Materials Letters 211 (2018) 43–47

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Materials Letters
journal homepage: www.elsevier.com/locate/mlblue

Biogenic synthesis, characterization, acute oral toxicity studies of

synthesized Ag and ZnO nanoparticles using aqueous extract of Lawsonia
inermis
N. Jayarambabu a, K. Venkateswara Rao a,⇑, V. Rajendar b,c,⇑,1
a
Centre for Nanoscience and Technology, Institute of Science and Technology, Jawaharlal Nehru Technological University Hyderabad, Telangana State 500085, India
b
Department of Physics, B.V. Raju Institute of Technology, Narsapur, Medak, Telangana State 502313, India
c
Department of Electronic Engineering, Yeungnam University, Gyeongsan-si, Gyeongsangbuk-do 38541, Republic of Korea

a r t i c l e i n f o a b s t r a c t

Article history: The Ag and ZnO nanoparticles are synthesized by a green method using Lawsonia inermis plant extract.
Received 3 August 2017 The phytochemical constituent (2-hydroxy-1, 4-napthoquionone) found in the leaf extract of Lawsonia
Received in revised form 19 September inermis plays an important role in the reduction and formation of Ag and ZnO nanoparticles. The biosyn-
2017
thesis of Ag and ZnO NPs was observed by the color change from colorless to dark brown and light brown.
Accepted 23 September 2017
Available online 23 September 2017
The FE SEM, TEM DLS analysis confirmed the average particle size of Ag and ZnO NPs as 100 nm, 20 nm,
28.5 nm and 100 nm, 20 nm, 38.4 nm respectively. Crystalline nature and presence of Ag and ZnO NPs
were confirmed by XRD. The FT-IR spectra of the Ag and ZnO authorized the presence of phyto con-
Keywords:
Biogenic synthesis
stituents as capping agent. From results of acute oral toxicity studies, it was found that Ag and ZnO
Lawsonia inermis NPs at 2000 mg/kg b.w have shown no mortality during the treatment period. The present study revealed
Nanoparticles the safer use of eco-friendly green synthesized Ag and ZnO NPs in the field of nano-biomedicine.
Biomaterials Ó 2017 Elsevier B.V. All rights reserved.
Acute oral toxicity studies

1. Introduction fic surface area compared to chemical route synthesized nanopar-

Nanotechnology, especially nanomaterials research has been
developing rapidly due to potential applications in numerous
areas, including biomedical, magnetics sciences, biosensors, opto-
electronics, catalysis and shown to be a promising antimicrobial
agent. The green synthesis of nanoparticles has acquired interest
as a relatively, sustainable, and environmentally eco-friendly
method for the production of nanoparticles [1–7]. The rationale
behind the use of plant leafs and fruits for the synthesis of Ag
and ZnO NPs are not only because the method is simple, easy
and faster but the synthesized particles were also stable, capped
by the biological compounds, reliable and cost effective than other
conventional methods [7–9]. The green method processes can
make use of plant or fruit extracts, enzymes, bacterial extracts,
fungi and replace dangerous chemicals. Green method NPs have
been shown to exhibit higher catalytic reactivity and greater speci-
ticles [10–12]. The more research is needed to improve the
production and application of green method synthesized nanopar-
ticles [13]. The Ag and ZnO NPs were known to be effective against
bacterial and viral mediated diseases and possess a huge potential
in treating various diseases like revascularization, retinal, cancer,
diabetes, hepatitis B, acquired human immunodeficiency
syndrome (AIDS), etc in the near future. The present work aims
in synthesis of Ag and ZnO NPs using Lawsonia inermis and to
explore its toxicity studies.
2. Synthesis of silver and zinc oxide nanoparticles
All the chemicals used in this method were analytical grade and
used as received without any further purification.
2.1. Lawsonia inermis plant leaf extract

⇑ Corresponding authors at: Department of Electronic Engineering, Yeungnam The Lawsonia inermis plant used in this experiment were pro-
University, Gyeongsan-si, Gyeongsangbuk-do 38541, Republic of Korea
vided from a local garden in JNTUH campus. 50 g of fresh leaves
(V. Rajendar).
E-mail addresses: kalagadda2003@gmail.com (K.V. Rao), rajendar.nano@gmail.
were washed several times for removing any adherent particles,
com (V. Rajendar). and placed in the beaker to heat at 80 °C for 40 min. Filtered the
1
http://orcid.org/0000-0002-2049-5085. solution with the help of whataman filter paper.

https://doi.org/10.1016/j.matlet.2017.09.082
0167-577X/Ó 2017 Elsevier B.V. All rights reserved.
44 N. Jayarambabu et al. / Materials Letters 211 (2018) 43–47
2.2. Synthesis of Ag nanoparticles
The 0.5 g of AgNO3 precursor dissolved in 50 ml water and the
filtrate of Lawsonia inermis leaf extract was added and stirred for
60 min at 50 °C. After stirring the system was maintained to room
temperature and the precipitate was collected. Finally the precipi-
tate was washed with absolute ethanol for several times and dried
in vacuum condition at 70 °C for 24 h.
2.3. Synthesis of ZnO nanoparticles
50 ml of filtered Lawsonia inermis leaf aqueous extract and 2 g
Zn(NO3)2 6H2O was dissolved. The salt was completely dissolved
in aqueous extract without any additional heat treatment. The
mixtures were covered with aluminium foil to avoid any photoin-
duced phenomenon and kept at room temperature. The color
change observed after 1 h, precipitate suggesting the formation
of a zinc oxide complex in suspension. The mixture compound
was dried at 120 °C in a oven, the powder was collected and
washed several times with distilled water to remove any residue
of the extract. The obtained product was subjected to an additional
heat treatment in air at 200 °C for 2 h.
2.4. Acute oral toxicity studies
The experiments were carried out according to the guidelines of
the Committee for the Purpose of Control and Supervision of
Experiments on Animals (CPCSEA), New Delhi, India on either sex
of Swiss albino mice weighing 20–25 g. The study was conducted
after getting approval from the Institutional animal ethical com-
mittee of CMR College of pharmacy, Hyderabad (approval no.:
IAEC/1657/CMRCP/T2/PhD-16/47). The selected Swiss albino mice
were divided into 3 groups of 3 animals in each. Test mice were
fasted for 4 h before dosing with free access to water only. The nor-
mal control group received distilled water (10 ml/kg) and the test
Fig. 1. XRD analysis (A) Ag NPs, (B) ZnO NPS. FTIR spectra (A) Ag NPs, (B) ZnO NPs & UV–Vis (A) Ag NPs, (B) ZnO NPs.
groups received 2000 mg/kg b.w of Ag and ZnO nanoparticles.
The ZnO NPs do not affect the acute oral toxicity and the
histopathological study. The biological synthesized ZnO NPs has
shown small particle size of NPs creates a large surface area per
unit mass. In this study 2000 mg/kg concentration of ZnO Nanopar-
ticles synthesized using Lawsonia inermis leaf extract has not
induced any toxicity during the treatment period. Immediately
after administration of dose, the mice were observed for the first
4 h for skin and fur changes, eye secretion, respiration, any behav-
ioral changes and death, if any, intermittently for the next 6 h, then
again at 24 h after dosing. Special attention was paid on the clinical
signs of toxicity including convulsions, tremors, salivation, nausea,
vomiting, diarrhea, lethargy, coma, etc. They were then kept under
observation up to 14 days to identify mortality, if any. The observa-
tions were made daily. In addition, the histopathological changes
have been examined as well [14]. After 2 weeks, mice were sacri-
ficed by cervical dislocation, tissues were collected and kept in
10% formalin solution and subjected for histopathology study [15].
3. Results and discussion
The number of braggs reflections can be seen with 2 theta val-
ues of 38.6, 42.8, 62.7 and 78.3 corresponding to the lattice planes,
which may be indexed to the (1 1 1), (2 0 0), (2 2 0) and (2 2 2) of
Ag NPs respectively [16]. The calculated average crystalline size
for Ag NPs is found to be 16 nm, which is close to the average par-
ticle size of 20 nm obtained from TEM analysis. The crystal struc-
ture of synthesized ZnO NPs was investigated by powder X-ray
diffraction analysis. The XRD patterns of pure ZnO calcinated at
200 °C for 4 h. the peaks at 31.82, 65.4, 36.6, 47.7, 62.9, 66.30,
67.5, 69.3 and 78.4 indexed to the (1 0 0), (0 0 2), (1 0 1), (1 0 2),
(1 1 0), (1 0 3), (1 1 2) and (2 0 1) crystal planes of ZnO respec-
tively. The average crystalline size of the sample was calculated
using Scherrer equation. The average crystalline size of ZnO NPs
calculated was found to be 24 nm.
 N. Jayarambabu et al. / Materials Letters 211 (2018) 43–47
The UV–Vis spectrum reveals the Ag nanoparticles formation by
exhibiting the typical Plasmon absorption at 458 nm which is indi-
cating the presence of spherical Ag nanoparticles [17]. It also clar-
ified the formation of ZnO nanoparticles by showing absorption at
382 nm. FT-IR analysis of Ag and ZnO NPs showed the intensive
peaks at 3217.3 cm 1 to 1650 cm 1 (aromatic C–H, amide bond
of proteins due to carbonyl stretch in proteins), 1300–800 cm 1
(C–C bond of aromatic or amide group-II, C–N aromatic and
aliphatic amines). The FT-IR spectrum of the biosynthesized Ag
nanoparticles showed an absorption peak exhibited at wavenum-
ber 600–500 cm 1 which is associated to the stretching mode of
Ag nanoparticles. The ZnO nanoparticles peak exhibited at
570 cm 1 (Fig. 1).
In order to investigate the morphology of the synthesized
nanoparticles, FESEM images were recorded with different magni-
fications. In Fig. 2 Ag nanoparticles with size of 100 nm are detect-
able showing leaf like structures. In case of ZnO nanoparticle
spherical structures were shown.
The TEM image was taken for the characterization of the size
and structure of Ag & ZnO nanoparticles. Fig. 2 shown confirms
the very uniform spherical shape of Ag nanoparticles. The average
particle size from TEM image was estimated to be 20 nm. In
synthesis process the plant compounds of Linalool a-terpineol,
EtherphenylvinylA 1,3-indandione, Eugenol, 2-hydroxy-1,4-
napthoquionone, Oxirane-tetradecyl, Hexadecanoic acid. These
compounds present in the Lawsonia inermis plant they act as a
capping agent and stabilizing agent. These factors mainly affect
on reduction and formation of the nanoparticles size. The TEM
image of ZnO nanoparticles showed the spherical like structure
with average size of 20 nm, which is inconsistent with that of
observed from FESEM images [18,19]. Due to the reason the Law-
sonia inermis plant extract compounds coated on surface of the
Fig. 2. FESEM images (A) Ag NPs, (B) ZnO NPs & TEM images (A) Ag NPs, (B) ZnO NPs. PSA analysis (A) Ag NPs, (B) ZnO NPs and Zeta potential (A) Ag NPs, (B) ZnO NPs.
45
ZnO NPs. From FESEM morphological studies ZnO NPs we did not
observed the spherical like structure it may be the plant compound
coated on ZnO nanoparticles. DLS analysis showed that the Ag and
ZnO NPs size was approximately 28.5 nm and 38.4 nm with low
polydispersity index (PDI) of 0.035 and 0.028. The measured size
of particles by DLS was moderately similar as compared to the
TEM and XRD analysis. The zeta potential value of dispersed
biosynthesized Ag and ZnO NPs was – 42.7 mV and 39.9 mV typi-
cally have degree of stability.
3.1. Acute oral toxicity and Histopathology studies
The animals appeared to be normal without any signs of toxic-
ity at a dose of 2000 mg/kg b.w administered orally and no mortal-
ity was observed up to 14 days [20]. No gross physiological
changes were observed in control and Ag and ZnO NPs treated mice
liver, brain, heart, and spleen. The liver hepatocytes appeared nor-
mal with no signs of inflammation and necrosis in Control and Ag
and ZnO NPs treated mice (Fig. 3A–C). Brain didn’t show any
abnormalities in astrocytes, nerve fibres and no demyelination
were observed in treated mice (Fig. 3D–F). The Control, Ag & ZnO
NPs treated Heart tissue showed normal cardiac fiber (Fig. 3G–I).
The Spleen tissue showing normal lymphatic follicle size in both
Ag and ZnO NPs as that of control group (Fig. 3J–L). Acute oral tox-
icity and histopathological parameters were noted in Table 1. This
study confirmed that Ag and ZnO NPs were nontoxic to the Liver,
brain, heart, and spleen within the treatment durations. In this
study Ag and ZnO nanoparticles were synthesized using Lawsonia
inermis leaf extract. Which is a antioxidant, anti-inflammatory
and acts as liver tonic apart from other uses. The phytoconstituents
present in the extract are majorly acting as the reducing agent and
capping agent for synthesis of Ag and ZnO nanoparticles was very
46 N. Jayarambabu et al. / Materials Letters 211 (2018) 43–47

Fig. 3. A, D, G & J indicates Control, B & C, E & F, H & I, K & L indicates Ag, ZnO treated liver, brain, heart & spleen respectively.

Table 1
Clinical parameters observed in control, Ag and ZnO NPs injected mice at 2000 mg/kg b.w.

S. Parameters Organ Control group 10 ml/kg Ag Nanoparticles treated 2000 mg/kg Zno Nanoparticles treated 2000 mg/
No kg
1 Histopathological Brain Normal brain tissues with astrocytes Normal brain tissues with astrocytes Normal brain tissues with astrocytes
changes and nerve fibres and nerve fibres and nerve fibres
2 Heart Normal cardiac fibre Normal cardiac fibre Normal cardiac fibre
3 Liver Normal liver with hepatocytes No inflammation in liver No inflammation in liver
4 Spleen Lymphatic follicles size appeared Lymphatic follicles size appeared Lymphatic follicles size appeared
normal normal normal
5 Clinical Skin and fur No change No change No change
abnormalities change
6 Urination Normal Normal Normal
7 Behavioral Tremors and Not observed Not observed Not observed
changes convulsions
8 Salivation Normal Normal Normal
9 Lethargy active active Active
10 Mortality Nil Nil Nil

safe so there is no inflammation seen in hepatocytes of liver of the
Ag and ZnO treated groups and looked similar to control group
liver. So, from this study it was proved that the biogenic synthesis
of Ag and ZnO nanoparticles are non-toxic to any of the vital
organs.
Acute toxicity studies do not detect effects on vital functions
like the cardiovascular, central nervous, and respiratory systems
and it is mainly performed to obtain an appropriate dose for phar-
macological activity and to find out the affected organs at the end
of the treatment. From the histopathological images, it was
reported that the Ag and ZnO Nanoparticles synthesized using
Lawsonia inermis leaf extract has not induced any toxicity during
the treatment period. From the Ag and ZnO treated liver sectioning
it was reported that the hepatocytes (the major mass of liver) are
without any inflammation and necrosis. If there is any inflamma-
tion, a high concentration of leukocytes will appear in the connec-
tive tissue of liver. Such signs of toxicity were not seen in the Ag &
ZnO treated mice and cell architecture is similar to that of control
mice. The brain image of Ag and ZnO nanoparticles treated mice
was similar to that of control mice without any degeneration of
neurons. The cardiac fiber of the heart tissue is normal without
any degeneration or fragmentation or hyalinization of muscle fiber
without congested blood vessels indicating that no toxicity is
observed in the heart of Ag & ZnO treated mice and is similar to
that of Control mice heart architecture. As the spleen is the direct
and indirect site for toxicity, there is no significant changes
observed in the spleen lymphatic follicle tissue and no loss of cel-
lularity was observed in both Ag and ZnO treated mice and is sim-
ilar to that of control mice spleen tissue indicating that the Ag and
ZnO nanoparticles synthesized using Lawsonia inermis are non
toxic from the histopathological study performed.
4. Conclusion
The achieved data revealed that Ag and ZnO NPs demonstrated
a high potential in toxicity studies. The size, surface area, crys-
talline nature and the phytochemical present in the Lawsonia iner-
mis aqueous leaf extract which acted as a capping agent, are the
factors responsible for the potential biomedical application. The
acute oral toxicity studies of biosynthesized Ag and ZnO nanopar-
ticles coated with active biocompounds of Lawsonia inermis leaf
extract for a period of 14 days showed no toxicity on tested mice.
Additionally, the histopathological assessment revealed that syn-
thesized Ag and ZnO NPs are safe, nontoxic and biocompatible with
potential in treatment of various diseases. This study aims to prove
 N. Jayarambabu et al. / Materials Letters 211 (2018) 43–47
that the biogenic synthesis of Ag and ZnO nanoparticles using Law-
sonia inermis leaf extract at the highest dose of 2000 mg/kg b.w
have not showed any toxicity during the study. The method
choosed was very economical because we used only precursor
compared to chemical method of synthesis where more than one
chemical is needed for synthesis. I also want to prove that the
nanoparticles capped with plant extract can be used in major ther-
apies because of its non-toxicity in acute oral toxicity studies. Tox-
icity results from animals will be crucial in definitively judging the
safety of medicinal plants if they are found to have sufficient
potential for development into pharmacological products.
Acknowledgement
One of the authors, N. Jayarambabu would like to thank CMR
College of Pharmacy, Hyderabad and CNST, IST, JNTUH for provid-
ing lab and instrumentation facility.
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