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In this article, we discuss research in the field of green nanotechnology published in the journal Biotechnology and Bioprocess Engineering. Here, zinc oxide nanocomposites (ZnO NPs) are synthesized using pomegranate peel and chemical precipitation. The prepared nanocomposite exhibits good antibacterial and antioxidant activities, indicating its potential in biomedical applications.
Photo Credit: George Dolgikh/Shutterstock.com
In the booming field of nanotechnology, the particle size of nanoparticles (NP) is about 100 nanometers. NPs are divided into four types: carbon, polymers, metals, metal oxides and other inorganic substances.
Due to their low toxicity, metal oxide NPs are widely used, of which ZnO NPs are used in many applications, including modern drug delivery systems. They are also used as external antibacterial agents in the preparation of ointments, food packaging, lotions, cloths and mouthwashes to avoid microbial load.
In particular, ZnO NPs are often found in cosmetics to protect the skin from harmful radiation. The US Food and Drug Administration (FDA, USA) accepts ZnO as a GRAS (Generally Recognized As Safe) material.
The cosmetics industry increasingly relies on natural compounds of plant origin, mainly plant extracts with antibacterial, antioxidant and photoprotective effects. Previous studies have confirmed that orange fruit and peel extracts can be used to synthesize ZnO NPs to exert antibacterial activity. In addition, beneficial phytochemicals, such as fruit flavonoids, alkaloids, carotenoids, terpenes, and tannins, can be used as nanocompositing agents to synthesize nanoparticle materials.
Punica granatum L. (Pomegranate) is an edible fruit of the pomegranate family (now part of the Lychee family). It is rich in natural polyphenols, flavonoids and proanthocyanidins. The peel of pomegranate fruit contains effective biologically active compounds, such as gallic acid, ellagitannin, ellagic acid, punicarin, pelargonidin, anthocyanin delphirin and luteolin.
The following details the method of synthesizing new PE-ZnO NPs using pomegranate ethanol extract (PE), and the subsequent use of human keratinocyte cell lines to analyze the antioxidant, antibacterial and cytotoxic properties in vitro.
Folin-Ciocalteu, 2, 2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), 1,1-diphenyl-2-picryl hydrazyl (DPPH) and Methyl thiazolyl diphenyl-tetrazolium bromide (MTT) , Fetal Bovine Serum (FBS), Trypsin-Ethylenediaminetetraacetic Acid (EDTA), Dulbecco's Modified Eagle Medium (DMEM) and Phosphate Buffered Saline (PBS) and Penicillin (100 Units/mL)/Streptomycin (100μg/ml).
Harvest healthy pomegranate fruits and wash them. The peel is cut and dried at room temperature, then ground into a coarse powder using an electronic grinder and stored in an incubator at 4°C. The extract is prepared, and then the combined pomegranate extract (PE) is used to analyze phenolic, antioxidant, and antibacterial activities.
Check the total phenol content and total flavonoid content of the collected PE.
The pomegranate-peel extract functionalized zinc oxide nanocomposites (PE-ZnO NPs) were synthesized by chemical precipitation method, and they were dried in an incubator at 40°C.
The formation of nanocomposites of the synthesized PE-ZnO NPs was analyzed by monitoring the metal plasma peaks in the UV-Vis spectrum calculated using a UV/Vis spectrophotometer, and their morphology was checked using a scanning electron microscope (SEM).
The ABTS· free radical scavenging method was used to analyze the antioxidant activity of PE-ZnO NPs. The DPPH free radical scavenging activity of PE-ZnO NPs was also evaluated.
The human keratinocyte (HaCaT) cell line is grown and maintained in Dulbecco's Modified Eagle Medium (DMEM), and subcultured every 2-3 days.
The MTT method was used to analyze the cytotoxicity and proliferation inhibition induced by synthetic PE-ZnO NPs.
Bacterial strains, namely Bacillus licheniformis (ATCC 1458), Bacillus cereus (ATCC 14579) and Escherichia coli (ATCC 15597) were used to determine the antibacterial activity of tetracycline as a positive control.
Use SPSS software package (SPSS, USA) to calculate all data as mean ± SD value.
Pomegranate peel extract functionalized zinc oxide nanoparticles (PE-zinc oxide nanoparticles) were synthesized, characterized and examined for biological properties. The analysis of biologically active compounds showed that PE contains quite a lot of polyphenols and flavonoids, which can be used to synthesize PE-ZnO NPs.
The pomegranate peel, which is usually discarded, was evaluated for total biological activity and used to synthesize PE-ZnO NPs. PE shows large amounts of polyphenols and flavonoids (Table 1).
Table 1. Total polyphenol and flavonoid content of pomegranate extract. Source: Singh et al., 2021.
#GAE: Gallic acid equivalent; ##QE: Quercetin equivalent. aMean ± SD (n = 3).
PE-ZnO NPs were synthesized by chemical precipitation, and the formation of nanocomposites was analyzed using UV-Vis spectroscopy (Figure 1A).
Figure 1. (A) UV absorption spectra of PE, ZnO NPs and PE-ZnO NPs. (B) FT-IR spectrum analysis of PE, ZnO NPs and PE-ZnNP. Image source: Singh et al., 2021.
FT-IR spectroscopy was used to determine the functional groups related to PE, ZnO NPs, and PE-ZnO NPs (Figure 1B).
According to the FT-IR results, PE functionalization with ZnO NPs was established. Compared with ZnO NPs, SEM imaging characterizes the nanostructures developed in PE-ZnO NPs. ZnO NPs are sheet-like and foam-like nanostructures (Figure 2A and 2B), while PE-ZnO NPs are nanoplate clusters (Figure 2C and 2D).
Figure 2. (A and B) This is a schematic diagram showing the shape of ZnO NPs. (C and D) Measurement of pomegranate-zinc oxide nanoparticles (PEZnO NPs) by SEM. Image source: Singh et al., 2021.
PE and PE-ZnO NPs showed enhanced ABTS and DPPH free radical scavenging activities in a concentration-dependent manner. The clearance activity percentages of PE and PE-ZnO NPs (1,000 μg/mL) were 95.1% and 95.2%, respectively (Figures 3A and 3B).
Figure 3. (A and B) DPPH and ABTS· radical scavenging activities of PE, ZnO NPs and ZnO-PE NPs. The result is the mean ± SD compared to ascorbic acid (AA), * p <0.05, # p <0.005. Image source: Singh et al., 2021.
The MTT method was used to analyze the cytotoxic activity to study the non-toxic concentration of PE-ZnO NPs in the HaCaT cell line. The results showed that PE and PE-ZnO NPs were not significantly toxic to HaCaT cells (Figure 4).
Figure 4. The effects of PE, ZnO NPs and PE-ZnO NPs on the viability of human skin epithelial keratinocytes (HaCaT). Cells were treated with different concentrations of PE-ZnO NPs (25, 50 and 100 µg/mL) for 24 hours, and then cell viability was assessed by MTT assay. Image source: Singh et al., 2021.
The colony forming unit (CFU) test of the antibacterial activity of different percentages of PE and PE-ZnO NPs against Bacillus cereus, Bacillus licheniformis and E. coli showed that PE-ZnO NPs (1.0 mg/mL) treatment showed extensive antibacterial activity Activity (Figure 5).
Figure 5. Antibacterial efficiency of PE, ZnO NPs and PE-ZnO NPs against Bacillus cereus, Bacillus licheniformis and Escherichia coli. Colony forming units (CFU/mL) of PE, ZnO NPs and PE-ZnO NPs. Image source: Singh et al., 2021.
ZnO NPs synthesized by functionalizing the bioactive compounds in the ethanol extract of pomegranate peel exhibit strong free radical scavenging and antibacterial activities. The results of this study indicate that these organometallic-based nanoparticles are a valuable nanomaterial with potential applications as multi-target antibacterial agents in pharmaceuticals and cosmetics.
Read on: Nanotechnology and Green Technology: How can the two go hand in hand?
Singh, M., Lee, KE, Vinayagam, R., Kang, SG (2021) Antioxidant and antibacterial analysis of functionalized zinc oxide nanocomposites with pomegranate peel extract. Biotechnology and Bioprocess Engineering, 26, pp. 728-737. Website: https://link.springer.com/article/10.1007/s12257-021-0211-1.
Megan graduated from the University of Manchester with a Bachelor of Science degree. In genetics, and decided to pursue a master's degree. Because of her passion for combining science with content creation, she earned a degree in Science and Health Communication. As part of the learning, Megan worked with Jodrell Bank Discovery Center as a digital marketing assistant, producing content and updating parts of his website. In her spare time, she likes to travel, exploring the culture and history of each place-including local cuisine. Her other interests include embroidery, reading novels and practicing Japanese.
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