Green Synthesis of Zinc Oxide Nanoparticles (ZnO-

NPs) by Pterolobium hexapetalum (Roth) Santapau

& Wagh Aqueous Leaf Extract and Its Assessment
of Biocompatible, Antibacterial, DPPH Radical
Scavenging, Anticancer and Larvicidal Activities: An
Effective Eco-Friendly Approach
Settu Loganathan 
Sri Sivasubramaniya Nadar College of Engineering
Kumar Manimaran 
Sri Sivasubramaniya Nadar College of Engineering
Karuvelan Mutamimurugan 
Sri Sivasubramaniya Nadar College of Engineering
Dhakshinamoorthy Gnana Prakash 
(

[email protected]
)
Sri Sivasubramaniya Nadar College of Engineering
Rajakannu Subashini 
Sri Sivasubramaniya Nadar College of Engineering

Research Article

Keywords: Green Synthesis, Biocompatibility, Antibacterial Activity, Breast Cancer, Lethal Concentration

Posted Date: July 1st, 2022

DOI: https://doi.org/10.21203/rs.3.rs-1793777/v1

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Abstract
Pterolobium hexapetalum aqueous leaf extract (P. hexapetalum-ALE) was utilized as a reducing agent to
synthesize zinc oxide nanoparticles (ZnO-NPs) and it’s forms of white precipitate. The synthesized P.
hexapetalum - ZnO-NPs (Ph-ZnO-NPs) were characterized by UV-Vis, XRD, IR, EDAX, and HR-TEM with
SAED pattern. UV-visible spectra results showed an absorbance peak at 280 and 370nm for Ph-ALE and
synthesized Ph-ZnO-NPs. The HR-TEM result shows a spherical in shape with a size of 10-93 nm by Ph-
ZnO-NPs. X-ray powder diffraction (P-XRD) spectra confirmed the crystalline nature of the synthesized
ZnO-NPs. Hemolytic activity of the synthesized ZnO-NPs was carried out against Human RBCs and it’s
found to be 0.6% at 100 µg/mL. The synthesized Ph-ZnO-NPs appeared excellent antibacterial and
antioxidant activity against P. aeruginosa (17.8mm) and DPPH radical scavenging activity
(IC50:118.14µg/mL). Ph-ZnO-NPs exposed superior anticancer activity against breast cancer cell line
(MCF-7) with an IC50 value of 22.91 µg/mL. Additionally, the synthesized Ph-ZnO-NPs exhibited an
excellent larvicidal activity with LC50 & LC90 values of 4.178 and 16.157 mg/mL on Cx. quinquefasciatus
larvae at 24h inspection. Thus, plant-based synthesized Ph-ZnO-NPs exhibit significant biological
activities, and hence it can be considered a potential source for the development of innovative drugs.

Introduction
The success of nanotechnology has altered the scope and output of research owing to its assorted
various applications [1]. Nanoparticles have been synthesized by various techniques, like as, physical,
chemical and biological. Biological synthesis (microbes, algae, fungi and plants) gains more importance
because of their it’s less toxic, low cost, safe and simple when compared to other synthesis methods [2].
Metal oxide nanoparticles (MONPs) have been widely utilized for therapeutic purposes in the past many
years. A few of the MONPs such as Fe3O4, TiO2, CuO and ZnO have been explored by different biological
activities. Amid these NPs especially, ZnO-NPs also have been potential applications in the field of
medicine like drug delivery and also antibacterial, antioxidant, anticancer and larvicidal applications. In
recent times, several studies have documented the biosynthesis of ZnO-NPs such Knoxia sumatrensis [3],
Garcinia cambogia [4], Labeo rohita [5], Spondias pinnata [6].

In everyday life, adherence and multiplication activity of bacterial strains cause extremely irresistible
sicknesses among humans as well as animals. The important complication is the improvement of
microbial resistance against commercial antibiotics in the medicinal zone [7]. Subsequently, to overcome
the problem, there is a need for the development of a new drug. Medicinal plants with nanotechnology are
fundamental sources to annihilate the microorganism. In earlier document, the synthesized ZnO-NPs
using Conyza canadensis; Bioflavonoid rutin compound displayed significant antibacterial activity
against human bacterial pathogens [8, 9].

Oxidative pressure was generated when there is an unreasonable free radical formation or low
antioxidant resistance, which prompts a change in biomolecules causing morphology and functional
changes [10]. Antioxidants are very important in the performance of entire bio-systems. Numerous
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medicinal plants with nanotechnology have been explored to find the inventive application as
antioxidants drugs [11].

Information on mosquito involvement in environment and control is of opportune significance to battle

the spread of numerous mosquito-borne diseases [12]. Since mosquito-borne sicknesses now don’t have
effective vaccines or medicinal drugs. The utilizing of synthetic insect sprays such as, diflubenzuron,
organophosphates, organochlorine and carbamates has been considered a valuable technique to
manage insects. Utilization of these insecticides develops resistance in mosquitoes, and they are unsafe
and destructive [13]. Resistance to mosquitoes, the insecticides used against mosquitoes are risky to
other’s lives on the earth, hence there is a necessity to find the eco-friendly, drugs. In the current study, we
explored the synthesis of ZnO-NPs using Pterolobium hexapetalum aqueous leaf extract and characterize
the NPs using various techniques. Furthermore, we examined the, in vitro studies such as hemolytic,
antibacterial, antioxidant, anticancer and larvicidal activities of Ph-ZnO-NPs.

Materials And Methods

Collection and Extraction

Pterolobium hexapetalum leaves were collected in September-2021 from Paali hills, Thoppur, (latitude-
11.941996 and longitude-78.054291), Dharmapuri district, Tamil Nadu, India. The Ph leaves were
cleaned, washed with distilled water (D. H2O) and then dried for fourteen days in room temperature. The
dried leaves were powdered in a sterilized electrical blender. Plant powder (10g) was weighed and added
in 100mL D. H2O and then it was heated for 30mins at 70 ºC. The Whatman No.1 filter paper was used
for extract filtration and stored at 4 ºC by further assessment.

Green Synthesis of ZnO-NPs

The method followed by Vijayakumar et al. [14] with little modification was used in this study. 80 mL of
2M zinc acetate dehydrate solution was added into 20mL of Ph-ALE and then stirred for 3 h at 80 ºC.
After 1 hour the NaOH solution (2 M) was added drop by drop to adjust the pH level at 12. The change in
color and formation of white precipitates, indicate the formation by ZnO-NPs. The white precipitate was
obtained and dried. After, the dried materials were calcinated for muffle furnace (350 °C at 3 h).

Characterization Study

The formation of Ph-ZnO-NPs was confirmed by Ultraviolet visible spectroscopy (LI-UV-7000  wavelength

at 200-800nm). Crystalline nature was analyzed by X-Ray Diffraction (XRD-Rigaku, Japan) and 2 theta
ranges in 20º-80º. Functional groups present in the Ph-ZnO-NPs were identified using Fourier transform
infrared spectroscopy (FT-IR-Perkin Elmer-L1600300, Liantrisant, USA). Further the NPs size and
morphology was analyzed through high-resolution-transmission electron microscopy (HR-TEM-FEI
Tecnai, F30).

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Hemolytic Assay 

Methodology of Rajapriya et al. [15] with slight modification has been used for this study. Blood sample
(1st author) was obtained and citrated to avoid coagulation.  3mL of blood was centrifuged at 1600
rotate per minutes (RPM) for seven minutes. The plasma was discarded and the pellets containing Red
Blood Cells (RBCs) were saved. Further, the RBCs were cleaned with sterile phosphate-buffered saline for
5 times (PBS-6mL, pH-7.4). The RBCs were diluted with PBS solution. The different dosages of Ph-ZnO-
NPs (50 and 100µg/mL) were mixed  with RBCs by vortex. All the test samples were kept in an incubator
at 25 °C for 1h. Triton X 100 (2%) was used as a positive and negative control for PBS.  The test samples
were then centrifuged at 3000 rpm for 5mins and 0.1 µL of supernatant was obtained. The absorbance
was recorded using a UV-Vis Spectrophotometer at 545nm. The hemolysis percentage was calculated as
shown below.

Antibacterial Study

The synthesized Ph-ZnO-NPs were demonstrated of Bacillus cereus, Enterococcus faecalis,

Pseudomonas aeruginosa and Escherichia coli using disk diffusion method of Loo et al. [16]. The
bacterial cultures were swabbed on Petri-plates and then dried for 10 min. The synthesized Ph-ZnO-NPs
dosages of 25 and 50µg were used in this study along with Chloramphenicol used as a positive control
(30μg/disc). The tested plates were incubated for 24h at 37 ºC and the bacterial growth was measured in
millimeters (mm).

Antioxidant Study

DPPH assay of the synthesized Ph-ZnO-NPs were determined as described by Shimada et al. [17] In brief,
five hundred (500 µL) of DPPH solution (0.1M) dissolved in methanol was added to different
concentrations of synthesized Ph-ZnO-NPs (20-100 µg/mL) and transferred to an incubator (30mins).
Ascorbic acid was used as a positive control. After 30 min the mixed solution absorbance was measured
at 517nm. The inhibition percentage was calculated as below,

A0-Control (ascorbic acid), A1-ZnO-NPs.

Anticancer Activity
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Cell culture & Viability 

Breast cancer cell-line (MCF-7) was collected from NCCS-Pune, India. The MCF-7 cell line was grown in
Dulbecco with DMEM medium (Sigma Aldrich, USA). Cell viability assay was reported by Mosmann, [18].
Ninety-six well plates (3×103cells/wells) were used for the seeding of MCF-7 cells and it is incubated at
37 °C, for 24h with different concentrations of ZnO-NPs (6.5 to 100 µg/mL) to treat the cancer cells
during the incubation period. Cell viability was calculated (MTT 10 µL for 4 h at 37° C) after 24h
exposure. Dimethyl Sulfoxide (DMSO) was used for dissolving the treated cells. The ELISA instrument
(2.0-Epoch-USA) was used to measure (OD-540nm-reference: 630nm) crystal formation. The treated cell
morphology images were captured and its calculation is given below.

Larvicidal Activity

Ae. aegypti, An. stephensi and Cx. quinquefasciatus 4th instar larvae were collected from the Centre for
Medical Entomology (CRME), Madurai, Tamil Nadu and then preserve it for study by providing feed. The
assay was trailed by World Health Organization WHO, [19] a standard protocol with few changes
according to the technique for Loganathan et al. [3]. Larvae (4th instar) of twenty numbers were added
from all paper cups (200mL). The concentration of Ph-ALE and synthesized Ph-ZnO-NPs (5-25 mg/L)
was included. The die larva had been counted after 24h post treatment then mortality % was obtained
from average of n=3. Abbott, [20] was used in correction of larval mortality.

Statistical Analysis

The data was analyzed by mean ± SD. Larval mortality was calculated probit analysis for finding out
LC50, LC90 and chi-square values using SPSS software.

Results
Visual Observation and UV-Vis spectroscopy analysis

Ph-ALE in synthesized ZnO-NPs was affirmed by the color changes (yellow to white precipitate). Fig. 1a.
showed that the UV-Vis absorbance peak at 280 by Ph-ALE. The synthesized Ph-ZnO-NPs illustrated a UV-
Vis absorption peak at 370nm due to Surface Plasmon Resonance (SPR), which confirms the synthesis
of ZnO-NPs (Fig. 1). 

FT-IR

FT-IR spectra of Ph-ALE and synthesized Ph-ZnO-NPs appeared the presence of seven (07) functional
groups (Fig. 2 a&b). The characteristic peak at 3447.02 cm-1 is related  to O-H group (Alcohols, phenols)
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followed by 1429.47 cm-1 can be attributed to the OH (Carboxylic acids), 865.14 cm-1 represents CH (1,2,4-
trisubst benzenes), 557.73 cm-1 can be assigned to the C-C=O (Aldehydes), 498.26 cm-1 linked to the NO2
group (Nitro compounds), 457.99cm-1 for C-O-Cgroup (Ethers) and the strong characteristic peak at
434 cm-1   (Table. 1) indicate a formation ZnO-NPs.

XRD study

XRD analysis (Fig. 3). displayed that the 11 diffraction values were presented at 2 theta ranges with
indexed of 31.83º(100), 34.50º(002), 36.32º(101), 47.53º(102), 56.72º(110), 62.94º(103), 66.57º(200),
68.06º(112), 69.19º(201), 72.84º(004), and 77.05º(202) by the synthesized Ph-ZnO-NPs. These results
noticed the configuration of ZnO-NPs and its hexagonal phase (JCPDS-NO:89-0511) and also the
synthesized NP’s average crystalline size of 60nm (Scherrer's formula). 

HR-TEM with SAED Pattern and EDAX

The synthesized Ph-ZnO-NPs shape and size were checked by HR-TEM and it disclosed the spherical and
few particles were a cluster in shape with an average size of 10-93 nm Fig. 4a. The EDAX spectra of Ph-
ZnO-NPs proved the strong Zn signal value as well as another element of Oxygen was present as shown
in Fig. 4b. 

Hemolytic activity

Biocompatible study of the synthesized Ph-ZnO-NPs was done to determine the hemolytic activity
against human RBCs. In this study, Ph-ZnO-NPs appeared less than 5% of hemolytic activity. Fig.5,
exhibited the treated images of the synthesized ZnO-NPs against Human RBCs. This study, indicates that
the concentration of the synthesized ZnO-NPs (50 and 100µg/mL) was increased while gradually
increasing the lysis percentage. The highest lysis percentage was observed from 0.6% at a higher
concentration of 100µg/mL followed by 0.4% at 50 µg/mL. From these outcomes, we propose that the
synthesized Ph-ZnO-NPs can be utilized in the domain of biomedical applications.

Antibacterial activity

The synthesized Ph-ZnO-NPs proved the strong antibacterial activity using human pathogens. A total of
two dosages were used in this study, such as 25 and 50 ug/mL (Fig. 6). The highest zone of inhibition
was seen in P. aeruginosa (17.8mm), followed by E. coli (15.0mm) and E. faecalis (14.5mm) at a higher
concentration of 50 µg/mL (Table.2). A certain level zone of inhibition was noted by plant extract to
chosen pathogens. 

Antioxidant potential

Ph-ZnO-NPs were tested against the DPPH assay and it expressed superior antioxidant activity. Ascorbic
acid (standard) and synthesized ZnO-NPs showed the maximum inhibition percentages of 57.10%;

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53.45% was noticed from 125µg/mL dosages as shown in Fig.7 with IC50 values of 102.98
µg/mL; 118.14 µg/mL respectively. 

Anticancer activity

For IC50 value was obtained by the synthesized Ph-ZnO-NPs and it was found to be 22.91µg/mL against
the MCF-7 cell line. This study confirmed excellent anticancer activity against MCF-7 cell line. The cell
viability of MCF-7 cells was decreased with the increasing concentration of Ph-ZnO-NP & the treated
images (Fig. 8a,b). 

Larvicidal activity

Larval bioassay was performed for Ph-ALE and Ph-ZnO-NPs against Ae. ageypti, An. stephensi and Cx
.quinquefasciauts (4th instar) larvaes at 24h surveillance. In this study, the synthesized ZnO-NPs proved
tremendous larvicidal activity. The maximum mortality percentage (100%) was observed in Cx.
quinquefasciatus treated with synthesized Ph-ZnO-NPs at a higher dosage (25 mg/mL). Though, the
various concentrations of Ph-ALE revealed a low mortality percentage obtained by Cx.
quinquefasciatus at a higher concentration of 25mg/mL Ph-ALE. Results noticed that the mortality
percentage was higher when increasing the synthesized ZnO-NPs dosages. For the LC50 and LC90 values
were recorded in Cx. quinquefasciatus (7.640 and 53.165 mg/mL) by Ph-ALE at 24h exposure. Similarly,
synthesized Ph-ZnO-NPs have shown LC50 and LC90 values of 4.178 and 16.157 against Cx.
quinquefasciauts (Table.3). 

Discussion
Ph-ALE of the synthesized ZnO-NPs was affirmed by the color changes (yellow to white precipitate). In
concurrence with our results, Azadirachta indica and Costus igneus [21, 22].The green synthesis of the
Ph-ZnO-NPs was confirmed by UV-Vis spectroscopy & its absorption peak at 370nm due to Surface
Plasmon Resonance (SPR). Similarly, the synthesized ZnO-NPs using aqueous leaf extract by Cynara
scolymus [15]. The functional groups like as, alcohols, phenols, carboxylic acids, 1,2,4-trisubst benzenes,
aldehydes, nitro compounds, ethers and the strong characteristic peak at 434 cm− 1 indicate a formation
ZnO-NPs [23]. Similarly, the ZnO-NPs synthesized by Momordica charantia & Solanum nigrum leaf extract
displayed the presence of six & five functional groups [24, 25]. XRD analysis demonstrate that the
presence of 11 diffraction value at 2 theta ranges with crystalline size of 60nm (Scherrer's formula),
which was comparable earlier documented on ZnO-NPs by Cassia fistula and Spondias pinnata [26, 6]
The result from HR-TEM analysis of the synthesized Ph-ZnO-NPs displayed same results from
synthesized ZnO-NPs by D. tortuosa leaf extract [27]. The EDAX elemental analysis of the synthesized Ph-
ZnO-NPs was revealed that the strong Zn signal value were present which is very similar to Abdelmigid et
al. [28].

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A biocompatible study of the synthesized Ph-ZnO-NPs displayed less than 5% of hemolytic activity was
recorded. According to 5% of hemolysis is recommended by biomaterials [15]. The related work was
observed from ZnO-NPs by Costus igneus [22].

The common mechanisms of action for antibacterial activity proposed to date are direct damage of ZnO-
NPs to the cell membranes finally cells died [29]. An earlier report, registered varying degrees of
antibacterial activity according to the type of pathogens, synthesis method, and dose depending on ZnO-
NPs [28]. In the present study, Ph-ZnO-NPs authonticated strong antibacterial activity and also the best
zone of inhibition was perceived by P. aeruginosa (17.8mm). Similarly, the synthesized ZnO-NPs of Beta
vulgaris [30]. In another report, Cassia alata leaf extract in synthesized ZnO-NPs indicated dose-
dependent activity [31]. Moreover, the synthesized ZnO-NPs of bioflavonoid rutin displayed moderate
antibacterial activity [9]. The present data acted as potential activity against the experimental bacterial
pathogens and can be utilized as another source of drugs.

Antioxidant is a substance. It can be protect the oxidation of additional compounds or neutralize free
radicals and also it based medicine formulations are utilized by the prevention as well as treatment of a
lot of complex diseases [32]. The present study the antioxidant assay displayed various levels of
antioxidant activity of Ph-ZnO-NPs. Similarly, the synthesized ZnO-NPs using Knoxia sumatrensis; Carica
papaya leaf extract appeared excellent DPPH activity with IC50 values of 95.80µg/mL & 104.9 µg/mL [3,
33]. The high antioxidant performance of Ph-ZnO-NPs makes them very useful in therapeutic application.

Breast cancer cell line (MCF-7) was test at different concentrations (6.5 to 100µg/mL) of Ph-ZnO-NPs
utilized MTT assay at 24h observation. This result depicts the dose-dependent anticancer activity.
Similarly, the synthesized ZnO-NPs by Pongamia pinnata leaf extract [34]. In Fact, the action mode of
ZnO-NPs against cancer cell lines may induce ROS species and then finally cells are damaged. In
previously, more exploration revealed that the cytotoxicity impact of ZnO-NPs utilizing the biosynthesis
approach against the MCF-7 cell line has significant anticancer activity [4, 35]. This study indicates that
the synthesized Ph-ZnO-NPs were performed effectively, and they could be used as an anticancer drug in
near future.

Nano-pesticide is an agrochemical amalgamation used to overcome the human health problem caused
by regular pesticides [36]. Presently, we have investigated the larvicidal effect of Ph-ALE and Ph-ZnO-NPs
against Ae. ageypti, An. stephensi and Cx .quinquefasciauts (4th instar) larvaes at 24h examination.
Commonly, the fourth instar larvae are recommended to be containing more immune competency than
the younger larvae [37] Several types of materials such as, surfactants, organic polymers and minerals
which fall in the nanometer size have been utilized for nano-pesticides formulation [38]. In this study, the
synthesized Ph-ZnO-NPs proved significant larvicidal activity. Results noticed that the larvae toxicity was
proposed a concentration-dependent manner for tested species. Similar concentration-dependent
larvicidal efficacy was noticed from synthesized ZnO-NPs using Cuscuta reflexa [39]. In recent times, the
larvicidal activity of synthesized ZnO-NPs using Elettaria cardamomum and Pleurotus djamor indicated
superior larvicidal activity against Cx. quinquefasciauts larvae [13, 40]. In this investigation, the

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synthesized ZnO-NPs show potential larvicidal action and the results can be useful to synthesize nano-
based mosquito drugs, due to being less hazardous to the environment.

Conclusion
In this study, ZnO-NPs were effectively synthesized using P. hexapetalum aqueous leaf extract as a
biological material. The characteristic for UV-Vis absorption peak at 370nm proved the configuration of
ZnO-NPs. XRD pattern indicates the presence of 11 diffraction values. The HR-TEM result shows
spherical nanoparticles with few particle cluster shapes. FT-IR study revealed the functional groups
presence in the synthesized ZnO-NPs due to enormous secondary metabolites related to NPs synthesis.
Further, antibacterial & antioxidant activity of Ph-ZnO-NPs displayed predominant activity against P.
aeruginosa as well as DPPH assay. In addition, the anticancer activity of Ph-ZnO-NPs expressed
significant activity against MCF-7 cell-line. Besides, the synthesized Ph-ZnO-NPs revealed strong
larvicidal activity against Cx. quinquefasciatus larvae. Thus, this plant medicated synthesis of ZnO-NPs
can fill in as a compelling pharmacological tool in biomedical area for different applications like beauty
care products, drug transporters, and coatings in clinical gadgets.

Declarations
Acknowledgements The authors acknowledge Sri Sivasubramaniya Nadar College of Engineering,
Kalavakkam for providing Post-Doctoral Fellowship (SSN-PDF) (Ref. No. SSN CE PDF/2021). The authors
also thank the Department of Chemical Engineering, Sri Sivasubramaniya Nadar College of Engineering,
Chennai– 603110, Tamil Nadu, India, for providing infrastructural facility. 

Availability of Data and Materials All the data presented in the manuscript are the original work of the
authors.

Conflict of Interest 

The authors declare that there are no conflicts of interest regarding the publication of this paper.

Author Contributions SL and DGP conceived and designed the research. SL Lab related work & KMM
hemolytic work. SL and DGP data analysis. The manuscript corrected for SL, DGP, KM & RS. all authors
read and approved the final manuscript.

Compliance with Ethical Standards

Conflict of interests: The authors have no conflict of interests to declare.

Ethical Approval Not applicable.

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Tables
Table 1 FT-IR analysis of the synthesized Ph-ZnO-NPs.

Wave number Intensity Group Functional

(CM-1)/Vibrations Compound Group

3447.02/ Stretch Medium O-H Alcohols, Phenols

1429.47/bending Medium OH Carboxylic acids

865.14/ Stretch Very Strong CH 1,2,4-trisubst benzenes

557.73/ Stretch Strong C-C=O Aldehydes

 

498.26/ Stretch Medium/ NO2 Nitro compounds

Strong

457.99/ bend Medium/ C-O-C Ethers

  Strong    

       
434.02/stretch Strong Cl-C=O  

Acid chlorides
 

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Table 2 Antibacterial activity of the synthesized Ph-ZnO-NPs.

Microorganisms   Zone of inhibition (mm)  

Positive Control Plant Extract Zinc ZnO-NPs

 (Chloramphenicol)
(25 (µg/mL) nitrate

  25 50 (µg/mL)
(µg/mL)

B. cereus 19.4 ± 0.8 7.0 ± 0.5 7.5 ± 0.5 10.8 ± 0.5 11.0 ± 0.9

E. faecalis 15.3 ± 0.8 6.0 ± 0.1 00 ± 00 12.3 ± 0.3 14.5 ± 0.7

P. aeruginosa 16.8 ± 0.6 8.0 ± 0.3  00 ± 00 13.9 ± 1.6 17.8  ± 0.6

E. coli 19.8 ± 1.1  7.5 ± 0.57 00 ± 00 12.0 ± 0.5 15.0 ± 0.4

Value are given as Data (Mean ± SD) (n=3) represent the zone of bacterial growth inhibition (mm).

Table 3 Larvicidal activity of the Ph-ALE and the synthesized Ph-ZnO-NPs against 4th in-star larvaes of
Aedes aegypti, Anopheles stephensi and Culex quinquefasciatus at 24h exposure.

Types of Samples LC50 (mg/mL) LC90 (mg/mL) χ2 Df

Larvae (LCL-UCL) (LCL-UCL)

Aedes Plant 14.274 (11.157- 113.814 (58.256- 1.59 13

extract 18.847) 570.691)
Aegypti
ZnO-NPs 8.982 (6.840-10.868) 43.563 (30.782-83.337) 1.939 13

Anopheles          
stephensi
Plant 13.688 (10.433- 125.801(60.651- 2.523 13
extract 18.299) 815.782)

ZnO-NPs 6.247 (3.919-8.084)  36.338(25.622-73.086) 4.234 13

Culex          
quinquefasciaus
Plant 7.640 (4.978-9.762) 53.165 (33.752- 3.289 13
extract 143.884)

ZnO-NPs 4.178 (2.511-5.542) 16.157 (13.160-22.128) 6.499 13

LC50: Lethal concentration kills 50% of the exposed larvae, LC90: Lethal concentration kills 90% of the
exposed larvae, LCL: Lower confidence limit, UCL: Upper confidence limit, χ2 Chi-square value, df; degrees
of freedom.

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Figures

Figure 1

UV-visible absorbance spectra of a P. hexapetalum-aqueous leaf extract (Ph-ALE), and b Synthesized P.
hexapetalum-ZnO-NPs (Ph-ZnO-NPs).

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Figure 2

a FT-IR analysis of Ph-ALE b Synthesized Ph-ZnO-NPs.

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Figure 3

XRD patterns of the synthesized Ph-ZnO-NPs.

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Figure 4

a HR-TEM morphological analysis of the synthesized Ph-ZnO-NPs. b SAED pattern by the synthesized Ph-
ZnO-NPs.

Figure 5

Hemolytic activity of the synthesized Ph-ZnO-NPs. Control and various concentrations (50 and
100µg/mL) of the synthesized Ph-ZnO-NPs treated on Human RBCs. The positive control was used for
Triton X 100 (2%). The RBCs morphology was captured by light microscope (magnification 40x).

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Figure 6

Antibacterial activity of the synthesized Ph-ZnO-NPs a B. subtilis b E. faecalis c P. aeruginosa, and d E.

coli.

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Figure 7

Antioxidant activity of the synthesized Ph-ZnO-NPs. (a) DPPH radical scavenging activity. The values are
expressed as mean ± SD values and analyzed by Two-Way analysis of variance (ANOVA). Asterisk (**, ***)
indicates significant different among treatments with respect to control (P < 0.01 and P < 0.001).

Figure 8

a MTT assay confirming the anticancer effects of the synthesized Ph-ZnO-NPs against breast cancer cell
line (MCF-7). The values are expressed as mean ± SD values and analyzed by One - way analysis of
variance (ANOVA). Asterisk (***) indicates significant different among treatments with respect to control
(P < 0.001).

b The anticancer activity (MCF 7 cell-line) of the synthesized Ph-ZnO-NPs. Control and various
concentrations by the synthesized Ph-ZnO-NPs (6.25-100µg/mL). The MCF-7 cell morphology was
captured from a confocal microscope (340 pixels).

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