# Exploiting in vitro potential and characterization of surface modified zinc oxide nanoparticles of isodon rugosus extract: their clinical potential towards hepg2 cell line and human pathogenic bacteria

Aisha Siddiquah, Syed Salman Hashmi, Sadaf Mushtaq, Sullivan Renouard, Jean Philippe Blondeau, Rashda Abbasi, Christophe Hano, Bilal Haider Abbasi

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Aisha Siddiquah, Syed Salman Hashmi, Sadaf Mushtaq, Sullivan Renouard, Jean Philippe Blondeau, et al.. Exploiting in vitro potential and characterization of surface modified zinc oxide nanoparticles of isodon rugosus extract: their clinical potential towards hepg2 cell line and human pathogenic bacteria. EXCLI Journal, 2018, 17, pp.671 - 687. 10.17179/excli2018-1327. hal-02622821

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Original article:

# EXPLOITING *IN VITRO* POTENTIAL AND CHARACTERIZATION OF SURFACE MODIFIED ZINC OXIDE NANOPARTICLES OF *ISODON RUGOSUS* EXTRACT: THEIR CLINICAL POTENTIAL TOWARDS HEPG2 CELL LINE AND HUMAN PATHOGENIC BACTERIA

Aisha Siddiquah¹, Syed Salman Hashmi¹, Sadaf Mushtaq¹, Sullivan Renouard²,
Jean Philippe Blondeau³, Rashda Abbasi⁴, Christophe Hano², Bilal Haider Abbasi¹,²*

¹ Department of Biotechnology, Quaid-i-Azam University, Islamabad-45320, Pakistan

² Laboratoire de Biologie des Ligneux et des Grandes Cultures (LBLGC), UPRES EA 1207, Université d'Orléans, Chartres, France

³ Conditions Extrêmes et Matériaux: Haute Température et Irradiation (CEMHTI) CNRS UPR3079, 1D avenue de la Recherche Scientifique, 45071 Orléans, France

⁴ Institute of Biomedical & Genetic Engineering (IBGE), Sector G-9/1, Islamabad, Pakistan

* Corresponding author: Bilal Haider Abbasi, Tel & Fax: 0092-51-90644121, Mobile: 0033-767-97-0619, E-mail: bhabbasi@qau.edu.pk

http://dx.doi.org/10.17179/excli2018-1327

This is an Open Access article distributed under the terms of the Creative Commons Attribution License
(http://creativecommons.org/licenses/by/4.0/).

ABSTRACT

Little is known about biogenically synthesized Zinc oxide nanoparticles (ZnONPs) from *Isodon rugosus*. Synthesis of metal oxide NPs from essential oil producing medicinal plants results in less harmful side effects to the human population as compared to chemically synthesized NPs. In this article, we report biogenic synthesis of ZnONPs from *in vitro* derived plantlets and thidiazuron (TDZ) induced callus culture of *Isodon rugosus*. Synthesized NPs were characterized using UV-spectra, XRD, FTIR, SEM and EDX. Furthermore, the NPs were evaluated for their potential cytotoxic (against HepG2 cell line) and antimicrobial (against drug resistant *Staphylococcus epidermidis*, *Bacillus subtilis*, *Klebsiella pneumoniae* and *Pseudomonas aeruginosa*) activities. Pure crystalline ZnONPs with hexagonal and triangular shapes were obtained as a result of callus extract (CE) and whole plant extract (WPE), respectively. ZnONPs showed potent cytotoxic and antimicrobial potential. The antimicrobial and cytotoxic activities of ZnONPs were found to be shape and surface bound phytochemicals dependent. CE mediated hexagonal ZnONPs showed superior anti-cancer and antimicrobial activities as compared to WPE mediated triangular shaped ZnONPs. It is concluded that biogenic ZnONPs have incredible potential as theranostic agents and can be adopted as useful drug delivery system in next generation treatment strategies.

**Keywords:** Nanotoxicity, ZnONP, pH, surface adaptation, characterization, *Isodon rugosus*

**Abbreviations:** ZnONPs: Zinc oxide nanoparticles, CE: Callus extract, WPE: Whole plant extract, C-ZnONPs: Callus derived zinc oxide nanoparticles, W-ZnONPs: Whole plant derived zinc oxide nanoparticles, MS: Murashige and Skoog medium, TDZ: Thidiazuron, TPC: Total phenolic content, TFC: Total flavonoid content, DW: Dry weight, PGRs: Plant growth regulators, UV-vis: Ultra violet-visible, FTIR: Fourier-transform infrared, XRD: X- ray diffraction, SEM: Scanning electron microscopy, EDX: Energy dispersive X-rays, HepG2: Human hepatocellular carcinoma cell line, DMEM: Dulbecco's Modified Eagle's medium, SRB: Sulforhodamine B, TCA: Trichloroacetic acid, ROS: Reactive oxygen species, DMSO: Dimethyl sulfoxide, CFU: Colony forming unit
INTRODUCTION

In recent years, chemotherapy, radiation and surgery are among the main strategies to treat cancer. The problem with such strategies is they are highly uneconomical. Production of nanomedicine through green route is an economic alternative to the aforementioned strategies (Gowda et al., 2014). Plant extracts have the potential to produce nanoparticles (NPs) having specific size, shape, and composition. Green nano-biotechnology utilizes the reducing and stabilizing agents present in the plant extracts through an alternative bottom-up approach that results in formulation of stable and biocompatible NPs (Parveen et al., 2016). Bio-synthesis of NPs follows three main phases: selection of suitable solvent, a benign and eco-friendly reducing agent and nontoxic capping agent. Alteration in the aforementioned agents results in controlled synthesis for preparation of NPs with specific morphological features. Controlled synthesis of zinc oxide (ZnO) NPs with unique features like selectivity, cell imaging, bio-sensing, drug/gene delivery and enhanced cytotoxicity may serve as an alternate anticancer strategy (Lewinski et al., 2008; Sur et al., 2010). ZnONPs have been categorized as “GRAS” (generally recognized as safe) substance by FDA owing to their biocompatible nature, thus, making them applicable for drug delivery (Jamdagni et al., 2016). ZnONPs possess properties of semiconductors due to their components (Zn and O) which belong to d-block (2nd group) and p-block (6th group) of the periodic table, respectively. The logic for this is that ZnONPs have high band gap (3.37 eV) and exciton binding energy (60 meV) (Neumark et al., 2007). Therefore, it can tolerate detectable electric fields, adequate temperatures, and high power activities.

The efficacy of ZnONPs has been evaluated both independently and in complexes with organic compounds. Conjunction of NPs with cells biologically activates the cellular proteins to minimize the toxicity (Abbasi et al., 2017; Riaz et al., 2018; Balaji and Gothandam, 2016; Huang et al., 2007). The

generation of cellular reactive oxygen species (OH*, O₂*, HO₂*) from the surface of ZnONPs is believed to be a major component of its cytotoxicity, yet the mechanism of cytotoxicity is not fully understood. Overproduction of these highly active free radicals has been shown to instigate oxidative stress in macrophages which in turn elicit apoptosis due to insufficient anti-oxidative protective mechanism (Willet, 2002).

Previous studies investigated the *in vitro* cytotoxicity of ZnONPs by exploiting variety of mammalian cell lines (Xia et al., 2008; Yuan et al., 2010; Heng et al., 2011). Moreover, the impact of shape and size of ZnONPs on their cytotoxic potential has also been explored. Nagarajan and Kuppusamy (2013) studied ZnONPs with different shapes (hexagonal and triangular) regardless of their specific surface area. Practically, variations in capping agents (secondary metabolites) bound to the surface of NPs, influence their physiochemical action within cell culture media resulting in different levels of cytotoxicity (Bogutska et al., 2013). Alternatively, surface charge ratio of NPs also play considerable role in cytotoxicity, as positively charged NPs were shown to possess a higher cytotoxicity. Outer layer of the cancer cell holds negatively charged phospholipids (Abercrombie and Ambrose, 1962). The electrostatic interaction between positively charged ZnONPs and negatively charged phospholipids on cell surface affects cellular uptake and intracellular localization of ZnONPs (Rasmussen et al., 2010; Sultana et al., 2017).

Hepatocellular carcinoma (HCC) is the fifth most common malignant tumor, with a poor survival rate of 10% (overall 5 years). HepG2 (Human hepatocellular carcinoma) cell line has been used extensively to study HCC and drug metabolism due to its phenotypic stability (Hanley et al., 2008). Shelf-life of conventional single drug moiety is usually limited to few minutes and requires repeated injections (Wang et al., 2009). However, capping of NPs strengthen shelf-life of the drugs up to several hours, thus in-
creasing their availability to target cancer
cells. The objective of the current study was
to assess the *in vitro* cytotoxic potency of bi-
ologically synthesized hexagonal and trian-
gular ZnONPs against HepG2 cell line and
drug resistant bacteria.

# MATERIAL AND METHODS

Preparation of Isodon rugosus plant extract

Isodon rugosus seeds, acquired from the Plant Cell Culture Laboratory, Department of Biotechnology, Quaid-e-Azam University, Islamabad, were germinated under sterile conditions. Callus culture was established on MS medium (Murashige and Skoog, 1962) and maintained in growth room at 25±2 °C for 16/8 hrs (light/dark) photoperiod. After 40 days, explants fragments were shifted to MS medium supplied with various concentration of TDZ (1, 2, 3, 4, 5 mg/l) for callus induction (Figure 1A). Extract of Callus (CE) and *in vitro* derived whole plant (WPE) was prepared by adding 10 g of freshly har-

vested callus and plant sample separately in-
to 500ml flask. 100 ml of distilled water was
added to each flask and boiled. After over-
night incubation at 40 °C, the broth was fil-
tered carefully. These extracts were further
used for the bio-reduction of Zn⁺ to Zn²⁺.

Biological synthesis of ZnONPs

Synthesis of NPs depends on type and
concentration of plant extract, metallic salt,
response time, pH and temperature (Dwivedi
and Gopal, 2011). Zinc Acetate Dihydrate
(C4H6O4Zn) was used as a precursor solvent.
0.02 M solution of zinc acetate (50 ml) was
mixed with 1 ml sample extract separately
(1:50) and was stirred constantly. pH of the
medium was maintained at 12 by adding 2M
Sodium Hydroxide (NaOH) drop-wise. The
reduction was attributed to phenolics, terpe-
noids, polysaccharides and flavones present
in the extract. White and pale precipitates
were seen for CE and WPE, respectively.
The resulting precipitates were washed three

<figure><img src="image_2.png" /><figcaption>**Figure 1:** (A) Thidiazuron induced callus from stem explant, (B) Estimation of phenolic and flavonoid content in callus extract, (C) UV-Vis spectral analysis of ZnONPs, (D) Optical observation of biologically synthesized ZnONPs using *Isodon rugosus* extract, (à) Thidiazuron induced callus extract, (b') reaction mixture of callus derived ZnONPs, (ć) plant extract, (é) reaction mixture of plant derived ZnONPs. After completion of the reaction, presence of white precipitate indicates the synthesis of ZnONPs.</figcaption></figure>
times (first with ethanol and then twice with
distilled water). Samples were centrifuged at
6000 rpm for 15 min and incubated over-
night at 60 °C for drying. The powdered
samples of ZnONPs obtained after drying
were stored until further use.

Detection of phytochemicals

The Folin–Ciocalteu reagent (FCR) method, also called the gallic acid equivalence method (GAE), was used to measure the total phenolic content in CE and WPE (Singleton et al., 1999). This reagent does not only measure phenolic content but is also used to measure any reducing substance in the sample extract. Folin's reagent assist to detect amine and sulfur containing compounds, which are mainly involved in reduction and stabilization of NPs. In brief, 20 µl of sample extract (CE and WPE) was mixed with 90 µl of FC reagent. After 5 min of incubation, sodium carbonate (Na₂CO₃, 6 % w/v) was added. Absorbance was measured (in triplicates) at 725 nm (Huang et al., 2007).

To measure the level of flavonoids in CE
and WPE, we followed the Willet's method
of estimation with some alteration. 20 µl ali-
quot of sample extract was mixed with 10 µl
of potassium acetate (1 M) and 10 µl of 10 %
Aluminium chloride (AlCl3). Samples were
incubated for 30 min, followed by addition
of 160 µl distilled water. Absorbance was
detected at 415 nm (Thermo Fisher Scien-
tific, model 4001/4). Quercetin was used to
make the calibration curve. Calculation of
TFC was carried out in triplicate (Willet,
2002).

Structural and optical characterization of
ZnO NPs

Characterization of the synthesized NPs
for their configuration, diameter and surface
modification was performed by XRD, EDX
and SEM analysis. Optical properties of
ZnONPs were characterized through HALO
DB-20 UV-absorption spectra with the
wavelength range of 300-700 nm. Samples
were sonicated for 30 min and the aqueous

solution was subsequently evaluated at room
temperature for the optical band gap. Crys-
talline nature of powdered samples was
evaluated by X-ray diffraction analysis
(Shimadzu-6000), operating at 30 kV and 40
mA. Elemental compositions and size of the
synthesized NPs was characterized using
SEM (SIGMA model, MIRA3 TESCAN)
operated at an accelerating voltage of 10 kV
and EDX detector. All the apprehensible
functional groups involved in capping and
reduction of ZnO NPs were identified using
FTIR (Bruker V70).

In vitro cytotoxicity screening against
HepG2 cell line

Cell culture

Human hepatocellular carcinoma cells (ATCC HB-8065) were cultured in DMEM containing 10% Fetal calf serum (FCS), supplemented with 2 mM L-glutamine, 1 mM Na-pyruvate, 100 U/mL penicillin, 100 µg/mL streptomycin at 37 °C in a humidified 5 % CO₂ atmosphere. Cell harvesting was done with 0.5 mM trypsin/EDTA at room temperature for 1 min.

Cell viability assay

ZnONPs as well as CE and WPE were
evaluated for their cytotoxic activity against
HepG2 cell line through SRB assay as previ-
ously described by Arooj et al. (2015). Na-
noparticles were suspended in deionized wa-
ter and extracts were dissolved in DMSO for
cytotoxicity screening. HepG2 cells (> 90 %
confluency) were seeded in a 96-well plate at
a density of 12000 cells/well and allowed to
adhere for 24 hrs at 37 °C. Subsequently,
cells were treated with 200 µg/ml NPs as
well as callus and plant extract for 24 hrs.
Cells were fixed by adding 50 % pre-chilled
TCA and incubated at 4 °C for 1 hr, followed
by rinsing with deionized water thrice. The
resulting plates were then air dried and cells
were stained with 0.01 % SRB dye followed
by incubation for 30 min at room tempera-
ture and washing with 1% acetic acid for the
removal of unbounded dye. Untreated cells
and DMSO were used as negative and doxo-
rubicin (34 µM) was used as positive control
in the experiment. Blanks representing background optical density consisted of sample only and media only controls. Photographs were taken using Olympus CK2 light microscope equipped with digital camera. Experiment was repeated twice with triplicates for each sample. SRB dye was solubilized by adding 100 µl of 10 mM Tris (pH 8) into each well at room temperature for 5 min. Absorbance values were analysed at 565 nm wavelength using Microplate reader (Platos R 496, AMP) (Supplementary Table 1). Percentage viability relative to untreated sample was calculated by using the following formula:

$$ \text{Cell viability (\%)} = \frac{\text{Absorbance of sample} - \text{Absorbance of sample control}}{\text{Absorbance of untreated cells} - \text{Absorbance of media only}} \times 100 $$

Percentage inhibition was calculated by formula:

$$ \text{Cell inhibition (\%)} = 100 - \text{Cell viability (\%)} $$

### Microbial susceptibility test

Investigation of four multiple drug resistant bacterial strains i.e., *Bacillus subtilis* (ATCC 6051), *Pseudomonas aeruginosa* (ATCC 9027), *Klebsiella pneumoniae* (ATCC 70068) and *Staphylococcus epidermidis* (ATCC 12228) was carried out for biological activities of ZnONPs. Microbial susceptibility testing was performed by agar disc diffusion technique. Bacterial broth was evenly dispensed on nutrient agar plate at density of $1 \times 10^8$ CFU/ml, previously refreshed in shaking incubator at 37 °C, 200 rpm in nutrient agar broth (Oxoid-CM0003). Zinc Acetate Dihydrate (2 mM) and Amoxicillin (standard antibiotic) (10 µg/ml) were added in the experiment as negative and positive controls respectively. Samples were dissolved in deionized water prior to experiment. Each sterile filter disc placed on culture plate, was loaded with 10 mg/ml of sample and incubated for 24 hrs at 37 °C. Zones of inhibition (mm) were measured afterwards.

Antibacterial assay was performed in triplicates.

### Statistical analysis

All statistical measurements were performed in triplicates and analysed by Pareto analysis of variance (ANOVA) with Duncan's multiple range test (Duncan, 1955). Values in text and in figures were statistically analysed as mean ± SD (standard deviation). All the graphical analyses were done by using OriginPro software. The probability was considered to be significantly different at the level of P< 0.05 (95 %).

## RESULTS AND DISCUSSION

### Phytochemical investigation

A number of primary and secondary metabolites (phytochemicals) have been reported to perform primal role in the reduction of metal ions to NPs. The selection of plants for the purpose of NPs biosynthesis is mostly based on their capacity to produce important secondary metabolites that plays their respective role as capping and reducing agents. These capping and reducing agents stabilize the NPs, thus helping them to avoid agglomeration. Ponarulselvam et al. (2012) selected *Catharanthus roseus* for the evaluation of vincristine and vinblastine as phyto-reducing agents. Both were selected due to their potential nematicidal activities. Similarly, Akhtar et al. (2016), reported that plectranthoic acid inhibits proliferation of prostate cancer cells by the activation of 5'AMP-activated kinase (AMPK). Present study reveals the maximum production of TPC (11.3 mg g⁻¹ DW) and TFC (0.54 mg g⁻¹ DW) when treated with TDZ (2.0 mgL⁻¹) in case of CE. The TPC and TFC for WPE were 6.1 mg and 0.5 mg g⁻¹ DW, respectively (Figure 1B). Presence of sufficient amount of TPC and TFC shows that *Isodon rugosus* can prove efficient in ZnONPs biosynthesis.

### UV-Vis analysis of ZnO NPs

Optical characterization of the ZnONPs was carried out using UV-Vis absorption spectrophotometer in the range of 300-700
nm. Figure 1C shows characteristic spectra
of ZnONPs synthesized using CE and WPE
of *Isodon rugosus*. Dispersed ZnONPs ab-
sorbed the radiations approximately at 362
nm in case of CE mediated ZnONPs (C-
ZnONPs) and 382 nm in case of WPE medi-
ated ZnONPs (W-ZnONPs). Narrowing of
the peaks confirms the reduction of zinc ace-
tate into ZnONPs. Indramahalakshmi (2017)
and Jamdagni et al. (2016) also reported sim-
ilar characteristic peaks for ZnONPs. The
band gap energy of C-ZnONPs and W-
ZnONPs was 3.692 keV and 3.691 keV re-
spectively, representing characteristic ab-
sorption peaks of ZnONPs. This may be due
to the presence of different fractions of sec-
ondary metabolites in both CE and WPE,
which in turn resulted in shape variation in
ZnONPs. Similar findings were previously
reported by Manokari et al. (2016).

XRD results

X-ray diffraction determines phase purity
(crystalline nature), structural properties,
thickness of thin films and the atomic ar-
rangement of amorphous materials. In XRD,

X-rays penetration provide information
about overall structure of the material under
observation. Diffraction pattern given in
Figure 2A and 2B shows several peaks in the
range of 30-80° which corresponds to 100,
002, 101, 102, 110, 103, 200, 112, 201, 004
and 202 crystal planes, respectively. The da-
ta revealed crystalline hexagonal structure of
ZnONPs.

SEM and EDX results

SEM images of the biosynthesized
ZnONPs are shown in Figure 2 (C, D). It is
observed that alkaline pH results in shape-
dependent synthesis of ZnONPs. Higher pH
results in availability of large number of
functional groups for capping and stabiliza-
tion of NPs. The exact mechanism, however,
is difficult to grasp due to involvement of
large amounts of diverse phyto-reducing me-
tabolites. Current study revealed that C-
ZnONPs were irregular hexagonal shaped
(Figure 2C) while W-ZnONPs were roughly
triangular (Figure 2D). The stability and
morphology of ZnONPs were increased by
the combined effect of pH and reducing

<figure><img src="image_3.png" /><figcaption>**Figure 2:** XRD and SEM analysis of ZnONPs where (A) XRD image of C-ZnONPs, (B) XRD image of W-ZnONPs, (C) SEM image of C-ZnONPs and (D) SEM image of W-ZnONPs</figcaption></figure>

**Abbreviations:** C-ZnONPs: callus derived naoparticles, W-ZnONPs whole plant derived nanoparticles
agents (primary and secondary metabolites of samples extract). Previous findings suggested that the adjustment of pH was compulsory for the stability of ZnONPs. The current findings are well supported by the findings of Song et al. (2009) and Amin et al. (2012). Ghodake et al. (2010) reported similar findings that alkaline pH is responsible for hexagonal and triangular shaped NPs. Altering pH results in modification of the surface charge upon the secondary metabolites which alternatively affects their binding capacity and reduction potential of metal ions during the synthesis of NPs (Kumar and

Yadav, 2008; Pereira et al., 2015). Sathishkumar et al. (2010) confirmed that alkaline pH plays a major role in shape and size variations of NPs. Additionally, alterations in shape and size of NPs may also occur due to other parameters like reaction time, salt concentration, localization of NPs, and change in functional molecules.

The chemical composition of ZnONPs was confirmed by EDX-ray spectrum. The EDX profile exhibits strong signals of zinc and oxygen in both C-ZnONPs and W-ZnONPs (Figure 3A, B), (Table 1).

<figure><img src="image_4.png" /><figcaption>**Figure 3:** EDX analysis ZnONPs showing the purity of zinc and oxygen ions in the samples (A) C-ZnONPs, (B) W-ZnONPs</figcaption></figure>

<table><caption>**Table 1:** Atomic percentage of zinc and oxygen in ZnONPs as obtained from EDX analysis</caption><caption>EDX analysis of CE and WPE</caption><thead><tr><th>Element</th><th>Weight</th><th>Atomic %</th></tr></thead><tbody><tr><td>Hexagonal ZnO NPS</td><td></td><td></td></tr><tr><td>OK</td><td>24.41</td><td>56.89</td></tr><tr><td>Zn K</td><td>75.59</td><td>43.11</td></tr><tr><td colspan="3">Peaks possibly omitted : 0.275,1.745,3.692 keV</td></tr><tr><td>Triangular ZnO NPs</td><td></td><td></td></tr><tr><td>OK</td><td>21.89</td><td>53.37</td></tr><tr><td>Zn K</td><td>78.11</td><td>46.63</td></tr><tr><td colspan="3">Peaks possibly omitted : 0.274,1.739, 3.691 keV</td></tr><tr><td>Total</td><td>100</td><td></td></tr></tbody></table>
FTIR analysis

The FTIR spectra of ZnONPs (CE/WPE)
was observed in the range of wave-numbers
500-3500 cm⁻¹ to identify the functional
groups involved in the reduction and cap-
ping/stabilization of the synthesized NPs
(Figure 4). FTIR analysis confirmed the
presence of Zn-O bond in both C-ZnONPs
and W-ZnONPs due to the presence of peaks
at 893.71 cm⁻¹ and 886.04 cm⁻¹, respectively
(Pinto and Nazareth, 2016). ZnONPs showed
broad bands at 3375.54 cm⁻¹ (C-ZnONPs)
and 3377.88 cm⁻¹ (W-ZnONPs) which was
attributed to the stretching vibrations of hy-
drogen bond (O-H) as reported by Thiruma-
valavan et al. (2013). Spectra at 1216.79
cm⁻¹ (C-ZnONPs) and 1216.81 cm⁻¹ (W-

ZnONPs) confirm C-N stretch bond of ali-
phatic amines. Peaks at 2360.81 cm⁻¹ (C-
ZnONPs) and 2359.57 cm⁻¹ (W-ZnONPs)
represent asymmetric C=O stretching
(Saikh et al. 2017).

These findings clearly demonstrate the
attachment of primary and secondary metab-
olites to the ZnONPs. Electrostatic forces be-
tween the negatively charged groups of or-
ganic molecules and positively charged zinc
ions are responsible for the binding of these
functional groups. This interaction makes
these NPs a perfect candidate for various ap-
plications such as drug delivery (Wang et al.,
2013) and study of biological interactions
(Song et al., 2009).

<figure><img src="image_5.png" /><figcaption>**Figure 4:** FTIR spectra of powdered samples of (A) C-ZnO NPs, (B) W-ZnO NPs</figcaption></figure>
*In vitro cytotoxicity screening against
HepG2 cell line*

Suforhodamine B assay was used to screen *in vitro* cytotoxicity of extracts and NPs using HepG2 cell line. Ionic strength and pH of NPs depend upon formation of hydroxyl (OH⁻) ions which directly influence the repulsion and attractions of particles (Peng et al., 2017). Cells were treated with 200 µg/ml of ZnONPs (C-ZnONPs and W-ZnONPs), CE and WPE for 24 hours. Our results suggested that CE showed slightly higher cytotoxicity (29.47 %) as compared to WPE (31 %). Morphological changes were also observed in both samples i.e., cells became long and fibrous. On the other hand, cell viability in C-ZnONPs was lower (23.92 %) as compared to W-ZnONPs (35 %). Morphologically, cells became small and round upon treatment of C-ZnONPs while long and fibrous cells were observed in the presence of W-ZnONPs. Najim et al. (2014) also reported morphological changes induced by cytotoxic ZnONPs on different cell lines (U937, SH-SY5Y, differentiated SH-SY5Y, and Hs888Lu). Morphological changes in cells may affect their metastatic process including substrate attachment, migration and invasion (Brandhagen et al., 2013). DMSO (1 %), used as a negative control showed slight cytotoxicity and doxorubicin (positive control) showed high cytotoxicity at 34 µM concentration (Figures 5, 6).

Akhtar et al. (2012) reported cytotoxicity of ZnONPs in HepG2 cells due to production of reactive oxygen species. In spite of unique physical and chemical properties of metallic NPs, quantum size and shape also assist in the field of therapeutics (Khan et al., 2017). In present study, the surface charge ratio of ZnONPs was pH dependent to absorb various chemical clusters (-ZnOH²⁺, -ZnOH, -ZnO⁻). In this regard, it was hypothesized that surface orientation of ZnONPs allows

them to conjugate with protein targets or
chemical groups. Therefore, ZnONPs pos-
sess cytotoxicity towards cancer cells (Fig-
ures 5, 6). Literature suggests formation of
ROS as leading cause of cell death by apop-
tosis (Figure 7).

Our present findings suggest configura-
tional role of ZnONPs towards cytotoxicity.
It was established that callus derived hexag-
onal NPs (C-ZnONPs) showed high cell in-
hibition (% viability 23.92 %) as compared
to whole plant derived triangular NPs (W-
ZnONPs) in which cell viability was 35 %
(Supplementary Table 2). From FTIR and
SEM data, we concluded that this may be
due to difference in surface charge of sec-
ondary metabolites which eventually control
the bonding of NPs with cancer cells. Suresh
et al. (2018) supported similar results. They
reported that biologically synthesized hexag-
onal ZnONPs exhibit good cell inhibition
and viability against Daltons Lymphoma As-
cites (DLA).

Also, *in vitro* derived callus extract (CE)
showed cell viability of 29.47 % while whole
plant extract (WPE) showed 31 % cell via-
bility (Figure 6). The current findings are in
agreement with the results of Kamalanathan
and Natarajan (2018). They reported that po-
tency of callus was better than wild leaf ex-
tract sample against human breast cancer cell
line MCF-7. Literature review proved pres-
ence of commercially important anticancer
phenolics and triterpenoids i.e., plectranthoic
acid, limonene, rosmarinic acid, caffeic acid
(Abdel-Mogib et al., 2002; Akhtar et al.,
2016; Hossan et al., 2014; Hafidha et al.,
2018; Krishna et al., 2017). To the best of
our knowledge very little data is available
that compares the cytotoxicity of CE, WPE,
C-ZnONPs and W-ZnONPs against HepG2
cell line.
<figure><img src="image_6.png" /><figcaption>**Figure 5:** Cytotoxic effects of ZnONPs and extracts on HepG2 cells upon 24 hour treatment with 200 µg/ml concentration. Untreated cells, DMSO (solvent) and Doxorubicin (34 µM) were included as controls. Microscopic images of HepG2 cells (treated and untreated). Magnification = 200X, Scale = 100 µm. (A) Callus extracts (CE). (B) Callus derived ZnONPs (C-ZnONPs). (C) Untreated HepG2 cells. (D) Whole plant extracts (WPE). (E) Whole plant derived ZnONPs (W-ZnONPs). (F) DMSO 1% (negative control). (G) Doxorubicin 34 µM (positive control)</figcaption></figure>
<figure><img src="image_7.png" /><figcaption>**Figure 6:** Percentage viabilities and inhibition of cells relative to untreated control (Mean ± SD). Each sample was studied in triplicates and experiment was performed twice.</figcaption></figure>

<figure><img src="image_8.png" /><figcaption>**Figure 7:** Tentative graphical representation of ZnONPs cytotoxicity towards HepG2 cell line. ZnONPs induce cytotoxicity by morphological changes, loss of membrane integrity, cell shrinkage, and reduced cell density which are characteristic features of apoptotic cell formation.</figcaption></figure>
**Effect of ZnONPs on bactericidal activity**

Literature reported the increased emer-
gence of drug resistant bacteria in the clinical
field directs the scientific community to look
for alternatives of antibiotics to minimize the
risk from bacterial infections. Essential oils
produced from *Isodon rugosus* consist of
complex mixture of volatile molecules such
as sesquiterpene, amorphene, germacrene-D,
triterpenoids, plectranthoic acid A and B, ac-
etyl plectranthoic acid and plectranthadiol.
These volatiles compounds potentially ex-
hibit bactericidal property against various
human pathogenic bacteria. Similarly
Hussain et al. (2017) and Faleiro (2011) re-
ported the effectiveness of essential oils
against human pathogenic bacteria. The bac-
tericidal action of essential oils is linked to
its hydrophobicity, resulting in increased cell
permeability and subsequent leakage of cell
constituents. Permeability of ZnONPs and
their capability to bind with the cell mem-
brane generates reactive oxygen species
(ROS), thus enhancing the efficacy against

drug-resistant bacterial strains (Abinaya et
al., 2017). Our results (Figure 8A, B) were in
accordance with the data reported by Prema-
nathan et al. (2011). According to them, the
effects of ZnONPs were more potent against
Gram-positive bacterial strains as compared
to Gram-negative strains. Our evaluation of
potency of ZnONPs also revealed more po-
tent bactericidal activity against Gram-
positive S. epidermidis and B. subtilis as
compared to Gram-negative, K. pneumoniae
and P. aeruginosa, respectively (Table 2).

It was hypothesized that Gram-positive
bacteria possess specific cell wall compo-
nents that help in embedding ZnONPs within
the cellular wall. The presence of ZnONPs
within the walls results in cellular disintegra-
tion and leakage of internal components. In
case of Gram-negative bacteria, no such em-
bedding is observed hence only a small
amount of surface bound ZnONPs causes
partial leakage of internal contents.

<figure><img src="image_9.png" /><figcaption>**Figure 8:** Antibacterial evaluation of Plant derived ZnONPs and extract (A-WPE) and Callus derived (B-CE) ZnONPs and extract analysed by disc diffusion method, showing zone of inhibition against two Gram-positive (*Staphylococcus epidermidis*, *Bacillus subtilis*) and two Gram-negative bacteria (*Klebsiella pneumoniae*, *Pseudomonas aeruginosa*). Zone of inhibition were measured in mm, taking sample concentration 10 mg/mL. Notes: Values (mean ± SD) indicates of three experiments</figcaption></figure>

**Abbreviations:** Abs, amoxicillin antibiotic; WPE, whole plant extract; W-ZnONPs, whole plant derived zinc oxide nanoparticles; CE, callus extract; C-ZnONPs, callus derived ZnONPs; Zn acetate, zinc acetate dihydrate salt; SD, standard deviation
<table><caption>**Table 2:** Antibacterial activity of CE, WPE, C-ZnO NPs and W-ZnO NPs against two Gram-positive (*Staphylococcus epidermidis*, *Bacillus subtilis*) and two Gram-negative (*Klebsiella pneumoniae*, *Pseudomonas aeruginosa*) bacteria</caption><thead><tr><th rowspan="3">Bacteria tested</th><th colspan="6">Zone of inhibition (mm)</th></tr><tr><th rowspan="2">Zn acetate salt (2 mM) (mean ± SD)</th><th colspan="2">Amoxicillin anti-biotic (10 µg/ml) (mean ± SD)</th><th rowspan="2">CE (10 mg/ml) (mean ± SD)</th><th rowspan="2">C-ZnONPs (10 mg/ml) (mean ± SD)</th><th rowspan="2">WPE (10 mg/ml) (mean ± SD)</th><th rowspan="2">W-ZnONPs (10 mg/ml) (mean ± SD)</th></tr><tr><th>CE</th><th>WPE</th></tr></thead><tbody><tr><td colspan="8"><strong>Gram positive</strong></td></tr><tr><td><em>Staphylococcus epidermidis</em></td><td>---</td><td>20 ± 1.21</td><td>21 ± 1.08</td><td>8 ± 0.65</td><td>9 ± 0.98</td><td>9 ± 0.78</td><td>8 ± 0.91</td></tr><tr><td><em>Bacillus subtilis</em></td><td>---</td><td>23 ± 2.28</td><td>22 ± 1.06</td><td>10 ± 1.18</td><td>11 ± 1.58</td><td>8 ± 0.69</td><td>10 ± 1.01</td></tr><tr><td colspan="8"><strong>Gram negative</strong></td></tr><tr><td><em>Klebsiella pneumoniae</em></td><td>---</td><td>20 ± 0.87</td><td>21 ± 0.98</td><td>7 ± 0.57</td><td>9 ± 0.95</td><td>---</td><td>7 ± 0.63</td></tr><tr><td><em>Pseudomonas aeruginosa</em></td><td>---</td><td>18 ± 1.23</td><td>16 ± 0.82</td><td>8 ± 0.22</td><td>10 ± 0.34</td><td>---</td><td>8 ± 0.69</td></tr></tbody></table>

**Abbreviations:** CE, callus extract; C-ZnONPs, callus derived ZnONPs; WPE, whole plant extract; W-ZnONPs, whole plant derived zinc oxide nanoparticles; Zn acetate, zinc acetate dihydrate salt; SD, standard deviation

Our findings endorse the idea of biocidal effect of ZnONPs due to combination of various mechanisms such as ROS production in response to zinc ions within the bacteria. Minor sensitivity of *S. epidermidis*, *B. subtilis* compared to *K. pneumoniae* and *P. aeruginosa* was due to fact that the outer layer of Gram-positive bacteria is tightly packed due to assembly of lipopolysaccharide (LPS) and peptidoglycans. Furthermore, rational estimation of decline in sensitivity may be due to reduced stability of O₂⁻ materialization at the surface and the decrease of pH value in the medium (Tang and Lv, 2014) (Figure 9).

## CONCLUSIONS

Our results conclude that *Isodon rugosus* possesses essential bioactive compounds responsible for therapeutic properties of the plant against microbial and cancer diseases. The FTIR analysis revealed that capping of ZnONPs was due to the phenolics, flavonoids, amino acids and amides that were

collectively involved in stabilizing ZnONPs. SEM and EDX confirmed the hexagonal and triangular shapes of C-ZnONPs and W-ZnONPs, respectively. The hexagonal C-ZnONPs were more effective against Gram-positive bacterial strains as compared to Gram-negative bacterial strains, while triangular W-ZnONPs were found less effective. The biocompatibility, stability and morphology of ZnONPs generally depend upon the pH at which they were synthesized. Cytotoxicity of ZnONPs against HepG2 cell lines was found to be morphology and surface chemistry dependent. Based on enhanced biocompatibility, improved solubility and less toxicity, the efficacy of ZnONPs synthesized from *Isodon rugosus* in the field of medicine could play a significant role in future. In the light of current research, ZnONPs could be developed as a potential next generation cancer treatment strategy. In order to do so, further research must be carried out in order to resolve the issue of cancer, a long-term health hazard.
<figure><img src="image_10.png" /><figcaption>**Figure 9:** Diagrammatic representation of ZnONPs cytotoxicity towards human pathogenic bacterial strains depending upon many factors such as shape (transformation of particles as function of pH), production of ROS, dysfunction of bacterial membrane, effect of oxidative stress on DNA, protein, mitochondria (cell death), electrostatic forces, charge surface area, ultimately causing death of microbes</figcaption></figure>

***Compliance with ethical research standard***

All the experimental work reported in
this article was performed in accordance
with principles of ethical research that com-
plies with all applicable legislation. All the
authors certify that there is no conflict of in-
terest over any content of this article.

REFERENCES

Abbasi BH, Anjum S; Hano C. Differential effects of in vitro cultures of Linum usitatissimum L. (Flax) on biosynthesis, stability, antibacterial and antileishmalarial activites of zinc oxide nanopartilces: a mechanistic approach. RSC Advances. 2017;7:15931.

Abdel-Mogib M, Albar HA, Batterjee SM. Chemistry of the Genus Plectranthus. Molecules. 2002;2:271-301.

Abercrombie M, Ambrose EJ. The surface properties of cancer cells: a review. Cancer Res. 1962;22:525-48.

Abinaya C, Mayandi J, Osborne J, Frost M, Ekstrum C, Pearce JM. Inhibition of growth of S. epidermidis by hydrothermally synthesized ZnO nanoplates. Master Res Express. 2017;4:075401.

Akhtar MJ, Ahamed M, Kumar S, Khan MAM, Sal-
man JA, Alrokayan A. Zinc oxide nanoparticles selec-
tively induce apoptosis in human cancer cells through
reactive oxygen species. Int J Nanomed. 2012;7:845-
57.

Akhtar N, Syed DN, Khan MI, Adhami VM, Mirza B, Mukhtar H. The pentacyclic triterpenoid, plectranthoic acid, a novel activator of AMPK induces apoptotic death in prostate cancer cells. Oncotarget. 2016; 7:3819-31.
Amin M, Anwar F, Janjua MR, Iqbal MA, Rashid U. Green synthesis of silver nanoparticles through reduction with Solanum xanthocarpum L. berry extract: characterization, antimicrobial and urease inhibitory activities against Helicobacter pylori. Int J Mol Sci. 2012;8:9923-41.

Arooj S, Nazir S, Nadhman A, Ahmad N, Bakhtiar M, Ahmad I, et al. Novel ZnO:Ag nanocomposites induce significant oxidative stress in human fibroblast malignant melanoma (Ht144) cells. Beilstein J Nanotechnol. 2015;6:570-82.

Balaji K, Gothandam KM. Cytotoxic effect on cancerous cell lines by biologically synthesized silver nanoparticles. Braz Arch Biol Technol. 2016;59.

Bogutska KI, Sklyarov Yu P, Prylutskyy YuL. Zinc and zinc nanoparticles: biological role and application in biomedicine. Ukrainica Bioorg Acta. 2013;1:9-16.

Brandhagen BN, Tieszen CR, Ulmer TM, Tracy MS, Goyeneche AA, Telleria CM. Cytostasis and morphological changes induced by mifepristone in human metastatic cancer cells involve cytoskeletal filamentous actin reorganization and impairment of cell adhesion dynamics. BMC Cancer. 2013;13:1-15.

Duncan DB. Multiple range and multiple F tests. Biometrics. 1955;11:1-42.

Dwivedi AD, Gopal K. Plant-mediated biosynthesis of silver and gold nanoparticles. J Biomed Nanotechnol. 2011;7:163-4.

Faleiro ML. The mode of antibacterial action of essential oils. In: Méndez-Vilas A (ed.): Science against microbial pathogens: communicating current research and technological advances (pp 1143-56). Badajoz: Formatex Research Center, 2011.

Ghodake GS, Deshpande NG, Lee YP, Jin ES. Pear fruit extract-assisted room-temperature biosynthesis of gold nanoplates. Colloids Surf B Biointerfaces. 2010;75:584-9.

Gowda R, Jones NR, Banerjee S, Robertson GP. Use of nanotechnology to develop multi-drug inhibitors for cancer therapy. J Nanomed Nanotechnol. 2014;4: 6.

Hafidha RR, Husseinb SZ, MalAllahc MQ, Abdulamird SA, Abu Bakar F. A high-throughput quantitative expression analysis of cancer-related genes in human HepG2 cells in response to limonene, a potential anticancer agent. Curr Cancer Drug Targets. 2018; 18:1-9.

Hanley C, Layne J, Punnoose A, Reddy KM, Coombs I, Feris K, et al. Preferential killing of cancer cells and activated human T cells using zinc oxide nanoparticles. Nanotechnology. 2008;19:295103.

Heng BC, Zhao X, Tan EC, Khamis N, Assodani A, Xiong S, et al. Evaluation of the cytotoxic and inflammatory potential of differentially shaped zinc oxide nanoparticles. Arch Toxicol. 2011;85:1517-28.

Hossan MDS, Rahman S, Anwarul Bashar ABM, Jahan R, Abdullah Al-Nahain A, Rahmatullah M. Rosmarinic acid: a review of its anticancer action. J Pharm Pharma Sci. 2014;3:57-70.

Huang X, Jain PK, El-Sayed IH, El-Sayed MA. Gold nanoparticles: interesting optical properties and recent applications in cancer diagnostics and therapy. Nanomedicine (London). 2007;2:681-93.

Hussain I, Khan AU, Ullah R, Alsaid MS, Salman S, Iftikhar S, et al. Chemical composition, antioxidant and anti-bacterial potential of essential oil of medicinal plant Isodon rugosus. J Essent Oil Bear Pl. 2017; 20:1607-13.

Indramahalakshmi G. Characterization and antibacterial activity of zinc oxide nanoparticles synthesized using Opuntia ficus indica fruit aqueous extract. Asian J Phys Chem Sci. 2017;3(2):1-7.

Jamdagni P, Khatri P, Rana JS. Green synthesis of zinc oxide nanoparticles using flower extract of Nycantanthes arbor-tristis and their antifungal activity. KSU Riyadh. 2016;30:168-75.

Kamalanathan D, Natarajan D. Anticancer potential of leaf and leaf-derived callus extracts of Aerva javanica against MCF-7 breast cancer cell line. J Cancer Res Ther. 2018;14:321-7.

Khan I, Saeed K, Khan I. Nanoparticles: Properties, applications and toxicities. Arab J Chem. 2017;epub. https://doi.org/10.1016/j.arabjc.2017.05.011

Krishna PS, Bhaskar D, Sathyamurthy B. In vitro studies on the effect of Citrus limon Linn in neuroblastoma Sh-Sy5y cell lines. J Med Plants Stud. 2017; 5:197-202.

Kumar V, Yadav SK. Plant-mediated synthesis of silver and gold nanoparticles and their applications. J Chem Technol Biotechnol. 2008;84:151-7.

Lewinski N, Colvin V, Drezek R. Cytotoxicity of nanoparticles. Small. 2008;41:26-49.

Manokari M, Shekhawat MS. Zinc oxide nanoparticles synthesis from Moringa oleifera Lam. Extracts and their characterization. World Sci News. 2016;55: 252-62.
Murashige T, Skoog F. A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant. 1962;15:473-97.

Nagarajan S, Kuppusamy KA. Extracellular synthesis of zinc oxide nanoparticle using seaweeds of Gulf of Mannar, India. J Nanobiotechnol. 2013;11:39.

Najim N, Rusdi R, Hamzah AS, Shaameri Z, Zain MM, Kamarulzaman N. Effects of the absorption behaviour of ZnO nanoparticles on cytotoxicity measurements. J Nanomater. 2014;10:694737.

Neumark G, Gong Y, Kuskovsky I. Doping aspects of Zn-based wide-band-gap semiconductors. In: Kasap S, Capper P (eds): Springer handbook of electronic and photonic materials (pp 843–54). New York: Springer, 2007.

Parveen K, Banse V, Ledwani L. Green synthesis of nanoparticles: Their advantages and disadvantages. In: Sharma NN, Gaol FL, Akhtar J (eds.): 2nd International Conference on Emerging Technologies: Micro to Nano 2015 (ETMN-2015) (pp 020048-1-020048-7). College Park, MD: American Institute of Physics. (AIP Conference Proceedings, Vol. 1724).

Peng C, Zhang W, Gao H, Li Y, Tong X, Li K, et al. Behavior and potential impacts of metal-based engineered nanoparticles in aquatic environments. Nanomaterials. 2017;7:21.

Pereira L, Mehboob F, Stams LJ, Mota MM, Rijnaarts HHM, Alve MM. Metallic nanoparticles: microbial synthesis and unique properties for biotechnological applications, bioavailability and biotransformation. Crit Rev Biotechnol. 2015;1:114-28.

Pinto GM, Nazareth R. Green synthesis and characterization of zinc oxide nanoparticles. J Chem Pharm Res. 2016;6:427-32.

Ponarulselvam S, Panneerselvam C, Murugan K, Aartha N, Kalimuthu K, Thangaman S. Synthesis of silver nanoparticles using leaves of Catharanthus roseus Linn G Don and their antiplasmodial activities. Asian Pac J Trop Biomed. 2012;2:574-80.

Premanathan M, Karthikeyan K, Jeyasubramanian K, Manivannan G. Selective toxicity of ZnO nanoparticles toward Gram-positive bacteria and cancer cells by apoptosis through lipid peroxidation. Nanomedicine. 2011;7:84-192.

Rasmussen JW, Martinez E, Louka P, Wingett DG. Zinc oxide nanoparticles for selective destruction of tumor cells and potential for drug delivery applications. Expert Opin Drug Deliv. 2010;9:1063-77.

Riaz R, Hashmi SS, Khan T, Hano C, Giglioli-Guivarc'h N, Abbasi BH. Melatonin stimulated biosynthesis of antimicrobial ZnONPs by enhancing bio-reductive prospective in callus cultures of Catharanthus roseus var. Alba. Artif Cells Nanomed Biotechnol. 2018;epub. https://doi.org/10.1080/21691401.2018.1473413.

Sathishkumar M, Sneha K, Yun YS. Immobilization of silver nanoparticles synthesized using Curcuma longa tuber powder and extract on cotton cloth for bactericidal activity. Bioresour Technol. 2010;20: 7958-65.

Shaikh SS, Saraf MS, Agrawal YK. Green synthesis, characterization of metal antibacterial nanoparticles and removal of fluoride from waste water. Adv Bio-tech Micro. 2017;2(3):555590.

Singleton VL, Orthofer R, Lamuela-Raventos RM. Analysis of total phenols and other oxidation substrates and antioxidants by means of folin-ciocalteu reagent. Methods Enzymol. 1999;299:152-78.

Song JY, Jang HK, Kim BS. Biological synthesis of gold nanoparticles using Magnolia kobus and Diopyros kaki leaf extracts. Process Biochem. 2009;10: 1133-8.

Sultana F, Barman J, Kalita MC. Biogenic synthesis of ZnO nanoparticles using Polygonum chinense leaf extract and their ntibacterial activity. Int J Naut Archaeol (IJNA). 2017;11:155-65.

Sur I, Cam D, Kahraman M, Baysal A, Culha M. Interaction of multi-functional silver nanoparticles with living cells. Nanotechnology. 2010;21:10.

Suresh J, Pradheesh G, Alexramani V, Sundrarajan M, Hong SI. Green synthesis and characterization of zinc oxide nanoparticle using insulin plant (Costus pictus D. Don) and investigation of its antimicrobial as well as anticancer activities. Adv Nat Sci Nanosci Nanotechnol. 2018;9:8.

Tang ZX, Lv BF. MgO nanoparticles as antibacterial agent: preparation and activity. Braz J Chem Eng. 2014;31:591.

Thirumavalavan M, Huang KL, Lee JF. Preparation and morphology studies of nano zinc oxide obtained using native and modified chitosans. Materials. 2013; 9:4198-212.

Wang H, Wingett D, Engelhard MH. Fluorescent dye encapsulated ZnO particles with cellspecific toxicity for potential use in biomedical applications. J Mater Sci Mater Med. 2009;20:11-22.
Wang R, Billone PS, Mullett WM. Nanomedicine in action: an overview of cancer nanomedicine on the market and in clinical trials. J Nanomater. 2013;12: 629681.

Willet WC. Balancing life-style and genomics re-
search for disease prevention. Science. 2002;296:695-
8.

Xia T, Kovochich M, Liong M, Mädler L, Gilbert B, Shi H, et al. Comparison of the mechanism of toxicity of zinc oxide and cerium oxide nanoparticles based on dissolution and oxidative stress properties. ACS Nano. 2008;2:2121-34.

Yuan JH, Chen Y, Zha HX, Song LJ, Li CY, Li JQ, et al. Determination, characterization and cytotoxicity on HELF cells of ZnO nanoparticles. Colloids Surf B Biointerfaces. 2010;76:145-50.