Journal of Molecular Structure

Manuscript Draft

Manuscript Number: MOLSTRUC-D-18-01755

Title: Green synthesized ZnO nanoparticles using Sutherlandia Frutescens

plant extract: Photocatalytic and biological applications

Article Type: Research Paper

Keywords: Green synthesis; Sutherlandia frutescens; photocatalysis; ZnO

nanoparticles; biological studies
Highlights (for review)

Highlights
ZnO nanoparticles were synthesised for the first time using Sutherlandia Frutescence.
ZnO nanoparticles were photocatalytically active against MB degradation
The nanoparticles were potent against both gram-negative and gram-positive strains
The bacterial effect was also investigated against real water samples
These particles showed an enhanced activity against A549 lung cancer cells
Cover letter
Click here to view linked References

University of Limpopo
Department of Chemistry, School of Physical and Mineral Sciences
Private Bag X1106, Sovenga, 0727, South Africa
Tel: (015) 268 2205, Cell: 0748067910, Email: nomso.hintsho-mbita@ul.ac.za

The Editor, Journal of Molecular Structure

8th June 2018
Dear Editor

Please find enclosed a copy of our manuscript titled “Green synthesis of ZnO nanoparticles using Sutherlandia
Frutescens plant extract: Photocatalytic and biological applications” by LM Mahlaule-Glory, Z Mbita, M.M
Mathipa, N Mketo, B Ntsendwana, NC Hintsho-Mbita submitted for consideration for publication in the
Journal of Molecular Structure

Justification for publication

Recently, green synthetic approaches have received a lot of attention in the materials industry since they
use sources that are environmentally friendly. This is due to the high costs that are associated with
nanoparticle preparation and the harsh solvents normally used as reducing agents for the preparation of
these materials. The use of plant extracts affords the nanomaterials an opportunity to have traits that could
be incorporated onto the nanoparticles during synthesis thus to enhance their use in the biomedical
industry as they are mostly used in the catalytic industry. The use of green synthesized zinc oxide
nanoparticles has been documented, but the use of these materials for photocatalytic and biomedical
applications is still in its infancy.

In this study, we have demonstrated for the first time the use of a traditional medicinal plant, Sutherlandia
Frutescence for the synthesis of any nanomaterial. Photocatalytic, antibacterial and cytotoxicity studies
were conducted. We have demonstrated a positive and enhanced effect of this material for both
antibacterial and anticancer activity. We believe that the Journal of Molecular Structure is the appropriate
journal to get this simple and important message through to a large audience who are interested in this type
work.
The authors declare that this is our original work, no parts of this work have been plagiarised. The work
has not been submitted elsewhere for publication and will not be submitted elsewhere whilst it is still
under consideration for publication in this journal. Finally, the authors declare that there are no competing
interests

We look forward to your response

Regards
Dr NC Hintsho-Mbita
Department of Chemistry,
University of Limpopo
nomso.hintsho-mbita@ul.ac.za
Manuscript
Click here to view linked References

Green synthesized ZnO nanoparticles using Sutherlandia Frutescens plant

extract: Photocatalytic and biological applications

L M Mahlaule-Glory a,b, Z Mbitac, M.M Mathipa d, N Mketoe,

B Ntsendwanaf, NC Hintsho-Mbitaa,b*

a
Department of Chemistry, University of Limpopo, Sovenga, Polokwane, 0727, South
Africa

b
DST/NRF Centre of Excellence in Strong Materials, South Africa

c
University of Limpopo, Sovenga, Polokwane, 0727, South Africa
Department of Biochemistry, Microbiology and Biotechnology

d
University of Limpopo, Sovenga, Polokwane, 0727, Limpopo Agro-food technology
station, South Africa

e
Department of Chemistry, College of Science Engineering and Technology,
University of South Africa, Private Bag X3, Johannesburg, South Africa.

f
Nanotechnology and Water Sustainability Research Unit, Science Campus,
University of South Africa, Roodepoort, Johannesburg, 1710, South Africa

*Corresponding author:

Email: nomso.hintsho-mbita@ul.ac.za Tel: +27 (0) 15 268 2205 Postal address:

Department of Chemistry, University of Limpopo, Private Bag X 1106, Sovenga 0727,
Polokwane, South Africa
Abstract
The quest for eco-friendly synthetic routes for the development of multifunctional
nanoparticles has reinforced the use of plant extracts as replacement solvents.
Amongst the various nanoparticles, ZnO nanoparticles have emerged as a preferred
candidate for various biological applications. In this study, ZnO nanoparticles were
successfully synthesized by the solvothermal method using Sutherlandia frutescens
extract. The morphological, structural, elemental and functional properties were
evaluated using TEM, SEM, SAED, EDS and FTIR techniques. The ZnO nanoparticles
exhibited various morphological shapes (spherical, hexagonal) with a particle size
distribution ranging from 5-25 nm. The FTIR results showed the presence of intrinsic
absorption bands below 1000 cm−1(fingerprint region) which emanate from inter-
atomic vibrations formation and thus confirming the formation of the ZnO
nanoparticles. Furthermore, it was observed that the phytochemical functional groups
present in the Sutherlandia frutescens plant extract were successfully incoporated into
the ZnO nanoparticles indicating that the ZnO plant possess improved biological
properties. The elemental mapping revealed strong ZnO peaks as confirmed by the
EDS technique. The photocatalytic activity of the Sutherlandia frutescens derived ZnO
towards Methylene Blue dye as the target pollutant resulted in a 45% degradation
efficiency with a fast kinetic rate of 0.024 min-1 within 75 min reaction time. In addition,
ZnO nanoparticles displayed exceptional biomedical activity with an antiproliferative
effect of 93% against human lung cancer cells (A549) owing to the phytochemicals
obtained from the Sutherlandia frutescens plant extract. These nanoparticles were
also potent against gram negative (Escherichia coli, Pseudomonas aeruginosa) and
gram positive (Staphylococcus aureus, Enterobacter faecalis) strains using model
pollutants and real water pollutants.

Keywords: Green synthesis, Sutherlandia frutescens, photocatalysis, ZnO

nanoparticles and biological studies.
Introduction
Nanomaterials are particles characterised with a nanoscale dimension within the
range of 1-100 nm [1]. They possess unique physicochemical attributes as compared
to their bulk counterparts, thus enhancing their optical, chemical, mechanical and
biological properties [2]. The choice of a suitable synthetic route or procedure to
prepare these nanomaterials has a major effect on their resultant properties for their
desired applications. These include sol gel, hydrothermal, laser ablation and many
more [3]. However, these methods are time consuming, extremely costly and they
require use of toxic chemicals, which can be detrimental to the environment. This has
necessitated a paradigm shift from the physical and chemical processes towards a
cheap, eco-friendly and bioprocessed route. These green approaches involve the use
of plant extracts, bio-waste materials and microbe-mediated synthetic methods [4].
They have been successfully used in the preparation of different dimensional
nanostructures such as metal oxides and carbon nanomaterials [5, 6].

Currently, metal oxides have been used extensively in various applications due to their
high surface area and high surface energy, which offer easy fabrication and formation
of nanoparticles [7]. Some of the most common metal oxides include Zinc Oxides
(ZnO); Nickel Oxide (NiO), Magnesium Oxides (MgO), Titanium dioxides (TiO2), and
Iron (III) Oxide (Fe2O3). Amongst these, ZnO and TiO2 are the most widely used
nanomaterials in heterogeneous photocatalysis due to their attractive electrical,
optical, mechanical and morphological properties [8]. They both possess a wide band
gap (3.3 eV) with high excitation energy (60 eV) [9, 10] and a large surface area.
However, the use of ZnO is more advantageous since it exhibits a higher absorption
efficiency than TiO2, thus increasing its photocatalytic degradation activity against
organic pollutants such as methylene blue (MB). Furthermore, ZnO consists of a high
photo-stability under ambient conditions, therefore, allowing prolonged reaction time
during the photodegradation processes [11, 12].

Moreover, ZnO nanoparticles have also been reported to possess exceptional

biological properties such as antibacterial, anticancer and antioxidant activities [11,
13–16]. Their biomedical properties are due to their narrow particle size distribution
with smaller particle sizes [17]. Since, ZnO nanoparticles are less toxic as compared
to TiO2, they are regarded as suitable biocompatible materials [2].
Biocompatible compounds are used to obtain biomolecules from plants via green
synthesis [18]. Rathnasamy et al [2] reported the enhancement of photocatalytic
activity of ZnO nanoparticles towards the degradation of methylene blue using the
Carica papaya leaf extract. Jamdagni et al [4] also reported on the ability of
Nyctanthes arbor-tristis flower extract to strengthen the antifungal activity of ZnO
nanoparticles against five pathogenic fungi. In light of these, all the above mentioned
plant extracts have been used either to enhance the biomedical applications of these
materials or to assist their photocatalytic activity performance.

Sutherlandia Frutescens (Sf) also known as bush cancer tea, is an important member
of the Fabacea family [19]. It is an overwhelming and multipurpose Southern African
medicinal plant. The plant has been reported to be a safe tonic for diverse human
health conditions and provides immune system support [19]. It has been documented
to reduce mental stress and to cleanse blood [20–22]. The presence of
phytochemicals such as cycloartane glycosides, flavonols, tripenoid, canavanine,
pinitol and gaba, in this plant [19,20,23,24] has propelled the investigation of the
Sutherlandia frutescens plant extract as a suitable reducing and capping agent for the
synthesis of ZnO nanoparticles. Thus, this study focuses on the synthesis of
Sutherlandia frutescens derived ZnO nanoparticles and evaluation of their
photocatalytic and biological activities. To the best of our knowledge, Sutherlandia
frutescens has not yet been used in the green synthesis of nanoparticles.
1. Experimental Methods
2.1 Materials and Reagents
Sutherlandia Frutescens tea was purchased from a local store in Polokwane, South
Africa. Methylene Blue (MB), Sodium hydroxide (NaOH) pellets, Hydrochloric acid
(HCl) 37% and Zinc Sulphate hexahydrate (ZnSO4.7H2O) were purchased from Sigma
Aldrich, Germany. Nutrient agar and nutrient broth was purchased from Chart Biomed,
Germany and the European Institute for Speciation Analysis (EVISA) United Kingdom,
respectively. The four bacterial strains Escherichia Coli (E coli), Staphylococcus
aureas (S. aureus), Pseudomonas aeruginosa (P. aeruginosa) and Enterobacter
faecalis (E. faecalis) were obtained from the Department of Biochemistry, Microbiology
and Biotechnology, University of Limpopo, South Africa. Tap, pond and sewage water
were collected at the University of Limpopo, South Africa while the river water was
collected from the Mokolo River in Limpopo, South Africa.

2.2 Extraction process of Sutherlandia frutescens

Sutherlandia frutescens (Sf) was extracted using the tea infusion method [25]. Briefly,
about 10 g of Sutherlandia frutescens was added into boiling deionised water in a 500
mL volumetric flask. The mixture was then heated for 15 minutes at 80 °C until a milky
yellow mixture was obtained. The mixture was allowed to cool and then filtered using a
70 cm diameter Munktell filter paper. The obtained extract was kept at 4 oC in the
fridge till further analysis.

2.3 Synthesis of ZnO Nanoparticles

About 4.069 g of ZnSO4.7H2O was poured into a 100 mL flask. An aliquot of 50 mL of
the extract was added to the Zinc salt. The mixture was boiled for 1 hour and allowed
to cool at room temperature, before it was transferred into a crucible. The crucible was
placed in an oven and left to dry for overnight at 80 °C. The product was filtered (0.45
µm whatman filter paper) and washed several times with deionised water to remove
impurities. The product was calcined at 700 oC for 2 hours in the furnace and allowed
to cool at room temperature.
2.4 Characterization techniques
The particle size distribution and surface morphology were analysed using
Transmission electron microscopy (TEM) and Scanning Electron Microscopy (SEM).
The diffraction planes were investigated using the Selected Area Electron Diffraction
(SAED). The presence of functional groups on the formed ZnO plant was evaluated
using Fourier-Transform Infrared Spectroscopy (FTIR) while the elemental analysis
was analysed by the Energy Dispersive X-ray (EDS) Spectrometer.

2.5 Photocatalytic activity of ZnO nanoparticle against MB dye

ZnO nanoparticles were used as a photocatalyst towards the degradation of the MB
dye. About 120 mg of the ZnO nanoparticle was added to 500 mL of the MB solution
containing an initial concentration of 20 ppm. Prior to light irradiation, the mixture was
stirred continuously for an hour in the dark to reach the adsorption-desorption
equilibrium between the dye and the photocatalyst. The solution was then exposed to
light source irradiation of 40 W LED for 120 min and aliquots of 5 mL were withdrawn
for every 15 min time interval. The aliquots were then filtered using the 0.45µm
membrane filter, collected and taken for analysis. The percentage of dye degradation
was calculated using the formula below:

(%) Degradation = (Ao - At)/A0 x 100 [2]

Where A0 is the initial absorbance and At is the final absorbance. The photocatalytic
reaction rate was calculated using this formula:

In (Ao/At)= kt [26]

Where Ao is the initial absorbance, At is the final absorbance, k being the degradation
kinetic of the photocatalyst and t is the irradiation time.

2.6 Antibacterial Activity of Sutherlandia frutescens ZnO nanoparticles

2.6.1 Treatment and analysis of the model pollutants using plate count method
The antibacterial activity of Sutherlandia frutescens ZnO nanoparticles against the four
bacterial strains: E coli, S. aureus, P. aeruginosa and E. faecalis was determined
using the plate count method [27].

2.6.2 Treatment and analysis of various water samples containing natural

pollutants using tempo reader
Four water types emanating from different sources (tap, river, pond and sewage) were
used to test the presence of bacterial strains (E. coli, E. faecallis) and total population
of microorganisms (aerobic count). For the preparation, aliquots of 10 mL of each
water type were diluted with peptone water (broth) and 100 mL of sterile distilled
water. About 1 mL of the mixture was added to the media before treatment. The
inoculation was performed using two concentrations (0.005 mg/mL and 0.05 mg/mL)
and incubated at 37 oC for 24 hrs (E. coli, E. faecalis) and 48hrs for Aerobic. count.
The mixtures were loaded onto the count cards and incubated for 24 hrs at 37 oC for
E. coli and E. faecalis while the Aerobic. count was incubated for 48 hrs.

2.7 Anticancer activity of Sutherlandia frutescence ZnO nanoparticles using the

Fluorescence Activated Cell Sorting (FACS) Cell Viability test and
fluorescence microscopy

2.7.1. Cell Viability Using the Muse® Cell Analyzer

Cells were cultured in six well plates overnight then washed with sterile 1X PBS and
treated with various concentrations (125; 250; 500 and 1000 µg/mL) of ZnO
nanoparticles for 24 hours in appropriate cell culture conditions against the human
lung cancer cell lines (A549). After trypsinisation, about 20 L (1 × 106 cells) of
harvested cells were added to 380 L Count and Viability reagent (Merck-Millipore,
Germany). The cell viability assay was performed using the Muse® Count & Viability
reagent (Merck-Millipore), following the manufacturer’s instructions. The results were
analysed using the Muse® Cell Analyser Count and Viability software module.
2.7.2. 4′, 6-diamidino-2-phenylinode (DAPI) staining to assess
The A549 cells were seeded and treated with ZnO plant nanoparticles as mentioned
above. After the 24 hour treatment incubation, the cells were washed twice with
1XPBS and then fixed in 4% paraformaldehyde. The cells were then stained
with (DAPI) for 15 minutes at room temperature and then captured under the
fluorescence microscope (Nikon Instruments Inc., USA) using a digital camera.

3. Results and discussion

3.1 Vibrational analysis and Mechanism of formation of Sutherlandia
frutescence ZnO nanoparticles
The FTIR results were taken at room temperature within the range between 500 cm-1
to 4500 cm-1 to determine the groups found in the pure ZnO, ZnO plant and
Sutherlandia frutescence plant extract. Metal oxides are known to exhibit absorption
bands in the fingerprint region (i.e. below 1000cm-1), which comes from the interatomic
vibrations [10]. The FTIR results showed vibration peaks in the fingerprint region at
900 cm-1 and 1000 cm-1 for pure ZnO and Sutherlandia frutescence derived ZnO
nanoparticles respectively, indicating the formation of ZnO nanoparticles. It was also
observed that the vibration bands of ZnO plant shifted to higher wavenumbers due to
the presence of phytochemicals. This is in agreement with the results obtained by
several authors who reported the formation of ZnO nanoparticles in that region [2, 17,
23]. When observing the plant extract, several phytochemicals identified in the ZnO
plant were also identified in the extract. This shows that there was a successful
incorporation of functional groups from the plant to the ZnO nanoparticles. The
medium peaks at 1400 cm-1 were attributed to C=C groups, while the broad weak
absorption band at 1600 cm-1 were due to N-H groups. The intense broad peak
displayed between 2900 cm-1 and 3500 cm-1 consisted of several phytochemical
groups, which could be ascribed to OH, NH2, H3CO and CH aromatic stretching bands.
With the incorporation of these phytochemicals, it is expected that the ZnO plant will
demonstrate an enhanced biological activity.
 Figure 1: FTIR spectra of ZnO pure, ZnO plant and Sutherlandia frutescence plant
extract.

It has been reported that the phytochemicals present in the plant extract are the main
determining factor on how the biomolecules interact with the Zn salt [17, 25]. To
explain the interaction, the ZnSO4.7H2O salt was mixed with the Sutherlandia
frutescence plant extract, which acted as a reducing agent by balancing the acidity
and basicity in the aqueous solution. Thereafter, the plant extract provided three main
functions (i) neutralization of the free radicals, (ii) donating the hydrogen atom and (iii)
releasing a Zn metal ion in a solution. Thereafter, the Zn2+ was able to attach to the
polyphenols from the Sutherlandia frutescence plant extract through electrostatic
interaction. After the chelation of the metals to the ligands, a complex was formed.
This was confirmed by FTIR (Fig.1) as shown above where some of the functional
groups found in the bioactive compounds were identified in the ZnO plant, thus
enhancing the ZnO properties.
3.2 Morphology, SAED and particle size distribution of Sutherlandia
frutescence ZnO nanoparticles
The surface morphology of the as ZnO plant was studied using TEM and SEM. As
shown in Fig. 2 (a-b), the ZnO plant exhibited particles with spherical and hexagonal
shapes. However, there was a noticeable agglomeration. In addition, the SAED
showed typical ZnO miller indexation planes confirming the crystallinity of the
synthesised nanoparticles (Fig.2c). These results correlate with work reported by
Matinise et al [17] where the structure of ZnO nanoparticles exhibited hexagonal
wurtzite structure. The particle size distribution of the ZnO nanoparticles ranged
between 5-25 nm as shown in Fig 2 (d). The most dominant particle size was 15 nm,
which agrees with reports that showed that these types of material range between 12-
36 nm [2, 4]. The different particle sizes obtained can be attributed to different
parameters during the synthetic procedure which could affect the particle size
distribution of the composite. .

Figure 2: (a-b) TEM at various magnifications (c) SAED and (d) Particle size
distribution of Sutherlandia frutescence ZnO
The SEM results also showed that the ZnO plant exhibited hexagonal wurtzite
structure and this correlated with morphological structures (Fig. 3a). From the EDS
(Fig. 3c), the ZnO plant exhibited two major intense peaks of Zn and O constituting
37.24% and 25.80 % weight percentage respectively. These were also identified as
the major elements using electron mapping. In addition, minimal elemental traces of
Silica and Calcium were also observed. The presence of these elements is attributed
to the phytochemicals and various essential elements from the plant which act as
minerals or nutrients to plants [29,30].

Figure 3: (a) SEM, (c) EDS and mapping (b, d, e, f) analysis of ZnO nanoparticles.
*(b)-Zinc, (d)-Oxygen,(e)- Carbon and (f) -Sulphur
3.3 Photocatalytic activity of Sutherlandia frutescence derived ZnO
nanoparticles
The photocatalytic activity of the ZnO plant towards MB was evaluated within 120 min
reaction time as shown in Fig.4 (a). The intensity in the absorbance declined as the
reaction time increased, indicating that degradation of MB had occurred. This is due to
the fact that when the light source reacts with ZnO nanoparticles, it generates
electrons and holes that eventually form hydroxyl radicals, which are highly oxidative
and thus can degrade the dye to form less harmful products such CO2 and H2O [4,31].
This degradation mechanism is represented in Fig.4 (d) and verified by the reactions
below:

ZnO + hv→ ZnO + e + OH- [6]

-
OH+ OH- +O2 + MB → H2O + CO2 [4]

The maximum photo-degradation efficiency of 45% was reached within 75 min as

demonstrated in Fig 4 (b). The degradation rate was found to be 0.024 min-1 with
28.88 min half-life. It was observed that the degradation rate between 0 min to 15 min
was faster as shown in Fig 4 (c) whereas the degradation kinetics from 15 min was a
bit slower. This could be due to the fact that the photocatalyst reached saturation at 75
min even though the reaction continued and degraded 40% at 120 min. This differs
with Rathnasamy et al [2] and Rana et al [26] where the irradiation period was longer
and provided complete degradation of the pollutant. This variation could be attributed
to the fact that as time increased, there was interaction of MB with the ZnO active
surface thereby concealing the ZnO surface from light excitation leading to low
concentration of OH radicals responsible for complete mineralisation of MB. In
addition, this could be the effect of the embedded phytochemicals from Sutherlandia
frutescence, which reduced the optical activity while enhancing the biological activity
of the ZnO particles. The biomolecules might have affected the surface area of the
material, forcing the material to emit low radiant energy. This lead to a low reflectance
and thus affecting the photocatalytic activity of ZnO plant nanoparticles.
Figure 4: (a) UV-Vis absorption spectra of photocatalytic degradation of MB using ZnO
nanoparticle, (b) percentage degradation of MB, (c) the kinetic rate constant and (d)
the schematic image of the photocatalytic process of ZnO NPs.

3.4 Antibacterial effect of Sutherlandia frutescence ZnO particles on various

strains using model pollutants
Four bacterial strains (E. coli; S. aureas; E. faecalis and P. aeruginosa) were treated
with ZnO nanoparticles at different concentrations of 0.005, 0.01, 0.02, and 0.05
mg/mL (Fig. 5). The results showed that ZnO plant exhibited a high bacterial growth
reduction suggesting a high potency against all the bacterial strains. This was more
evident against gram-negative bacteria such as P. aeruginosa and E-coli. This could
be due to the biomolecules obtained from Sutherlandia frutescens that have
previously been reported to possess a highly bactericidal effect against gram negative
strains [18, 20]. Furthermore, the narrow particle size distribution also influenced the
activity of the ZnO nanomaterial [10].
Figure 5: Antibacterial activity of (a) ZnO pure and (b) ZnO plant against various
bacterial strains

In contrast, the ZnO pure nanoparticles were found to be dosage dependent on E.

faecalis (Fig. 5 (a)). The higher the concentration, the lower the bacterial growth. The
bacterial activity for the other three strains were inactive, indicating that ZnO pure
possess less bactericidal activity as compared to ZnO plant nanoparticles. In Fig. 5(b)
the ZnO plant was more effective at the lowest concentration of 0.005 mg/mL against
E.coli and thereafter it appeared to have reached its optimum. The effect of ZnO plant
nanoparticles against the S. aureas strain was found to be dosage dependent. Upon
increasing the concentration of the ZnO plant nanoparticles, the bacterial growth was
further decreased. Lastly, against E. faecalis, there was no bacterial inhibition after the
0.01mg/ml treatment. This is due to the fact that E. faecalis is a gram positive strain
and thus resistant due to the formation of spores which allows the bacterial strains to
survive under extreme conditions [32–35]. The phytochemicals from the plant played a
major role on the enhancement of the bacterial properties of ZnO plant. (Fig 5 b).

3.5 Antimicrobial activity of Sutherlandia frutescence ZnO particles against real

water pollutants
Real water samples were investigated from four different sources, namely; pond, river,
tap and sewage. Fig. 6 (a) represents the bacterial counts after inoculating against E.
coli, E. faecallis and the aerobic count before treatment. The E. faecallis exhibited and
68 000 bacterial counts in pond and sewage water respectively. The aerobic counts
for all the strains before the treatment were found to be 490 000, 580 000, 7 300, 11
000 for pond, sewage, river and tap water, respectively. After treatment, all the
selected bacterias (E. coli and E. faecalis) were completely inhibited to 0% bacterial
growth, which could be due to the enhancement of ZnO nanomaterial from the
obtained phytochemicals. However, the aerobic counts increased to 34 000 and 91
000 for both the tap and river water, respectively. This could be due to some of the
elements contained in the ZnO plant, which could serve as nutrients for unidentified
bacterial strains, thus, enhancing their growth instead of inhibiting it.

Figure 6: Real water treatment against bacterial strains using ZnO nanoparticle

A detailed ZnO nanoparticle mechanism towards cell membrane damage remains

unclear but several authors have come up with proposed mechanisms. Mohanraj et al
[36] and Kaviyarasu et al. [29] suggested that when ZnO nanoparticles come into
contact with bacterial strains during treatment, they start by attacking the cell
membrane and penetrate the bacteria and continue to attack the enzymes and DNA
which damages the cytoplasmic membranes leading to cell death [37]. In this study,
we also believe that this is the mechanism that resulted in bacterial growth inhibition
(Fig. 7).The bactericidal property of ZnO nanoparticles is influenced by the surface
area, stability of the material as well as the solubility in growth medium, which provides
retention time between the bacteria and the nanoparticles [34]. The surface area
produces free radicals when interacting with bacteria and disrupts the intercellular
contents. Thus, when the particle sizes are small, the surface area tends to be large,
therefore, assisting with the interaction between the nanoparticles and the bacteria.
The secondary phytochemicals then act as capping agents and inhibits the growth of
microorganism by prohibiting the exotoxins in bacteria [36] and thus enhancing the
bacteriostatic property of ZnO nanoparticles.

Figure 7: Schematic representation of mechanism of action for ZnO nanoparticles

3.6 Anticancer effect of Sutherlandia frutescence ZnO against A549 lung

cancer cell lines
The in-vitro cytotoxic effect of ZnO plant nanoparticles was evaluated against the
human lung cancer lines, A549 cell lines. Our results showed that the activity of the
ZnO plant nanoparticles was concentration dependent. As shown in Fig. 8, the
exposure of the A549 cells to Sutherlandia frutescence-derived ZnO nanoparticles at
different concentrations (62.5 µg/ml, 125 µg/ml, 250 µg/mL and 500 µg/mL) reduced
the cell viability after 24 hours. At lower concentrations (62.5 µg/mL and 125 µg/mL),
there was a decrease in cells of more than 50%, while at higher concentrations (250
µg/mL-1000 µg/mL) there was a drastic decrease in cell viability to 25%, 15% and 7%
respectively. The treatment with pure ZnO, the viability was slightly reduced after the
first treatments (61.5 ug/ml – 250 ug/ml). Thereafter, with the 500 μg/ml treatment
there was a 50% reduction in the viability. These results indicate that indeed the
phytochemicals from the Sutherlandia Frutescence enhanced the biomedical
properties of ZnO nanoparticles. Furthermore, they concur with results reported by
Vijayakumar et al [33] and Hanley et al [38] where ZnO nanoparticles reduced cell
proliferation of cancer cells which lead to a decrease of the viability of the cells.

Figure 8: Cell viability of (a) ZnO pure and (b) ZnO plant nanoparticles against human
lung cancer cell (A549)

The viability profiles of ZnO plant (Fig. 9) clearly show that the cell death observed
were dose-dependent. A maximum cell death obtained was 93.4 % at the 1000 µg/mL
concentration. To understand the mode of cell death, morphological analysis was also
conducted.
 Figure 9: Viability profile of A549 under ZnO nanoparticle as treatment

3.7 Morphological characterisation of A549 before and after treatment with ZnO
nanoparticles at different concentrations
The effect of the ZnO plant nanoparticles on A549 cell was further confirmed using the
fluorescence microscopy. As shown in Fig. 10, morphological epithelial characteristics
of the A549 cells were maintained in the untreated control cells [Fig 10 (a)], pointed in
white arrows while minimum normal cell death, apoptosis [39,40](red arrows) was also
observed. The number of the cells undergoing apoptosis increased as the
concentration of ZnO plant increased (Fig. 10 b-f). These nanoparticles induced one of
the common morphological characteristics apoptosis, which is nuclear condensation
and cell shrinkage. The cell numbers also decreased as the concentration of the ZnO
plant nanoparticles increased. This increased activity is due to the presence of
flavonols and canavine which possess highly effective antiproliferative activity against
cancer cells [40].In principle, the nanomaterials with particle size smaller than 25 nm
exhibit better biological properties. Hence, the 15 nm particle size of ZnO plant
nanoparticles obtained played an important role in enhancing the biological activities.
This further confirms that these green synthesized ZnO nanoparticles are suitable
bionanomaterial for use in various biomedical applications.
Fig. 10: ZnO treatment against A549 cells taken at different concentrations:
(a) untreated,(b) 62.5 µg/mL, (c) 125 µg/mL, (d) 250 µg/mL, (e) 500 µg/mL and
(f) 1000 µg/mL.
Conclusion
The ZnO nanoparticles consisting of hexagonal wurzite structure were successfully
synthesised with the extraction from Sutherlandia frutescens plant. The ZnO plant
showed photocatalytic activity towards MB leading to optimum photodegradation of
45% and 0.024 min-1reaction rate within 75 min reaction time. Furthermore, the
Sutherlandia frutescence synthesized ZnO nanoparticles exhibited bactericidal effects
against the different strains and against microorganisms from the real water samples.
An enhanced antiproliferative effect against A549 lung cancer cell line was observed.
This shows the ZnO nanoparticles derived from the Sutherlandia frutescence plant
extract can be used in various medical applications..

Acknowledgements
The authors would like to thank the DST/NRF Centre of Excellence in Strong materials
for funding and the Department of Chemistry at the University of Limpopo for
Research facilities.

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Graphical Abstract