Modeling the impact of early interventions on the transmission dynamics of coronavirus infection [version 1; peer review: awaiting peer review]

Research Article
Revised

Green synthesis and characterization of zinc oxide nanoparticles using bush tea (Athrixia phylicoides DC) natural extract: assessment of the synthesis process.

[version 2; peer review: 1 not approved]

PUBLISHED 14 Jan 2022

Author details Author details

OPEN PEER REVIEW

REVIEWER STATUS

Introduction

Materials with a diameter of less than 100 nm are classified as nanoparticles. These particles have a reduced size associated with their high surface/volume ratio that increases as their size decreases (Ohno et al., 2010). They are considered as the borderline between single molecules and bulk materials and present more properties compared to their bulk counterpart (Mansoori and Soelaiman, 2005). Nanoparticles are globally synthesized for their various properties, such as antimicrobial, anti-inflammatory, wound healing, catalytic, magnetic, optical, and electronic properties, that have put them at the forefront of a wide variety of studies (Gunalan et al., 2012; Jamdagni et al., 2018).

Nanoparticles have been incorporated into numerous consumer industries such as industrial, health, food, space, chemical, and cosmetics, necessitating a green and environmentally responsible strategy for their production (Rao and Gautam, 2016). Among all nanoparticles, metal oxides and dioxides such as zinc oxide, silver, gold and titanium dioxide have received copious consideration because of their multiple properties and applications (Dobrucka and Długaszewska, 2016). Numerous physicochemical methods of the synthesis of nanoparticles such as laser ablation, microwave irradiation and vapour deposition have been reported to date (Satyanarayana and Reddy, 2018). The physical and chemical methods involve forces of condensation, dispersion, or fragmentation of bulk particles into nanoparticles (Dhandapani et al., 2014; Krupa and Vimala, 2016). Hence, chemical methods often require toxic chemicals that are harmful to the environment due to the difficulty of removing them from the nanoparticles after synthesis, thus a new, safe and cost-effective method is needed (Dhandapani et al., 2014).

Synthesis of nanomaterials through biological systems assisted by some biotechnological tools is an emerging field of nanotechnology called green nanotechnology (Shinwari and Maaza, 2017). Plants, diatoms, fungi, yeast, algae, bacteria, and human cells have been used to reduce metal ions into nanoparticles. Their proteins and other metabolites have been well reported to have a reductive capacity that can transform metal ions into metal nanoparticles (Dobrucka and Długaszewska, 2016; Parveen et al., 2016). The biological synthesis of nanoparticles provides more advantages than chemical and physical ones (Kharissova et al., 2013). Numerous metal oxide nanoparticles, such as TiO2, CuO, and ZnO have been produced by total green chemistry. Among them ZnO, an n-type semiconductor, has gained interest owing to its easy production, cost-effectiveness, and safety of synthesis and usage (Agarwal et al., 2017). Several studies have successfully been led to synthesize ZnO nanoparticles using different organisms such as bacteria, fungi, algae, and plants (Agarwal et al., 2017).

Among all biological systems, plant phytosynthesis of nanoparticles using plants has shown great potential. Plant-mediated nanoparticle synthesis is simple, eco-friendly, and provides antibacterial assets (Gunalan et al., 2012; Iravani et al., 2015; Thema et al., 2015). A variety of metabolites such as terpenoids, polyphenols, sugars, alkaloids, phenolic acids and proteins have been reported to have metal ion reduction assets (Parveen et al., 2016). Several studies dedicated to the green synthesis of ZnO nanoparticles using plant extracts as capping or reducing agents have shown the use of different plant aerial parts, such as leaves and fruits of different species such as Aloe vera, Hibiscus sabdariffa, Allium sativum, Allium cepa, Petroselinum crispum, Moringa oleifera and Camellia sinensis, for the synthesis of nanoparticles (Mahendiran et al., 2017; Matinise et al., 2017; Senthilkumar and Sivakumar, 2014; Stan et al., 2015). Bush tea, mostly known as a medicinal tea plant in southern Africa where it originates has high concentrations of phenolic compounds such as tannins and flavonoids (Lerotholi et al., 2017). However, data explaining the synthesis processes of nanoparticles using this plant are lacking. Hence, the objective of this study was to contribute to the explanation of the compounds induced in the synthesis process of ZnO nanoparticles using Athrixia phylicoides leaf extract.

Methods

Material

Leaves of bush tea (A. phylicoides DC) were used to reduce zinc nitrate hexahydrate. Analytical grade Zn (NO3)2.6H2O of 99% purity was purchased from Sigma-Aldrich, South Africa. Bush tea leaves were harvested from the wild in Thohoyandou (22.8785°S; 30.4818°E) in the Limpopo province, South Africa. Following the harvest, the leaves were washed with deionized water and freeze-dried for 72 hours at -50°C at a pressure of 0.32 Kpa, hereafter they were ground and kept for further usage.

Material preparation

Extract preparation

Ten grams of ground bush tea leaves were weighed and mixed with 300 ml of deionized water. The mixture was heated at 60°C for 30 minutes until the water changed to a dark green colour. After centrifugation using a Hermle Labortecnik GmbH Z 216-M benchmark centrifuge at 4000 rpm for 10 minutes, the mixture was filtered twice using Whatman filter paper number 1, and the extract was kept in an airtight container in a fridge at ≈4°C for analysis and ZnO nanoparticles synthesis.

Synthesis of ZnO nanoparticles

In this study, zinc nitrate hexahydrate [Zn (NO3)2.6H2O] was used as the precursor. One gram of the precursor was mixed with 25 ml of A. phylicoides extract. The mixture was kept on a magnetic stirrer at 300 rpm at 60°C for 30 minutes then left to cool down at room temperature for 12 hours, a precipitate was observed. The mixture was centrifuged for 15 minutes at 4000 rpm. The supernatant was collected and transferred to LC-MS vials for analysis and the precipitate was dried at 60°C for one hour then annealed at 800°C for two hours. The obtained powder was then kept for characterization.

Bush tea compounds profiling and identification

The determination and profiling of different compounds present in the extract before the synthesis as well as the supernatant after synthesis were performed using Liquid chromatography quadrupole time-of-flight mass spectrometry (LC-Q-TOF-MS) using a Bruker impact II (Germany). After peak integration and Pareto scaling, the liquid chromatography–mass spectrometry (LC-MS) data were transformed into buckets using the Bruker Compass data analysis programme version 4.3.110 (https://www.bruker.com/en/). Peaks were determined using real mass, MS/MS, and retention time (RT). The accuracy of the mass and MS/MS spectral data was compared to the Kyoto standard Encyclopaedia of Genes and Genomes (KEGG) and ChemSpider databases using the MetFrag 2.2 online software (Tete et al., 2020). Principal component analysis (PCA) and T-tests were performed using MetaboAnalyst 4.0.

ZnO nanoparticle characterization

The characterization of the obtained ZnO nanopowders was done using X-ray diffraction (XRD), Ultraviolet-visible spectroscopy (UV-Vis), Fourier-transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDS). The crystallite size of ZnO nanoparticles was estimated using the modified Scherrer equation:

L=Kλβcosθ

where

λ

is the X-ray wavelength, β the peak width at half maximum weight, K = 0.9, the Scherrer constant (Monshi et al., 2012).

Results

Assessment of the synthesis process

Evaluation of extract composition relative to the synthesis of ZnO nanoparticles using LC-Q-TOF-MS

The crude extract from bush tea leaves and the supernatant after the synthesis of ZnO nanoparticles were investigated. The differences between the composition of the crude extract and the supernatant after synthesis is represented in Figure 1. The results from the PCA co-variance of data show that two distinct groups were observed from the three principal components with 85.1%, 8.6% and 2.1% respectively for principal components 1, 2 and 3. The compounds (represented in red) resulting from the supernatant after synthesis of ZnO nanoparticles clustered together following the Y-axis of the PCA while the compounds of crude extract were on the Z-axis. The differences observed are due to the reaction between the plant extract and the precursor to form ZnO nanoparticles. The synthesis of ZnO nanoparticles involves a reaction between the plant extract and the precursor resulting in the reduction of Zn+2 ions into ZnO nanoparticles (Hussain et al., 2019).

7352c2f1-3c13-43d8-bea0-11de03720bde_figure1.gif

Figure 1. Principal component analysis of liquid chromatography quadrupole time-of-flight mass spectrometry (LC-Q-TOF-MS) peak intensities of 10 bush tea (Athrixia phylicoides DC.) leaf extracts before and after ZnO nanoparticle synthesis.

Figure 2 shows the different compound peaks observed using LC-Q-TOF-MS analysis. The dissection of observed spectra into compounds produced 100 different peaks for the crude extract (Figure 2a) and 84 peaks for the supernatant after synthesis (Figure 2b). The reduction in the number of compounds confirms that the synthesis took place and secondary metabolites from A. phylicoides DC. extract has reacted with the precursor reducing Zn2+ ions into ZnO nanoparticles.

7352c2f1-3c13-43d8-bea0-11de03720bde_figure2.gif

Figure 2. Compound dissection (a) before the synthesis process (100 peaks) (b) after the synthesis process (84 peaks).

Bush tea extract compounds identification before and after synthesis

Different peaks identified, after chromatogram dissection (Figure 2), from LC-Q-TOF-MS revealed the presence of several compounds in both the crude extract and the supernatant after ZnO nanoparticle synthesis, with a reduction of the compound’s amount in the supernatant collected after synthesis. Thus, revealing the presence of an interaction between the precursor and the extract mainly by the oxidation, reduction or degradation of the phytochemical compounds that occur during nanoparticle formation (Jeevanandam et al., 2016). Table 1 presents the secondary metabolites investigated for both the crude bush tea extract and the supernatant solution after synthesis, respectively.

Table 1. Liquid chromatography quadrupole time-of-flight mass spectrometry (LC-Q-TOF-MS) bush tea extract compounds identified before ZnO nanoparticles synthesis using MetFrag software (KEGG and ChemSpider databases, 50 ppm).

Compound name Formula RT [sec]
1 (+)-7-Isojasmonic acid C12H18O3 294
2 (6Z,9Z,12Z)-Octadecatrienoic acid C18H30O2 543.6
3 10-Oxo-11,15-phytodienoic acid C18H28O3 357.6
4 13-hydroxy-9Z,11E-octadecadienoic acid C18H32O3 652.2
5 17-Hydroxylinolenic acid C18H30O3 340.8
6 1-O,6-O-Digalloyl-beta-D-glucose (tannin) C20H20O14 351
7 3,6-Anhydroglucose C6H10O5 228.6
8 3-hydroperoxy-4-phenyl-pentan-1-ol/Loliolide C11H16O3 309
9 3-tert-Butyl-5-methylcatechol C11H16O2 426
10 4-Heptyloxyphenol C13H20O2 282.6
11 4”-Hydroxyacetophenone C8H8O2 71.4
12 4-Hydroxyestradiol-17beta C18H24O3 443.4
13 5,7,3′-Trimethoxy-6,4′,5′-trimethoxyisoflavone C18H16O8 690
14 7-Hydroxy-2”,4”,5”-trimethoxyisoflavone C18H16O6 726
15 Naringenin 7-O-beta-D-glucoside C21H22O10 435.6
16 17-Hydroxylinolenic acid C18H30O3 372.6
17 Adenine C5H5N5 783
18 alpha-Curcumene C15H22 609.6
19 5S-Hydroperoxy-18R-HEPE C20H30O5 274.8
20 Atropaldehyde C9H8O 345
21 Scullcapflavone II C19H18O8 462.6
22 Cinnamaldehyde C9H8O 354
23 Cisapride C29H27N3O3 115.8
24 Coumaric acid/Caffeic Aldehyde C9H8O3 285
25 Coumarin C9H6O2 76.8
26 D-Norvaline C5H11NO2 48
27 Homovanillate/Dihydrocaffeic acid C9H10O4 288.6
28 Lancerin C19H18O10 348.6
29 Lophophorine/Stovaine C13H17NO3 55.8
30 Mallotophenone C21H24O8 432
31 Malonyldaidzin C24H22O12 207.6
32 Melampodin A C21H24O9 399.6
33 Montanol C21H36O4 513.6
34 Myrcene/(E)-beta-Ocimene C10H16 321.6
35 Nafenopin glucuronide C26H30O9 291
36 Neocnidilide/4-Hexyloxyphenol C12H18O2 421.8
37 Pentalen-13-ol/Nonylphenol C15H24O 411
38 Petasin/Cafestol C20H28O3 558.6
39 Pinosylvin C14H12O2 276.6
40 Quinestrol C25H32O2 232.2
41 Traumatic acid C12H20O4 403.8
42 Tricin C17H14O7 379.8
43 Umbelliferone/4-Hydroxycoumarin C9H6O3 209.4
44 4”-Hydroxyacetophenone C8H8O2 1.19

Table 2 present the compounds identified from the supernatant after synthesis of ZnO nanoparticles. The secondary metabolites investigated present a reduced number compared to the ones from the crude extract, thus revealing that a reaction has taken place between bush tea natural extract metabolites and the precursor resulting in the formation of ZnO nanoparticles.

Table 2. Liquid chromatography quadrupole time-of-flight mass spectrometry (LC-Q-TOF-MS) bush tea extract compounds identified after ZnO nanoparticles synthesis using MetFrag software (KEGG and ChemSpider, 50 ppm).

Compound name Formula RT [sec]
1 Indanone C9H8O 348.6
2 Mallotophenone C21H24O8 432.6
3 Melampodin A C21H24O9 399.6
4 Sterigmatocystin C18H12O6 268.8
5 Umbelliferone C9H6O3 211.8
6 Salicylate C7H6O3 182.4
7 Resolvin E2 C20H30O4 265.8
8 Scullcapflavone II C19H18O8 463.8
9 Myrtenol C10H16O 306
10 3-tert-Butyl-5-methylcatechol C11H16O2 427.8
11 (+)-7-Isojasmonic acid C12H18O3 404.4
12 Traumatic acid C12H20O4 91.2
13 4-Heptyloxyphenol C13H20O2 282.6
14 4,4”-Dihydroxystilbene C14H12O2 276.6
15 1,3-Diphenylpropane C15H16 309.6
16 Geranyl hydroquinone C16H22O2 781.2
17 Syringin C17H24O9 232.8
18 3-Hydroxybenzaldehyde C7H6O2 280.2
19 6-Hydroxyluteolin 7-glucoside C21H20O12 256.2
20 6-Methoxyaromadendrin 3-O-acetate C18H16O8 388.8
21 Adenine C5H5N5 72.6
22 9S-hydroxy-10E,12Z,15Z-octadecatrienoic acid C18H30O3 372.6
23 9E-Heptadecenoic acid C17H32O2 337.2
24 Carboxymethyloxysuccinate C6H8O7 81
25 Coumarin C9H6O2 219
26 Pent-7alpha-Hydroxykaur-16-en-19-oic acid C20H30O3 319.8
27 Etherolenic acid C18H28O3 357.6
28 Icariin C33H40O15 240

Assessment of the implication of bush tea compounds in ZnO nanoparticles synthesis

In this study, compound identification was carried out using Bruker data analysis and data profiling tools. The KEGG and ChemSpider databases were consulted to find the name and the chemical formula of each identified compound. The different compounds with mass to ratio (m/z) values as well as their retention time (in seconds) were shown with a variable importance in progression (VIP) score plot (Figure 3). The concentration of eight compounds were found to be high in the crude extract compared to the supernatant after synthesis of ZnO nanoparticles where their concentrations were low.

7352c2f1-3c13-43d8-bea0-11de03720bde_figure3.gif

Figure 3. Variable importance in progression (VP) score plot of different compounds found in the bush tea crude extract before synthesis and the supernatant after synthesis of ZnO nanoparticles.

Table 3 present the various compounds that were involved in the synthesis process of ZnO nanoparticles including five flavonoids and two polyphenol compounds, as well as one aromatic compound, which highly reacted with the precursor to form ZnO nanoparticles. Studies have shown that the synthesis of nanoparticles using plant extracts involves terpenoids, flavonoids, alkaloids and phenolic acid, which act as reducing, capping, and stabilizing agents (Kuppusamy et al., 2016).

Table 3. Identified compounds reported having mostly interacted with the precursor to form ZnO nanoparticles.

Compound name Formula Type
Naringenin 7-O-beta-D-glucoside C21H22O10 Flavonoid
Scullcapflavone II C19H18O8 Flavonoid
Mallotophenone C21H24O8 Polyphenol
6-Methoxyaromadendrin 3-O-acetate C18H16O8 Flavonoid
2-Phenylacetamide C8H9NO Polyphenol group
7-Hydroxy-2”,4”,5”-trimethoxyisoflavone C18H16O6 Flavonoid
Coumarin C9H6O2 Aromatic
Malonyldaidzin C24H22O12 Flavonoid

ZnO nanoparticles characterization

XRD analysis

The XRD analysis was done to confirm the crystallinity of the synthesized ZnO nanoparticles using a Bruker AXS (Germany) D8 advance X-ray diffractometer. Figure 4 presents the XRD pattern of the ZnO nanoparticles. The crystallinity of the powder resulting from the synthesis using A. phylicoides DC extract. The peaks (100), (002), (101), (102), (110), (103), (200), (112), (201), (004) and (202) are lattice planes. The diffraction peaks reveal that the synthesized ZnO nanoparticles are essentially crystalline, in accord with the ICDD #897102 in the wurtzite structure (Noman et al., 2020). The same results have been observed by the green synthesis of ZnO nanoparticles using Ocimum basilicum (Salam et al., 2014) and Agathosma betulina (Thema et al., 2015). The average crystallite size of obtained ZnO nanoparticles calculated using the modified Scherrer equation was approximately 24.53 nm.

7352c2f1-3c13-43d8-bea0-11de03720bde_figure4.gif

Figure 4. X-ray diffraction pattern of ZnO nanoparticles.

Fourier-transform infrared spectroscopy

The PerkinElmer Frontier FTIR spectrometer was used to perform FTIR analyses using Potassium bromide (KBr) (Potassium bromide) optics. The presence of ZnO nanoparticles was confirmed by the peak at 479 cm−1 as shown in Figure 5. The other observed peaks are attributed to the phytochemical components present in the extract solution. The peak at 1113 cm−1 is attributed to the C-O stretching of primary alcohols. The peak at 1427 cm−1 corresponds to the O-H bending of the carboxylic acid. The peak observed at 2351 cm−1 is attributed to the O=C=O stretching of carbon dioxide. The FTIR spectra of bush tea extract, presented in Figure 6, show the presence of carboxylic acid bonding, primary alcohol stretching as well as the intramolecular hydrogen bond.

7352c2f1-3c13-43d8-bea0-11de03720bde_figure5.gif

Figure 5. Fourier-transform infrared spectra of ZnO powder annealed at 600°C.

7352c2f1-3c13-43d8-bea0-11de03720bde_figure6.gif

Figure 6. Fourier-transform infrared spectra of Bush tea leaf extract.

UV-Vis analysis

UV-Vis analyses were performed at a resolution of 1 nm at 250–800 nm wavelength range using a PerkinElmer Lambda 650S UV-Vis spectrometer. The absorption of ZnO nanoparticles is observed in the wavelength range of 250–400 nm (Kolekar et al., 2011). The measured peak at 380 nm (as shown in Figure 7) reveals the presence of ZnO nanoparticles with a band gap energy of 3.11 eV, smaller than the bulk ZnO of 3.37 eV. Thus, the presence of hexagonal wurtzite structures in the analysed samples is indicated, in accordance with the XRD results.

7352c2f1-3c13-43d8-bea0-11de03720bde_figure7.gif

Figure 7. Ultraviolet-visible spectra of as-synthesized ZnO nanoparticles.

SEM and EDS analyses

A JEOL JSM-7500F field-emission scanning electron microscope (FE-SEM) coupled with a JXA-8230/SXEDS/EDS/WDS energy-dispersive X-ray spectrometer (EDS) was used to get the morphology and the purity of the ZnO nanoparticles. SEM results are represented in Figure 8. The image shows quasi-spherical shaped ZnO nanoparticles agglomerated together. The EDS confirmed the presence of Zn and O. These findings are supported by Nethavhanani (2017) using natural extracts of Aspalathus linearis as a reducing agent (Nethavhanani, 2017).

7352c2f1-3c13-43d8-bea0-11de03720bde_figure8.gif

Figure 8. (a) Scanning electron microscopy image and (b) Energy-dispersive X-ray spectra of ZnO nanoparticles.

Discussion

Understanding the process of nanoparticles synthesis using the green route is key to the efficiency of the process and the outcome. Following the lack of data on chemical interactions of plant extracts with different metals to form nanoparticles, this study aimed to investigate the interaction of compounds with zinc nitrate to form ZnO nanoparticles. The identification of plant metabolites was performed using LC-MS tools by means of different databases such as KEGG, ChemSpider or Metfrag (Cecilia et al., 2020). Henceforth, the differences in the extracts resulting from the synthesis of ZnO nanoparticles were shown by means of PCA and the VIP score plot. Bush tea leaves contain a high percentage of flavonoids and tannins, apart from non-structural carbohydrates, proteins, fatty acids, and minerals, such as calcium, magnesium, phosphorus, potassium, sodium, iron, manganese, zinc, copper, aluminium, sulphur and fluoride (Lerotholi et al., 2017). Hence, the synthesis process resulted in the complete use of some metabolites as shown in Figure 2. The supernatant recorded low quantities of 8-C-Glucosylnaringenin/Naringenin 7-O-beta-D-glucoside, Scullcapflavone II, Mallotophenone, 6-Methoxyaromadendrin 3-O-acetate, 2-Phenylacetamide, 7-Hydroxy-2″,4″,5″-trimethoxyisoflavone, Coumarin, Malonyldaidzin (Figure 3). A variety of metabolites, such as terpenoids, polyphenols, sugars, alkaloids, phenolic acids, and proteins can reduce metal ions into nanoparticles (Marslin et al., 2018). Flavonoids, polyphenols as well as an aromatic compound interacted most with the precursor to form ZnO nanoparticles (Table 2). UV-Vis is a wonderful tool for the examination of the size and the shape of nanoparticles (Raut Rajesh et al., 2009). The analysed samples show the presence of a wurtzite structure at 380 nm. These findings are supported by (Krupa and Vimala, 2016) who reported the synthesis of ZnO nanoparticles absorbing light at 368 nm. The wavelength of 380 nm corresponds to the bulk band-edge of 3.2 eV for ZnO (Kolekar et al., 2011).

Conclusion

In this study, bush tea metabolites were screened to understand their interaction with metal ions to form nanoparticles. The LC-MMS peaks in both the crude extract before ZnO nanoparticles synthesis and the supernatant after synthesis revealed a significant difference, shown by the PCAs. Different flavonoids, polyphenols and an aromatic compound were found to react with zinc nitrate to form zinc nanoparticles. The FTIR as well as the XRD and UV-Vis analyses confirmed the formation of ZnO nanoparticles with a hexagonal wurtzite structure.

Data availability

All data underlying the results are available as part of the article and no additional source data are required.

Source link