In vitro antibacterial and cytotoxic activity of zinc oxide, iron oxide and silver nanoparticles synthesised from Artemisia afra

The synthesis of nanoparticles using medicinal plants is a potential pathway for developing environmentally friendly drugs with minimal side effects. The aim of this study was to isolate heptadecyl-trans-p -coumarate from the medicinal plant, Artemisia afra , utilise it and its extract to synthesise silver nanoparticles (AgNPs), zinc oxide nanoparticles (ZnONPs) and iron oxide nanoparticles (Fe 2 O 3 NPs) that were evaluated for antibacterial and cytotoxic activity. AgNPs synthesised from the coumarate and extract were mostly spherical with an average size of 12 nm and 29 nm, respectively. ZnONPs were mostly rods, plates and spheres with average sizes of 31 nm (extract) and 22 nm (coumarate). Fe 2 O 3 NPs were hexagons and spheres with average sizes of 31 nm (extract) and 24 nm (coumarate). Nanoparticles improved the antibacterial activity of the extract and coumarate against Escherichia coli, Pseudomonas aeruginosa, Chromobacterium violaceum and Staphylococcus aureus . Shape of nanoparticles influenced activity; rod-shaped ZnONPs and platelet-like Fe 2 O 3 NPs synthesised using the extract exhibited better antibacterial activity. Spherical ZnONPs synthesised using the coumarate and spherical AgNPs showed greater cytotoxicity. The results suggest a synergistic effect between the metal nanoparticles and capping agents. Overall, this study confirms the use of Artemisia afra for the biosynthesis of silver, zinc oxide and iron oxide nanoparticles.


INTRODUCTION
Nanotechnology is the synthesis, design and application of materials and devices with engineered shapes and sizes at the nanoscale level that are used in applications such as medicine, catalysis and environmental chemistry. 1According to Sahoo et al. 2 , nanoparticle-bound drugs have an extended half-life in vivo, longer circulation times and convey a high concentration of potent drugs to the target sites.This is due to the reduced particle sizes and high surface to volume ratios. 3anoparticle-bound drugs are easily suspended in liquids and are able to penetrate deep into organs and tissues.Therefore, there is ongoing research on the development of new nanoparticle-bound drugs that are more effective, have reduced side effects and reach their targets.
Artemisia annua (sweet wormwood or qinghao), is a shrub from the Asteraceae family, that has been used in Chinese traditional medicine to kill the malaria parasite, Plasmodium falciparum due to the presence of the highly oxygenated sesquiterpene, artemisinin, in its extract. 4This plant is currently receiving much attention due to its potential activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).6] Preliminary studies on the crude extract of A. afra have shown it to possess antibacterial activity against Mycobacterium aurum and Mycobacterium tuberculosis, antihistaminic and narcotic analgesic activities. 7he exploitation of medicinal plants for the synthesis of metal and metal oxide nanoparticles for drug delivery is considered as green synthesis. 8Plant secondary metabolites have the potential to reduce metal ions in solution, resulting in the subsequent formation of nanoparticles.−10 The combination of metal oxide nanoparticles and phytocompounds are documented to enhance the therapeutic activity of the nanostructure against a wide range of microbial infections. 11Iron is an important transitional metal due to its magnetic properties that can be exploited in a variety of applications such as sensors, catalysis, environmental and biomedical remediation. 12For biomedicine, iron oxide nanoparticles are frequently coated with hydrophilic and biocompatible coatings such as polymers that increase the thickness and the radii up to 100 nm, which causes damping of the magnetic resonance signal. 13The use of phytocompounds, which are biocompatible and yield nanoscale magnetic iron oxide nanoparticles with functionalized surfaces, is a viable alternative.
In this study, we aim to isolate and identify the major secondary metabolite from the extracts of A. afra, which would then be used to reduce silver, zinc oxide and iron oxide salts to their nano sizes.The antibacterial and cytotoxic activities of the extract, isolate and synthesised nanoparticles will be compared and evaluated.To date, there have been no reports on the use of A. afra for the synthesis of metal nanoparticles.

Collection of plant samples and preparation of aqueous extract
The leaves from Artemisia afra were purchased from the Durban Muthi Market, KwaZulu-Natal.The leaves were dried at room temperature for two weeks and crushed using a food processor.Approximately 50 g of crushed leaves was weighed and transferred into a 1000 mL beaker containing 500 mL distilled water, and refluxed at 90 °C for 20 minutes.The extract was cooled and filtered twice through Whatman No. 1 filter paper and stored in a refrigerator at 4 °C until further use.

Isolation and purification of compound from Artemisia afra
The ground leaves from A. afra (1 kg) were extracted with methanol (MeOH) for three days using an orbital shaker as described by

Synthesis of nanoparticles using the aqueous extract and isolated compound
Nanoparticles were synthesised according to the method described by Singh, Singh and Rawat 15 for zinc oxide nanoparticles (ZnONPs), El-Kassas, Aly-Eldeen and Gharib 8 for iron oxide nanoparticles (Fe 2 O 3 NPs) and Jyoti, Baunthiyal and Singh 16 for silver nanoparticles (AgNPs).The colour changes of the solution or appearance of a precipitate indicated the formation of nanoparticles.Nanoparticles were only synthesised using the isolated compound and aqueous extract as these particles may be used in biomedical applications.

Silver nanoparticles (AgNPs)
Nanoparticles were synthesised according to the method described by Jyoti, Baunthiyal and Singh. 16An aqueous solution of silver nitrate (50 mL, 1 mM) was added dropwise (2 mL per minute) to the aqueous plant extract (50 mL).The solution was stirred overnight at room temperature using a magnetic stirrer.A color change from orange to grey-black was observed.The suspended AgNPs were centrifuged at 5000 rpm for 30 minutes, washed several times with deionized water, and dried in an oven for eight hours at 50 °C.For synthesis of AgNPs using the compound, 10 mg of the compound was dissolved in 10 mL of MeOH and reacted with 10 mL of AgNO 3 .

Zinc oxide nanoparticles (ZnONPs)
A solution of zinc chloride (50 mL, 0.4 M) was added to the aqueous plant extract (50 mL) drop-wise.The solution was stirred at room temperature for 2 h.The pH of the solution was adjusted to 11 by adding a solution of NaOH (1.0 M) drop-wise with constant stirring for 2 h at room temperature.The resulting precipitate was obtained after centrifugation at 5000 rpm for 30 minutes, washing several times with deionized water, and drying in an oven at 100 °C for 2 h.

Antibacterial activity
The antibacterial activity of the nanoparticles synthesised from the aqueous extract of Artemisia afra and isolated compounds were tested against a series of bacterial strains including the Gramnegative Escherichia coli ATCC 35218 and ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Klebsiella pneumonia ATCC 700603 and Chromobacterium violaceum ATCC 12472, and Gram-positive Staphylococcus aureus ATCC 43300 and ATCC 29213 and Enterococcus faecalis ATCC 51299 and ATCC 29212, using the well diffusion method. 17Molten and cooled nutrient Mueller Hinton (MH) (20 mL) agar media was poured into a sterilized petri dish and left overnight at room temperature.Agar plates were bored using a sterile gel borer to make wells of 3 mm diameter.Nanoparticles suspended in distilled water, extract or phytocompound dissolved in 10 % DMSO was loaded into wells (40 μL or 20 μL, 10 mg mL −1 ) in triplicate.Ampicillin (10 μg per disc) was used as a positive control while sterile water and DMSO was used as negative controls.The disc was placed on Muller Hilton agar plates inoculated with each microorganism.The plates were incubated at 37 °C for 24 hours.After the incubation period, the zone of inhibition was measured (mm).

Cell viability and cytotoxicity testing
The cancerous MCF7 (breast adenocarcinoma) and A549 (human lung adenocarcinoma), and non-cancerous HEK239 (human embryonic kidney) cell lines were obtained from the American Tissue Culture Collection (ATCC) (Virginia, USA).Cells were grown in 25 mL tissue culture flasks in Dulbecco's modified Eagle's minimum essential medium (DMEM) supplemented with foetal bovine serum (FBS) (10 %) with an antibiotic mixture of penicillin (100 μg mL −1 ) and streptomycin (100 μg mL −1 ).At approximately 85 % confluence, the cells were harvested using 0.25 % trypsin and seeding in a 25 mL flask.Cells at a seeding cell density of 1.8 × 10 5 cells per well were plated into a 96-well plate containing 200 μL of medium.The cells were incubated at 37 °C in 5 % CO 2 and 95 % air with 80 % humidity, overnight (eight hours).Thereafter, the medium was removed and freshly prepared medium (100 μL) was added.
Test samples (1 mg) were dissolved or suspended in 20 % DMSO and was further diluted with media to make a total volume of 4 mL.Different concentrations (30, 75, 125 and 250 μg mL −1 ) were added in triplicate to the cells and incubated for 48 hours at 37 °C.The medium was again removed and a volume of 20 μL of 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) solution was added.The solution was incubated at 37 °C for 3 hours.Thereafter, DMSO (100 μL) was added to dissolve the crystal and incubated for 10 minutes.The absorbance was measured at the wavelength of 570 nm and the percent viability was calculated.Standard anticancer drugs, 5-fluorouracil (5FU) and doxorubicin, were used as positive controls and untreated cells as a negative control.The cell viability was calculated using the following equation: Cell viability (%) = (A sample /A untreated cells ) × 100 (1)

Structural elucidation of isolated compound from A. afra
The compound was isolated as a dark brown solid (23 mg).The The compound had a similar backbone to the coumarate previously isolated from Ipomoea sepiari. 18The 1 H NMR spectrum indicated that there is a chain with 16 methylenes.With the data obtained, it can be concluded that the compound is heptadecyl-trans-p-coumarate (Figure 1).

Characterisation of nanoparticles
Three metal and metal oxide nanoparticles (ZnONPs, Fe 2 O 3 NPs and AgNPs) were successfully synthesised using the aqueous extract and isolated compound (heptadecyl-trans-p-coumarate) from A. afra.A colour change caused by excitation of the surface plasmon resonance (SPR) was indicative of the formation of nanoparticles. 19igure 2 shows the UV-Vis absorption spectra of ZnONPs, Fe 2 O 3 NPs and AgNPs synthesised from the aqueous extract and isolated compound, respectively.1] The UV-Vis spectrum of AgNPs synthesised from the coumarate (413 nm) exhibits a blue shift

RESEARCH ARTICLE
Bongisiwe Shelembe, Roshila Moodley and Hafizah Chenia 121 S. Afr.J. Chem., 2023, 77, 119-125 https://journals.co.za/content/journal/chem/ compared to those synthesised from the aqueous extract (429 nm), which is in agreement with that in literature, indicative of particle shape and size differences with both peaks being relatively broad, indicative of large particle size distribution. 9,22he morphology and size of the nanomaterial prepared using the aqueous extract and isolated coumarate were analysed using transmission electron microscopy (TEM) and are presented in Figure 3.The average size represented by the histogram, also represented particle size distribution.
The images demonstrated agglomeration of ZnONPs (Figure 3 (1a, 1b)) prepared using the aqueous extract with a variety of shapes, dominated by spheres, which coagulated to form rods, hexagons and rectangular nanoparticles.Particle sizes (from 150 particles) ranged from 5 to 80 nm, with an average size of 31 nm.Nanoparticles synthesised from the coumarate (Figure 3 (1c, 1d)) showed agglomerated hexagons, triangles, rods and spheres with smaller (6 nm) and larger (48 nm) particles.
Silver nanoparticles synthesised using the aqueous extract showed spherical and irregular shapes with a wide, fairly dispersed, particle size range (Figure 3 (2a, 2b)).The particle size distribution ranged from 4 nm to 24 nm, with an average size of 13 nm.Silver nanoparticles synthesised using the coumarate showed spherical nanoparticles with particle sizes ranging from 25 to 75 nm, with an average size of 29 nm, indicating larger and more monodispersed particles than those synthesised using the aqueous extract thereby supporting the blue shift observed from the UV-Vis data.
The TEM images (Figure 3 (3a, 3b)) of Fe 2 O 3 NPs synthesised using the aqueous extract, showed the presence of lattice fringes (insert Figure 3 (3a)), which represents the crystalline nature and the distance between the planes.A mixture of irregular platelet-like structures and hexagons with a size range of 5-80 nm and average size of 31 nm was detected.Nanoparticles synthesised using the coumarate, showed agglomerated spheres and rods (Figure 3 (3c, 3d)) and end-capping by phytocompounds (insert Figure 3 (3c)).A 28 nm nanoparticle was capped with phytocompounds that were 2 nm in size.The average particle size was 24 nm and their sizes ranged from 5-85 nm.
The scanning electron microscopy (SEM) coupled with energy dispersive x-ray (EDX) spectroscopy (Figure 4) was further used to confirm the morphology of the synthesised nanoparticles.The selected area electron diffraction (SAED) pattern for ZnONPs synthesised from the aqueous extract (Figure 4 (1a)) showed reflections arranged    101), ( 101), ( 102) and (103) planes, corresponding to zincite of ZnO (Table 1).The SAED pattern of AgNPs synthesised using the aqueous extract and coumarate (Figure 4 (2a-c)) showed bright spots arranged in circular rings, which can be indexed to reflections from the (111), ( 200), (220), (311) and (222) planes from the calculated lattice spacing (d) presented (Table 2).The planes correspond to the face centred cubic (fcc) structure of silver. 16,23 e SEM micrographs (Figure 4 (3a)) confirmed the hexagon platelet-like particles of Fe 2 O 3 NPs synthesised using the aqueous extract.The SAED pattern (Figure 4 (3b)) showed bright spots making up a ring.This supports the TEM image and showed the presence of lattice fringes that represent the crystalline nature and the distance between the planes.The calculated reciprocal of the radius from the SAED pattern was used to identify the crystalline phase of the Fe 2 O 3 NPs.The obtained d-values for the nanoparticles synthesised using the extract (in nm) were 0.33634, 0.2631, 0.25196, 0.2212 and 0.1644 corresponding to (104), (110), (113), ( 116) and (018) lattice planes, while those synthesised using the coumarate (in nm) were 0.2524, 0.2587, 0.2151, 0.1682 and 0.1615 corresponding to (110), (104), (113), ( 116) and (018) lattice planes of a α-Fe 2 O 3.

2
The EDX spectrum (Figure 5) of the synthesised nanomaterials confirmed the presence of zinc in ZnONPs (Figure 5a and 5b), iron and oxygen in Fe 2 O 3 NPs (Figure 5c and 5f) and silver in AgNPs (Figure 5d and 5e).Carbon and oxygen are due to scattering caused by phytocompounds capping the particles, the source of nitrogen was the starting material and gold came from the sample coating used to prevent charge of the specimen.The EDX data of the synthesised Fe 2 O 3 NPs showed the effect of fluorescence caused by excitation from the Cu k-α energy = 8.046 keV since it has higher energy than Fe, which is 7.112 keV.

Antibacterial activity
Antibacterial activity of ZnONPs, Fe 2 O 3 NPs, and AgNPs was tested against both Gram-negative (E. coli and C. violaceum) and Grampositive (S. aureus and P. aeruginosa) bacterial strains.The activity of the test samples was determined by measuring the zones of inhibition using a standard agar-well diffusion method and results are presented in Table 3.The displayed results are only for the test samples that showed inhibitory potential.The aqueous extract and coumarate alone showed no antibacterial activity against all bacterial strains tested except for C. violaceum, indicating anti-quorum sensing potential.Reducing metals with both the coumarate and aqueous extract showed better activity.The antibacterial activity of the nanoparticles (capping agent, average size) was found to be in decreasing order of Fe 2 O 3 NPs (extract, 31 nm) > ZnONPs (extract, 31 nm) > AgNPs (coumarate, 29 nm) > ZnONPs (coumarate, 25 nm) ~ AgNPs (extract, 13 nm) > Fe 2 O 3 NPs (coumarate, 22.4 nm).
ZnONPs and Fe 2 O 3 NPs capped with the aqueous extract exhibited  better antibacterial activity against both the Gram-negative and Grampositive strains tested, compared to the aqueous extract alone, with Fe 2 O 3 NPs having the best activity overall.The antibacterial activity of ZnONPs synthesised using the coumarate showed better activity compared to the coumarate alone for all bacterial strains tested whilst Fe 2 O 3 NPs had no effect on the coumarate.Previous studies on the effects of various shapes of ZnONPs on toxicity of bacteria showed rods and wires to influence the mechanism of internalization and penetrated the cell walls easier than spherical nanoparticles. 24This was confirmed by the improved antibacterial activity of ZnONPs capped with the extract that were rods and platelets compared to ZnONPs capped with the coumarate that were spheres and platelets.Iron is one of the essential elements involved in metabolic processes such as respiration, photosynthesis and gene regulation in aerobic organisms.At high concentrations, bacteria tend to absorb iron and reduce it, which in turn, reacts with H 2 O 2 forming a hydroxyl radical.The radical damages proteins and nucleic acids of the cell membrane of the bacteria. 25Fe 2 O 3 NPs synthesised using the extract produced irregular platelet-like structures and hexagons whilst those synthesised using the coumarate produced a cluster of agglomerated particles with rods; shape affected the activity more than size as the smaller particles capped with the coumarate were less active than the larger ones capped with the extract.
Silver nanoparticles have previously been shown to have good antibacterial activity.In this study, AgNPs improved the antibacterial activity of the extract and coumarate for all bacterial strains tested.AgNPs synthesised with the aqueous extract demonstrated antibacterial potential towards the Gram-negative bacteria, E. coli and C. violaceum, whilst AgNPs synthesised with the coumarate were active against all bacterial strains tested.
Overall, Fe 2 O 3 NPs (extract, sizes up to 90 nm), ZnONPs (extract, sizes up to 80 nm) and AgNPs (coumarate, sizes up to 75 nm) were more active than ZnONPs (coumarate, sizes up to 22.5 nm), AgNPs (extract, sizes up to 24 nm) and Fe 2 O 3 NPs (coumarate, sizes up to 48 nm).The results indicate that larger particles have more antibacterial activity than smaller ones.
According to Alswieleh and Alghamdi 11 , the size of the nanoparticles allows them to penetrate the cell wall of the bacteria and interact with cellular elements.][28][29] Our results show shape or morphology to influence antibacterial activity more than size for ZnONPs, Fe 2 O 3 NPs and AgNPs against the bacterial strains tested.Therefore, our results agree with those that demonstrate that AgNPs undergo a shape-dependent interaction with bacteria such as E. coli. 30

Cell viability and cytotoxicity testing
Three cell lines (human ambryonic kidney (HEK293), human lung adenocarcinoma (A549) and human breast adenocarcinoma (MCF-7)) were used to evaluate the cytotoxicity of the aqueous extract, isolated compound (coumarate) and their nanoparticles (ZnONPs and AgNPs), using the MTT cell viability assay.Fe 2 O 3 NPs did not exhibit cytotoxic activity against cancerous cells.Untreated cells were used as  3. Antimicrobial susceptibility (zone of inhibition (mm)) (mean ± SD, n=3) of the aqueous extract of A. afra, isolated coumarate and their synthesised silver nanoparticle (AgNPs), iron oxide nanoparticles (Fe 2 O 3 NPs) and zinc oxide nanoparticles (ZnONPs) against Gram-negative and Gram-positive bacterial strains at different concentrations (50 and 100 µg mL −1 6 indicate that all the test samples reduced cell viability for most of the cell lines. The coumarate displayed selective cytotoxicity towards the cancerous A549 without being cytotoxic against the non-cancerous HEK293 and with moderate cytotoxicity against the cancerous MCF-7 cell lines.When treated with the extract, cell viability of both cancerous and non-cancerous cell lines was greater than 80 %, at low concentrations.Cell growth did not go below 50 % in all cases at high concentrations, thereby indicating moderate cytotoxicity.These results confirm the antagonistic effects of inactive compounds present in the extract that inhibit the cytotoxic activity of active compounds, in this case the coumarate.A possible explanation for the activity of the coumarate could be that the chain enhances its lipophilicity and hence its permeability whilst the cells provide an environment for hydrolysis of the ester into a weak acid that improves activity. 31Previous studies have also shown coumarins to have good anticancer activity against MCF-7 and Caco-2 cell lines. 31gNPs synthesised using the coumarate displayed non-selectivity with it being highly cytotoxic against the cancerous A549 and non-cancerous HEK293 and moderately cytotoxic against the cancerous MCF-7 cell lines.AgNPs synthesised using the extract also exhibited non-selectivity with good cytotoxicity against all cell lines tested.ZnONPs synthesised using the coumarate were not as active as the AgNPs against the cancerous cell lines.However, at low concentrations they were more selective towards the cancerous A549 (cell viability < 20%) than non-cancerous HEK293 (cell viability > 75 %) cell lines.ZnONPs synthesised using the extract were mostly inactive against the cancerous cell lines.
Contrary to antibacterial activity, ZnONPs synthesised using the coumarate that were mostly spherical were more cytotoxic than the rod shaped ZnONPs synthesised using the extract.Similarly, AgNPs synthesised using the extract and coumarate that were mostly spherical were highly cytotoxic.

CONCLUSION
In this study, heptadecyl-trans-p-coumarate was isolated for the first time from A. afra.The biomolecules in A. afra reduced and stabilized silver, zinc and iron salts to form their respective AgNPs, ZnONP and Fe 2 O 3 NPs.ZnONPs and Fe 2 O 3 NPs synthesised with the extract showed better antibacterial activity than those synthesised with the coumarate; AgNPs synthesised with the coumarate showed better antibacterial activity than those synthesised with the extract.The results indicate that larger particles have more antibacterial activity than smaller ones and that shape was a determinant of activity more than size.AgNPs capped with the coumarate showed better cytotoxic potential against A549 (human lung adenocarcinoma) compared to the biomolecule alone.Synergistic effects were observed for cytotoxicity between the synthesised nanoparticles and the active biomolecules.Overall, this study confirms that the medicinal plant species, A. afra, could be used for the biosynthesis of AgNPs, ZnONPs and Fe 2 O 3 NPs.It also provides a scientific basis for the ethno-medicinal use of the plant to treat various ailments

1 H
NMR spectrum showed two resonances that integrated to two protons each, at δ H 7.4 (H-5/9) and 6.8 (6/8), indicative of a p-substituted benzene ring.Furthermore, two doublets that integrated to one proton each, with resonances at δ H 7.6 (H-3) and 6.3 (H-2), were indicative of an olefenic group.The resonances between δ H 1.25-1.06indicated methyl and methylene groups.The resonance at δ H 1.25 integrated to 26 protons, indicating 13 methylene groups.The 13 C NMR spectrum showed a characteristic resonance of a carbonyl at δ C 167, which correlated with the proton at δ H 4.1 (H-1') in the HMBC experiment.The GC-MS spectrum gave a molecular ion peak at m/z 401, which corresponds to the molecular formula C 26 H 42 O 3 .

Table 1 .
Structural parameters of synthesised ZnONPs

Table 2 .
Structural parameters of synthesised AgNPs ). Ampicillin was used as a positive control Afr.J. Chem., 2023, 77, 119-125 https://journals.co.za/content/journal/chem/ the negative control and 5-FU as the positive control.The results in Figure