2.1 Chemicals

Poly-ɛ-caprolactone (Mw-80,000, Sigma Aldrich, UK), Porcine Gelatin (Gel strength 300-Type A, Sigma Aldrich, USA), Dulbecco’s Modified Eagle Medium (DMEM), Phosphate-buffered saline (PBS) (Hi-Media, India), Acetic acid, Ethanol (Analytical reagent), 2,2,2 Trifluoroethanol (TFE), Acridine Orange (AO), Ethidium bromide (EtBr) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), and DPPH (diphenyl-1-picrylhydrazyl) (SRL, India).

2.2 Processing of Melia dubia leaves

MD leaves were cleaned, shade dried for 3 weeks, then made into powder using a mechanical blender. To obtain ethanolic plant extract, 25 g of leaf powder was added in 0.5 L of 70 % ethanol and stirred for 3 h in a shaker. For extraction, the stirred mixture was placed in an ultrasonic water bath at 60 ℃ for 60 min. The extract was then filtered, concentrated and stored at 4 °C. The GCMS analysis of the extract was performed to identify the presence of phytochemical using the mass spectral patterns stored in NIST library (Konappa et al., 2020).

2.3 Electrospinning of nanofiber

Nanofiber was fabricated by ESPIN-Nano, Physics Instrument Co., Chennai, India. PCL (8 %), gelatin (4 %) and plant extract (25 %) (respect to PCL Wt. %) were added to the TFE and kept in stirring condition for 14 h. The polymer mixture was taken in a plastic syringe (5 mL) and 13 kV high voltage was applied. The polymer solution at the needle orifice begun to stretch and the nanofibers were collected on the collector that was kept 14 cm away from the tip of the needle.

2.4 Characterization of synthesized nanofiber

The morphology was identified using HR-SEM (Thermo Scientific Apreo S), Functional groups of the nanofiber were identified using FTIR (Shimadzu, IR Tracer 100), crystalline nature of the compound was determined using XRD (PANalytical, Netherlands), and the wettability of NFs was determined by contact angle analysis (KYOWA DMS-401, Japan), Mechanical property of the NFs was analysed using tensile strength tester (Instron, USA).

2.5 In vitro drug release

50 mg of the plant extract incorporated nanofiber was submerged in 3 mL of PBS (pH 7.4). At different time interval, 3 mL of PBS was taken from the release medium, at the same time replaced with 3 mL of fresh PBS. The different concentration of standard was prepared using plant extract and absorbance was measured at 268 nm.

2.6 Antioxidant assay

The antioxidant activity of NF was examined at a fixed time point. For that 10 mg of PCL/Gel, PCL/Gel/MD NFs are added to 5 mL of 50 µm DPPH (dissolved in methanol) solution and kept in dark for 30 min. The antioxidant activity of the NF was analyzed using UV spectrophotometer at 514 nm. Antioxidant activity was evaluated using equation (1).

A- denotes absorbance of DPPH solution and A1-denotes absorbance of nanofibers immersed DPPH solution.

2.7 Antibacterial studies

PCL/Gel/MD NFs antibacterial activity was analyzed by disc diffusion method. The clinical pathogens (Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), Methicillin-Resistant Staphylococcus aureus (multidrug-resistant S. aureus) and Pseudomonas aeruginosa (P. aeruginosa)) were obtained from Department of Microbiology, SRM Institute of Medical College Hospital and Research Center, Kattankulathur, Chengalpattu. The bacterial cultures were (1.5 × 10⁸ CFU/mL) spread on Muller Hinton Agar (MHA). Then UV sterilized PCL/Gel and PCL/Gel/MD NFs (30 × 30 mm) were placed on MHA and incubated at 36 ± 1 °C for 24 h.

2.8 Cell culture

3.1 GCMS analysis

GCMS analysis of MD plant extract demonstrated the presence of numerous bioactive compounds that enhances the wound healing process (Fig. 1). The detected compounds are presented in Table 1. The bioactive compounds like squalene, vitamin E, octadecanoic acid, ethyl ester, azafrin, heneicosane, palmitic acid, ethyl ester and 4-epi-cubedol has antioxidant, antibacterial and anti-inflammatory properties (Brigelius-Flohé, 2006; Eswaraiah et al., 2020; Islam et al., 2018; Rosselli et al., 2019; Vanitha et al., 2020). Presence of these bioactive compounds confirms that plant can be suitable for enhancing the wound healing process.

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Fig. 1

GC–MS spectrum of ethanolic extract of Melia dubia leaf extract.

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3.2 HR-SEM analysis

The structure and morphology of plant extract-loaded nanofiber were investigated using HRSEM. Fig. 2a & c shows the random orientation of electrospun nanofiber with a bead-free, continuous, smooth, narrow size distribution and also no particulate aggregates were found indicating no phase separation occurred during electrospinning after the incorporation of plant extract. The mean diameter for PCL/Gel and PCL/Gel/MD NFs (Fig. 2b & d) was found to be 319 ± 104 and 256 ± 89 nm, respectively. Previous study reports incorporation of Gymnema sylvestre and Henna plant extract into polymer solution decreased the fiber diameter due to higher charge density and conductivity (Ramalingam et al., 2021; Yousefi et al., 2017). PCL/Gel/MD NFs were found to be entangled compared to PCL/Gel NFs. Rosa et al. (2017) reported the entangled NFs helps in improving the nutrition infiltration, new cell integration, oxygen permeation, long-term medication release and wound exudate clearance (Rosa et al., 2017).

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Fig. 2

HRSEM images and histogram plot of (a&b) PCL/Gel and (c&d) PCL/Gel/MD NFs (Magnification-5000X). Insert represents higher magnification image of nanofibers ((Magnification- 15000X).

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3.3 Functional group analysis NFs

The chemical bonding of PCL/Gel NFs and PCL/Gel NFs with MD plant extract was determined by FTIR analysis as given in Fig. 3a. The peaks obtained at 2942 and 1723 cm⁻¹ in PCL/Gel nanofiber represents asymmetric –CH₂ and C = O stretching vibration that corresponds to PCL. N–H stretching associated with the peaks at 3309 cm⁻¹, C = O amide I stretching at 1648 cm⁻¹, N–H bending associated at 1652 cm⁻¹ and stretching at 1040 cm⁻¹ associated with C = O corresponds to gelatin. The sole difference between PCL and gelatin is the NH₂ and OH stretchings (Chong et al., 2015). In-plant extract, stretching at 3043 cm⁻¹ represents O–H stretching and 1032 cm⁻¹ represents the C-O stretching. In PCL/Gel/MD NFs, the plant extract band was overlapped in PCL/Gel NFs at the wavenumber of 3043, 2612, 1211, 1052, 795 and 673 cm⁻¹.

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Fig. 3

a) FTIR spectrum of plant extract, PCL/Gel/ MD NFs, PCL/Gel NFs, gelatin, and PCL, b) XRD spectrum of plant extract, gelatin, PCL and PCL/Gel/MD NFs, c) contact angle analysis of PCL/Gel and PCL/Gel/MD NFs, d) cumulative drug release percentage of PCL/Gel/MD NFs.

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3.4 XRD analysis

Fig. 3b depicts the PCL/Gel & PCL/Gel/MD NFs XRD spectrum. In PCL peaks at 2θ = 21.7° and 24.8° indicate the plane of 110 and 200, respectively indicating the crystalline nature of PCL. The plant and gelatin exhibits the amorphous nature (Fig. 3b). The peak intensity of PCL/Gel/MD NFs is lesser than PCL which is due to the incorporation of plant extract. Gautam et al. (2013) stated that incorporating the amorphous components decreased the intensity and semi-crystalline nature of PCL NFs (Gautam et al., 2013).

3.5 Water contact angle analysis

The hydrophilic property of the nanofibers was analysed using water contact angle analysis. The water contact angle was found to be 51.16 ± 5.26° and 22.93 ± 4.3° for PCL/Gel and PCL/Gel/MD NFs, respectively (Fig. 3c). The wettability of MD plant extract loaded nanofibers was found to be increased when compared to PCL/Gel nanofibers; this can enhance the absorption of wound ooze outs and keeps a moist environment around the wound bed (Yeum et al., 2016). Unalan et al. (2019) reported that the existence of polar phytocomponents and hydrophilic groups in the plant enhances the hydrophilicity of the PCL/Gel NFs (Unalan et al., 2019). The wettability of NFs exhibits a dynamic role in the adhesion and proliferation of the fibroblast cells.

3.6 In-vitro drug release

Fig. 3d demonstrates the drug release profile of PCL/Gel/MD NFs. About 75.9 % of plant extract was entrapped in NFs. In the first 2 h of incubation, initial burst release was observed which might be due to the dissolution of plant extract present in the surface of the nanofibers. After that, a continuous and sustained drug release was observed up to 72 h, releasing about 74 % of the drug from the initial concentration. The extended drug release of PCL/Gel/MD NFs is due to slow degradation of gelatin entangled with PCL (Xue et al., 2014). The initial burst release of the plant extract helps to reduce the bacterial colonization (Ramalingam et al., 2019b).

3.7 Mechanical strength analysis

Mechanical properties of the NFs plays important role in tissue engineering applications. The incorporation of plant extract into polymer increased their mechanical strength (Table 2). Raghavendra et al. (2019) reported that incorporation of plant extract increased tensile strength, toughness and tensile modulus in nanofibers (Ramalingam et al., 2019a). Selvaraj et al. (2017) fabricated fenugreek incorporated nanofiber with enhanced mechanical property compared to silk fibroin (Selvaraj and Fathima, 2017). Small fiber with densely packed fibrillar structure with more entanglements enhanced the mechanical properties, in case of PCL/Gel NFs alignment of NFs with increased fiber diameter decreased the mechanical property (Lim et al., 2008).

3.8 Antioxidant assay

The excess amount of oxidative stress leads to impaired wound healing. The antioxidants helps to control the oxidative stress in wound site thereby accelerating the wound healing process (Fitzmaurice et al., 2011; Krochmal-Marczak et al., 2021). The antioxidant property of nanofibers was evaluated by antioxidant assay (DPPH) shown in Fig. 4a & b. The DPPH solution exhibits maximum absorbance at 514 nm. PCL/Gel and PCL/Gel/MD NFs exhibited the DPPH scavenging activity of 16.65 ± 0.18 and 49.27 ± 0.53 %, respectively. The antioxidant activity of the PCL/Gel NFs, might be due to the presence of gelatin (radical scavenging peptide) (Mendis et al., 2005). GCMS and FTIR analysis of the MD extract confirms presence of phenolic compounds/groups. Kanthaswamy et al. (2019) fabricated quinone-based chromenopyrazole antioxidant-laden silk fibroin NFs, reduced oxidative stress and enhanced the dense growth of the fibroblasts (Kandhasamy et al., 2019).

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Fig. 4

a) & b) antioxidant activity of PCL/Gel and PCL/Gel/MD NFs after 30 min incubation, c) Antibacterial activity PCL/Gel and PCL/Gel/MD NFs against Gram-positive and negative organisms.

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3.9 Kirby-Bauer disc diffusion analysis

The antibacterial effect of the nanofibers was assessed using disc diffusion method (Fig. 4c). PCL/Gel NFs has no antibacterial activity against the wound pathogens. Zone of inhibition for MD plant extract incorporated NFs against E. coli, S. aureus, multidrug-resistant S. aureus, and P. aeruginosa were 18.2 ± 1.5, 26.5 ± 2.0, 23.0 ± 0.9 and 28.1 ± 1.2 mm, respectively (excluding fiber diameter). The ethanolic extract of MD leaves have terpenoids, phenolic derivatives, lipophilic compounds and limonoids as bioactive compounds which possess antibacterial activity against clinical pathogens (Goswami et al., 2020; Thangavel et al., 2019). Bioactive compounds inhibit bacterial growth by forming complex with cell wall, extracellular matrix and soluble proteins (El-Zayat et al., 2021). Zheng et al. (2005) reported unsaturated fatty acids exhibited antibacterial activity by inhibiting the bacterial enoyl-acyl carrier protein reductase (Zheng et al., 2005).

3.10 Scratch assay

The cells were scratched artificially in the center, causing the cells to migrate to the damaged location. The pace, pattern and direction of cell migration may all be analyzed using this assay (Kandhasamy et al., 2019). By densitometry analysis, the percentage of wound healing or the cell migration percentage was calculated. These values were comparable to PCL/Gel nanofiber levels. When compared to PCL/Gel NFs, nanofiber containing plant extract enhanced the healing rate, which was observed over a longer period of up to 24 h (Fig. 5a). The mean percentage of cell migration achieved for PCL/Gel was 87.74 % and 97.13 % for the PCL/Gel/MD NFs.

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Fig. 5

a) Wound healing assay of PCL/Gel NFs and PCL/Gel/MD NFs, b) MTT analysis of TCPS, PCL/Gel and PCL/Gel/MD NFs at 24 and 48 h. Two way ANOVA was performed. p > 0.05 is considered as nonsignificant (ns) c) Confocal image of PCL/Gel and PCL/Gel/MD NFs after 48 h (scale bar- 50 µm).

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3.11 MTT assay and confocal analysis

Biocompatibility of PCL/Gel and PCL/Gel/MD NFs was assessed by MTT assay and confocal analysis. Compared to control (TCPS), the PCL/Gel and PCL/Gel/MD NFs extract-treated wells showed increased color (purple) intensity. It indicates that PCL/Gel/MD NFs positively enhance the proliferation of L929 cells at 24 and 48 h. Fig. (5b&c) indicates that the plant extract has proliferating ability and no cytotoxicity. MD ethanolic extract enhances collagen synthesis and hexosamine in wound surfaces (Thangavel et al., 2019). Ajmal et al. (2019) reported that gelatin and flavonoid content in the nanofiber enhanced fibroblast proliferation (Ajmal et al., 2019).