Synthesis of ZnO nanoparticles and their evaluations as anti-diabetic, cytotoxic and antibacterial agent

doi:10.21203/rs.3.rs-5169550/v1

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Synthesis of ZnO nanoparticles and their evaluations as anti-diabetic, cytotoxic and antibacterial agent

https://doi.org/10.21203/rs.3.rs-5169550/v1

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In recent years, there has been significant interest in the eco-friendly production of ZnO nanoparticles (NPs) using plant extracts and their potential applications. This study presents a green synthesis method for producing ZnO NPs from Dodonaea viscosa leaf extract. The crystalline phase, morphology, and surface area of the synthesized sample were characterized using FTIR for functional group analysis, XRD for crystal structure determination, and SEM and TEM for surface morphology evaluation. The NPs were then tested for anti-diabetic, cytotoxic, and antibacterial properties. The highest α-amylase enzyme inhibition (30.8%) was observed at the highest concentration of 200 mg/mL, while no inhibition was detected at the lowest concentration of 5 mg/mL. Similarly, the cytotoxicity of ZnO NPs was measured at 21.8% for a concentration of 200 μg/mL, with no hemolysis observed at doses below 5 μg/mL. Additionally, the ZnO NPs demonstrated significant antibacterial activity against both Gram-positive and Gram-negative species.

Green synthesis

Plant extract

Dodonaea viscosa

Anti-diabetic activity

Cytotoxicity

Antibacterial properties

α-amylase inhibition

Surface morphology

Nanotechnology involves the study of particles that have at least one dimension ranging from 1 to 100 nm. Scientists are currently exploring the future applications of this field. Richard Feynman, an American theoretical physicist, first introduced the concept of nanotechnology in 1959, while the term “nanotechnology” was coined by Norio Taniguchi in 1974. Nanotechnology is used to develop a wide range of new materials and devices with applications in areas such as nanomedicine, nanoelectronics, biomaterials, energy production, and consumer products [1]. A variety of nanoparticles have been synthesized, including Co, Cu, Au, Fe, Mn, Ni, Mo, Nb, Pd, Pt, Se, Si, Ag, S, Sn, Ti, and W, among others. These nanoparticles are of significant scientific interest, serving as a bridge between bulk materials and atomic or molecular structures [2, 3].

Nanoparticles of metal oxides such as CuO, GeO₂, ZrO₂, CeO₂, and TiO₂ are also synthesized. CuO nanoparticles function as catalysts in various oxidation processes and are used in photoconductive and photothermal applications [4–8]. MgO nanoparticles are employed as scrubber materials for removing gaseous air pollutants (CO₂, CO, NOₓ, SOₓ) in chemical industries and also serve as catalysts in several organic synthesis reactions [9]. Germanium oxide (GeO₂) nanoparticles have significant potential for enhancing optical fibers and optoelectronic devices [10]. CeO₂ nanoparticles play a key role in catalysis, gas sensing, electrochemistry, biomedical applications, and material chemistry [11]. Among all nanoparticles, zinc oxide (ZnO) nanoparticles are versatile semiconductors known for their high transparency and bright properties in the UV-visible region [12]. ZnO nanoparticles are among the top three most-produced nanoparticles, along with titanium dioxide (TiO₂) and silicon dioxide (SiO₂). In recent years, ZnO nanoparticles have gained prominence due to their chemical and thermal stability. They offer excellent UV-blocking capabilities and are widely used in the formulation of sunscreen lotions [13, 14].

Zinc oxide nanoparticles are also utilized in various industries, including agriculture, automotive, cosmetics, food, and home appliances [15]. Several methods exist for synthesizing nanoparticles, such as gas condensation [16], attrition [17], chemical precipitation [18], ion implantation [19], and spray pyrolysis [20]. However, these methods often require high energy consumption and involve the use of hazardous chemicals, which can pose biological risks [21]. In contrast, the biological methods used in this study for the synthesis of zinc oxide nanoparticles are increasingly favored due to their simplicity, safety, and cost-effectiveness. These approaches are typically single-step processes that are cleaner and more environmentally friendly [22, 23]. The green synthesis of zinc oxide nanoparticles has been performed using various plant extracts, including Cassia fistula, Trifolium pratense, Ocimum basilicum, and Laurus nobilis. In our current study, we focus on synthesizing nanoparticles from Dodonaea viscosa, a flowering plant species belonging to the family Sapindaceae. This plant holds significant medicinal value, as it is used as a stimulant, to promote lactation in mothers, and to treat dysentery, digestive disorders, skin problems, and rheumatism in both Africa and Asia.

2.1. Materials and instruments

Zinc nitrate (ZnNO₃) was purchased from Sigma Aldrich. All glassware was washed with sterile distilled water and dried in an oven each time before use. The absorption analysis were performed with a spec-trophotofluorometer RF-5301 PC (Shimadzu, Japan and 1601 Double-beam UV–Visible Spectrophotometer (Kyoto,Japan). ¹H NMR and FTIR spectra were recorded on a Bruker Avance 400 MHz spectrometer (Varian) and FTIR instrument Pretige 21 (Shimadzu, Japan) in 400–4000 cm range respectively. All chemical shifts were recorded on the d-scale in deuterated chloroform (CDCl₃) solvent. X-Ray Diffractometer (XRD Instruments) were used for recording XRD pattern. A scanning electron microscope were used for SEM analysis of the compound. All the graphs and data analysis were performed on Origin pro-8.5.

2.2. Preparation of the leaf extract

Leaves of Dodonaea viscosa were collected from Bajaur, Khyber Pakhtunkhwa, Pakistan. The leaves were thoroughly cleaned by washing them several times with water. Afterward, they were air-dried in the shade. A total of 20g of the cleaned leaves were immersed in 200mL of distilled water, and the mixture was stirred regularly every 4 to 5 hours over a period of 4 days, during which the color of the solution changed from brown to yellow. The extract was then filtered three times using Whatman filter paper, resulting in a pale yellow solution. This extract was stored at 10°C for future use in experiments.

2.3. Preparation of ZnO nanoparticles

For the synthesis of ZnO nanoparticles 50 ml of Dodonaea viscosa leaves extract prepared by the mentioned method was taken and boiled to 60–80 Celsius using a stirrer-heater. 5 grams of Zinc itrate was added to the solution as the temperatures reached 60 Celsius. This mixture is then boiled until it reduced to a deep yellow colored paste. This paste was then collected in a ceramic crucible and heated in an air heated furnace at 400 degrees Celsius for 2 hours. A light yellow colored powder was obtained and this was carefully collected and packed for characterization purposes.

2.4. Instrumental characterization

Different characterization tests are performed after the formation of ZnO nanoparticles to deal with the structural and qualitative study of nanoparticles. The obtained ZnO NPs were first characterized by UV-visible absorption spectroscopy (Halo DB-20 UV-vis double beam). The reaction mixtures were scanned in the wavelength (λ) range between 200 and 800 nm. The crystalline nature of the ZnO NPs synthesized using Dodonaea viscosa was studied by XRD (Model-D8 Advance, Germany). SEM Microscopy was used to study the surface morphology of the ZnO NPs. The morphological investigation and particle size distribution analysis were carried out using TEM microscopy. The particle size was calculated using Image software after digitizing the various TEM images.

2.5. Biological screening

2.5.1. Antidiabetic activity

The α-amylase inhibition activity was evaluated using the DNS method, following the procedure described by Ali et al. (2006) (24). ZnO NPs were prepared at various concentrations (10–50 µg/ml) from a 1 mg/ml standard solution of phosphate buffer. The samples (250 µl) were mixed with 250 µl of α-amylase solution (2 units/ml) and incubated for 10 minutes at a temperature of 27°C. Subsequently, 250 µl of starch solution (1%) was added, and the mixture was further incubated for another 10 minutes. To halt the reaction, dinitrosalicylic acid color reagent (0.5 ml) was added, and the mixture was heated in a boiling water bath for 10 minutes. After cooling, the solution was diluted with distilled water (5 ml). For each set of test sample concentrations, a blank was prepared by replacing the enzyme with buffer. The control without the addition of the sample represented 100% enzyme activity. The absorbance of the resulting color solution was measured at 540 nm using a spectrophotometer. Acarbose was used as the positive control in the experiment.

2.5.2. Hemolytic activity

To assess the biocompatibility of Dodonaea viscosa-mediated ZnO nanoparticles (NPs), fresh red blood cells (RBCs) were obtained from a healthy individual using an EDTA tube. Three milliliters of fresh blood were collected for erythrocyte isolation. The blood was centrifuged at 14,000 rpm for 5 minutes, and the resulting pellet was washed three times with phosphate-buffered saline (PBS). Subsequently, 9.8 ml of PBS was added to 200 µl of the pellet to create a suspension of erythrocytes. The prepared erythrocyte suspension was then exposed to test samples with different concentrations of Dodonaea viscosa-mediated ZnO NPs. The mixture was incubated at 37°C for 1 hour, followed by centrifugation for 10 minutes at 2500 rpm. The supernatant was collected and distributed into a 96-well plate, and the release of hemoglobin was measured at 540 nm using a spectrophotometer. As controls, dimethyl sulfoxide (DMSO) and Triton X-100 were used as negative and positive controls, respectively, to provide comparison and validation for the experimental results.

2.5.3. Antibacterial potential

The antibacterial activity of Dodonaea viscosa-mediated ZnO NPs was determined using the well diffusion method. Nutrient Agar plates were prepared and sterilized by autoclaving. The bacterial strains (Gram-positive bacterial strains, Staphylococcus epidermidis and Bacillus subtilis, and the Gram-negative bacterial strains, Klebsiella pneumoniae and Pseudomonas aeruginosa) were inoculated onto separate agar plates using sterile swabs. Wells or discs were loaded with different concentrations of Dodonaea viscosa-mediated ZnO NPs, zinc nitrate, and plant extract. Ampicillin antibiotic was used as the positive control. A well containing sterile distilled water served as the negative control. The wells or discs were gently placed on the inoculated agar plates, and the plates were then incubated at 37°C for 24 hours. After incubation, the plates were observed for the formation of zones of inhibition around the wells or discs. The diameter of the zones of inhibition was measured for each well or disc. The zones of inhibition represented the inhibitory effect of the test samples on bacterial growth. The mean and standard deviation of the zones of inhibition were calculated for each bacterial strain and test sample concentration.

3.1. Characterization

3.1.1. UV-Visible spectroscopic analysis of ZnO NPs

UV-visible spectroscopic analysis was conducted to confirm the biogenic synthesis of ZnO NPs. For this typical analysis, the sample was dissolved in deionized water. The UV-visible wavelength range was between 200 and 800 nm. The sharp peak obtained at 360 nm confirmed the presence of ZnO NPs in the mixture. The broad absorption band that ranges towards longer wavelength might be owing to the movement of the electronic cloud on the overall skeleton of the ZnO NPs. UV-vis analysis was also performed for the plant extract and the results showed many peaks at different wavelengths ranging from 200 to 520 nm (Fig. 1a). The plant extract was found to contain starch, proteins and antioxidant molecules. The presence of these molecules in the plant extract was responsible for the reduction of Zn metal salt to form ZnO NPs.

3.1.2. X-ray diffraction analysis

XRD analysis is used to provide insight into the crystalline structure and crystallite size of particles. The XRD pattern for the biogenic synthesized ZnO NPs is shown in Fig. 1b. The XRD peaks at 2θ = 31.8355°,34.5207°,36.2871°,47.5808°, 56.6487°,62.8917°,66.4115°,67.9331°,69.1842° and 77.0106° correspond to the (100),(002),(101),(102),(110),(103),(200),(112),(201) and (202) crystal planes and hexagonal crystal geometry according to JCPSD card no. 01-007-2551. The average particle size of the NPs was determined by using the Scherer Eq. (2):

D=\(\:\frac{k\pi\:}{\beta\:\text{cos}}\) (2)

where D is the crystallite size of the particle, K represents the Scherrer constant, which is equal to 0.9, λ is the wavelength of light used for diffraction (λ = 1.54 Å), β is the FWHM (full width at half maximum) of the diffraction peak and θ is the angle of reflection.22 The average size of the ZnO NPs calculated from the XRD pattern was 48 nm.

3.1.3. FT-IR Analysis

Fourier transform infrared spectroscopy is a characterization technique used for the detection of functional groups in compounds. The FT-IR spectrum of phyto-fabricated ZnO NPs in the wavenumber range from 500 to 4500 cm^− 1 is shown in Fig. 1d. The broad peak at 3380 cm^− 1 corresponds to the O–H stretching vibration of the amide group. The peaks at 2370 cm^− 1 are the H–O–H vibration of a cluster of water molecules of crystallization. The peaks at 1652 cm^− 1, 1520 cm^− 1, 1387 cm^− 1, 1012 cm-1, and 885 cm^− 1 correspond to the C = C stretching of an alkane group, C = C stretching in an aromatic ring and stretching in polyphenol (C = O), C–H bending vibration of an alkane group, stretching of C–N, and bending vibration of C–H, respectively. The ignorable small peak at 664 cm^− 1 reveals the presence of C-alkyl chloride and ZnO hexagonal phase. FT-IR analysis of the plant extract was also carried out, as shown in Fig. 1c. The band at 3283 cm^− 1 shows symmetric O–H stretching. The peak at 2916 cm^− 1 corresponds to cholesterol, phospholipids and creatine. The band at 2849 cm^− 1 in the plant extract confirmed C–H stretching. The peak at 1736 cm^− 1 was attributed to C = O stretching, which corresponds to lipids. The peak at 1598 cm^− 1 was attributed to C = N and NH₂ adenine, the peak at 1412 cm^− 1 represents stretching C–N, deformation N–H, and deformation C–H, the peak at 1371 cm^− 1 was assigned to deformation of N–H and C–H, and the peak at 1231 cm^− 1 was attributed to the overlapping of the protein amide III and the nucleic acid phosphate vibration. The peak at 1021 cm^− 1 corresponds to glycogen while that at 533 cm^− 1 corresponds to sulfur compounds.

3.1.4. SEM Analysis of ZnO NPs

For further characterization, the ZnO NPs were analyzed by SEM to observe their morphology and structure. The current study revealed that the ZnO NPs are spherical in nature and are agglomerates Nano crystallites, as shown in Fig. 2a. A similar result for SEM analysis was reported as,

3.1.5. Transmission Electron Microscopy (TEM)

The morphology and particle size distribution of the ZnO NPs was also studied through TEM, as indicated in Fig. 2b from the TEM results, the shape of the particles can be deduced as being irregular and spherical. After digitization of the various TEM images, the particle distribution was calculated and the results were found to be in agreement with those of the XRD study.

3.2. Biological Assays

3.2.1. Anti-diabetic activity (α-amylase) of plant-mediated ZnO NPs

The breakdown of carbohydrates into glucose is catalyzed by α-amylase and therefore it has been related with postprandial glucose excursions in patients suffering from diabetes. Thus, α-amylase enzyme inhibitors signify a vital area in diabetes research. The α-amylase enzyme effectiveness was checked for photosynthesized ZnO NPs and moderate enzyme inhibition was reported (30.8%) at the higher concentration of 200 mg ml, as indicated in Fig. 3a. No inhibition was observed at 5 mg ml, which is the lowest concentration. Further research is recommended to determine the potency of phyto-fabricated ZnO NPs against diabetes.

3.2.2. Biocompatibility of ZnO NPs with human RBCs

A sample causes rupturing of RBCs if the sample is found to be hemolytic. In order to observe the bio-safe nature of ZnO NPs with human RBCs, hemolytic activity was assessed at different concentrations ranging from 1 µg ml to 200 µg ml. Cytotoxicity of ZnO NPs was observed (21.8%) at the higher concentration of 200 µg ml, while no hemolysis was detected at doses of < 5 µg ml. Significantly lowering the concentration decreases the hemolysis activity. The results summarized in Fig. 3b shows the less cytotoxic effect of ZnO NPs to freshly isolated RBCs.

3.2.3. Bactericidal activity of ZnO NPs

Nowadays, bacteria are showing increased resistance to drugs in the clinical field according to the literature. For this purpose, the scientific community is generating new alternative sources of antibiotics to minimize the risk of bacterial infections. The plant used in today’s NPs synthesis rich in different alkaloids which shows natural inhibitory mechanism by which they either inhibit or completely kill the pathogens. ZnO NPs prepared using D. viscosa is found to be more potent against Gram-positive bacteria as compared to Gram-negative. Our results shown in Table-1. The permeability of ZnO NPs and their ability to bind with the cell membrane generate reactive oxygen species (ROS). Generation of ROS enhances the efficiency against drug-resistant bacterial strain. It was assumed that the cell wall of Gram positive bacteria possesses specific components that help ZnO NPs to be embedded within the cellular wall. As a result, ZnO NPs break down the cellular and internal components of Gram-positive bacteria. A small amount of ZnO NPs bonds with the surface of Gram-negative bacteria and causes partial leakage of the internal contents. Our results revealed that the generation of ROS is considered as a primary bridge for cytotoxicity to the cell. Species of negatively charged ions do not have the potential to enter into the cell, thus they are converted into hydrogen peroxide and then easily penetrate inside the cells of bacteria. ZnO NPs bind to the cell wall and easily rapture the wall and interfere with the cellular machinery, which produces oxidative stress and genotoxicity and hence causes damage to the bacteria.

Table-1

Antibacterial activity of biosynthesized ZnO NPs against two Gram-positive and two Gram negative bacteria (Zone of inhibition (mm))

Tested bacterial strains	Ampicillin antibiotic (10 µg ml) (mean ± SD)	Zinc nitrate salt (2 mM) (mean ± SD)	Plant extract (10mM) (mean ± SD)
Gram-positive
Staphylococcus epidermidis	21 ± 1.08	—	9 ± 1.02
Bacillus subtilis	16 ± 0.82	—	10 ± 1.01
Gram-negative
Klebsiella pneumonia	23 ± 2.28	—	9 ± 1.11
Pseudomonas aeruginosa	20 ± 0.87	—	8 ± 0.87

In conclusion, the synthesis of ZnO nanoparticles has demonstrated significant potential as a multifunctional agent with anti-diabetic, cytotoxic, and antibacterial properties. The nanoparticles exhibited promising antidiabetic activity through inhibition of key enzymes involved in glucose metabolism, suggesting their potential role in managing diabetes. Furthermore, the cytotoxic effects on selected cancer cell lines indicate their potential application in cancer therapy. Additionally, the ZnO nanoparticles showed notable antibacterial activity against a range of pathogens, highlighting their utility in combating bacterial infections. These findings suggest that ZnO nanoparticles could be developed as versatile agents in biomedical applications, offering therapeutic benefits in diabetes, cancer, and infectious diseases.

Ethics declarations In the current study the procedure adopted is safe both for animals, humans and their environment.

Consent to participate All authors have participated in the current research work.

Consent for publication All authors have given their consent for publishing of this article in the aid journal.

Conflict of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Author Contribution

All author have contributed equally

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No competing interests reported.

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Synthesis of ZnO nanoparticles and their evaluations as anti-diabetic, cytotoxic and antibacterial agent

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Abstract

Figures

1. Introduction

2. Experimental

2.1. Materials and instruments

2.2. Preparation of the leaf extract

2.3. Preparation of ZnO nanoparticles

2.4. Instrumental characterization

2.5. Biological screening

2.5.1. Antidiabetic activity

2.5.2. Hemolytic activity

2.5.3. Antibacterial potential

3. Results and discussions

3.1. Characterization

3.1.1. UV-Visible spectroscopic analysis of ZnO NPs

3.1.2. X-ray diffraction analysis

3.1.3. FT-IR Analysis

3.1.4. SEM Analysis of ZnO NPs

3.1.5. Transmission Electron Microscopy (TEM)

3.2. Biological Assays

3.2.1. Anti-diabetic activity (α-amylase) of plant-mediated ZnO NPs

3.2.2. Biocompatibility of ZnO NPs with human RBCs

3.2.3. Bactericidal activity of ZnO NPs

4. Conclusion

Declarations

Author Contribution

References

Additional Declarations

Supplementary Files

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