Aloe vera leaf extract as a sustainable route for silver nanoparticle synthesis with enhanced antimicrobial activity | Scientific Reports

Jul 02, 2025

Scientific Reports volume 15, Article number: 22481 (2025) Cite this article

This study shows how to easily make silver nanoparticles (AgNPs) using Aloe vera leaf extract a simple and green approach. This method offers a biocompatible, economical, and environmentally friendly method without hazardous chemicals. A central composite design (CCD) was used to find the best way to make nanoparticles, adjusting the amount of AgNO3, the pH, and the incubation time to see how they all worked together. Samples of the mixture were taken every hour to track the development of nanoparticles using the UV–Vis spectrophotometer. The reaction mixture gradually turned from white to a reddish-brown hue within the first 5 to 10 min, signaling the formation of AgNPs. We found that using 2.2185 mM of AgNO3, a pH of 11.91, and a 2.91-h incubation period produced the most AgNPs. Further tests, including UV–Vis spectroscopy, DLS, XRD, FTIR, SEM, and DSC, showed that these nanoparticles were generally spherical, with an average size of 68.79 nm. The sample also had a good PDI of 0.374, and the powder was mostly made up of a highly crystalline Ag–3C silver phase (70.8%). Antimicrobial studies revealed that the synthesized AgNPs exhibited significant inhibitory activity against Salmonella thypae, This research shows that Aloe vera leaf extract can be used to create AgNPs in an environmentally friendly way. These AgNPs show promise for fighting bacteria like E. coli and Staphylococcus aureus, as well as the fungus Aspergillus niger.

Nanotechnology is poised to revolutionize a vast array of industries. Its potential is being explored in fields ranging from medicine and environmental cleanup to advanced electronics and next-generation energy solutions1,2 Nanomaterials have many properties that distinguish them from bulk materials, such as a fraction of surface atoms, high surface energy, limitations, and reduced numbers of flaws that do not exist in bulk materials. Zinc, Copper3, gold, and silver nanoparticles have unique properties that give them anti-microbial effects. They interact with cellular membranes and disrupt the structure of the cell wall4,5. Silver has been well-known for a long time for its substantial toxicity against different types of microorganisms, such as bacteria and fungi6,7. There are a lot of different routes to synthesise silver nanoparticles, which include physical and chemical methods. However, these techniques apply toxic chemicals, mostly non-polar solvents, and they generally are energy intensive, time-consuming, and costly8. As a result, there is a great need to find a new way of synthesizing biocompatible nanoparticles that are cost-effective and sustainable9. The researchers are looking for new metal nanoparticles that can better combat these resistant bacteria10,11. The AgNPs exhibit good bactericidal action12 against both Gram-positive and Gram-negative bacteria4,13. Different scholars found that the silver nanoparticle binds itself to membranes and cell walls14,15.

Many research papers found that AgNPs have been found very effective against Aspergillus sp16, C. albicans17, C.tropicalis18, C. krusei19, and T. mentagrophytes20. Studies found that the Tulsi (Ocimum sanctum L.) mediated AgNPs showed high antifungal properties toward an opportunistic human fungal pathogen. Therefore, it has become a strong and fast-acting fungicide counter to a broad spectrum of fungi containing Aspergillus, Candida, and Saccharomyces21,22.

AgNPs incorporated into gels and creams are used for treating actions such as wound healing, burns, and microbial infection23. Medical applications of AgNPs include the medical device and implants which is a major sector. Also, they are included in the colloidal silver gel and, cotton and fabrics impregnated with silver and used in sporting equipments. Silver makes medical devices and implants better at healing wounds and burns. It also helps keep textiles, glass windows, and other surfaces clean and hygienic24,25.

Synthesis of nanoparticles using plant and plant leaves has more advantages than that of the microbial based method because in the plant-based synthesis technique, there is no complex process of maintaining the microorganism’s cell culture26,27. In plant mediated synthesis of metal nanoparticles the reaction rate, morphologies, and particle size growth could be maintained by varying the reaction conditions like pH, plant extract concentration, temperature, and the mixing ratio of the reactants28. Fundamentally, bio-mediated metal nanoparticle synthesis is carried out in extracellular or intracellular media. In the intracellular media, the reaction between the metal and biomaterials occurs inside the plant; on the other hand, extracellular synthesis takes place in vitro. Many research findings show that the extracellular production of nanoparticles by plant extracts has been better compared to the intracellular method of synthesis since it reduces the extraction and purification procedures11,29,30.

Aloe vera is a bush, juicy herb belonging to a Liliaceous family with turgid leaves hanging to stem in a rosette way (Fig. 1). This herb is known for its thick, fleshy leaves. The leaves have no stems, ending in a sharp tip, and have spiny edges31. Due to that aloe vera has been utilized for centuries for different therapeutic properties. Aloe Vera is well-known for reducing inflammation, boosting the immune system, and promoting cell growth32,33. Aloe Vera’s properties toward different kinds of infectious agents in terms of antiviral34, antifungal35,36, and antibacterial37 has also been reported. Since ancient times, Aloe vera gel has been used to treat skin cuts and burns5,38.

Olea Vera plant leaf8.

Many different natural substances, like anthraquinones and flavonoids, are found in Aloe vera and Aloe ferox. These substances are responsible for the plants’ medicinal effects. Aloe vera leaf extracts offer several benefits for synthesizing AgNPs. They are widely available, safe, and generally nontoxic. Plant extracts offer a green and efficient method for making AgNPs. The natural compounds within these extracts, including terpenoids and flavones quickly convert silver ions into stable nanoparticles39,40. Water-soluble compounds like flavones and organic acids play a particularly important role 41. Using Aloe vera extract demonstrates how this approach is effective, environmentally friendly, and scalable, eliminating the need for harsh conditions and toxic chemicals 42.

Kokila et al., 202543 found a way to create AgNPs using banana peels, and these nanoparticles turned out to be good at killing bacteria. It effectively fought against two drug-resistant bacteria, E. coli and K. pneumoniae, creating a 10.75 mm clear zone where bacterial growth was stopped. They were also tested against two other drug-resistant bacteria, S. aureus and B. subtilis. While Aloe vera has been previously used in AgNPs synthesis, further optimization is needed to maximize yields and control nanoparticle size. This study develops a new, environmentally friendly method for synthesizing AgNPs, using Aloe vera leaf extract as both a reducing and stabilizing agent. We’ll use response surface methodology (RSM) to optimize the process and find the ideal conditions for high yields and controlled nanoparticle characteristics. This research contributes to the development of sustainable silver nanoparticle production methods for potential antimicrobial applications.

The Aloe vera leaves, fresh from the uncultivated land around the Mugher River in Ethiopia, were carefully washed and then left to dry in the sunlight. Once dried, we prepared them for use by cutting them up and grinding them into a fine powder. Weighed 15 g of the fine powder and wrapped it with Whatman number 1 filter paper. The active compounds in Aloe vera were extracted using 200 mL of hexane solvent in a Soxhlet extractor at the boiling point temperature (69°C) for 2 to 6 h. After the Soxhlet extraction, hexane and the extractives were separated using a rotary evaporator (Model No. ML-E14-2050) at the boiling point of hexane. The isolated compound was characterized using FTIR and NMR spectroscopy. Using a Bruker BioSpin instrument, we performed proton and carbon-13 NMR spectroscopy to analyze our sample. The spectra were measured at 500 MHz and 125 MHz. Chemical shifts are given in ppm, using the solvent as a reference point. As a common practice, Chloroform was used as a solvent for the NMR analysis experiment44. Silver nitrate (AgNO3) and sodium hydroxide (NaOH), both from Sigma-Aldrich, were obtained from Fine Chemical General Trading Plc. in Addis Ababa, Ethiopia. Muller Hinton agar medium (BD Difco), Dextrose agar medium (BD Difco), and the microbial cultures (Aspergillus Niger sp., Escherichia coli, Salmonella typhi, and Staphylococcus) were obtained from the Department of Biotechnology, Addis Ababa Science and Technology University. All experiments were done using distilled water, except an antimicrobial study conducted using sterilized distilled water. The experimental framework for this thesis is schematically presented in Fig. 2 and the detailed description is presented in sections below.

The experimental framework of synthesis and characterization of AgNPs.

The repeatedly washed Aloe vera leaf was sun-dried and then chopped into small pieces using a kitchen knife, mortar and pestle. In a glass beaker, 20 g of the fine, homogenized pieces were heated in 100 mL of distilled water for 20 min at 60°C. The mixture was stirred during heating until the aqueous solution turned light yellow. After cooling, the extract was filtered through Whatman number 1 filter paper to remove solids. The resulting liquid was collected and stored at 4°C45,46.

Silver nitrate solutions were prepared at concentrations of 1 mM, 2 mM, 3 mM, 4 mM, and 5 mM. 5 mL of Aloe vera extract was added to each 50 mL solution of silver nitrate, and the mixture was stirred using a magnetic stirrer at 350 rpm for 1 min. The resulting mixtures were incubated in the dark (wrapped in aluminium foil) at a constant temperature of 60°C, maintained using a water bath, similar to the work done by47. During the incubation period, the bioreduction of Ag + ions to Ag was monitored using UV–Vis spectroscopy. Samples were collected every hour, and the absorbance at a specific wavelength was used to determine the extent of bioreduction. The effects of precursor concentration, pH, and incubation time were investigated to optimize the synthesis process. The pH was adjusted by adding a 0.1 M aqueous caustic soda solution. The reaction temperature was maintained at 60°C using a water bath.

After the reaction mixture was centrifuged (Pro-Analytical C2004) at 3000 rpm for 30 min to isolate the nanoparticles, they were washed three times with distilled water to remove any unreacted material. These washes and spins caused the AgNPs to form clumps. The nanoparticles were sonicated to reduce aggregation using an ultrasonic bath (Soniprep 150 Plu) at 60 W, 20 kHz for 15 min at ambient temperature. The suspension was then oven-dried for further characterization, as reported by Matebie et al., 202148.

The effects of pH, silver nitrate concentration, and incubation time on silver nanoparticle formation were studied using a three-level factorial design. Preliminary experiments were conducted for each individual parameter (OVAT), as detailed in Table 1. A central composite design (CCD) is used to study and identify the best interaction effect of experimental process conditions. The goal was to develop a model system that accurately predicts the maximum yield of silver48. Additionally, a system model was created to best represent the interaction between parameters with a minimal number of experimental runs49. Statistical optimization was used to achieve high yield. The Design of Experiments (DOE) and data analysis were conducted using Design-Expert statistical software (Version 12.0.0). The DOE focused on three parameters: aqueous concentration of AgNO3, pH, and stirring time, each with three levels. Based on the Face Centered Central Composite Design (FCCCD), 20 experimental runs were recommended. The response, "yield," was determined by estimating the area under the curve of the wavelength from 350 to 450 nm50,51 .

A) UV–Vis spectroscopy analysis

To understand the optical properties of the synthesized AgNPs, we used a UV–Vis spectrophotometer (Shimadzu UV-1800). This instrument measures how much light is absorbed by a substance across a range of wavelengths (300–800 nm in our case). By monitoring the reduction of silver ions to silver metal, we could track the formation of the AgNPs. The samples are prepared by mixing equal volumes of the AgNP solution and distilled water in quartz cuvettes, using distilled water as a reference. Absorption spectra were recorded at various time intervals, and the concentration of AgNPs was determined by analyzing the area under the curve of these spectra.

b) Particle size analysis

The particle size distribution (PSD) of the synthesized AgNPs was determined using Dynamic Light Scattering (DLS) via a Zetasizer (Nano-ZS90, Malvern, UK). DLS is a technique that measures the Brownian motion of particles in a suspension. The rate of Brownian motion is related to the size of the particles. DLS can determine the particle size distribution by analyzing the fluctuations in light scattering intensity. This analysis was performed to assess the uniformity of the synthesized AgNPs and to understand the influence of different synthesis parameters on the size distribution. The DLS measurements were performed at a controlled temperature of 25°C. The following parameters were set for the analysis: material refractive index of 0.56, dispersant refractive index of 1.330, material absorption of 4.27, and viscosity of 0.8872 cP. These parameters were specific to the synthesized AgNPs and the aqueous medium in which they were dispersed. For each measurement, the nanoparticle suspension was prepared for analysis. The DLS measurement was performed in triplicate, and 32 runs were performed for each measurement to obtain an average hydrodynamic diameter. The average hydrodynamic diameter was then calculated by taking the arithmetic average of the 32 runs.

c) X-ray diffraction (XRD) analysis

The percentages of crystallites and the phases of the synthesized AgNPs were determined by X-ray diffraction spectroscopy measurement method using Shimadzu XRD-6000/6100 model. The Machine was operated with Ni as a filter at a setting of 40 kV/30 mA Cu-kα (λ = 1.540598) with a 3 degree/min scan speed and with a scan range of 5–60° for wide angle diffraction (2θ), and a continuous scanning mode, the crystallinity of each sample was assessed based on the observed peak. Equation 2.1 was used to calculate the percentage of crystallites using the data generated by XRD analysis.

d) Fourier transmission-infrared spectroscopy (FTIRs) analysis

To study the possible functional groups that could result from the reduction and stabilization of silver nanoparticles; a purified and dried nanopowder was used FTIR analysis (Nicolet iS50, Thermo Scientific, USA). An FT-IR analysis was conducted. The instrument scanned the samples in diffuse reflectance mode, measuring the wavelengths of light from 4000 to 500 cm-1. The scan rate was 4 ms-1, and the resolution was 4 cm-1 31.

e) SEM-based morphological analysis

The surface details of the synthesized nanoparticles were examined using a JCM-6000Plus Versatile Benchtop SEM (Japan). The instrument was operated under the following conditions: working distance of 6–13 mm, accelerating voltage of 5–20 keV, and emission current of 75–80 A.

f) Differential scanning calorimeter (DSC) analysis

The thermal behavior of the synthesized AgNPs was investigated by Differential Scanning Calorimeter (SKZ1052B). Twenty milligrams of its powder were accurately weighed into the Aluminum crucible without seals and heated from 30 to 500°C at the heating rate of 10°C/min for 60 min 52.

A) Media preparation for the fungus strain

A growth medium (1.5 g nutrient broth, 5 g dextrose monohydrate, 50 ml distilled water) was prepared and sterilized in a conical flask by autoclaving (120 °C, 15 psi, 15 min). Aspergillus sp. fungi were then transferred from a pure culture to 10 ml of the growth medium in a test tube and incubated at 37 ± 1 °C for 72 h.

b) Media preparation for the bacterial strain

The process of making 500 ml of Muller Hinton Agar Medium involved dissolving 17.8 g of the powdered medium in 500 ml of distilled water. This mixture was then autoclaved at 121 °C for 15 min at 15 lbs pressure to sterilize it. However, before the antibacterial was carried out, three bacterial cultures: Escherichia coli, Salmonella thypae and Staphylococcus were refreshed in 10 ml/strain Muller Hinton Agar Medium.

c) Preparation of media plates

Fifteen milliliters of nutrient agar media for both fungus and bacterial strains were separately prepared and poured into Petri dishes. The media were autoclaved at 15 lbs pressure and 120 °C for 15 min. The organism suspension was vortexed to ensure it was well-mixed. A sterile wire loop was dipped into the suspension, then removed and used to inoculate the sterilized media, making sure the media temperature did not exceed 50 °C. The inoculated media were shaken well and 20 ml were poured into each petri dish (100 mm diameter, 10 mm height). The dishes were left to solidify. This procedure was carried out in a laminar airflow cabinet meeting the USP Class 100 (Class A) standards.

d) Agar well diffusion method

The antimicrobial activity of synthesized nanoparticles was evaluated by the well plate agar diffusion method53. Each plate received 15 ml of nutrient agar, and then we carefully spread 1 ml of a standardized test organism across the agar’s surface. A gentle swirl ensured even distribution. Finally, we used a sterile borer to create small, 6 mm-wide wells in the solidified agar. Each well was filled with 15 µl of nanoparticles. After letting the nanoparticles diffuse into the agar for an hour (NCCLS, 1993), we incubated the plates at 37 °C for 24 h to encourage any microbial growth. We maintained a sterile environment by working under a laminar air-flow cabinet, which meets USP class 100 (class A) standards. Then, we measured the size of the clear zones around each well to determine the antimicrobial activity.

Fourier Transform Infrared (FTIR) spectroscopy was used to analyze the phytochemicals present in Aloe vera leaf extract. Figure 3 show the FT-IR spectrum (between 4000 and 350 cm−1) of powder from Aloe vera leaf and the bioactive chemicals extracted from Aloe powder. Many peaks were portrayed within absorption spectra ranging 3264.29 to 513.28 cm-1. The absorption bands observed at 3264.29, 3008.00, 2922.68, 2854.16 cm-1 show the presence of OH and CH stretching bands of phenols, polyphenols and alcohols. The peak at 1743.41 cm⁻1 is caused by the C = O stretching in the triglyceride ester linkage and the 6-membered lactone. A peak at 1596.6 cm⁻1 shows the N–H bending of the amine group. The bending vibrations of CH₂ and CH₃ are clearly seen in the region around 1460.14 cm⁻1. The spectrum found at 1315.81 cm-1 represents sulfone (SO-stretching) and aromatic amine of the CN-stretching. The peaks at 1159.79 cm-1 suggest the presence of sugars, indicating an alcoholic group (C–O–C) and the bending of OH groups (CH2OH) within the phytoconstituents. The peaks formed at 1024.05 and 720.86 cm-1 show the presence of amines and aromatics or benzene derivatives, whereas the peak 513.28 cm-1 represents the presence of Alkyl halides.

FTIR spectrum of compound in the Aloe vera leaf.

To further understand the makeup of the aloe vera leaf extract, we used NMR spectroscopy in addition to FT-IR. Table 2 and Fig. 4 show the chemical shifts (δ) and assignments for the main signals in the 1H NMR spectrum, while Table 3 and Fig. 5 present the corresponding information for the 13C NMR spectrum. According to an analysis of the ACD/NMR processor, compounds such as fatty alcohol, acetyl chloride, oleyl amine, azelaic acid, n-exanodoic acid, n-hexane decanoic acid, linoleic acid, aleyl alcohol, oxane, palmitoleic acid, squalene, tetradecanoic acid 1-metyl ester, and triglyceride were found in the extract of Aloe vera. The research report are in congruence with the present study’s findings44. The NMR spectroscopy analysis also confirms the presence of the same organic chemicals, which were identified with the FT-IR analysis.

Proton NMR of active compounds isolated from Aloe Vera leaf.

13C NMR of active compounds isolated from Aloe Vera leaf.

Using Aloe vera extract (Fig. 6a) as a reducing agent, we observed a distinct color change in the silver nitrate (AgNO3) solution, indicating the formation of silver nanoparticles. The solution progressed from clear to a yellowish-brown, eventually becoming a dark brown color (Fig. 6c) as the silver ions (Ag + , Fig. 6b) were converted to silver metal. Meanwhile, the separate control solution of AgNO3 and the bio-extract showed no color change over time. The color change results from metal nanoparticles’ surface plasmon resonance (SPR) properties54. The metal nanoparticles contain free electrons. These electrons create the SPR absorption band by vibrating in sync with light waves55.

Color dispersion of solution (a) Aloe vera plant extract, (b) AgNO3 aqueous solution, (c) Final silver dispersion formed after reduction.

Chan & Don56 described that silver ion reduction to silver metal has occurred due to the presence of a bio-chemical reducing agent in the plant extract. Logaranjan et al.57 also suggested how the free ions from the active organic chemicals found in the Aloe extract reduce Ag+ in AgNO3 solution into nano-sized Ag metal.

Along with the visible color change when the plant extract was added to the silver ions, we used UV–Vis spectroscopy to confirm the formation of silver nanoparticles. The UV–visible absorption peak between 350 and 500 nm is a signature feature of AgNPs58. In this experiment, the highest absorption was observed at 450 nm. The UV–visible spectra also provide information about the morphology of the biosynthesized silver nanoparticles. The synthesized AgNPs in this study are isotropic and spherical, as indicated by the clear, single surface plasmon peak seen in Fig. 7.

Absorption spectra of AgNPs formed in an extract of Aloe vera leaf.

To find the ideal conditions for turning silver nitrate into silver nanoparticles, we experimented with different combinations of pH, silver nitrate concentration, and reduction time, using a three-level factorial design. A central composite design was used to carry out the study and identify the best combination of experimental process conditions to synthesize and develop a model system for achieving the highest silver yield48. This design also helped to develop a system model that accurately reflects the interactions between the parameters with a minimal number of experimental runs49. Table 4 displays the RSM-CCD for three parameters, with the yield of AgNPs as the outcome. The highest yield, 118.13, was recorded at the center point (run 17). The lowest yield, 14.15, was recorded at run 18.

The following equation is coded in order to represent the relationship between yield and the concentration of AgNO3, incubation time, and pH (range 8–12) of the solution during the reaction.

A model was built to predict AgNPs yield (Y) based on the concentration of AgNO3 (A), incubation time (B), and pH (C). This model, shown in Table 5, was analyzed using ANOVA to assess its fit to the experimental data. A "Prob > F" value of less than 0.05 generally indicates a significant model. The model’s extremely low p-value (< 0.0001) demonstrates its high significance, meaning it is very unlikely the results are due to chance. The analysis revealed that the concentration of AgNO3, incubation time, and pH (A, B, and C), along with the interactions between AgNO3 and time (AB), and AgNO3 and pH (AC), as well as the squared effects of AgNO3 and time (A2 and B2) all had a significant impact on AgNPs yield. An F-test confirmed the model’s significance (F-value = 103.57), suggesting only a 0.01% chance the results are random. The lack-of-fit F-value (2.37) was small, indicating the model’s good fit. Finally, the Adeq Precision of 32.926, well above the desired 4, shows a strong signal and further supports the model’s reliability. This model helps explore the design space. Furthermore, the variability and data fit statistics of a predicted with a real response were checked by the coefficient of determination (R2) 59,56. An R2 value close to 1 means the model does a good job of predicting the results we saw in the experiments. Ideally, this value should fall between 0 and 1, and the closer it is to 1, the better the fit. The Predicted R2 of 0.9528 is in reasonable agreement with the Adjusted R2 of 0.9798, and the variation is due to independent variables in the synthesis of AgNPs.

In this study, a 3D surface response plot was used to visualize the regression equation from the experimental model. This plot helps to examine how each factor interacts and identify the best conditions for maximizing AgNPs yield in biosynthesis. The plot shows two independent variables and the third variable represents the response of the experiment.

Figure 8, 9, 10, shows the interaction effects of the concentration of AgNO3, pH of the solution and incubation time on the yield of AgNPs. According to Fig. 8, as the Concentration of AgNO3 increases from 1 to 2.2185 mM and the pH of the solution increases from 8 to 12, the yield of AgNPs increases, whereas the AgNO3 gradually turns 2.285 mM, and the yield of AgNPs starts decreasing. The U-shape 3D-response surface also suggests an optimization condition in the biosynthesis of AgNPs with a decisive effect on AgNO3 concentration. AgNO3 concentration instantly has recognizable effects during the Ag + reduction to the silver particles. The formation of AgNPs are accelerated by increasing the silver nitrate (AgNO3) concentration. This suggests that the higher concentration of silver ions (Ag +), along with the availability of bioactive reducing agents (enzymes and proteins), facilitates the reduction reaction and allows it to reach the optimal equilibrium for AgNPs synthesis more rapidly 60,21.

Plot of AgNPs yield, pH and AgNO3 concentration.

Plot of AgNPs yield, stirring time and AgNO3 concentration.

Plot of AgNPs yield, pH, and stirring time.

There is also a significant interaction effect between stirring time and AgNO3 concentration. Figure 9 indicates that as the time of exposure and the AgNO3 concentration increase from 1 to 2.91 h and 1 to 2.2185 mM, respectively, the yield of AgNPs increases due to enough exposure time for the metal ions to reduce to AgNPs. Regarding this,47 observed an increase in the yield of AgNPs with an increased reaction/incubation time reducing agent.

Figure 10 illustrates the connection between pH, stirring time, and silver nanoparticle yield. A higher pH generally leads to more nanoparticles. This happens because the higher pH changes the electrical charge in the solution, allowing the silver ions to bind more easily to the biomolecules. Since silver ions are positive, they are drawn to the negative charges on the biomolecules.

A residual study evaluated the model’s validity and how close a model approximation is to the real experimental data. Figure 11 presents the main tools important for diagnosing the model validation model. The residuals vs. predicted plot, as well as the residuals vs. experimental run, showed a random scatter pattern. The residuals are evenly spread across both positive and negative values, ranging from -3 to + 3. Therefore, the model generated is adequate for analyzing and evaluating the optimization experiment.

Normal plot of Residuals and versus the experimental run.

Moreover, experimental model validation was made by analyzing the predicted values and the experimental values of AgNPs yield. A graph from Fig. 12 shows a linear relationship between the predicted versus experimental results of AgNPs yield, and the correlation graph indicates that a predicted model fits well with the actual results with an error of R = 0. 989.

The comparison plot of experimental and predicted values of silver nanoparticle yield.

The model was successfully created. It reveals a connection between the variables that influence AgNPs yield production. All trials were repeated three times to ensure the accuracy and reliability of the CCD model of RSM and to better understand the response. An experiment was conducted to analyze the optimization results and confirm the developed model based on the optimal conditions for AgNPs synthesis. The quadratic model predicted a AgNPs yield of 126.037 unit at the optimum conditions (pH = 11.6198, AgNO3 concentration = 2.2185 mM, and incubation time = 2.91 h). Experimental verification was done using triplicate at the same optimal conditions. Accordingly, AgNPs yield of 121.88 ± 1.87 unit was obtained from experimental work.

1. Dynamic light scattering (DLS) analysis

Dynamic Light Scattering (DLS) of Aloe vera-synthesized silver nanoparticles (AgNPs) is controlled by various physical properties such as ionic strength, charge on the surface, aggregation of the particles, and conditions of the synthesis process. These parameters have a direct effect on the hydrodynamic diameter, polydispersity index (PDI), and particle size distribution as shown in experiments with AgNPs61.The size distribution of AgNPs was analysed by dynamic light scattering. According to Fig. 13, the colloidal solution contains AgNPs with average sizes of 68.79 nm. The synthesized AgNPs had a polydispersity index (PDI) value of about 0.374. Our result is well below the acceptable limit of 0.7 for PDI values, which means our sample has a narrow particle size distribution62. The concentration of AgNPs is a key factor in determining the size and shape of silver nanoparticles. While higher amounts tend to favor larger particles and varied shapes, and lower amounts smaller, uniform particles, the precise outcome is a result of many interacting factors.

Size distribution of AgNPs obtained using Malvern Zetasizer; nano ZS.

2. X-ray diffraction pattern of AgNPs

The X-ray diffraction pattern of the synthesized AgNPs is shown in Fig. 14. The intense peak with no secondary diffraction peaks corresponding to the diffraction pattern of AgNO3 and their side products indicates the silver element’s and its crystals’ existence. Interestingly, the powder contains 70.814% crystallite particles which Ag highly dominated–3C silver crystalline phase. The broad shape of the diffraction spectra illustrates the presence of crystalline silver grains with small sizes in the XRD measurement range63,64. XRD analysis shows several strong diffraction peaks at 2θ = 38.06, 43.95, 64.37, 77.43 which corresponds to Bragg reflections of (111), (200), (220) and (311) planes respectively. Each Brags reflection is attributed to the presence of face-centered cubic symmetry (FCC) Ag–3C silver crystalline arrangement with lattice parameters of a = 4.0855 Å and calculated density 10.506 g/cm3. Besides the cubic crystal phase, there is also an indication of the small amount of hexagonal structure of silver nanocrystals found in the powder.65 suggested that the formation of cubic and hexagonal crystal phases of AgNPs is due to the rate of diffusion reaction and growth rate of the individual crystalline. The higher amount of the FCC of Ag-3C silver metal crystal contributes higher stability to the AgNPs powder clusters66. XRD analysis confirms the FCC crystalline structure, nanosize range, and phase purity of biosynthesized AgNPs. These properties form the basis of their structural stability for their uses in plant-based antibacterial nanomaterials, with controlled crystallite dimensions and lattice structure maximizing their targeting of microorganisms67.

X-ray diffraction pattern of AgNPs mediated using extract of Aloe Vera leaf and 2.5 mM of silver nitrate solution.

3. Fourier transform infrared spectroscopy (FTIR) analysis

The FTIR results (Fig. 15) show us the fingerprint of the dried AgNPs created using Aloe vera. It’s clear from the peaks that the natural compounds in the Aloe vera extract were essential for both making and keeping the nanoparticles stable. Table 6 adds more detail about these functional groups, and Fig. 15 helps us visualize them.

FTIR spectroscopy of synthesized AgNPs using an extract Aloe vera leaf.

4. Morphological analysis of AgNPs

To get a closer look at the silver nanoparticles, we used a powerful microscope called a scanning electron microscope (SEM). The images (Figure 16) show that the nanoparticles are mostly spherical in shape, which is consistent with what others have found27 and53.

Scanning electron microscopy image of silver nanoparticle synthesized with an Aloe Vera plant pulp leaf extract.

5. Differential scanning calorimeter (DSC) analysis of synthesised silver nanoparticles

The thermal behaviour of a biosynthesized AgNPs powder was depicted by Differential scanning calorimeter (DSC) analysis. It can be seen from Fig. 17 that the DSC thermogram of synthesized AgNPs reveals an endothermic peak from 30 to 88.9°C reveals the evaporation of water from the surface AgNPs. Another endothermic peak at 186, 241.5 and 327.3°C indicates the phase change of organic chemicals bound to AgNPs during the reduction reaction while the exothermic peak at 206.1°C indicates the decomposition of organic materials. This result shows that the AgNPs mediated by Aloe vera are thermally stable up to 206.1°C. Analysis of DSC data shows that silver nanoparticles created using Aloe vera are quite stable when heated. They exhibit specific temperature changes – an endothermic shift related to the loss of moisture and an exothermic shift indicating the breakdown of the organic coating. This thermal behavior suggests these nanoparticles could be really useful in delicate medical and environmental applications where precise temperature control is important68.

Differential Scanning calorimeter thermograph of biosynthsized silver nanoparticles.

Figure 18 reveals that AgNPs with small concentrations can penetrate the fungal, indicating that even a small amount of nanoparticles can be enough for microbial control. The trend of the data from the plotted graph illustrates that as concentration increases, the inhibition zone also increases, even though the increasing effect of antifungal activity was highly demonstrated as concentration increased from 2 to 6 mg/ml.

The comparison of AgNPs concentration effect on the antifungal property.

The antifungal properties AgNPs have novel properties which could be due to morphological, size, and physiological changes and the particles are highly reactive as it produce Ag ions. In contrast, the bulk metallic silver is relatively unreactive. According to Medda et al.31, AgNPs has a severe effect on the transporting system of microorganism cells, and further dysfunction of ion efflux.

Aloe vera-based AgNPs created an inhibition zone. This zone was effective against multidrug-resistant human pathogens. It showed activity against Gram-positive bacteria, like Staphylococcus aureus, and Gram-negative bacteria, such as Salmonella typhi and E. coli. For each bacterial strain, we saw a clear area where growth was stopped, ranging from 8 to 19 mm in size. Figure 19 shows the effect of different concentrations of AgNPs on each bacterial strain. It showed a magnificent inhibition capacity (19 mm) against Staphylococcus aureus followed by S. typhi whereas a test against Escherichia coli showed a minimum inhibition zone relative to other strains. It can stop bacteria from growing, and researchers have several ideas about how this happens. Tippayawat et al. (2016)69 found that the bacteria’s outer layers (cell wall and proteins) play a key role, affecting how the nanoparticles can attach and penetrate.

The comparison of AgNPs concentration effects on its antibacterial activity.

Nanoparticles attach themselves to a cell wall and membrane, increasing membrane cell permeability and intracellular leakage, with the subsequent deactivation of enzymes and denaturing of DNA molecules70,71. The current study deduces that E. coli is highly resistant to AgNPs due to its thick layer of peptidoglycan relative to Salmonella Typhi and S. aureus. It work by disrupting the bacteria’s internal systems. They trigger a chain reaction of damage, affecting the bacteria’s proteins, cell walls, and DNA. This ultimately stops the bacteria from functioning and leads to its death. Biosynthesized AgNPs show antibacterial effects against both Gram-positive (e.g., Staphylococcus aureus, Bacillus subtilis) and Gram-negative bacteria (e.g., Escherichia coli, Klebsiella pneumoniae), with inhibition zones ranging from 5.5 mm to 39 mm depending on the strain and synthesis method72.

Recently, biosynthesis of AgNPs using plant extracts has appeared as a novel approach due to the broad availability of plant extracts and the bio­degradability of active metabolite components. In the present study, a robust, simple, cheap, and eco-friendly biosynthesis of AgNPs using an Aloe vera leaf extract was successfully achieved via the reduction of AgNO3. For the first time, the process conditions for the biosynthesis of AgNPs using an Aloe vera leaf extract were optimized using the response surface methodology. After performing DOE based on central composite design (CCD), the statistical optimization model revealed that 2.2185 mM of AgNO3 for 2.91 h at pH 11.91 is the optimum condition to synthesize a higher yield of AgNPs.

The formation of it was first characterized by the colour change of the reaction solution from white to a reddish-brown colour for the first 5–10 min of the incubation time. The colour change was due to the surface plasmon resonance band properties of silver nanoparticles. The UV–Vis spectra revealed the appearance of the surface plasmon resonance band in the range of 350 to 500 nm. The morphology of the particles was identified with scanning electron microscopy, and the particles were arranged spherically, cluster-to-cluster. Analyses from DLS revealed that the average size of particles was 68.79 nm, with a good polydispersity index value (PDI) of 0.374. The formation of face-centred cubic structured Ag was also demonstrated by using X-RD analysis. The DSC analysis report also indicated good thermal stability. The antimicrobial study revealed that the synthesized silver nanoparticle had a strong antimicrobial effect against Staphylococcus aureus, Escherichia coli, Salmonella thypae and fungus species of Aspergillus Niger. According to the outcome of this study, the synthesized biogenic AgNPs can treat multi-drug-resistant microbes. For the next studies, it would be good to check how temperature affects the nanoparticles, and also see if they stay stable for a long time.

All data generated or analysed during this study are included in the manuscript. Any other information available from the corresponding author on reasonable request.

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The authors are grateful to Ethiopia, ASTU, and AASTU for supplying the facilities and technical assistance required. The authors also thank the staff of Adama Science Technology University Material Science and Engineering Department, Ethiopia’s Adama for XRD analyses.

This research received no specific funding from any agency, public or private.

Department of Chemical Engineering, Institute of Technology, University of Gondar, Gondar, Ethiopia

Tebelay Liknaw

Department of Chemical Engineering, College of Biological and Chemical Engineering, Sustainable Energy Centre of Excellence, Addis Ababa Science and Technology University, Addis Ababa, Ethiopia

Yohannes Belay

Department of Chemical Engineering, School of Mechanical, Chemical and Material Engineering, Adama Science and Technology University, 1888, Adama, Ethiopia

R. Ramesh

Chemical and Energy Engineering Programme Area, Faculty of Engineering, Universiti Teknologi Brunei, Tungku Highway, Gadong, BE1410, Brunei

Reddy Prasad D.M.

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R. Ramesh performed the visualization, conceptualization, and supervision; Reddy Prasad reviewed and edited the whole article.; Tebelay Liknawa contributed to experimentation, data collection, and Yohannes Belay data analysis and software.

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Liknaw, T., Belay, Y., Ramesh, R. et al. Aloe vera leaf extract as a sustainable route for silver nanoparticle synthesis with enhanced antimicrobial activity. Sci Rep 15, 22481 (2025). https://doi.org/10.1038/s41598-025-05070-5

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DOI: https://doi.org/10.1038/s41598-025-05070-5

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