Green Synthesis of Iron and Zinc Nanoparticles from Centella Asiatica, Lawsonia Inermis, Ocimum Tenuiflorum and Assessing their Biological Activity for Wound Healing Properties

Wound healing is a complex and dynamic biological process involving a coordinated sequence of events, including hemostasis, inflammation, proliferation, and tissue remodeling. Any disruption in these phases, particularly prolonged inflammation or microbial infection, can delay healing and lead to chronic wounds (Guo & DiPietro, 2010; Eming et al., 2014). Conventional therapeutic strategies, including synthetic anti-inflammatory drugs and antibiotics, are widely used to manage wound-related complications; however, their prolonged use is often associated with adverse effects such as cytotoxicity, antimicrobial resistance, and delayed tissue regeneration (Boateng & Catanzano, 2015; Frykberg & Banks, 2015). This has prompted increasing interest in the development of alternative, biocompatible, and multifunctional therapeutic systems. In recent years, nanotechnology has emerged as a promising approach in biomedical applications, particularly in wound management. Nanoparticles exhibit unique physicochemical properties, such as high surface area-to-volume ratio, enhanced reactivity, and improved penetration ability, which enable them to interact effectively with biological systems (Rai et al., 2009; Zhang et al., 2016). Among various nanomaterials, metal nanoparticles such as iron and zinc have gained considerable attention due to their intrinsic antimicrobial, antioxidant, and anti-inflammatory properties (Wang et al., 2017; Raghupathi et al., 2011). Zinc, an essential trace element, plays a critical role in cell proliferation, collagen synthesis, and immune regulation, making it particularly important in wound healing processes (Lin et al., 2018). Similarly, iron-based nanoparticles are known to facilitate oxygen transport and cellular metabolism, contributing to tissue repair and regeneration (Mahmoudi et al., 2011). Green synthesis of nanoparticles using plant extracts has emerged as an eco-friendly and sustainable alternative to conventional physical and chemical methods. This approach utilizes bioactive phytochemicals such as flavonoids, phenolics, alkaloids, and terpenoids as natural reducing and stabilizing agents, eliminating the need for toxic reagents (Iravani, 2011; Ahmed et al., 2016). Plant-mediated synthesis not only enhances the biocompatibility of nanoparticles but also imparts additional therapeutic properties due to the synergistic effects of phytoconstituents (Shankar et al., 2004; Singh et al., 2018). Medicinal plants such as Centella asiatica, Lawsonia inermis, and Ocimum tenuiflorum have been extensively studied for their pharmacological activities. Centella asiatica is well known for its wound healing potential, attributed to compounds such as asiaticoside and madecassoside, which promote collagen synthesis and angiogenesis (Gohil et al., 2010; James & Dubery, 2009). Lawsonia inermis possesses strong antimicrobial and anti-inflammatory properties due to the presence of lawsone and other phenolic compounds (Habbal et al., 2005). Ocimum tenuiflorum (Tulsi) is recognized for its antioxidant, antimicrobial, and immunomodulatory activities, primarily due to its rich content of eugenol, flavonoids, and phenolic acids (Prakash & Gupta, 2005; Pattanayak et al., 2010). The incorporation of these plant extracts in nanoparticle synthesis enhances their therapeutic efficacy through a synergistic mechanism. In addition to nanoparticles, natural biomaterials such as Aloe vera have been widely used in wound healing applications due to their moisturizing, anti-inflammatory, and antimicrobial properties. Aloe vera contains bioactive compounds such as polysaccharides, anthraquinones, and glycoproteins that promote fibroblast proliferation, collagen deposition, and epithelialization (Hamman, 2008; Chithra et al., 1998). Furthermore, Aloe vera can act as an effective carrier matrix for nanoparticles, improving their stability, controlled release, and bioavailability (Surjushe et al., 2008). The integration of plant-mediated nanoparticles with Aloe vera-based formulations represents a novel nano-herbal approach for wound healing. Such systems combine the advantages of nanotechnology with the therapeutic potential of medicinal plants and natural biomaterials, resulting in enhanced biological activity and reduced toxicity (Mittal et al., 2013; Thakkar et al., 2010). Despite significant progress in this field, there remains a need for comprehensive evaluation of the biological properties of such formulations, including their antioxidant, anti-inflammatory, antimicrobial, and hemocompatibility profiles. Therefore, the present study focuses on the green synthesis of iron and zinc nanoparticles using aqueous extracts of Centella asiatica, Lawsonia inermis, and Ocimum tenuiflorum, followed by their incorporation into an Aloe vera-based nano-herbal formulation. The synthesized nanoparticles were characterized and evaluated for their biological activities relevant to wound healing, including antioxidant, anti-inflammatory, antimicrobial, and toxicity assessments. The study aims to provide insights into the synergistic effects of plant-derived phytochemicals and metal nanoparticles, highlighting their potential as safe and effective alternatives for wound healing applications.

MATERIALS AND METHODS

2.1 Materials and Preparation of Nano-Herbal Formulation

Fresh, mature, and disease-free leaves of Lawsonia inermis, Ocimum tenuiflorum, and Centella asiatica were collected, washed thoroughly with distilled water, shade-dried, and finely powdered. Aqueous extracts were prepared by boiling 10 g of plant powder in 250 mL distilled water for 15–20 min, followed by filtration using Whatman No. 1 filter paper. The filtrates were stored at 4°C until further use. These extracts are known to contain bioactive phytochemicals such as flavonoids, phenolics, and tannins, which act as natural reducing and stabilizing agents in green nanoparticle synthesis (Iravani, 2011; Ahmed et al., 2016). Iron and zinc precursor solutions were prepared using ferrous chloride (FeCl₂) and zinc sulfate (ZnSO₄), respectively. Green synthesis of nanoparticles was performed by the dropwise addition of plant extract into heated metal salt solutions under constant magnetic stirring. The formation of nanoparticles was confirmed by a visible color change due to surface plasmon resonance. The synthesized nanoparticles were collected by centrifugation at 6000 rpm for 15 min, washed repeatedly with distilled water, and dried. Fresh Aloe vera gel was extracted, homogenized, and blended with the synthesized nanoparticles to obtain a uniform nano-herbal formulation. The formulation was dried and later rehydrated prior to experimental applications to improve stability and usability.

2.2 Phytochemical Quantification

The phytochemical composition of the plant extracts and synthesized formulations was evaluated by quantifying total flavonoid and alkaloid contents using established spectrophotometric methods. Total flavonoid content was determined using an aluminum chloride-based colorimetric assay, which relies on the formation of a stable flavonoid–aluminum complex (Chang et al., 2002). Briefly, different concentrations of the sample extracts (50–200 µL) were mixed with the reagent system and incubated under controlled conditions to allow color development. The absorbance of the resulting solution was measured at 415 nm using a UV–Visible spectrophotometer. The flavonoid content was calculated from a standard calibration curve and expressed as equivalent units, where increased absorbance corresponds to higher flavonoid concentration. Total alkaloid content was estimated using a colorimetric method involving the formation of a chromogenic complex with specific alkaloid-detecting reagents (Harborne, 1998). Sample aliquots (50–200 µL) were treated with the reagent system and incubated to allow complete reaction. The absorbance was recorded at 570 nm using a UV–Visible spectrophotometer. Alkaloid concentration was calculated using a standard curve, providing an estimate of the total alkaloid content in the samples.

2.3 Nanoparticle Synthesis and Nano-Herbal Formulation

Fresh, mature, and disease-free leaves of Lawsonia inermis, Ocimum tenuiflorum, and Centella asiatica were collected and thoroughly washed with distilled water to remove surface impurities. The plant materials were shade-dried at ambient temperature and finely powdered. Aqueous extracts were prepared by boiling 10 g of each powdered sample in 250 mL of distilled water for approximately 15–20 min, followed by filtration through Whatman No. 1 filter paper. The filtrates were stored at 4°C for subsequent use. These plant extracts are known to contain diverse phytoconstituents such as flavonoids, phenolics, and tannins, which function as reducing and stabilizing agents during green nanoparticle synthesis (Iravani, 2011; Ahmed et al., 2016). Iron and zinc precursor solutions were prepared using ferrous chloride (FeCl₂) and zinc sulfate (ZnSO₄), respectively. The green synthesis of nanoparticles was carried out by the gradual addition of plant extract into the heated metal salt solution under continuous magnetic stirring. The reaction was monitored visually, and the formation of nanoparticles was indicated by a distinct color change attributed to surface plasmon resonance. The synthesized nanoparticles were separated by centrifugation at 6000 rpm for 15 min, followed by repeated washing with distilled water to remove unreacted constituents. The purified nanoparticles were then dried and stored for further applications. Fresh Aloe vera gel was extracted, homogenized, and incorporated with the synthesized nanoparticles to prepare a uniform nano-herbal formulation. The formulation was dried under controlled conditions and later reconstituted prior to experimental use to ensure consistency and stability.

2.4 Characterization of Nanoparticles

The synthesized nanoparticles were characterized using a combination of analytical techniques to determine their morphology, functional groups, and optical properties Surface morphology and particle size distribution were examined using Scanning Electron Microscopy (SEM) (Goldstein et al., 2003). Dried nanoparticle samples were mounted on carbon-coated stubs and sputter-coated with a thin layer of gold to improve conductivity. The samples were then observed under SEM at suitable magnifications, and images were analyzed to assess particle shape, size, and distribution. The presence of well-defined and uniformly distributed particles confirmed successful nanoparticle synthesis. Fourier Transform Infrared (FTIR) spectroscopy was employed to identify functional groups involved in the reduction and stabilization of nanoparticles (Stuart, 2004). The dried nanoparticle samples were mixed with potassium bromide (KBr) and compressed into pellets. Spectral analysis was performed in the range of 4000–400 cm⁻¹. Characteristic absorption peaks corresponding to hydroxyl, carbonyl, and amine groups were identified, indicating the involvement of plant-derived biomolecules in nanoparticle formation.UV–Visible spectroscopy was used to confirm nanoparticle formation and evaluate their optical properties (Kelly et al., 2003). The nanoparticle suspension was diluted appropriately and scanned over a wavelength range of 200–800 nm. The appearance of characteristic absorption peaks due to surface plasmon resonance provided confirmation of nanoparticle formation and insights into particle size and distribution.

2.5 DPPH Radical Scavenging Assay

Antioxidant activity was evaluated using the DPPH radical scavenging assay (Brand-Williams et al., 1995). A 1 mL DPPH solution in methanol was added to varying concentrations of samples (50–250 µL). The mixtures were incubated in the dark for 30 min at room temperature. Absorbance was measured at 520 nm, and the percentage inhibition of DPPH radicals was calculated.

2.6 Anti-inflammatory assay

2.6.1 Protein Denaturation Assay

Anti-inflammatory activity was assessed using the protein denaturation assay (Mizushima and Kobayashi, 1968). Reaction mixtures containing phosphate buffer (pH 7.4), bovine serum albumin (BSA), and sample extracts were incubated and heated at 70°C. After cooling, absorbance was measured at 680 nm. The percentage inhibition of protein denaturation was calculated.

2.6.2 Human Red Blood Cell (HRBC) Membrane Stabilization Assay

The HRBC membrane stabilization assay was performed to evaluate anti-inflammatory activity (Shinde et al., 1999). Fresh human blood was processed to obtain RBC suspension. Samples at varying concentrations were incubated with RBCs under hypotonic conditions. After incubation and centrifugation, absorbance of the supernatant was measured at 540 nm. The percentage inhibition of hemolysis was calculated.

2.7 Toxicity analysis

2.7.1 Blood Clotting Index (BCI) Assay

Hemostatic activity was evaluated using the Blood Clotting Index (BCI) assay (Li et al., 2008). Fresh human blood (200 µL) was mixed with test samples and incubated to allow clot formation. Distilled water was added to lyse non-clotted RBCs, and absorbance was measured at 540 nm. Lower absorbance indicated better clotting ability.

2.7.2 Hemolysis Assay for Hemocompatibility

Hemocompatibility was assessed using the hemolysis assay (ISO 10993-4, 2017). RBC suspensions (2%) were treated with different sample concentrations. PBS and distilled water were used as negative and positive controls, respectively. After incubation and centrifugation, absorbance was measured at 540 nm. Percentage hemolysis was calculated to determine cytotoxicity.

2.8 Anti-Microbial activity

2.8.1 Minimum Inhibitory Concentration (MIC) Assay

Antimicrobial activity was evaluated using the broth dilution method (Andrews, 2001). LB broth was prepared, sterilized, and inoculated with bacterial cultures. Test samples were added at varying concentrations and incubated at 37°C for 24 h. Optical density was measured at 600 nm, and percentage inhibition was calculated.

2.8.2 Agar Well Diffusion Method

The antibacterial activity was further confirmed using the agar well diffusion method (Bauer et al., 1966). LB agar plates were prepared and inoculated with bacterial cultures. Test samples were introduced into wells, and plates were incubated at 37°C for 24 h. Zones of inhibition were measured to evaluate antimicrobial efficacy.

3. Results and Discussion

3.1 Extraction and Quantification of Phytochemical

3.1.2. Quantification of Flavonoids

The flavonoid quantification results (Table 1; Fig. 1) demonstrated a clear concentration-dependent increase across all plant extracts. Among the samples, Ocimum tenuiflorum (Tulsi) and Centella asiatica (Vallarai) exhibited comparatively higher flavonoid content at elevated concentrations, whereas Lawsonia inermis (Henna) showed a consistent increase throughout the tested range. The enhanced flavonoid levels at higher concentrations suggest improved extraction efficiency and availability of polyphenolic compounds. These findings are significant, as flavonoids are known to contribute to antioxidant and anti-inflammatory responses, which are critical in wound healing processes. The comparatively higher values observed in Tulsi and Vallarai indicate their stronger phytochemical potential, supporting their selection for nanoparticle synthesis.

Table 1. Total flavonoid content of plant extracts (mg RE/g extract)

Figure 1. Concentration-dependent increase in flavonoid content of plant extracts.

3.1.2 Total Alkaloid Content

Table 2. Total alkaloid content of plant extracts (mg AE/g extract).

Figure 2. Variation in alkaloid content with concentration.

As shown in Table 2 and Fig. 2, alkaloid content increased steadily with concentration for all samples. Vallarai exhibited the highest alkaloid levels, followed by Tulsi and Henna. The elevated alkaloid content suggests strong antimicrobial and pharmacological potential, as these compounds are known to interfere with microbial metabolism and inflammatory pathways. The higher initial and final values observed in Vallarai indicate its superior bioactive composition, making it particularly relevant for biomedical applications. Overall, the phytochemical results confirm that all three plant extracts are rich in therapeutic compounds, with concentration playing a key role in their availability.

3.2 Synthesis and Characterization of Nanoparticles

The green synthesis of iron and zinc nanoparticles using aqueous extracts of Lawsonia inermis, Ocimum tenuiflorum, and Centella asiatica was successfully achieved, as evidenced by visual, spectroscopic, and microscopic analyses. The formation of nanoparticles was initially indicated by a distinct color change in the reaction mixture upon addition of plant extracts to the metal precursor solutions. This change is attributed to the reduction of metal ions into nanoscale particles mediated by plant-derived phytochemicals such as flavonoids, phenolics, and proteins, which also act as stabilizing agents.

3.2.2 UV–Visible Spectroscopy Analysis

The UV–Visible spectroscopic analysis (Fig. 3a–b) confirmed the successful formation of nanoparticles. Zinc nanoparticles exhibited a characteristic absorption peak in the range of 320–380 nm, corresponding to band-gap absorption of ZnO nanoparticles. In contrast, iron nanoparticles showed a broad absorption band between 250 and 350 nm. The increase in absorbance intensity indicates progressive nanoparticle formation and improved dispersion stability. The broad peak observed in FeNPs suggests polydispersity and possible formation of mixed iron oxide phases, which is commonly reported in green synthesis approaches.

3.2.3 Fourier Transform Infrared (FTIR) Analysis

FTIR analysis (Fig. 4a–b) was performed to identify functional groups responsible for nanoparticle synthesis and stabilization. The spectra revealed a broad peak around 3400 cm⁻¹ corresponding to O–H stretching vibrations of phenolic compounds. Peaks near 2920 cm⁻¹ indicate C–H stretching, while bands observed at 1600–1650 cm⁻¹ correspond to C=O stretching of carbonyl groups. Additional peaks in the regions of 1400 cm⁻¹ and 1000–1100 cm⁻¹ were attributed to C–N and C–O stretching vibrations, respectively. The presence of distinct peaks below 600 cm⁻¹ confirms metal–oxygen bonding (Zn–O and Fe–O), validating nanoparticle formation. These results clearly indicate the involvement of plant-derived biomolecules in reducing and capping the nanoparticles.

3.2.4 Scanning Electron Microscopy (SEM) Analysis

SEM analysis (Fig. 5a–b) revealed that the synthesized nanoparticles predominantly exhibited spherical to irregular morphologies with noticeable agglomeration. The particle size ranged approximately between 20 and 100 nm. Zinc nanoparticles appeared relatively smaller and more uniformly distributed compared to iron nanoparticles, which showed a higher tendency toward aggregation. This aggregation behavior may be attributed to high surface energy and the presence of biological capping agents derived from plant extracts. The observed morphology confirms the successful green synthesis of nanoparticles and reflects the influence of phytochemicals on particle formation and stabilization.

3.3 Anti-Inflammatory Activity

3.3.1 HRBC Membrane Stabilization Assay

The HRBC assay results (Table 3) demonstrated that iron nanoparticles exhibited higher membrane stabilization compared to zinc nanoparticles. Henna-mediated FeNPs showed the highest stabilization (64.3%), indicating strong anti-inflammatory potential. The incorporation of Aloe vera significantly enhanced the activity, with inhibition values reaching up to ~90% (Fig. 9). This enhancement may be attributed to the synergistic interaction between nanoparticles and Aloe vera, which improves membrane integrity and reduces hemolysis.

Table 3. HRBC membrane stabilization (%) of nanoparticles.

3.3.2 Protein Denaturation Assay

The protein denaturation assay (Table 4; Fig. 10) showed that FeNPs exhibited higher inhibition compared to ZnNPs at elevated concentrations. Tulsi-based FeNPs demonstrated the highest inhibition (74.8%). The results indicate that nanoparticles effectively stabilize proteins under stress conditions, thereby reducing inflammatory responses.

Table 4. Percentage inhibition of protein denaturation.

Figure 10. Protein denaturation inhibition profile.

3.4 Antioxidant Activity (DPPH Assay)

The antioxidant results (Table 5 and Table 6; Fig. 11) showed a concentration-dependent increase in radical scavenging activity. Vallarai-mediated nanoparticles exhibited the highest activity (71.5%), indicating strong antioxidant potential. Interestingly, Aloe vera formulations showed slightly reduced activity, likely due to dilution or interaction effects within the gel matrix.

Table 5. DPPH radical scavenging activity of iron nanoparticles.

Table 6. DPPH activity of Aloe vera-based formulations.

Figure 11. DPPH radical scavenging activity.

3.5 Toxicity Analysis

3.5.1 Hemostatic Activity (BCI Assay)

The BCI results (Table 7 and Table 8; Fig. 12) indicated that lower concentrations exhibited better clotting efficiency. Zinc nanoparticles showed comparatively better hemostatic performance than iron nanoparticles. Higher concentrations resulted in reduced clotting efficiency, emphasizing the importance of dose optimization.

Table 7. Blood clotting index (%) of nanoparticles.

Table 8. Blood clotting index (%) of Aloe vera formulations.

Figure 12. Blood clotting index comparison.

3.5.2 Hemocompatibility (Hemolysis Assay)

The hemolysis assay (Table 9 and Table 10; Fig. 13) demonstrated that Vallarai-mediated nanoparticles exhibited the lowest hemolysis (2.6%), indicating superior biocompatibility. Aloe vera incorporation improved hemocompatibility across all samples, likely due to its membrane-protective properties.

Table 9. Percentage hemolysis of nanoparticles.

                                    Table 10. Percentage hemolysis of Aloe vera formulations.

Figure 13. Hemolysis percentage of samples.

3.6 Antimicrobial activity

3.6.1 MIC Assay

The MIC results (Table 11 and Table 12; Fig. 14) revealed moderate antibacterial activity, with Henna showing relatively higher inhibition. Aloe vera formulations demonstrated enhanced activity, particularly at higher concentrations.

Table 11. MIC-based bacterial inhibition (%) of nanoparticles.

Table 12. MIC-based inhibition of Aloe vera formulations.

Figure 14. Bacterial growth inhibition (OD600).

3.6.2 Agar Well Diffusion Assay

Table 13. Zone of inhibition (mm) of nanoparticle formulations.