Green synthesis and toxicological evaluation of zinc oxide nanoparticles utilizing Punica granatum fruit Peel extract: an eco-friendly approach

Ali Mohammadi; Negin Hashemi; Mohaddeseh Ghassabzadeh; Ali Sharafi; Alireza Yazdinezhad; Hossein Danafar

doi:10.1038/s41598-025-05544-6

. 2025 Jul 1;15:20853. doi: 10.1038/s41598-025-05544-6

Green synthesis and toxicological evaluation of zinc oxide nanoparticles utilizing Punica granatum fruit Peel extract: an eco-friendly approach

Ali Mohammadi ^1,², Negin Hashemi ^1,², Mohaddeseh Ghassabzadeh ^1,², Ali Sharafi ¹, Alireza Yazdinezhad ^3,⁴, Hossein Danafar ^1,^2,^✉

PMCID: PMC12215352 PMID: 40596273

Abstract

The green synthesis approach for nanoparticle production offers several advantages over traditional physical and chemical methods. Notably, it avoids the use of hazardous chemicals and acts as a cost-effective and eco-friendly process. This study focuses on the green synthesis of zinc oxide nanoparticles (ZnO- NPs) using the Punica granatum fruit peel extract. Punica granatum fruit peel extract was chosen for the environmentally friendly synthesis of ZnO nanoparticles due to its rich composition of bioactive compounds, including polyphenols, flavonoids, and tannins, which function as natural reducing and stabilizing agents. The morphological, structural, and optical observations were confirmed through various techniques such as X- ray diffraction (XRD), UV-Vis spectroscopy, field emission scanning electron microscopy (FE- SEM), Fourier- transform infrared spectroscopy (FT- IR), EDAX and dynamic light scattering (DLS). To further investigate the impact of synthesized nanoparticles, hemolysis and MTT (3-[4, 5- dimethylthiazol- 2- yl]− 2, 5-diphenyl tetrazolium bromide) assays were conducted. The morphology of the resulting nanoparticles was found to be spherical and homogeneous. DLS results show the hydrodynamic size of nanoparticles with a Z-average of 187 nm with polydispersity index (PdI) of nanoparticles to be 0.298 and zeta potential of nanoparticles is -17.6 mV. The results showed that green synthesis using Punica granatum fruit peel extract resulted in significantly higher cell viability in culture compared to traditional chemical synthesis. This finding highlights the project’s aim to emphasize the advantages of the green synthesis approach on HFF-2 cell lines. These results offer powerful evidence for the potential capability of green synthesis ZnO nanoparticles with Punica granatum fruit peel extract opens up a new avenue of research in this area.

Keywords: Zinc oxide, Green synthesis, Punica granatum fruit Peel extract, Chemical synthesis, HFF2 cell lines

Subject terms: Cancer, Materials science

Introduction

Nanotechnology acting an exciting part in the parts of computing, power generation, optics, drug delivery, and environmental sciences¹. In the beginning of nanotechnology, many nanoscale devices have been established using many approaches, such as physical, chemical, and green approaches. So far, green nanoparticle synthesis is a tool of choice that can be simply organized and planned^2–8. There are many problems of conventional methods for the synthesis of nanoparticles, including long term processing, high cost, laborious procedures, and in particular the use of toxic compounds. The applicable.

study has been directed to eco-friendly and wild synthesis procedures for the construction of nanoparticles due to these limitations^9,10.To attain more effectiveness and diminish the adverse impacts on material, scientists were compelled to turn to green chemistry by employing nanotechnology¹¹. Nanostructures of various metal oxides have been synthesized, demonstrating utility across multiple applications. Outstanding to their numerous tenders in many mechanical fields, complete examination into metal oxide nanoparticles has been focused in the previous period¹². Between these, with complicated benefits, Zinc oxide nanoparticles (ZnO-NPs) are thrilling inorganic materials. ZnO-NPs can be used in several segments, such as energy preservation, textiles, electronics, healthcare, catalysis, cosmetics, semiconductors, and chemical sensing^13–17. ZnO, characterized by a broadband gap of 3.3 eV, is noteworthy for its versatility¹⁸. It is employed in diverse technologies, including solar cells, gas sensors, and dye degradation processes. ZnO-NPs have been synthesized using chemical and physical techniques, such as Sol-Gel processes, precipitation, hydrothermal synthesis, arc discharge, and pulsed laser ablation^13,19,21. Recently, green synthesis has gained more attention as a viable alternative due to its environmental friendliness and great benefits. This approach employs plant extract materials that can be easily sourced from plants, thus avoiding the need for detrimental chemical reagents²². An extract is a concentrated substance derived from a plant or other organic material, achieved through distillation, pressing, or boiling with a solvent, typically water or alcohol. These extracts encapsulate the active compounds present in plants, which contribute to the therapeutic effects associated with medicinal herbs²³. The fruit peel extract of Pomegranate (Punica granatum) was employed as a green stabilizer in the preparation of ZnO-NPs, which were subsequently subjected to a series of characterization tests²⁴. Pomegranate (Punica granatum) peel consists of a complex system of internal membranes and is regarded as approximately 26–30% of the fruit’s overall weight. This structure is notable for its high concentration of phenolic compounds, which includes various flavonoids, such as anthocyanins, catechins, and other complex flavonoid structures, as well as hydrolyzable tannins like gallic acid, ellagic acid, punicalin, and punicalagin²⁵. In cancer treatment, studies have indicated that Punica granatum demonstrates striking anticancer activity against various cancer cell lines, including bladder cancer (T24), cervical cancer (HeLa), breast cancer, prostate cancer, thyroid cancer, and colon cancer²⁶. Antioxidants are molecules known to inhibit or moderate the reactions associated with free radicals and mitigate cellular damage²⁷. Among these compounds, phenolic components of Punica granatum fruit peel extract are gaining increased attention for their potential as effective antioxidants that support optimal physiological function²⁸. Their mechanisms of action include: preventing the generation of free radicals, neutralizing oxidizing agents, converting harmful free radicals into less detrimental forms, inhibiting the formation of toxic byproducts, interrupting the cascade of free radical-induced damage, facilitating cellular repair processes, enhancing the body’s intrinsic antioxidant defenses by disrupting the harmful effects associated with free radicals, antioxidants play a crucial role in protection cells and tissues against oxidative stress, which leads to a variety of health benefits²⁹. Punica granatum fruit peel extract was chosen for the environmentally friendly synthesis of ZnO NPs due to its rich composition of bioactive compounds, including polyphenols, flavonoids, and tannins, which function as natural reducing and stabilizing agents. When compared to other plant or fruit extracts, Punica granatum fruit peel extract provides superior control over the size and morphology of nanoparticles, resulting in more uniform and stable ZnO-NPs. Furthermore, it exhibits remarkable biocompatibility, displaying low toxicity towards Human Foreskin Fibroblast-2 cells (HFF-2 cell line) and reduced hemolytic activity, which makes it particularly suitable for biomedical applications. Recent studies have highlighted considerable interest in ZnO-NPs because of their diverse and advantageous characteristics. Specifically, these nanoparticles demonstrate high stability, biodegradability, versatile surface chemistry, and intrinsic biocompatibility. These attributes make them suitable for various applications, including antibacterial, antineoplastic, and immunogenic functions. The structure and characteristics of the nanoparticles were analyzed through various analytical techniques. Transmission Electron Microscopy (TEM) shows the shape of ZnO-NPs, and DLS was employed for particle size analysis³⁰. To determine the structural features, FTIR was utilized³¹, while Vis-UV was used for nanoparticle identification and analysis³². Additionally, XRD provided insights into the structural properties³³, and FE-SEM was used for detailed structural examination of the particles³⁴. For further assessment, we also performed the MTT assay and blood compatibility tests³⁵. This study aims to produce ZnO-NPs utilizing an extract derived from Punica granatum fruit peel extract and to evaluate their cytotoxic effects on HFF-2 cells³⁶ in comparison to ZnO-NPs synthesized through chemical methods. Herein, recent studies have reported that ZnO-NPs synthesized with Punica granatum fruit peel extract show improved size control, higher dispersion stability, and enhanced biocompatibility compared to chemically synthesized counterparts. In their study, Abdelmigid et al. demonstrated that biosynthesized ZnO-NPs using Punica granatum fruit peel extract exhibited superior antibacterial properties and favorable cytocompatibility profiles³⁷. Similarly, research conducted by Fouda et al. revealed that ZnO-NPs derived from Punica granatum fruit peel extract possessed improved catalytic activity and exhibited dose-dependent antimicrobial effects³⁸. These findings underscore the potential of Punica granatum fruit peel extract as an effective bio reductant in the synthesis of nanoparticles. The biosynthetic method, which employs plant extracts like pomegranate peel, presents significant environmental benefits compared to traditional chemical synthesis techniques. Conventional nanoparticle synthesis frequently relies on hazardous chemicals, solvents, and high-energy procedures, leading to environmental contamination and potential health risks. In contrast, plant extracts are renewable and non-toxic, typically containing abundant natural compounds that facilitate the reduction and stabilization of nanoparticles without the necessity for deleterious chemicals or intensive energy requirements. These results indicated that the use Punica granatum fruit peel extract as a green synthesis material contributes to the biocompatibility and safety of ZnO-NPs.

Method and materials

Materials

FBS, Acetone, HNO3, (CH3COO)2Zn.2H2O, Ethanol, sulfoxide (DMSO) and 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) (MTT) were acquired from Sigma Aldrich (St. Louis, USA). All solvents were of analytical grade, purchased from Merck. HFF2 cell line was obtained from the Pasteur Institute in Iran.

Methods

Plant collection and identification

The plant Punica granatum with voucher name 2705 was purchased from reputable centers and identified and approved by the pharmacognosy group of the school of pharmacy Zanjan university of medical sciences. The fruit peel is separated and washed with water, followed by a rinse with distilled water to remove impurities. Then it was dried in an oven at 40 degrees Celsius for two days.

Extraction

The dried herbs were crushed using a mortar and pestle. 150 g of the resultant fine powder were placed in a funnel. Then, 70% ethanol was added to a depth of 2 cm above the powder. After a day, the extract was drained from the funnel. The extraction process was repeated once more from the ethanol addition step. After repeating the steps three times, the attained extract was transferred to a rotary evaporator to remove the alcohol. Ultimately, the gummy residue was collected and utilized for nanoparticle preparation with the green synthesis approach.

Green synthesis of ZnO nanoparticles

In the synthesis of ZnO-NPs, 4 g of zinc acetate dihydrate were first dissolved in 25 mL of deionized water, yielding a 0.5 M solution. This solution was then heated to a temperature of 70 °C while being stirred continuously with a magnetic stirrer. Following this, 2.5 mL of Punica granatum fruit peel extract was introduced gradually in a dropwise manner. The pH of the resulting mixture was subsequently adjusted to 11 using a 1 M sodium hydroxide (NaOH) solution. The reaction was conducted under reflux conditions with ongoing stirring for a duration of 24 h (h). Upon completion of the reaction, the resultant white precipitate was isolated through centrifugation at 10,000 rpm for 10 min. The precipitate was then washed three times with deionized water and dried using freeze-drying techniques to obtain powdered ZnO-NPs.

Chemical synthesis of ZnO nanoparticles

For comparative purposes, ZnO-NPs were synthesized using a chemical precipitation method. Initially, a solution containing 2 g of zinc acetate dihydrate was prepared in 15 mL of deionized water. In a separate container, 8 g of NaOH were dissolved in 10 mL of deionized water. This NaOH solution was then gradually introduced to the zinc acetate solution while maintaining constant stirring for a duration of five minutes. Following this, 100 mL of ethanol was added dropwise, resulting in the formation of a white precipitate. This precipitate was then isolated through centrifugation at 10,000 rpm for 10 min. The resultant ZnO-NPs were washed three times with distilled water and subsequently dried in an oven at 40 °C.

Characterization of nanoparticles

UV-Vis analysis

UV-Vis spectroscopy was utilized to examine the synthesis of nanoparticles and to assess their optical properties. This analytical technique measures the absorption wavelengths of various compounds, facilitating the identification of different substances. Each material exhibits a unique absorption wavelength, which is determined by its specific absorption mechanism. Given their distinct characteristics, nanoparticles are typically excited at particular wavelengths, resulting in maximum absorption. Prior to the analysis, the spectrophotometer was calibrated using deionized water. Subsequently, the absorption profiles of both green and chemically synthesized nanoparticles were recorded within a wavelength range of 200 to 800 nm. To estimate the optical band gap energies, Tauc plots were constructed based on the absorbance data. The photon energy hνh\nuhν was calculated from the corresponding wavelengths using the equation:

where E is the photon energy in electron volts (eV) and Inline graphic is the wavelength in nanometers (nm). Assuming a direct allowed electronic transition (as is typical for ZnO), the Tauc relation:

was applied, where Inline graphic is the absorption coefficient (proportional to absorbance), is the photon energy, A is a constant, and E_g is the optical band gap energy. Absorbance values were used as a proxy for , and the Tauc plots of vs. were plotted for each sample. The band gap energy E_g was determined by extrapolating the linear portion of the Tauc plot to the x-axis intercept, corresponding to the energy at which Inline graphic 0.

FT-IR analysis

A potassium bromide pellet was synthesized to identify functional groups and verify the presence of phytochemicals associated with stabilization. This process involved examining the bonds formed in nanoparticles and analyzing the vibrations of their chemical bonds. To achieve this, the dry powder of the sample was mixed with potassium bromide powder at a ratio of 1:100. The resultant mixture was then compressed into thin discs using a hydraulic press. These discs were subsequently placed in a Fourier Transform Infrared (FT-IR) spectrometer, where their spectra were recorded at ambient temperature, spanning a frequency range from 500 to 4000 cm⁻¹.

XRD analysis

The X-ray diffraction pattern of the zinc oxide nanoparticles was acquired using a Bruker AXS Advance 8D diffractometer, employing Cu Kα radiation (λ = 1.542 Å) within a scanning range of 10 to 80 degrees. X-ray diffraction (XRD) serves as a specialized tool for assessing the crystalline structure and phase purity of nanoparticles. The operational principle of the XRD instrument involves irradiating the sample with X-rays and subsequently examining the diffraction or reflection pattern produced.

DLS analysis

Dynamic Light Scattering (DLS) is a technique utilized to ascertain the particle size distribution and the polydispersity index (PDI) by analyzing the random fluctuations in the intensity of light scattered from a suspension or solution. To accurately evaluate the nanoparticle size, the sample was diluted to minimize viscosity. Following this, the absorbance of control samples was measured at a wavelength of 633 nm with a spectrophotometer. Ultimately, the particle size was assessed at room temperature using the DLS instrument. The measurement of zeta potential is employed to assess both the surface charge and colloidal stability of nanoparticles. In this study, the zeta potential of the synthesized ZnO-NPs was analyzed utilizing a zeta sizer.

FE-SEM analysis

This method is employed to investigate the surface morphology and dimensions of the nanoparticles. The morphological characteristics of the zinc oxide nanoparticles were evaluated utilizing FE-SEM at an accelerating voltage of 30 kV. Additionally, the occurrence and distribution of carbon, nitrogen, oxygen, and silver atoms within the formulations were examined through Energy Dispersive X-ray (EDX) analysis.

Hemolysis assay

The hemolysis test was performed to evaluate the blood compatibility of ZnO-NPs synthesized by the aforementioned modalities. Fresh whole blood was collected from a volunteer and kept in a heparinized tube to prevent clotting. The blood was used within 2 h. 5 mL of human red blood cells (RBCs), were prepared and divided equally into two test tubes. After introducing the PBS buffer to the samples, they were centrifuged at 2000 rpm for 10 min. The supernatant was subsequently discarded, and the pellet, which contains the RBCs, was washed with PBS buffer five times. ZnO-NPs synthesized using Punica granatum fruit peel extract was prepared at various concentrations (50, 100, and 200 ppm) in PBS buffer and incubated with RBCs at 37 °C for 24 h using a shaker incubator. Finally, the microtubes underwent centrifugation at 14,000 rpm for 15 min. Following this, 100 µL of supernatant from each microtube were collected. The absorbance of hemoglobin was measured at a wavelength of 540 nm using a UV-Vis spectrophotometer. The percentage of hemolysis for each drug concentration was then calculated using the specified formula. For the controls, vials containing water and PBS buffer served as the positive and negative controls, respectively.

All the above-mentioned steps were also performed for chemically synthesized ZnO-NPs.

MTT assay

Cell viability was assessed after treatment with an external agent using a colorimetric assay. In viable cells, yellow tetrazolium crystals are reduced and cleaved by the enzyme succinate dehydrogenase, leading to the formation of insoluble purple formazan crystals. The formazan precipitate was dissolved in DMSO, and the intensity of the resulting color was measured using a spectrophotometer. The intensity of this color is directly related to the quantity of viable cells present. In this investigation, the HFF2 cell line was obtained from the Pasteur Institute in Iran. These cell lines were stored at -80 °C for use in experiments throughout the study. The suitable culture medium for it consists of DMEM and 10% FBS. To accomplish the MTT assay, 7,000 HFF-2 cells were deposited into each well of a 96-well plate, followed by the addition of 100 µL of the specific culture medium. The plates were then incubated at 37 °C for 24 h. After the incubation period, the growth medium was discarded, and three concentrations (15, 30, and 60 µg/mL) of nanoparticles, synthesized using both green and chemical methods, were introduced into the wells. The volume was adjusted to 100 µL with the specific culture medium. The plate was placed in a shaking incubator set at 37 °C for 24 h to allow for proper cell growth. After this incubation period, we carefully removed the culture medium and washed the cells with PBS to ensure a clean environment. Next, we added 100 µL of fresh culture medium to each well to maintain optimal conditions for the cells. Following this, we introduced 10 µL of MTT indicator into each well, and once again placed the plate in the shaking incubator at 37 °C, this time for 4 h. After the incubation, we added 100 µL of DMSO to each well and shook the plate for 10 min to ensure thorough mixing. Finally, we measured the absorbance in all wells at a wavelength of 570 nm using an ELISA reader to assess the results.

Results

UV-Vis and band gap analysis

The Vis-UV spectra of ZnO-NPs prepared with green and chemical synthesis and the extract are shown in Fig. 1B. The ZnO-NPs sample shows a distinct absorption peak around 230 nm, attributed to the intrinsic band gap absorption of ZnO due to electron transitions from the valence band to the conduction band. In contrast, the plant extract and Zn@NPs Green method samples exhibit broader absorption bands, indicating the presence of organic phytochemicals and surface modifications introduced during the green synthesis. The visible-ultraviolet absorption spectrum of the Zn@NPs chemical method is shown in the wavelength range of 200 to 800 nm. The UV-Vis absorption spectrum for Zn@NPs Green method showed an absorption peak at 280 and 360 nm. Previous studies have identified peaks in the wavelength range of 289 to 385 nm as the surface plasmon resonance (SPR) of ZnO-NPs. The volume of plant extracts and other reaction conditions can also cause changes in the SPR bands of ZnO-NPs. ZnO-NPs exhibited a characteristic peak at 360 nm, which is related to Zn-O, and this peak is also observed in the diagram of the chemical synthesis of nanoparticles but is not present in the diagram of the extract. To further analyze the optical band gap energies, Tauc plots were generated assuming a direct allowed transition, i.e., plotting Inline graphic vs. , as shown in Fig. 1B. From the extrapolation of the linear region of the plots, the estimated band gap energies for Zn@NPs chemical method, Zn@NPs Green method, and plant extract are 5.51, 5.34, and 4.99 eV, respectively. The slight red-shift (band gap narrowing) observed in the Zn@NPs Green method sample compared to bare Zn@NPs chemical method suggests a successful surface functionalization with Punica granatum fruit peel extract. The presence of phytochemicals possibly induces defect states or surface plasmon resonance effects that reduce the energy required for electronic transitions. This band gap tuning not only confirms the effective green synthesis but also implies potential for enhanced photocatalytic or biomedical performance due to altered light absorption characteristics.