0% READ
FULL TEXT
INTRODUCTION
The skin is the largest organ of the human body and plays a vital role as the first line of defense against environmental pollutants, Ultraviolet (UV) radiation, and microbial pathogens. Continuous exposure to these external stressors can impair skin function, leading to infections, inflammation, acne, and premature aging. As a result, maintaining skin health has become a major focus within both the pharmaceutical and cosmetic industries. Topical delivery systems, particularly creams, are widely preferred due to their ease of application, ability to deliver active ingredients locally, and improved patient compliance. In addition to providing hydration and occlusion, creams serve as effective carriers for therapeutic agents such as antioxidants and antimicrobial compounds (James and Dubery, 2009; Mishra et al., 2000).
Recent advances in nanotechnology have significantly influenced the development of modern skincare formulations by enhancing the stability, penetration, and bioavailability of active ingredients. Among various nanomaterials, Zinc Oxide (ZnO) nanoparticles have attracted considerable attention because of their broad-spectrum antimicrobial activity, effective UV protection, anti-inflammatory properties, and favorable biocompatibility. However, conventional synthesis methods for ZnO nanoparticles often involve toxic chemicals and high energy requirements, raising concerns regarding environmental safety and potential skin toxicity. In contrast, green synthesis approaches using plant extracts offer a sustainable and environmentally friendly alternative. Phytochemicals present in plant extracts act as natural reducing and stabilizing agents, while also contributing additional biological functionality to the nanoparticles (Raghupathi et al., 2011; Shukla et al., 1999).
Withania somnifera (Ashwagandha) and Centella asiatica (Gotu Kola) are well-recognized medicinal plants widely used in traditional medicine systems. Ashwagandha contains withanolides, which exhibit notable antioxidant, anti-inflammatory, and antimicrobial activities (Zhang et al., 2007). Similarly, C. asiatica is rich in triterpenoids such as asiaticoside and madecassoside, compounds known to promote wound healing, enhance collagen synthesis, and support skin regeneration. Beyond their therapeutic benefits, these plant extracts play a key role in the green synthesis process by facilitating nanoparticle formation and stabilization, thereby enhancing the biological performance of the final formulation (Babayevska et al., 2022; Sharma et al., 2020).
The antimicrobial activity of ZnO nanoparticles is attributed to multiple mechanisms, including the generation of Reactive Oxygen Species (ROS), disruption of microbial cell membranes, and the release of Zn²⁺ ions that interfere with essential enzymatic functions. These properties make ZnO nanoparticles particularly suitable for topical applications aimed at preventing infections, supporting wound healing, and protecting the skin barrier. When combined with herbal extracts, ZnO nanoparticles offer a synergistic approach that enhances formulation performance while aligning with the increasing demand for natural, safe, and sustainable skincare solutions (Padalia et al., 2015).
The present study focuses on the green synthesis of ZnO nanoparticles using extracts of Withania somnifera and Centella asiatica, followed by their incorporation into an oil-in-water cream formulation. The developed formulations were evaluated for physicochemical properties, antimicrobial activity, spreadability, and stability. This work highlights a promising herbal nanotechnology-based approach for the development of effective and sustainable topical skincare formulations.
MATERIALS AND METHODS
Materials
Withania somnifera and Centella asiatica extracts were kindly provided by Kapiva Ayurveda (Bangalore, India). Sodium hydroxide pellets and zinc acetate were procured from NR Chemicals, Bangalore. Stearic acid, cetyl alcohol, glycerin, liquid paraffin, beeswax, borax, triethanolamine, and methyl paraben were of analytical grade and used as received without further purification.
Pre-formulation Studies
Pre-formulation studies were conducted to evaluate the organoleptic properties, solubility, and pH of Withania somnifera (Ashwagandha) and Centella asiatica extracts. These assessments were performed to evaluate the suitability of the extracts for green synthesis of zinc oxide nanoparticles and their incorporation into topical cream formulations.
Method Development for HPLC Analysis
A High-Performance Liquid Chromatography (HPLC) method was developed for the quantitative estimation of withanolides using a C18 column (4.6 × 250 mm) maintained at 27ºC. The mobile phase consisted of phosphate buffer (prepared by dissolving 0.14 g of KH₂PO₄ and adding 1 mL of Orthophosphoric Acid (OPA) to 1000 mL of distilled water) and acetonitrile. Methanol was used as the diluent. The sample solution was prepared by dissolving 50 mg of Withania somnifera extract in methanol, while the standard solution was prepared using 5 mg of withanolide, followed by sonication and mild heating to ensure complete dissolution. Chromatographic analysis was carried out at a flow rate of 1.5 mL/min with an injection volume of 20 µL, and detection was performed at 227 nm.
Formula for Assay
Formulation of Zinc Oxide Nanoparticles
Zinc Oxide (ZnO) nanoparticles were initially synthesized by mixing 100 mL of a 0.1 M zinc acetate solution with varying amounts of Centella asiatica and Withania somnifera extracts. The reaction mixture was subjected to controlled alkaline precipitation by the dropwise addition of sodium hydroxide solution until a pH of 8 was achieved. The mixtures were continuously stirred at room temperature for 3 hr and subsequently centrifuged at 3000 rpm to separate the formed nanoparticles. The resulting light yellow precipitate was collected and dried at 60ºC for 4-6 hr. Various formulation parameters, including plant extract ratios, sodium hydroxide concentration, and drying time, were refined to achieve maximum nanoparticle yield (Iqbal et al., 2019; Singh et al., 2018; Singh et al., 2016).
Identification of ZnO Nanoparticles
The formation of ZnO nanoparticles was confirmed using UV-Visible spectrophotometry. A measured quantity of the dried nanoparticles was dispersed in deionized water and sonicated for 10 min to obtain a uniform suspension. The sample was scanned over a wavelength range of 200-800 nm using deionized water as the blank. The characteristic absorption peak corresponding to ZnO nanoparticles was recorded to confirm nanoparticle formation.
Zinc Oxide Nanoparticle Formulation
Based on preliminary studies, ZnO nanoparticles were synthesized using a 0.5 M zinc acetate solution. This solution was prepared by dissolving 27.44 g of zinc acetate dihydrate in 200 mL of distilled water and adjusting the final volume to 250 mL. A 2 M sodium hydroxide solution was prepared by dissolving 8 g of sodium hydroxide pellets in 70-80 mL of distilled water and making up the volume to 100 mL. For nanoparticle synthesis, 2.5 mL of the 0.5 M zinc acetate solution was mixed with 1, 1.5, or 2 g of each plant extract and stirred at room temperature for 2 hr. The sodium hydroxide solution was then added dropwise until the reaction mixture reached pH 8. The mixture was further stirred for 1 hr, during which a light yellow precipitate formed, indicating nanoparticle formation. The precipitate was collected by centrifugation at 3000 rpm, transferred to a Petri dish, and dried at 60ºC for 6 hr.
Drug Loading Efficiency of ZnO Nanoparticles
Drug loading efficiency was defined as the ratio of withanolides associated with the ZnO nanoparticles to the total amount of withanolides used during formulation. The quantities of free (unloaded) and entrapped (loaded) withanolides were determined using High-Performance Liquid Chromatography (HPLC), a sensitive and selective analytical technique. Higher loading efficiency indicates effective incorporation of bioactive compounds into the nanoparticles, enabling reduced nanoparticle dosage while maintaining adequate therapeutic levels.
Formula for loading efficiency
In vitro Drug Release Studies of ZnO Nanoparticles
The in vitro release of withanolides from ZnO nanoparticles with high loading capacity was studied using the membrane diffusion method. The extract-loaded ZnO nanoparticles were placed in a dialysis membrane and immersed in 100 mL of Phosphate-Buffered Saline (PBS, pH 7.4) containing 10% v/v methanol as the receptor medium to maintain sink conditions. The receptor medium was maintained at 37ºC under continuous stirring using a magnetic stirrer. At predetermined time intervals, 5 mL aliquots were withdrawn and replaced with an equal volume of fresh receptor medium to maintain constant volume. The collected samples were analyzed for withanolide content using High-Performance Liquid Chromatography (HPLC).
FTIR STUDIES
Fourier-Transform Infrared (FTIR) spectroscopy was employed to analyze the chemical characteristics of ZnO nanoparticles and their compatibility with excipients used in the skincare cream formulation. FTIR spectra of the ZnO nanoparticles and ZnO nanoparticles incorporated with excipients were recorded using a Bruker ATR Alpha spectrophotometer at an ambient temperature of 25.0±0.5ºC. The samples were placed directly on a Zinc Selenide (ZnSe) crystal plate, and spectra were recorded over the wavenumber range of 4000-400 cm⁻¹.
Particle Size Analysis and Zeta Potential Measurement
The average particle size of the prepared ZnO nanoparticles was determined using the Dynamic Light Scattering (DLS) technique. A small volume of the nanoparticle dispersion was diluted with double-distilled water to minimize multiple scattering effects. The diluted sample was transferred to a clean cuvette and analyzed using a particle size analyzer (Horiba SZ-100) at 25ºC. The mean particle diameter was recorded in nanometers (nm), and the Polydispersity Index (PDI) was determined to assess the uniformity of particle size distribution. The surface charge of the ZnO nanoparticles was measured by zeta potential analysis based on Electrophoretic Light Scattering (ELS). The nanoparticle dispersion was diluted with double-distilled water in a 1:10 ratio to ensure appropriate conductivity and reduce signal interference. The diluted sample was loaded into a disposable zeta cell and analyzed using the same instrument (Horiba SZ-100) at 25ºC after proper calibration. Zeta potential values were recorded in millivolts (mV).
Scanning Electron Microscopy (SEM) Analysis
The morphology and surface characteristics of the green-synthesized ZnO nanoparticles were examined using Scanning Electron Microscopy (SEM). For sample preparation, the synthesized nanoparticles were dispersed in methanol, an evaporating solvent, and a thin layer of the dispersion was placed onto a clean glass slide. The solvent was allowed to evaporate at room temperature, and excess liquid was removed using blotting paper prior to SEM analysis.
Formulation of Skincare Cream
Two topical cream formulations with different base compositions were prepared using the prepared ZnO nanoparticles exhibiting high withanolide entrapment efficiency. The formulations were designed to evaluate the influence of different cream bases on physicochemical properties, stability, and performance (Table 1).
| Sl. No. | Ingredients | Quantity for Formulation 1 | Quantity for Formulation 2 |
|---|---|---|---|
| ZnO NP’s | 1 g | 1 g | |
| Stearic acid | 6 g | -- | |
| Cetyl alcohol | 1.5 g | -- | |
| Glycerin | 3 mL | -- | |
| Methyl paraben | 0.1 g | -- | |
| Triethanolamine | 0.75 g | -- | |
| Bees wax | -- | 6 g | |
| Liquid paraffin | -- | 18 mL | |
| Borax | -- | 0.3 g | |
| water | 35.1 mL | 6 mL | |
| Linseed oil | 1 mL | 1 mL | |
| Rose water | Q.S | Q.S |
Formulation 1: Stearic Acid-Based (Simple emulsion Type)
The cream was prepared by separately melting the oil phase, consisting of stearic acid and cetyl alcohol, and heating the aqueous phase containing glycerin, triethanolamine, and methyl paraben to 70ºC. Zinc oxide nanoparticles were dispersed into the aqueous phase, after which the oil and aqueous phases were combined at 40ºC under continuous stirring to form a uniform emulsion. Linseed oil and rose oil were subsequently incorporated, and the resulting cream was allowed to cool and stored at room temperature until further evaluation.
Formulation 2: Creamy Base with Emulsifying wax
The cream was prepared by separately heating the oil phase, consisting of beeswax and liquid paraffin, and the aqueous phase containing borax and methyl paraben to 70ºC. Zinc oxide nanoparticles were dispersed into the aqueous phase, after which the oil and aqueous phases were combined at 40ºC under continuous stirring to form a uniform emulsion. Linseed oil and rose oil were subsequently incorporated, and the prepared cream was allowed to cool and stored at room temperature until further evaluation.
Evaluation of Skincare Cream
Organoleptic Evaluation
The prepared cream formulations were evaluated for organoleptic properties, including color, odor, and physical state. The appearance of each formulation was assessed visually for color uniformity and surface smoothness and graded accordingly. Additional parameters such as homogeneity, texture, after-feel, type of smear, and ease of removal were also examined.
Spreadability Studies
The spreadability of the cream formulations was determined using two standard glass slides. A fixed amount of cream was placed on one slide and covered with a second slide. A weight of 140 g was applied uniformly over the slides to facilitate even spreading of the cream over a distance of 5 cm. Excess cream was removed, and the lower slide was fixed in position. A 30 g weight was then attached to the upper slide using a pulley system, and the time required for the slide to move a distance of 5 cm was recorded. Spreadability was calculated based on the recorded time, with shorter times indicating better spreadability.
Where,
M = weight tied to the upper slide (30 g),
L = length of glass slide (5 cm),
T = time taken in seconds.
Homogeneity
The prepared cream formulations were evaluated for homogeneity by visual inspection and by touch to detect the presence of lumps, flocculates, or aggregates. Uniform appearance and smooth consistency indicated good homogeneity.
pH of the Cream
The pH of the cream formulations was measured using a calibrated pH meter. Prior to analysis, the pH meter was standardized using a suitable buffer solution. Approximately 0.5 g of the cream was dispersed in 50 mL of distilled water, and the pH of the resulting dispersion was recorded.
Viscosity
The viscosity of the cream formulations was determined using a Brookfield viscometer (DV-II+ Pro Model) equipped with an LV-64 spindle. The formulation was transferred into the viscometer adapter, and measurements were carried out by progressively increasing the rotational speed from 0.5 to 20 rpm. Viscosity values were recorded to assess the flow behavior and consistency of the formulations.
After-Feel
The after-feel of the cream was evaluated by applying a fixed quantity to the skin and assessing parameters such as emolliency, slipperiness, and the amount of residue remaining after application.
Ease of Removal
Ease of removal was assessed by washing the area of application with tap water after applying a fixed amount of cream and observing the extent to which the formulation could be removed without leaving residue.
Irritancy Test
An irritancy test was performed on the dorsal surface of the left hand. A 1 cm² area was marked, and the cream was applied to the site. The treated area was observed at regular intervals over a 24-h period for any signs of skin irritation, erythema, or edema.
Stability Testing
Stability studies were conducted in accordance with ICH guidelines. The cream formulations were stored in a stability chamber maintained at 40±2ºC and 75±5% relative humidity for a period of three months. At the end of the study period, samples were evaluated for changes in physical appearance, homogeneity, and viscosity.
In vitro Diffusion Study
In vitro drug diffusion studies were carried out using a dialysis membrane method. 1 g of cream was placed in a dialysis membrane and immersed in 100 mL of phosphate buffer (pH 7.4) contained in a 250 mL beaker. The diffusion medium was maintained at 37ºC and stirred at 300 rpm using a magnetic stirrer. At predetermined time intervals, 5 mL aliquots were withdrawn and replaced with an equal volume of fresh buffer to maintain sink conditions. The samples were analyzed using High-Performance Liquid Chromatography (HPLC) to determine the percentage of drug released.
Antimicrobial Susceptibility Test
The antibacterial and antifungal activities of ZnO nanoparticles and cream formulations were evaluated using the agar well diffusion method. ZnO nanoparticles synthesized using Withania somnifera and Centella asiatica were dispersed in distilled water to prepare solutions of 1 mg/mL concentration. Escherichia coli, Staphylococcus aureus, and Candida albicans were cultured in Luria-Bertani (LB), Brain Heart Infusion (BHI), and Sabouraud Dextrose Broth (SDB) media, respectively, and incubated for 24-48 hr prior to testing. Sterile agar plates were prepared, and wells were punched aseptically and exposed to UV light for 5-10 min. Microbial cultures were spread evenly on the agar surface, and 50 µL of each test sample was introduced into the respective wells. The plates were incubated at 35ºC for bacterial strains and 25ºC for the fungal strain for 24-48 hr. Zones of inhibition were measured to assess antimicrobial activity. The following samples were evaluated: (1) ZnO nanoparticles of Withania somnifera, (2) ZnO nanoparticles of Centella asiatica, (3) Cream containing ZnO nanoparticles, and (4) Cream containing herbal extracts only.
RESULTS AND DISCUSSION
Pre-formulation Studies of Herbal Extracts
Pre-formulation evaluation of Withania somnifera and Centella asiatica extracts confirmed their suitability for topical application and nanoparticle synthesis. The extracts exhibited skin-compatible pH values of 5.72 and 6.34, respectively, indicating minimal risk of irritation upon application. Both extracts showed good solubility in water and other polar solvents but were insoluble in chloroform, supporting their incorporation into aqueous-based topical systems. However, their hydrophilic nature necessitates a suitable carrier system to improve stability and controlled delivery, which was achieved through zinc oxide nanoparticle encapsulation.
Green Synthesis and Identification of Zinc Oxide Nanoparticles
Green synthesis of ZnO nanoparticles was successfully carried out using aqueous extracts of W. somnifera and C. asiatica as reducing and stabilizing agents. Dropwise addition of sodium hydroxide to achieve pH 8 resulted in the formation of a characteristic whitish-brown precipitate, confirming nanoparticle formation. The precipitate was separated by centrifugation at 3000 rpm and dried at 60ºC to obtain fine ZnO nanoparticle powder.
UV-visible spectroscopic analysis confirmed nanoparticle formation, with absorption peaks ranging between 364-374 nm for the preliminary trials. These absorption maxima correspond to band gap energies between 3.31-3.41 eV, consistent with nanosized ZnO particles. Among the trials, Trial 2 showed an optimal absorption peak at 364 nm, indicating effective nanoparticle formation. This confirms the successful synthesis and optical characteristics of ZnO nanoparticles.
Preparation of Zinc Oxide Nanoparticles
The amount of plant extract (1, 1.5, and 2 g) significantly influenced nanoparticle yield and intensity of precipitation. Higher extract concentrations produced more intense precipitates, indicating enhanced participation of phytochemicals in reduction and stabilization processes. This observation supports previous reports on plant-mediated green synthesis mechanisms (Iqbal et al., 2019; Singh et al., 2018; Singh et al., 2016).
Drug Loading Efficiency of ZnO Nanoparticles
Drug loading efficiency of ZnO nanoparticles was quantified using HPLC analysis (Table 2). Three formulation trials demonstrated drug loading efficiencies of 33.38%, 45.81%, and 62.89%, respectively (Table 4). The increasing trend indicates improved encapsulation of withanolides with increasing extract concentration. The formulation (Trial 3) showed the highest loading efficiency (62.89%), confirming effective entrapment of bioactive compounds within the ZnO nanoparticle matrix. HPLC chromatograms of all trials are shown in Figure 1, validating consistent peak resolution and quantification. confirms successful phytoconstituent loading.
| Trial No | Theoretical % | Practical % | % Drug loaded |
|---|---|---|---|
| 1 | 2.81 | 0.938 | 33.38 |
| 2 | 2.96 | 1.356 | 45.81 |
| 3 | 2.8 | 1.761 | 62.89 |
Optical Characterization by UV-visible Spectroscopy
The ZnO nanoparticles exhibited a prominent absorption peak at 359 nm in the UV-Visible spectrum. This corresponds to a calculated band gap energy of approximately 3.4 eV using Planck’s equation, confirming nanoscale ZnO formation. The band gap value directly influences antimicrobial and UV-protective properties of ZnO nanoparticles (Sirelkhatim et al., 2015).
In vitro drug release study of ZnO nanoparticle
In vitro release studies demonstrated sustained release of withanolides from ZnO nanoparticles over an 8-hr period. A total of 3.29 mg of withanolides was released from 17.6 mg of loaded drug (Table 3). The controlled release profile is attributed to strong binding of phytoconstituents within the ZnO matrix and their limited solubility in phosphate buffer. Sustained release is desirable for topical applications as it prolongs therapeutic action and reduces dosing frequency (Patil et al., 2019; Rajendran et al., 2010). Additionally, nanoparticle systems are known to improve skin retention and minimize systemic absorption (Kumar et al., 2021).
| Sl. No. | Time (mins) | % Withanolide content present in Formulation | Drug Dissolution from Pure Nanoparticles | Drug Diffusion from Nanoparticles in Cream | ||
|---|---|---|---|---|---|---|
| % Withanolide released | Withanolide released in mg | % Withanolide released | Withanolide released in mg | |||
| 1 | 15 | 1.76 | 1.30±0.11 | 0.23 | 1.23±0.11 | 0.13 |
| 2 | 30 | 1.76 | 3.01±0.13 | 0.53 | 2.61±0.15 | 0.46 |
| 3 | 60 | 1.76 | 4.03±0.21 | 0.71 | 4.37±0.21 | 0.77 |
| 4 | 120 | 1.76 | 6.64±0.23 | 1.17 | 6.42±0.23 | 1.13 |
| 5 | 180 | 1.76 | 10.3±0.32 | 1.81 | 8.23±0.18 | 1.45 |
| 6 | 240 | 1.76 | 12.5±0.27 | 2.2 | 12.32±0.32 | 2.17 |
| 7 | 300 | 1.76 | 14.3±0.23 | 2.52 | 15.51±0.26 | 2.73 |
| 8 | 360 | 1.76 | 14.7±0.24 | 2.59 | 15.90±0.67 | 2.8 |
| 9 | 420 | 1.76 | 15.9±0.32 | 2.81 | 16.42±0.78 | 2.64 |
| 10 | 480 | 1.76 | 18.6±0.31 | 3.29 | 117.6±0.37 | 3.02 |
FTIR studies: FTIR studies of ZnO NPs
The FTIR spectrum of the synthesized ZnO nanoparticles, recorded in the 4000-400 cm⁻¹ range to observe the various functional groups involved in the preparation of the nanoparticles. As seen in the FTIR spectra, the research shows the characteristic absorption peak corresponding to Zn-O stretching vibrations at 539 cm⁻¹ and is in agreement with known literature. The observation of functional groups, such as alkanes, alkenes, carbonyl, amide, carboxylic acid, and hydroxyl groups suggests the role of phytochemicals in reducing the zinc ions and further stabilizing the ZnO nanoparticles. Phytochemicals played a role in capping and preventing aggregation of the ZnO nanoparticles while observing different stretching bands in the various FTIR spectra (Gunalan et al., 2012).
SEM, Particle Size, and Zeta Potential Analysis
SEM images revealed ZnO nanoparticles with flake- and rod-like morphology and slightly rough surfaces (Figure 2). Some degree of aggregation was observed, which is common in green-synthesized nanoparticles. Zeta potential analysis showed a surface charge of -25.6 mV (Figure 3), indicating moderate colloidal stability due to electrostatic repulsion. Particle size analysis confirmed nanoscale dimensions with uniform distribution (Figure 3). Similar stability profiles have been reported for green-synthesized ZnO nanoparticles (Sharma et al., 2020).
Formulation and Evaluation of Skincare Cream
ZnO nanoparticle-loaded creams were successfully formulated using two bases: a stearic acid-based emulsion (Formulation 1) and a beeswax-paraffin base (Formulation 2). Both formulations exhibited good homogeneity, smooth texture, and no phase separation. Evaluation studies showed skin-compatible pH (5.18-5.70), moderate viscosity (25,000-40,000 cps), good spreadability, pleasant after-feel, ease of removal, and absence of skin irritation. Stability studies confirmed no significant changes after storage under accelerated conditions. Based on overall performance, Formulation 1 was selected for further in vitro diffusion and antimicrobial studies due to its lighter texture and superior spreadability.
In vitro Diffusion Study
The in vitro diffusion study was performed to evaluate the release behavior of bioactive constituents from the ZnO nanoparticle-loaded cream formulation. The diffusion profile demonstrated a gradual and sustained release of withanolides over a period of 8 hr, indicating effective encapsulation of the phytoconstituents within the ZnO nanoparticle matrix and their uniform distribution in the cream base. An initial moderate release observed during the early time points may be attributed to the diffusion of surface-associated phytochemicals, followed by a controlled release phase governed primarily by diffusion from the nanoparticle core. The sustained release pattern suggests strong interaction between the phytochemicals and the ZnO nanoparticles, likely resulting from phytochemical capping during the green synthesis process. In addition, the viscosity and structural properties of the cream base contributed to slower diffusion by creating a barrier to rapid drug migration into the receptor medium. The absence of a pronounced burst release indicates formulation stability and uniform drug distribution, which are desirable characteristics for topical delivery systems. Overall, the in vitro diffusion results confirm that incorporation of green-synthesized ZnO nanoparticles effectively modulates the release of herbal bioactives from the cream formulation. The controlled release behavior supports the suitability of ZnO nanoparticles as carrier systems for topical applications, offering prolonged availability of active constituents and improved formulation performance when compared to conventional herbal creams.
Antimicrobial Activity
The antimicrobial activity of green-synthesized ZnO nanoparticles and their corresponding cream formulations was evaluated against Staphylococcus aureus, Escherichia coli, and Candida albicans. The ZnO nanoparticle-based samples exhibited noticeably larger zones of inhibition compared to formulations containing only herbal extracts, indicating enhanced antimicrobial effectiveness (Table 4, Figure 4). This improvement suggests a synergistic interaction between ZnO nanoparticles and plant-derived phytoconstituents in suppressing microbial growth.
| Sl. No. | Sample | Zone of inhibition (mm) | ||
|---|---|---|---|---|
| E. coli | S. aureus | C. albicans | ||
| 1 | ZnO NP’s (Withania somnifera) | 36 | 26 | 12 |
| 2 | ZnO NP’s (Centella asiatica) | 35 | 25 | 10 |
| 3 | Cream with ZnO NP’s | 33 | 24 | 9 |
| 4 | Cream with herbal extract | 9 | 7 | 8 |
The superior antimicrobial performance of the ZnO nanoparticle-loaded formulations can be attributed to multiple complementary mechanisms. ZnO nanoparticles are known to generate reactive oxygen species, disrupt microbial cell membranes, and release Zn²⁺ ions, all of which interfere with essential cellular and metabolic functions. When combined with phytochemicals from Withania somnifera and Centella asiatica, which possess inherent antibacterial and antifungal properties, the nanoparticles exert a multi-target antimicrobial effect. Phytochemical capping during green synthesis is also likely to enhance nanoparticle stability and promote closer interaction with microbial cell surfaces, thereby improving antimicrobial activity.
Incorporation of ZnO nanoparticles into the cream base further contributed to sustained antimicrobial performance. The controlled release of bioactive constituents from the nanoparticle-loaded formulation ensured prolonged exposure of microorganisms to inhibitory concentrations. In contrast, creams containing only herbal extracts exhibited comparatively lower zones of inhibition, possibly due to rapid diffusion and reduced stability of the active constituents. The nanoparticle-based system therefore offers improved retention and gradual release of antimicrobial agents at the site of application.
Overall, the antimicrobial findings confirm that ZnO nanoparticle-loaded formulations provide superior microbial inhibition compared to conventional herbal formulations. These results support the potential of green-synthesized ZnO nanoparticles as
effective antimicrobial carriers for topical applications, offering enhanced efficacy through synergistic action and controlled delivery of herbal bioactives (Jayaseelan et al., 2012; Nagarajan and Kuppusamy, 2013; Padalia et al., 2015; Rajiv et al., 2013).
CONCLUSION
The present study successfully demonstrated the green synthesis of zinc oxide nanoparticles using aqueous extracts of Withania somnifera and Centella asiatica, highlighting an eco-friendly, sustainable, and chemically safe approach to nanoparticle fabrication. The use of plant-derived phytochemicals as natural reducing and stabilizing agents eliminated the need for hazardous chemicals, resulting in a biocompatible ZnO nanocarrier system well suited for topical applications.
The green-synthesized ZnO nanoparticles showed efficient encapsulation of phytoconstituents and were successfully incorporated into cream formulations with acceptable physicochemical properties, homogeneity, and storage stability. Notably, in vitro drug release studies demonstrated a controlled and sustained release of withanolides from the nanoparticle-loaded cream over an extended period. This release behavior confirms effective entrapment within the nanoparticle matrix and underscores the ability of the green-synthesized ZnO nanoparticles and cream base to regulate diffusion and maintain prolonged availability of bioactive compounds at the site of application, without an initial burst release.
Overall, the findings emphasize the dual advantage of green synthesis and nanotechnology in enhancing formulation performance, drug release control, and antimicrobial efficacy. This environmentally sustainable nanotechnological strategy offers a promising platform for the development of advanced herbal skincare and dermatological formulations. Further investigations, including extended stability studies and comprehensive biological evaluations, are warranted to support translational and commercial applications.