD, n = 3)
| Formulation | Baicalin EE (%) | Eugenol EE (%) |
| F1 | 98.2 ± 0.6 | 97.0 ± 1.0 |
| F2 | 96.8 ± 0.8 | 96.5 ± 0.5 |
| F3 | 95.9 ± 0.5 | 98.0 ± 0.7 |
| F4 | 98.0 ± 0.5 | 96.3 ± 0.6 |
| F5 | 96.5 ± 0.7 | 97.9 ± 0.7 |
| F6 | 95.7 ± 0.6 | 97.2 ± 0.9 |
| F7 | 98.5 ± 0.4 | 97.7 ± 0.8 |
| F8 | 96.7 ± 0.7 | 97.4 ± 0.8 |
| F9 | 95.6 ± 0.5 | 98.6 ± 0.6 |
Fig.7- Drug Content and Encapsulation Efficiency
3.7 Antimicrobial Activity
All scaffold formulations demonstrated antimicrobial activity against both S. aureus and E. coli (Table 7). Inhibition zones against S. aureus ranged from 15.63 ± 0.17 mm (F1) to 17.10 ± 0.30 mm (F9), while zones against E. coli ranged from 13.93 ± 0.73 mm (F1) to 16.00 ± 0.20 mm (F9). One-way ANOVA confirmed statistically significant differences among formulations for both organisms (p < 0.05). The larger inhibition zones observed for S. aureus compared to E. coli are consistent with the generally greater susceptibility of Gram-positive bacteria to chitosan and eugenol’s membrane-disrupting mechanisms. The progressive increase in antimicrobial activity with higher active ingredient loading demonstrates a concentration-dependent effect, with F9 exhibiting optimal antibacterial performance.
Table 7. Antimicrobial Activity of Scaffold Formulations (Zone of Inhibition in mm, Mean ± SD, n = 3)
| Formulation | S. aureus (mm) | E. coli (mm) |
| F1 | 15.63 ± 0.17 | 13.93 ± 0.73 |
| F2 | 16.47 ± 0.38 | 14.37 ± 0.55 |
| F3 | 16.77 ± 0.37 | 15.27 ± 0.25 |
| F4 | 16.03 ± 0.17 | 14.57 ± 0.25 |
| F5 | 16.80 ± 0.24 | 15.30 ± 0.20 |
| F6 | 16.97 ± 0.29 | 15.63 ± 0.21 |
| F7 | 15.83 ± 0.25 | 14.20 ± 0.20 |
| F8 | 16.67 ± 0.29 | 15.47 ± 0.25 |
| F9 | 17.10 ± 0.30 | 16.00 ± 0.20 |
Fig.8- Antimicrobial Activity of Scaffold Formulations
Fig.9- Antimicrobial Activity of Formulations using Disc method
3.8 In Vitro Drug Release
Drug release profiles demonstrated sustained release patterns for both baicalin and eugenol over the 12-hour study period (Table 8). Cumulative baicalin release at 12 hours ranged from 63.6 ± 1.4% (F1) to 79.9 ± 1.8% (F3), while eugenol release ranged from 75.8 ± 1.9% (F1) to 97.4 ± 2.3% (F3). The higher release rates observed for eugenol compared to baicalin reflect differences in molecular size, solubility, and matrix interactions. Formulation F3 exhibited the highest cumulative release for both compounds, correlating with its greater porosity and swelling capacity. Statistical analysis confirmed significant differences among formulations at all time points (p < 0.05), demonstrating clear concentration-dependent release behavior.
Table 8. Cumulative Drug Release at 12 Hours (%, Mean ± SD, n = 3)
| Formulation | Baicalin Release (%) | Eugenol Release (%) |
| F1 | 63.6 ± 1.4 | 75.8 ± 1.9 |
| F2 | 76.9 ± 1.7 | 90.1 ± 2.1 |
| F3 | 79.9 ± 1.8 | 97.4 ± 2.3 |
| F4 | 66.3 ± 1.4 | 80.8 ± 1.9 |
| F5 | 77.3 ± 1.8 | 92.1 ± 2.3 |
| F6 | 71.7 ± 1.7 | 88.0 ± 2.1 |
| F7 | 70.1 ± 1.6 | 85.1 ± 1.9 |
| F8 | 74.4 ± 1.7 | 91.0 ± 2.1 |
| F9 | 78.3 ± 1.8 | 94.4 ± 2.3 |
Fig.10- Cumulative Drug Release at 12 Hours
3.9 Stability Studies
Stability assessment over one month demonstrated acceptable retention of scaffold properties under both storage conditions. Under refrigerated conditions (5 ± 3°C), all formulations maintained physical appearance without visible changes, and parameter variations remained within analytical error. At room temperature (25 ± 2°C, 60 ± 5% RH), slight deepening of yellow coloration was observed in formulations with higher active loading (F3, F6, F9), accompanied by marginal reductions in flexibility. pH values decreased by 0.05-0.09 units, viscosity declined by 2-5 cP, and encapsulation efficiency decreased by 1-2% under room temperature conditions. These changes, while statistically detectable, remained within acceptable limits for pharmaceutical applications, with refrigerated storage providing superior stability preservation.
4. DISCUSSION
The present study successfully developed chitosan-based scaffolds incorporating baicalin and eugenol using a systematic factorial design approach, revealing distinct relationships between formulation composition and scaffold properties.
The slightly acidic pH values (5.60-5.93) align with optimal stability for chitosan-based systems and are suitable for topical wound healing applications (Szymańska & Winnicka, 2015). The progressive pH increase with higher baicalin concentrations likely reflects interactions between baicalin’s phenolic hydroxyl groups and chitosan’s protonated amine functionalities (Zhang et al., 2015). Viscosity measurements demonstrated concentration-dependent increases, suggesting enhanced intermolecular interactions within the chitosan matrix through hydrogen bonding between bioactive agents and the polymer backbone (Liu et al., 2019).
SEM analysis revealed progressive increases in pore size (52-78 μm) and interconnectivity with higher active concentrations, falling within ranges favorable for tissue engineering applications (Chen et al., 2018; Hutmacher, 2000). The inverse relationship between active loading and tensile strength reflects disruption of chitosan’s hydrogen bonding network, with eugenol exerting a plasticizing effect (Kamatou et al., 2012). Nevertheless, all formulations maintained adequate tensile strengths (>1.9 MPa) for wound dressing applications (Murphy et al., 2010).
Formulation F3 demonstrated superior swelling capacity, correlating with larger pore volume and reduced crosslink density, which benefits drug release through enhanced matrix hydration and diffusion pathway formation (Zhang et al., 2017). High encapsulation efficiencies (>95%) for both compounds confirmed excellent compatibility with the chitosan matrix (Smith et al., 2020).
The broad-spectrum antimicrobial activity against both S. aureus and E. coli validates the therapeutic potential, with concentration-dependent inhibition zones confirming additive contributions from baicalin and eugenol to chitosan’s inherent antimicrobial activity (Dash et al., 2011; Kaur et al., 2019). Sustained drug release profiles, with higher eugenol release rates reflecting molecular size and solubility differences, support wound healing applications requiring prolonged antimicrobial activity (Liao et al., 2019).
Response surface analysis revealed significant baicalin-eugenol interaction effects, with high baicalin combined with low eugenol (F6) producing suboptimal performance across multiple parameters. Stability studies confirmed acceptable property retention over one month, with refrigerated storage providing superior preservation.
Collectively, formulation F3, with its optimal balance of high porosity, superior swelling capacity, maximum drug release, and potent antimicrobial activity, represents the most promising candidate for advanced wound care applications. Future investigations should include in vivo biocompatibility assessment and clinical evaluation to establish therapeutic utility.
5. CONCLUSION
This study successfully developed and characterized chitosan-based scaffolds incorporating baicalin and eugenol for potential antimicrobial therapy applications. A 32 full factorial design enabled systematic evaluation of formulation variables and their interactions, providing a rational basis for optimization.
Key findings include: (1) all formulations exhibited slightly acidic pH values (5.60-5.93) suitable for topical application and chitosan stability; (2) viscosity increased proportionally with active ingredient concentration, reflecting enhanced matrix interactions; (3) SEM revealed porous architectures with interconnected pore networks favorable for tissue engineering applications; (4) FTIR confirmed successful incorporation of both bioactive agents without chemical incompatibility; (5) encapsulation efficiencies exceeding 95% validated excellent drug-matrix compatibility; (6) broad-spectrum antimicrobial activity was demonstrated against both Gram-positive and Gram-negative bacteria; and (7) sustained drug release profiles supported potential therapeutic applications.
Formulation F3, containing 3% w/v baicalin and 1.5% v/v eugenol, emerged as the optimal candidate, demonstrating the highest swelling ratio (265.9%), maximum cumulative drug release (79.9% baicalin, 97.4% eugenol at 12 hours), and superior antimicrobial efficacy. Response surface analysis revealed significant baicalin-eugenol interactions influencing scaffold performance, highlighting the importance of factorial design approaches in formulation development.
The developed chitosan-based scaffolds incorporating baicalin and eugenol represent a promising platform for localized antimicrobial therapy, combining the inherent bioactivity of chitosan with the therapeutic properties of natural bioactive compounds. Future research should focus on in vivo biocompatibility evaluation, wound healing efficacy studies, extended stability assessment, and clinical translation to fully realize the therapeutic potential of these novel antimicrobial scaffolds.
Conflict of Interest
The author declares no conflict of interest.
Acknowledgments
The author acknowledges the support and facilities provided by Shri Satsangi Saketdham Ram Ashram Faculty of Pharmacy for conducting this research.
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