<i>Garlic</i> Extract-Mediated Synthesis of ZnS Nanoparticles: Structural, Optical, Antibacterial, and Hemolysis Studies

Alnehia, Adnan; Al-Odayni, Abdel-Basit; Al-Hammadi, A. H.; Alramadhan, Safiah A.; Alnahari, Hisham; Saeed, Waseem Sharaf; Al-Sharabi, Annas

doi:https://doi.org/10.1155/2023/8200912

Journal of Nanomaterials

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Abstract Introduction Materials and Methods Results and Discussion Conclusion Data Availability Conflicts of Interest References Copyright Related Articles

Research Article | Open Access

Volume 2023 | Article ID 8200912 | https://doi.org/10.1155/2023/8200912

Garlic Extract-Mediated Synthesis of ZnS Nanoparticles: Structural, Optical, Antibacterial, and Hemolysis Studies

Adnan Alnehia,^1,2Abdel-Basit Al-Odayni,^3,4A. H. Al-Hammadi,¹Safiah A. Alramadhan,^3,5Hisham Alnahari,¹Waseem Sharaf Saeed,³and Annas Al-Sharabi²

Academic Editor: Abdelwahab Omri

Received12 Oct 2022

Revised13 Dec 2022

Accepted14 Dec 2022

Published12 Feb 2023

Abstract

The green synthesis of zinc sulfide nanoparticles (ZnS NPs)-mediated plant extract is gaining importance because of its simplicity, cost-effectiveness, and ecofriendly nature. In this work, ZnS NPs were synthesized using garlic extract as NPs facilitating agent, characterized by Fourier transform infrared, X-ray diffraction, scanning electron microscope, and UV–visible, then their antibacterial and hemocompatibility were assayed. Analysis revealed a cubic phase, 2.33 nm crystallite size, and a 3.75 eV optical bandgap. Bioactivity test against Staphylococcus aureus and Escherichia coli indicated dose-dependent potency closer to that of azithromycin standard drug and more efficient on S. aureus (Gram-positive) than E. coli (Gram-negative) bacteria. Biocompatibility test in terms of erythrocyte hemolysis, in reference to normal saline and water as minimal and maximal controls, confirmed nontoxic substance up to 100 μg/mL as the highest examined concentration and at which a lysis of 2.9% was detected. Therefore, it could be concluded that this biogenic method is effective in producing ZnS NPs with desirable properties for potential biomedical applications.

1. Introduction

Green chemistry for sustainable development has gained greater interest in the last few years with researchers aim to reduce hazards via developing of greener methods for nanomaterials biosynthesis using plants as one simple and ecofriendly approach [1]. In principle, the synthesis of nanomaterials demands a selection of (1) adequate solvent, (2) a good reducing agent, and (3) a good stabilizing agent. Although various chemical methods can be applied for the synthesis of NPs, bio-approach is considered safer, eco-friendly, and low cost. Therefore, plant-mediated synthesis of NPs has become an innovative industrial technique with plenty of benefits due to plant obtainability and uncostly, the method simplicity and scaling up possibility, and the NPs obtained size and shape tailorability [2, 3].

Plant-based biosynthesis of NPs basically involves the application of plant extracts to reduce and stabilize metal ions through some implicated plant phytocompounds like amino acids, alkaloids, enzymes, saponins, terpenoids, phenolics [4, 5], etc. ZnS is a promising semiconductor with a wide bandgap, which benefiting electronic, optoelectronic, and electrochemical devices [6]. ZnS NPs commonly show distinct features different from its bulk counterpart as a result of high surface-to-volume ratio [7]. It can be synthesized by various methods such as physical, chemical, and biological approaches [8–10]. While physical routes such as thermal evaporation [11], pulsed laser vaporization [12], and molecular beam epitaxy [13] and chemical methods like coprecipitation [14], sol–gel [9], sonochemical [15], and hydrothermal [10] are widely employed, biosynthesis is still in its advent. In addition, the conventional physical and chemical methods are expensive and release hazardous materials into the environment [16]; hence, to reduce such risk, biosynthesis has been considered the most reliable and safe method. Furthermore, green synthesis has become preferred as it is quick and suitable for large-scale production, with plant-based extract being most applicable for NPs size and shape control as driven by their phytochemical compounds [17–19]. Bioactivity studies on ZnS NPs have reported significant action against number of test bacteria, being more effective on Gram-positive bacteria, and nontoxic against human erythrocytes [20].

Many studies have illustrated the feasibility of using plant extracts (seed, leaves, and roots) as a capping agent in the synthesis of nanomaterials with certain properties and applications [21–23]. In this regard, ZnS NPs have been the subject of many plant-based biosynthesis studies, of which garlic extract-based was a promising route [20, 24, 25]. Garlic, Allium sativum, is a spice widely used as a food and as a medicinal agent. It is one of the most extensively researched vegetables. It has acquired a reputation as a formidable prophylactic and therapeutic medicinal agent in the folklore of many cultures. It is rich in several sulfur-containing phytoconstituents such as alliin, allicin, ajoenes, vinyldithiins, and flavonoids such as quercetin [26, 27], with aqueous extract having abundance of S-allyl mercapto cysteine and glucose, those may play an important role on NPs production via contribution on capping and stabilizing process [28]. Accordingly, garlic extract can be used as NPs synthesis facilitating agent, where allicin and other carbohydrates are the primary stabilizing moieties [29].

To the best of our knowledge, phytosynthesis of ZnS NPs using garlic-based aqueous extract, and their bioactivity studies, are not well documented. Therefore, the main objective of this work is to utilize garlic aqueous extract for the synthesis of ZnS NPs and to investigate their physicochemical and biological properties. The biogenic ZnS NPs were characterized for their structural properties using Fourier transform infrared (FTIR), X-ray diffraction (XRD), scanning electron microscope (SEM), and UV–visible (UV–vis) and for their bioactivity using antibacterial and hemolysis assays.

2. Materials and Methods

2.1. Materials

Zinc nitrate hexahydrate (Zn(NO₃)₂·6H₂O; ≥99%) and sodium sulfide hydrate flakes (Na₂S·xH₂O; 60%) were purchased from Himedia Laboratories GmbH (Einhausen, Germany). Mueller–Hinton Agar (MHA) was acquired from Sigma–Aldrich (Darmstadt, Germany). Garlic (A. sativum) bulbs were collected from a local market that usually sold after a few months of harvesting. Bacteria strains (Staphylococcus aureus and Escherichia coli) were kindly obtained as a gift from Al-Jarfi Medical Lab (Tamar City, Yemen). Normal saline (NS; 0.9 w/v% sodium chloride; pH 4.5–7.0) was collected from Pharmaceutical Solutions Industry (Jeddah, Saudi Arabia). Deionized water (dH₂O) was used wherever required.

2.2. Preparation of Garlic Extract

Garlic mature bulbs were obtained by local sellers at Thamar city, Yemen, in winter first 2022, cleaned and peeled before grinding. Extraction was carried out following a previously described protocol [7, 30] with some modifications. The peeled bulbs were ground using a houseware electric grinder into fine fragments. Next, 5 g of the obtained garlic matter was added into 100 mL dH₂O and mixed with constant stirring at 25°C for 60 min, then filtered to obtain the intended garlic extract.

2.3. Green Synthesis of ZnS NPs

The biosynthesis of ZnS NPs was carried out as described in literature [2, 7], with slight modification. Thus, stochiometric (0.02 mol) solutions of zinc nitrate (6 g in 25 mL) and sodium sulfide (2.6 g in 25 mL) were separately prepared by dissolving the corresponding salt in dH₂O with magnetic stirring for 15 min. The two solutions were then mixed together and to which 30 mL of the freshly prepared garlic extract was added, then allowed to stir at 25°C for 60 min. The obtained precipitate was then filtered, washed with dH₂O, and dried at 25°C for 48 hr. Finally, the obtained powder was further annealed at 100°C for 60 min. Figure 1 summarizes the overall workflow of the present study, including extract preparation, ZnS NPs synthesis, characterization techniques, and applications.

2.4. Characterization

The XRD pattern was recorded using an XD–2 X-ray diffractometer (Beijing Purkinje General Instrument Co., Ltd., Beijing, China) with CuKα radiation of λ = 1.5418 Å, over two-theta (2θ) 15°–75° and scanning rate of 0.02 min⁻¹. FTIR spectra were obtained by Nicolet iS10 FTIR spectrometer (Thermo Scientific, Madison, WI, USA) using an attenuated total reflection (ATR) accessory (diamond type) on the range of 650–4,000 cm⁻¹, reported as the average of 32 scans and a resolution of 4 cm⁻¹. Surface morphology was imaged using a JSM-7600F field-emission scanning electron microscope (FESEM) (Jeol Ltd., Tokyo, Japan), where the sample was sputter coated with gold before shoot. UV–vis spectrophotometer (U3900, Hitachi, Tokyo, Japan), coupled with Varian Cary-50 software, was employed for acquiring electronic spectra in the range of 200–800 nm at room temperature.

2.5. Biological Studies

2.5.1. Antibacterial Test

The antibacterial activity of ZnS NPs was carried out via the disc diffusion method [31, 32], against S. aureus and E. coli bacteria representing Gram-positive and Gram-negative bacteria types, respectively. Thus, the bacterial strains were individually cultured at 37 ± 1°C for 24 hr. The turbidity of the final nutrient broth was adjusted to achieve an absorbance of 0.075–0.1. In this route, 20 mL of sterilized MHA was poured into sterile petri plates; after solidification, the test bacteria were then spread over MHA plate. The test disks of 0.6 cm diameter were wetted by 20 μL on each side of the target ZnS NPs aqueous suspension (63 and 126 mg/mL). Then, the disks were dried and placed aseptically on the surface of bacterially seeded petri dishes, which were subsequently incubated at 37 ± 1°C for 21 hr. Azithromycin (AzM) and dH₂O were used as positive and negative control, respectively, and treated similarly and simultaneously with test samples. The inhibitory effect was assessed in terms of zone of inhibition (ZOI) by measuring the inhibition area in mm and compared with the controls as described in literature [33].

2.5.2. Hemolytic Assay

Hemolytic activity of the synthesized ZnS NPs was evaluated against human erythrocytes over a concentration range of 3.12–100 μg/mL. The assay was carried out as previously described [2, 34] with few modifications. Briefly, 5 mL of blood was taken from a healthy male volunteer (25 years old, B-positive blood group) after the provision of informed consent. The collected blood sample was transferred into ethylenediaminetetraacetic acid (EDTA) tube. Then, red blood cells (RBCs) were isolated using a typical procedure described elsewhere. Thus, EDTA-blood suspension was centrifuged for 10 min at 4,000 rpm, decanting the supernatant, and pellet was adequately washed with 0.9% NS solution.

The test erythrocytes suspension was diluted as 2% cells while test samples of ZnS NPs were prepared as 3.12–100 μg/mL in NS. Experimentally, 0.5 mL of the cell suspension was mixed with 0.5 mL of each test sample and immediately incubated at 37°C for 60 min. Afterward, solutions were centrifuged at 4,000 rpm for 10 min to remove cell depression, and the supernatant containing free hemoglobin was photometrically measured at 540 nm. Sterile NS and dH₂O were used as minimal and maximal hemolytic controls, respectively, and were experimentally treated as test samples. The hemolytic percentage was calculated based on Equation (1).where A_S, A_N, and A_P are the absorbance of the ZnS NPs, NS, and dH₂O, respectively.

2.6. Data Analysis

Data for bioactivity (antibacterial ZOI and hemolysis) from two-independent experiments were statistically analyzed using Microsoft Excel, Version 2019 (Microsoft Corporation, WA, USA) and reported as the mean and standard deviation. Presented figures and histograms were drawn in OriginPro 2018 software (Origin Lab, Northampton, MA, USA). ImageJ Version 1.52a (National Institute of Health, Bethesda, MD, USA) was used for counting of particle sizes from FESEM micrograph.

3. Results and Discussion

3.1. FTIR Analysis

Figure 2 illustrates the FTIR spectrum of the biosynthesized ZnS NPs. Due to the capacity limit of the ATR-FTIR instrument, the measuring range was limited to 650–4,000 cm⁻¹. In this range, indicative peaks of the NPs, as well as the traced organics involved in the NPs production, can be identified. The broad band centered at about 3,186 cm⁻¹ is characteristic of different hydroxyls (–OH, such as alcohols, phenols, and carboxylic acids) and amines (–NHn) functional groups. Peaks on the wavenumber (e.g., above 3,500 cm⁻¹) are commonly of nonhydrogen bonded OH, while broadness indicates contribution from carboxylic acids [24]. Peaks observed in the range of 1,800–2,600 cm⁻¹ are instrument-based backgrounds [35]. The absorption peak at 1,623 cm⁻¹ can be attributed to C=C stretching bands associated with alkene and aromatic functional groups [36, 37]. The absorption band at 1,310 cm⁻¹ coincides to C–C bond. Peak observed at 1,045 cm⁻¹ may indicate vinyl of allicin (the principle component in garlic extract) and C–O bonds [38]. Bonds at 802 and 702 cm⁻¹ could be assigned to C–O asymmetric stretching and ZnS bending bonds [39], respectively. As a result of the complex structure of capping agents sourced from the target plant, the peaks tend to broaden and combine with shoulders for different functional groups.

3.2. XRD Analysis

To inspect the purity and crystal nature of the biogenic ZnS particles, XRD analysis was performed, and the data obtained were depicted in Figure 3, compared with the standard spectra (JCPDS card no: 05-0566), and tabulated for the corresponding structural and geometry parameters in Tables 1 and 2. The results indicate cubic crystalline structure and, according to Scherrer’s formula [34, 40] computed in MDI Jade6 software, the average crystallite size (D_ave) was 2.33 nm. Additionally, the lattice parameters a, b, and c (5.357 Å), the unit cell volume (153.732 Å³), space group (F43m), and density (3.9 g/cm³) of synthesized sample were calculated following formula described elsewhere [40, 41]. These results are comparable to some other synthesized ZnS NPs [2, 9, 42, 43]. For example, the D value calculated by XRD method for ZnS NPs obtained by, e.g., sol–gel (29.0 nm) [9], Praepagen HY (an alkyl ammonium salt; 4.8 nm) [42], solid-state (4.6), glucose-mediated (5.3) [24], starch-capped (3.3 nm) [43], polyvinyl alcohol-capped (2.9 nm) [43], and uncapped (3.7 nm) [2] were close to obtained one of 2.3 nm.

3.3. SEM Analysis

The field-emission SEM measurement was carried out to get insight into the surface morphology of ZnS NPs. The obtained FESEM micrograph is shown in Figure 4, which demonstrates particles agglomerated in a nanoscale while particles were formed in a uniform manner of shape and size, i.e., cubes and spherical shapes and calculated averaged particle sizes of 30.7 ± 5.3 nm (n = 35).

3.4. UV–Visible Analysis

Zinc sulfide NPs have attracted great attention because of their distinct optical properties with broad applications in optoelectronic devices and biomedical science [44, 45]. As seen in Figure 5(a), the recorded UV–vis spectra revealed an absorption decrease as wavelength increases, showing an absorption edge at about 331 nm; however, the absorption value is exclusively dependent on various factors, including the structure of the material, the size of the NPs and the defects in grain structure. Figure 5(b) also depicts the bandgap energy as determined by Tauc’s plot (Equation (2)) to be 3.75 eV [46, 47].

(a)

(b)

In addition, the particle size for the synthesized ZnS NPs can be calculated using Brus’s equation (Equation (3)) [48, 49].where E_NP and E_g are the bandgap of nano and bulk (3.68 eV) semiconductors, respectively, e is the electronic charge, ϵ is the dielectric constant of materials (for ZnS, ϵ = 8.3), h is the Planck’s constant and r is the radius of the NPs in Nanometer, is the effective mass of the electron, m_e mass of an electron (for ZnS, ), is the effective mass of the hole, and m_h mass of hole (for ZnS, ). Accordingly, the calculated particle size was found as 4.4 nm and being in consistence with other calculations, including XRD method.

3.5. Antibacterial Activity

Figure 6 displays selected plate images for the antibacterial activity of the garlic extract-mediated synthesized ZnS NPs, 63 and 126 mg/mL, as assessed against S. aureus and E. coli bacteria species using disk diffusion method. The calculated average ZOI (diameters, mm) from two-independent experiments were collected in Table 3. It is observed that the NPs obtained in this way have comparable antibacterial activity to that of standard drug as assessed against the two bacteria types, more effective on S. aureus than E. coli those representing Gram-positive and Gram-negative bacteria, respectively. Furthermore, it was found that the activity of ZnS NPs has increased as its concentration increased while being, in all cases, still slightly lower than the effect of standard drug AzM. The higher activity of metal NPs against S. aureus compared to E. coli is a result of the differences in their cell wall structures. Although the peptidoglycan in Gram-negative bacteria (e.g., E. coli) is thinner than that in Gram-positive, the outer membrane in the former may play a role in being less susceptible for antibiotics [32, 36]. Typically, the bioactivity of certain materials is attributed to various factors like the nature of the material, their particle size, shape, surface charge, and application conditions (e.g., concentration and exposure time). Although the mechanism involved in the inhibitory activity is still under debate, it is believed that the electrostatic attraction between the bacterial cell and ZnS NPs (and released species, e.g., Zn²⁺, S²⁻) plays a pivotal role. Generally, the antibacterial mechanism can go through one or simultaneous mechanisms: (1) attachment of NPs into bacterial surface; (2) releasing of ions (i.e., Zn²⁺, S²⁻), which could penetrate into the cell; (3) formation of reactive oxygen species, with the overall action of being led to cell destruction and death.

The antibacterial activity of some other ZnS NPs reported in literatures are given in Table 4 for comparison [9, 15, 50–52]. The ZOI as well as the test method and sample concentration were tabulated as well. Although the ZOI are seemingly higher for bio-based ZnS NPs, the test conditions (concentration and operation, in particular) differ significantly making comparison difficult. However, in each case, authors usually use standard drug as a positive control, which experimentally analyzed simultaneously with test samples. Thus, such controls can facilitate the comparison of activities and explain the results.

3.6. Hemolytic Activity on Erythrocytes

The hemolytic effect of ZnS NPs on human RBCs was in vitro assessed over a NPs concentration range of 3.12–100 μg/mL and in reference to NS and dH₂O as negative and positive controls [53], respectively. Figure 7 illustrates the average hemolytic activity from two independent experiments. In principle, the assay is based on measuring the free hemoglobin, which is released as a result of lysis induced by test samples. As can be seen, the hemolysis of ZNS NPs up to 100 μg/mL is less than 2.9%, which indicate nonhemolytic material. Basically, substances with hemolysis of less than 2% are standardized as nonhemolytic, 2%–5% slightly hemolytic, while more than 5% hemolytic [53]. Herein, the lysis was considerably low, increased in a dose-dependent manner up to 2.9% at 100 μg/mL, being less than 2% up to 12.5 μg/mL and, therefore, indicating nontoxic ZnS NPs at the applied concentrations and analysis condition. Furthermore, the obtained results were close or slightly higher than that reported by other researchers for ZnS over alike concentrations [20]; however, authors investigated longer exposure time and thus detected increased hemolytic activity with extended incubation time. The hemocompatibility of erythrocytes toward ZnS NPs may ascribe a repulsive action of ZnS NPs by the cells as a result of their negative surface charge [20]. Nevertheless, such results provide preliminary data for further in deep investigation of its prospective biomedical application.

4. Conclusion

In this study, ZnS NPs were successfully synthesized first ever using garlic aqueous extract as a simple and green method. Analysis revealed a 2.33 nm crystallite cubic phase structure, 3.75 eV optical bandgap, and 30.7 nm nanoparticles by FESEM. The antibacterial test indicated comparable and dose-dependent activities, slightly below that for AzM standard drug and higher against S. aureus than E. coli. Hemolysis result, in reference to normal saline and deionized water as minimal and maximal controls, respectively, has shown negligible lysis up to 12.5 μg/mL that becomes as higher as 2.9% at 100 μg/mL. Therefore, it is concluded that the utilized bio-based method is an effective in production of ZnS NPs with desirable properties for prospective application in biomedical fields.

Data Availability

The data used to support the findings of this study are included within the article.

Conflicts of Interest

The authors declare that they have no conflicts of interest.

Acknowledgment

The authors extend their appreciation to the Deputyship for Research & Innovation, Ministry of Education in Saudi Arabia for funding this research work through the project no. (IFKSURG-2-1615). The authors are thankful to Dr. Abdullah Al-Jarfi and Dr. Morad G. S. Saleh Laps for the help with biological experiments.

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Copyright © 2023 Adnan Alnehia et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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