IEEE TRANSACTIONS ON NANOBIOSCIENCE, VOL. 19, NO.
4, OCTOBER 2020 633
Biogenic Synthesis of Zinc Oxide Nanoparticles
by Bryophyllum pinnatum and Its Acute Oral
Toxicity Evaluation in Wistar Rats
Abhilash D. Jadhao, Sudhir Shende, Pramod Ingle, Aniket Gade, Sunil W. Hajare, and Ranjit S. Ingole
Abstract — The evaluation of toxic effects of nanopar-
ticles (NPs) has become an important aspect of Nan-
otechnology research in the 21st century. The present
investigation deals with the green synthesis of biogenic zinc
oxide nanoparticles (ZnO-NPs) using Bryophyllum pinna-
tum leaves, their characterization and evaluation of acute
oral toxicity in Wistar rats. The characterization of synthe-
sized ZnO-NPs revealed maximum absorbance at 307 nm
on UV-Vis spectrophotometric analysis, NTA showed mean
size of particles and mode of the particles distribution as
128.2 nm and 12.6 nm, respectively. Zeta potential was found
to be −0.369 mV. The absorbance shown by FTIR at 3469,
1644, 1355 and 887 cm-1 indicates the involvement of bio-
molecules that are accountable for capping and stabilization
of ZnO-NPs. The XRD assessment further demonstrated the
crystalline nature of the ZnO-NP. The TEM analysis of the
synthesized ZnO-NPs revealed the presence of spherical
NPs with the mean size of 3.7 nm. The acute oral toxicity
evaluation in rat showed an approximate median lethal
dose to be more than 2000 mg/kg body weight. It is thus
concluded that biogenic ZnO-NPs showed absence of acute
oral toxicity symptoms at the doses employed in the present
study.
Index Terms — Acute oral toxicity, Biogenic, Bryophyllum
pinnatum, NTA, TEM, Zinc oxide nanoparticles.
I. I NTRODUCTION
A MAJOR contribution of Nanotechnology is the devel-
opment of new materials at the nanoscale i.e.
1 to 100 nanometer (nm) [1]. Nanoparticles (NPs) are usu-
ally particulate materials with at least one dimension in the
nanoscale, except in case of quantum dots, where even the
particles could be in zero dimensions [2]. Nanosystem are
considered as one of the most effective systems as they can
Manuscript received May 17, 2020; revised June 26, 2020 and
July 21, 2020; accepted July 28, 2020. Date of publication August 3,
2020; date of current version October 1, 2020. (Corresponding author:
Ranjit S. Ingole.)
Abhilash D. Jadhao and Ranjit S. Ingole are with the Department of
Veterinary Pathology, Post Graduate Institute of Veterinary and Animal
Sciences, Akola 444104, India (e-mail: ingoleranjit@rediffmail.com).
Sudhir Shende, Pramod Ingle, and Aniket Gade are with the Depart-
ment of Biotechnology, Sant Gadge Baba Amravati University, Amravati
444602, India.
Sunil W. Hajare is with the Department of Veterinary Pharmacology and
Toxicology, Post Graduate Institute of Veterinary and Animal Sciences,
Akola 444104, India.
Digital Object Identifier 10.1109/TNB.2020.3014023
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be encapsulated easily during formulation due to their small
size and can reach the site effectively with no trouble [3].
Now-a-days, nanotechnology has created a growing sense
of excitement in the field of life sciences in general and
biomedical devices in particular, as NPs possess unique phys-
ical, chemical, optical, electrical, and biological properties,
helping for easy maneuvered for desired applications [4], like
biosensor [5] and drug delivery [6].
Chemical reduction methods are widely used, however, they
may involve toxic reducing agent, stabilization agent or an
organic solvent for the synthesis of NPs, which can even inter-
fere with the toxicity studies performed for the nanoparticles,
whereas nanoparticle synthesis using a plant leaf extract is a
green synthesis method due to involvement of biomolecules
as reducing, stabilization agent and water as a solvent and is
an eco-friendly approach to produce NPs [7]. The presence
of toxic chemicals as the capping agent of NPs and organic
solvent in the chemical synthesis method hinders their use in
clinical applications. Whereas, NPs synthesized by biological
method in general and plants in particular are more biocom-
patible due to the presence of biomolecule as a r capping
agents and though they exhibit variations in shape and size.
NPs produced by plants possess a faster rate of synthesis as
compared to NPs produced by other organisms [8].
Synthesis of NPs by plants is considered as a reliable
method because of its environmental friendly nature. Available
literature revealed rapid and extracellular synthesis of noble
metal NPs using biological method [9], [10]. Bryophyllum
pinnatum is one of the traditional medicinal plants used in
India. The biochemical analysis of B pinnatum has revealed
the presence of secondary metabolites like phenols, flavonoids,
lipids, tannins, tri-terpenes, organic acids, glycosides etc.
Moreover, the plant is reported to possess many pharmacologi-
cal properties viz. antimicrobial, antihypertensive, antileishma-
nial, antidiabetic, analgesic and anticancer activity [11].
Among various metal NPs, zinc (Zn) and its oxide (ZnO)
is considered as the most interesting and promising metallic
nanomaterials, because of its versatile nature [12]. Zinc is
an essential nutrient element in animals, which serves as
an activator for several enzymes involved in transcription,
transport of proteins in the body [13]. ZnO-NPs has various
potential applications like antimicrobial agent [14], [15]
634
wound healing, anti-diabetic anti-inflammatory, wound healing
[16], [17], antioxidant [18] and optic properties, gastro-
protective, nephro-protective as well hepato-protective
properties [19].
Moreover, the acute oral toxic potential assessment of
nanoparticles is required to determine their adverse effects
that might occur due to accidental or deliberate short-term
exposure [20]. Results from acute oral toxicity test serve as
a guide in dosage selection for long-term toxicity studies to
be done on the experimental animals [21]. From the result
of an acute oral toxicity test, a conclusion can be made
on the toxicity status of the test substance. As substances
with LD50 below 5 mg/kg are classified to be highly toxic
while substances with LD50 above 15,000 mg/kg are termed
relatively harmless [22].
In the present study, biogenic synthesis of ZnO-NPs was
attempted using leaves extract of B. pinnatum. The ZnO-NPs
obtained were characterized by various nanotechnological
techniques and the acute oral toxicity was evaluated in female
Wistar rats.
II. M ATERIALS AND M ETHODS
A. Collection, Identification and Authentication of Plant
B. pinnatum leaves extract was used to synthesize the
ZnO-NPs. B. pinnatum leaves were collected from medici-
nal garden of Department of Veterinary Pharmacology and
Toxicology of Post Graduate Institute of Veterinary and Ani-
mal Sciences (PGIVAS), Akola, Maharashtra, India and was
identified from Dr. S. P. Rothe, Taxonomist, Department of
Botany, Meharbanu College of Science and Commerce, Akola,
Maharashtra, India.
1) Preparation of Leaf Extract of B. pinnatum: Fresh leaves of
B. pinnatum, 25 grams were collected and washed thoroughly
with plenty of distilled water from both surfaces to remove
the dust particles and then dried to remove residual moisture.
Leaves were then surface sterilized using an absolute alcohol.
The leaves were transferred to 250 ml conical flask with
100 ml of sterile distilled water and then heated for 30 min in
at 50◦C. The extract was then filtered with Whatman’s filter
paper no. 1 and further filtered using vacuum filter of pore size
0.2 μm. The final filtrate was stored at cool and dry place for
further use.
B. Biogenic Synthesis of ZnO-NPs by B. pinnatum
The previously prepared leaves extract 100 ml was heated
at 50◦ C for 10 minute and 100 ml of 1 mM of zinc acetate
solution was added to it and stirred for 5 minutes on magnetic
stirrer. Then 1 mM NaOH of 1000 μl was added drop-
wise under stirring condition. The reaction mixture was then
stand-alone for 30 min for complete reduction and precipitate
formation of NPs.
C. Characterization of Biosynthesized ZnO-NPs
The detection of ZnO-NPs was carried out by UV-Visible
Spectrophotometric analysis, and further characterization of
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IEEE TRANSACTIONS ON NANOBIOSCIENCE, VOL. 19, NO. 4, OCTOBER 2020
ZnO-NPs was done by Fourier-transform infrared spec-
troscopy (FTIR), Powder X-ray diffraction (XRD), Transmis-
sion electron microscopy (TEM), Zeta potential measurement,
and Nanoparticles Tracking and Analysis (NTA).
1) UV-Visible Spectrophotometric Analysis: UV-Visible
(UV-Vis) spectroscopy is the measurement of intensity and
wavelength of absorption of ultraviolet and visible light by
the sample. The absorption is observed in the form of peak,
when plotted as the function of wavelength with respect to
intensity of absorbed radiation. UV-Vis spectroscopy proves
to be one of the important and simple ways to confirm the
formation of NPs. Preliminary confirmation of biosynthesized
ZnO-NPs was done with the help of UV-Visible double beam
spectrophotometer (Shimadzu-UV 1700, Japan) operated at a
resolution of 1 nm by scanning the absorbance spectra from
200 to 800 nm of colloidal sample. The powdered precipitate
of ZnO-NPs was dissolved in sterile water and subjected
to UV-Vis analysis. The baseline correction was done by
precursor salt solution i.e. 1 mM of zinc acetate.
2) Nanoparticles Tracking and Analysis (NTA): The ZnO-NPs
were characterized by nanoparticles tracking and analysis
(NTA) system to find out the average size of the parti-
cles. NTA is a laser-based light-scattering system (LM-20,
NanoSight Pvt. Ltd., UK), in which particles suspended in
the liquid medium were injected into the LM viewing unit
and viewed in the closed proximity to the optical element.
Sample preparations and measurements were carried out at
controlled condition and analysis was performed by NanoSight
LM 20 using a beta version of NTA 2.3 software.
3) Fourier Transformed Infrared (FTIR) Spectroscopic Analy-
sis: FTIR spectra were recorded using an instrument, Alpha-T
FTIR, Brukeroptik GmbH, Germany, equipped with a room
temperature DTGS detector supported with the OPUS soft-
ware, for synthesized ZnO-NPs. The scans recorded were the
average of 100 scans on Attenuated Total Reflection (ATR)
diamond crystal of Alpha-T FTIR instrument. The crystal was
cleaned and the background was measured with the ATR unit,
the sample was placed on the crystal ensuring good contact and
the sample measurement was done in transmission mode. The
sample was scanned in 500 to 4000 cm−1 with a resolution
of 4 cm−1 for each sample.
4) Zeta Potential Measurement: The zeta potential of
ZnO-NPs was measured using a Zetasizer (ZS 90, 3000 Har-
monized system; Malvern Instruments, Malvern, UK) with
a zeta dip cell. For the sample analysis 30 μl of ZnO-NPs
colloidal solution was diluted in 970 μl of distilled water.
The surface charge of NPs was measured by Zetasizer ZS90.
The charge acquired by the particles is partially due to the
adsorption of ions in the solution. The surface charges give rise
to a potential distribution around the particles. Zeta potential
is expressed in milliVolts (mV) and usually falls in the range
of −70 mV to +70 mV.
5) Powder X-Ray Diffraction (XRD) Analysis: Powder XRD
analysis was performed to determine the crystalline structure
of NPs. The obtained Bragg’s reflection index was used to
determine the crystal structure of NPs. The diffraction pattern
gave an idea about the crystalline nature of NPs. The X-ray
diffraction study was done using powdered NPs subjected on
JADHAO et al.: BIOGENIC SYNTHESIS OF ZINC OXIDE NANOPARTICLES
TABLE I
D ESIGN OF ACUTE O RAL T OXICITY S TUDY
OF ZnO-NPs IN F EMALE R AT
a RigakuMiniflex II desktop X-ray diffractometer using CuKα
radiation (0.15418 nm). The scanning range (2θ ) was selected
from 10◦ to 80◦ . A scanning speed of 1◦ /min and a chart speed
of 20 mm/min were set for the precise determination of the
lattice parameters of the synthesized NPs.
6) Transmission Electron Microscopy (TEM) Analysis: Trans-
mission electron microscopy (TEM) was carried out to deter-
mine the shape and size of biosynthesized ZnO-NPs. Colloidal
sample was sonicated (Vibronics, VS 80) for around 15 min.
The infrared light was used for evaporation of a drop of
this solution which was loaded on copper grid coated with
carbon. The TEM analysis was carried out on JEOL model
JEM 2100 instrument operated at an accelerating voltage
of 200 Kv[23].
D. Evaluation of an Acute Oral Toxicity
of ZnO-NPs on Rats
The experiment was first approved from Institutional Ani-
mal Ethics committee (IAEC) of Post Graduate Institute of
Veterinary and Animal Sciences, Akola (Reg. No. 312/GO/
ReBi/S/2000/CPCSEA). The national guidelines of Committee
for Purpose of Control and Supervision of Experiments on
Animals (CPCSEA), Ministry of Environment, Forest and
Climate Changes, Government of India, New Delhi, India were
followed throughout the experimentation. Total six female
Wistar rats weighing about 130-150 g were procured from
CPCSEA registered breeder. All rats were maintained under
identical managemental, hygienic and stress free conditions
in laboratory animal house of Post Graduate Institute of
Veterinary and Animal Sciences, Akola, Maharashtra, India,
during experimental period (as shown in Table 1).
Acute oral toxicity study was conducted as per OECD
guideline 423 using six female Wistar rats for evaluation of
safety/toxic potential of ZnO-NPs. The NPs of ZnO at dose
rate of 300 and 2000 mg/kg body weight was administered
to rats and the animals were closely observed for behavioral
changes, clinical signs and mortality for next 24 hours. The
animals were further monitored for next 14 days to observe
behavioral changes, clinical signs and mortality. At the end
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635
Fig. 1. UV-Vis spectroscopic analysis: A) Experimental (ZnO-NPs
showing SPR peak at 307 nm) B) Control (Bryophyllum pinnatum leaf
extract) (inset figure a) the reaction mixture with cream colored precipitate
of NPs b) ZnO-NPs powder.
of 14th day, all the rats in each group were sacrificed by
anesthetizing in a jar containing cotton wool soaked in diethyl
ether inhalation. Blood was then collected from retro-orbital
plexus using capillary tube in K2 EDTA (1-2 mg/ml). After
detailed gross examination, tissues of visceral organs viz. lung,
liver, heart, kidney, intestine, spleen, stomach and brain were
collected in 10% formal saline solution and were processed
for routine histopathology procedures. Sections of 4 to 6 μ
were cut on rotary microtome and stained with H and E stain
for recording histopathological observations [24].
III. R ESULTS AND D ISCUSSION
A. Synthesis and Characterization of ZnO-NPs
The leaves extract and zinc acetate solution were added
along with the drop-wise addition of 1 mM NaOH under
stirring condition, it has been observed that, the reaction mix-
ture became yellowish and forming cream colored precipitate
(inset Fig. 1a and b). The precipitate was then collected by
centrifugation at 16000 rpm for 10 min at 4◦ C. Resultant
precipitate was vacuum dried at 30◦C and stored at room
temperature for further studies (inset Fig. 1b).
1) UV-Visible Spectrophotometric Analysis: When the
biosynthesized NPs was subjected to UV-Vis spectroscopic
analysis, the UV-Vis spectrum of synthesized NPs showed an
absorbance peak around 307 nm (Fig. 1), which is due to the
characteristic surface plasmon resonance (SPR) absorbance
peak for ZnO and its position depends on the particle size,
shape and dielectric constant of the medium [25], [26]. The
result obtained in the present study, corroborates with the
previous report by Tiwari and coworkers (2017) [27] in which
ZnO-NPs were synthesized from petal extracts of Rosa indica
demonstrating the absorbance at 360 nm and in another study
in which ZnO-NPs were synthesized from flower extract of
Cassia auriculata which showed absorbance at 368 nm [28].
The absorbance of NPs was found to be directly proportional
to the color intensity of the solution [29]. The broadness of
the peak indicates the particle size distribution for NPs. In the
636
Fig. 2. Nanoparticle tracking and analysis by Nanosight (LM-20)
showing ZnO-NPs (concentration 1.13 × 109 particles/ml) in the colloidal
suspension (inset Nanosight (LM-20) image showing 3D plot forZnO-NPs
distribution in sample).
present study, the shifting absorbance towards the lower
wavelength can be attributed to the synthesis of nanoparticles
of smaller size in the colloidal form.
2) Nanoparticles Tracking and Analysis (NTA): NTA was per-
formed for freshly prepared NPs by Nanosight LM-20 to deter-
mine their size and size distribution of NPs, and concentration
of NPs depending on the Brownian motion in suspension. The
mean size of the ZnO-NPs from NTA analysis was found to
be 128.2 nm, whereas the mode of the particles distribution
was 12.6 nm and concentration of the particles in the colloidal
suspension was found to be 1.13 × 109 particles/ml (Fig. 2).
The data obtained after NTA analysis corroborates with the
results obtained for the same NPs in the previous study
conducted by Tiwari et al. (2017) [27], for determining the size
of ZnO-NPs and the size of silver nanoparticles (AgNPs) [30].
NTA analysis is a very useful technique, which can be used
to understand the aggregation in the samples, which cannot be
seen in the other methods such as differential centrifugation
sedimentation and dynamic light-scattering technique [31].
In the present study, the large difference between mean and
mode of the particle size of ZnO-NPs can be attributed to
the aggregation of NPs which might have formed the large
aggregates and would have resulted in the higher average size
of NPs [32 ].
3) Fourier Transformed Infrared Spectroscopy (FTIR) Analy-
sis: The synthesized ZnO-NPs were also further characterized
by FTIR analysis to determine the functional group respon-
sible for capping and stabilization of ZnO-NPs. In FTIR
analysis, control spectra of precursor salt shows peaks at
3353, 1644 and 1348 cm−1 , the plant extract revealed the
major peaks at 3360, 2939, 1632, and 1045. 3360 peak can
be assigned to O-H stretch, H-bonded, alcohols or phenols
as functional groups, 2939 can be assigned to C-H stretch
of alkanes, 1632 can be assigned to N-H bent of primary
amines and 1045 can be assigned to C-N stretch of aliphatic
amines. ZnO-NPs demonstrate the absorbance at 3469, 1644,
1355 and 887 cm−1 , 3469 can be assigned to H-bonded
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IEEE TRANSACTIONS ON NANOBIOSCIENCE, VOL. 19, NO. 4, OCTOBER 2020
Fig. 3. Fourier Transform Infrared spectroscopy spectra of ZnO-NPs
(A) Control salt (Zinc nitrate) (B) Experimental (ZnO-NPs) and (C) Plant
extract (Bryophyllum pinnatum leaf extract).
O-H stretch of alcoholic or phenolic, 1644 can be assigned
to N-H bend of primary amines, 1355 can be assigned to
N-O symmetric of nitro compound and 855 can be assigned
to N-H wag of primary and secondary amines (Fig. 3).
On comparing FTIR spectra of control and ZnO-NPs, it was
found that a few bands present in the precursor salt and
plant extract disappeared at the time of ZnO-NPs; this clearly
indicates the involvement of O-H group of phenol or alcoholic
and primary or secondary amides responsible for the capping
and stabilization of ZnO-NPs. Functional groups detected by
the FTIR study corroborates with phytochemical analysis done
by Aprioku and Igbe (2017) [33] for aqueous B. pinnatum
leaf extract. The ZnO-NPs FTIR spectrum shows the pres-
ence of O-H stretch, H bonded of alcohols or phenol, N-H
bend of 1◦ amines, C-H rock of alkanes and N-H wag of
1◦ and 2◦ amines. Comparing all the three spectra clearly
shows involvement of phenolic and protein molecules from
the B. pinnatum leaf extract in the synthesis and stabilization
of ZnO-NPs.
4) Zeta Potential Measurement: The zeta potential of
ZnO-NPs was found to be −0.369 mV, which provides the
evidence that the fabricated NPs are less stable and likely
to form the aggregates (Fig. 4a). The common dividing line
between stable and unstable suspensions is generally taken at
either +30 or −30 mV. The particles with the zeta potential
values higher than ±30 mV are usually considered stable
and zeta potential values greater than ±15 mV and less
than ±30 mV, indicates a suspension at the threshold of
agglomeration. Flocculation or coagulation is most rapid when
the zeta potential is 0 mV ± 3 mV [34], [35].
Although the factors like conductivity, pH and concentration
of a formulating component of the sample has the influence
on zeta potential measurement. In spite of lower zeta potential
value, which is approaching almost zero, the NPs were found
to be moderately stable due to capping of biomolecules and
zeta value of the particles is due to the potential value
contributed by NPs and by capping agent together. Moreover,
the presence of capping agent prevents the aggregation of
NPs and thereby imparting them stability. Zeta potential
JADHAO et al.: BIOGENIC SYNTHESIS OF ZINC OXIDE NANOPARTICLES
Fig. 4. a) Zeta potential of ZnO-NPs showing zeta potential value
−0.369 mV b) XRD pattern of ZnO-NPs synthesized from Bryophyllum
pinnatum leaves extract showing face cubic centre (FCC) pattern.
measurement data supports the formation of aggregates, which
was also demonstrated by the NTA analysis.
5) Powder X-Ray Diffraction (XRD) Analysis: The powdered
XRD pattern for the synthesized ZnO-NPs was observed at
positions 36.33◦, 59.96◦, 68.13◦ and 69.18◦ of 2θ indicat-
ing the presence of (101), (103) and (201) facet of face
cubic centre region. These values showed resemblance with
JCPDS (Joint Committee on powder diffraction), standard file
no. 36-1451. This confirms the crystalline nature of ZnO-NPs
(Fig. 4b).
6) Transmission Electron Microscopy (TEM): The transmis-
sion electron microscopy (TEM) analysis of the synthesized
ZnO-NPs revealed the presence of spherical NPs with the
mean size of 3.7 nm and standard deviation 0.71 as shown
in Fig.5a & 5b.
The size evaluated by TEM analysis was smaller than
predicted by NTA [36], [37]. These two methods rely on
different physical principles and/or detection methods there-
fore it shows the difference in the results. The NTA probes
the hydrodynamic radius which is always larger than core
diameter, whereas TEM analyzes dry particles, i.e. the metallic
core only. In addition, TEM is a number based method that
has a bias towards the smaller particle sizes compared to
NTA, which is a number based method but fails to probe
the weakly scattering particles (below 10-20 nm) and is thus
mostly measuring the NPs aggregates which may clarify some
of the differentiation in size measurements.
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637
Fig. 5. Transmission Electron Microscopic analysis a) TEM image of
ZnO-NPs b) Histogram showing average particle size distribution of
ZnO-NPs.
B. Acute Oral Toxicity Study
Acute toxicity study of ZnO-NPs was studied to assess the
efficacy and safety of single high oral dose of ZnO-NPs using
aqueous leaves extract of B. pinnatum. Prior to dosing for
acute toxicity study, rats were fasted for 4 hrs with adlib water
supplement. Initially one female rat was selected for sighting
study and was given ZnO-NPs @ 300 mg/kg body weight
single oral dose and was observed for 24 hrs. However, the ani-
mal was normal healthy without showing any clinical signs
for the period 24 hrs. Hence, another female rat was given
single oral dose of ZnO-NPs @ 2000 mg/kg body weight.
During first 24 hrs of oral treatment rat showed symptoms
of lethargy appearance only however, subsequently animal
showed normal behavior. Hence main study was followed by
administering ZnO-NPs @ 2000 mg/kg body weight single
oral dose in four animals and animals were observed for
24 hrs and for subsequent 14 days. There were no toxic
symptoms or mortality or moribund stage observed with single
acute limit dose level of 2000 mg/kg body weight in any of
the rat during 14 consecutive days of observation. Animal
showed normal feed and water consumption for 14 days.
Similar acute toxicity study of ZnO-NPs were also conducted
the by Srivastav et al. (2016) [38] having diameter 13 to 68 nm
reported lethal dose greater than 2000 mg/kg body weight in
rats which corroborates with the present findings. Contrary to
the present findings, previous study of Ko et al. (2015) [39]
recorded treatments related clinical signs as salivation, abdom-
inal distension, fur loss and weakness in female rats given
ZnO-NPs @ 2000 mg/kg repeatedly for 14 days and suggested
irritation of oral mucosa and gastrointestinal mucosa might be
the reason.
The haematobiochemical parameters of three studies (Sight-
ing study @300 mg/kg; Sighting study @ 2000 mg/kg
and Main study @ 2000 mg/kg body weight) accessed at
14 days post treatment revealed within the normal physiolog-
ical range [40].
On 14th day, histopathological observations of lung, liver,
heart, kidney, stomach, intestine and spleen revealed normal
histological appearance in rats given ZnO-NPs @ 300 mg/kg
and @ 2000 mg/kg body weight (Fig. 6a-6f). The absence
of toxicity symptoms suggest that ZnO-NPs were non-toxic
638
Fig. 6. Section of a) Lung showing normal bronchiole, alveoli and lung
parenchyma (H & E × 100) b) Liver showing central vein, portal triad
and normal hepatic cord (H & E × 100) c) Kidney showing normal renal
tubules, glomeruli and renal parenchyma (H & E × 100) d) Stomach
showing normal gastric glands (H & E × 100) e) Intestine showing normal
finger like villi with abundant goblet cells (H & E × 100) f) Spleen showing
well defined red and white pulp with abundant lymphoid population
(H & E × 40).
and were well tolerated at the doses employed in this study.
From the present observations it implies that the approximate
median lethal dose of ZnO-NPs is greater than 2000 mg/kg
body weight in rats. Hence, ZnO-NPs were considered to be in
Acute Toxic Category 5 in the Globally Harmonized System
as per OECD guideline 420.
IV. C ONCLUSION
This green synthesis approach shows that the environmen-
tally renewable leaf extract of B. pinnatum can be used as
an effective stabilizing as well as reducing agent for the
synthesis of ZnO-NPs and also to eliminate the use of expen-
sive chemicals, consumption of less energy and to produces
environmentally well-disposed products and byproducts. Acute
toxicity study showed absence of toxicity symptoms and
suggested that ZnO-NPs were nontoxic and was well tolerated
at the doses employed in this study.
C ONFLICT OF I NTEREST
The authors declare no conflict of interest, financial or
otherwise.
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IEEE TRANSACTIONS ON NANOBIOSCIENCE, VOL. 19, NO. 4, OCTOBER 2020
R EFERENCES
[1] J. Sivakumar, C. Premkumar, P. Santhanam, and N. Saraswathi, “Biosyn-
thesis of silver nanoparticles using Calotropis gigantean leaf,” Afr. J.
Basic Appl. Sci., vol. 3, no. 6, pp. 265–270, 2011.
[2] C. Vidya, S. Hiremath, M. N. Chandraprabha, I. Venugopal, A. Jain,
and K. Bansal, “Green synthesis of ZnO nanoparticles by Calotropis
gigantea,” Interfaces J. Current Eng. Technol., vol. 4, pp. 118–120,
Apr. 2013.
[3] S. P. Vyas and R. K. Khar, “Drug delivery to bone marrow,” in Targeted
and Controlled Drug Delivery, Novel Carrier Systems, S. P. Vyas and
R. K. Khar, Eds. New Delhi, India: CBS Publisher Distributor, 2002,
pp. 562–585.
[4] S. Iravani, “Green synthesis of metal nanoparticles using plants,” Green
Chem., vol. 13, no. 10, pp. 2638–2650, 2011.
[5] K. Xu, Y. Chen, T. A. Okhai, and L. W. Snyman, “Micro optical sensors
based on avalanching silicon light-emitting devices monolithically inte-
grated on chips,” Opt. Mater. Express, vol. 9, no. 10, pp. 3985–3997,
Oct. 2019.
[6] D. Fixler, R. Tirosh, T. Zinman, A. Shainberg, and M. Deutsch,
“Differential aspects in ratio measurements of [Ca2+ ]i relaxation in
cardiomyocyte contraction following various drug treatments,” Cell
Calcium, vol. 31, no. 6, pp. 279–287, Jun. 2002.
[7] J. K. Patra and K.-H. Baek, “Green nanobiotechnology: Factors affecting
synthesis and characterization techniques,” J. Nanomaterials, vol. 2014,
pp. 1–12, 2014, Art. no. 417305
[8] V. N. Kalpana and V. Devi Rajeswari, “A review on green synthesis,
biomedical applications, and toxicity studies of ZnO NPs,” Bioinorganic
Chem. Appl., vol. 2018, pp. 1–12, Aug. 2018, Art. no. 3569758
[9] S. Shende, D. Rathod, A. Gade, and M. Rai, “Biogenic copper nanopar-
ticles promote the growth of pigeon pea (Cajanus cajan L.),” IET
Nanobiotechnology, vol. 11, no. 7, pp. 773–781, May 2017.
[10] A. S. Jahagirdar, S. Shende, A. Gade, and M. Rai, “Bioinspired synthesis
of copper nanoparticles and its efficacy on seed viability and seedling
growth in mungbean (Vigna radiata L.),” Current Nanoscience, vol. 16,
no. 2, pp. 246–252, Mar. 2020.
[11] K. N. Gaind and R. L. Gupta, “Alkanes, alkanols, triterpenes and sterols
of Kalanchoe pinnata,” Phytochemistry, vol. 11, no. 4, pp. 1500–1502,
Apr. 1972.
[12] Y. Cao, B. Liu, R. Huang, Z. Xia, and S. Ge, “Flash synthesis of flower-
like ZnO nanostructures by microwave-induced combustion process,”
Mater. Lett., vol. 65, no. 2, pp. 160–163, Jan. 2011.
[13] W. Maret, “Metals on the move: Zinc ions in cellular regulation and in
the coordination dynamics of zinc proteins,” BioMetals, vol. 24, no. 3,
pp. 411–418, Jun. 2011.
[14] A. A. Seyedeh et al., “Antibacterial activity of zinc oxide nanoparticles
on Staphylococcus aureus,” Int. J. Adv. Biotechnol. Res., vol. 7, no. 3,
pp. 1569–1575, 2016.
[15] H. A. Atef et al., “Efficacy of zinc oxide nanoparticles and curcumin
in amelioration the toxic effects in aflatoxicated rabbits,” Int. J. Current
Microbiol. Appl. Sci., vol. 5, no. 12, pp. 795–818, Dec. 2016.
[16] A. Alkaladi, A. Abdelazim, and M. Afifi, “Antidiabetic activity of zinc
oxide and silver nanoparticles on streptozotocin-induced diabetic rats,”
Int. J. Mol. Sci., vol. 15, no. 2, pp. 2015–2023, Jan. 2014.
[17] M. S. Ågren, “Studies on zinc in wound healing,” Actadermato-
Venereologica, Suppl. (Stockh), vol. 70, no. 154, pp. 1–36, Jan. 1990.
[18] S. Soren, S. Kumar, S. Mishra, P. K. Jena, S. K. Verma, and P. Parhi,
“Evaluation of antibacterial and antioxidant potential of the zinc oxide
nanoparticles synthesized by aqueous and polyol method,” Microbial
Pathogenesis, vol. 119, pp. 145–151, Jun. 2018.
[19] S. A. E. Bashandy, A. Alaamer, S. A. A. Moussa, and E. A. Omara,
“Role of zinc oxide nanoparticles in alleviating hepatic fibrosis and
nephrotoxicity induced by thioacetamide in rats,” Can. J. Physiol.
Pharmacol., vol. 96, no. 4, pp. 337–344, Apr. 2018.
[20] C. Clemedson et al., “MEIC evaluation of acute systemic toxicity-part
VII. Prediction of human toxicity by results from testing of the first 30
reference chemicals with 27 further in vitro assays,” Alter Lab Animals,
vol. 28, no. 1, pp. 161–200, 1999.
[21] D. G. Maheshwari and N. K. Shaikh, “An overview on toxicity testing
method,” Int. J. Pharma. Technol., vol. 8, no. 2, pp. 3834–3849, 2016.
[22] T. A. Loomis and A. W. Hayes, Loomis’s Essentials of Toxicology, vol. 4.
New York, NY, USA: Academic, 1996, pp. 208–245.
[23] D. Baishya, N. Sharma, and R. B. Green, “Synthesis of silver nanopar-
ticle using Bryophyllum pinnatum (Lam.) and monitoring their antibac-
terial activities,” Arch. Appl. Sci. Res., vol. 4, no. 5, pp. 2098–2104,
2012.
JADHAO et al.: BIOGENIC SYNTHESIS OF ZINC OXIDE NANOPARTICLES
[24] L. G. Luna, Manual of Histologic Staining Methods of the Armed Forces
Institute of Pathology, 3rd ed. New York, NY, USA: McGraw-Hill, 1968,
pp. 124–125.
[25] S. Kapoor, “Preparation, characterization, and surface modifica-
tion of silver particles,” Langmuir, vol. 14, no. 5, pp. 1021–1025,
Mar. 1998.
[26] J. Zhu, S. Liu, O. Palchik, Y. Koltypin, and A. Gedanken, “Shape-
controlled synthesis of silver nanoparticles by pulse sonoelectrochemical
methods,” Langmuir, vol. 16, no. 16, pp. 6396–6399, Aug. 2000.
[27] N. Tiwari, R. Pandit, S. Gaikwad, A. Gade, and M. Rai, “Biosynthesis
of zinc oxide nanoparticles by petals extract of Rosa indica L., its
formulation as nail paint and evaluation of antifungal activity against
fungi causing onychomycosis,” IET Nanobiotechnology, vol. 11, no. 2,
pp. 205–211, Mar. 2017.
[28] P. Ramesh, A. Rajendran, and M. Sundaram, “Green syntheis of zinc
oxide nanoparticles using flower extract Cassia auriculata,” J. Nanosci.
Nanotechnol., vol. 1, no. 1, pp. 41–45, 2014.
[29] A. Nawaz, G. Khan, A. Hussain, A. Ahmad, A. Khan, and M. Safdar,
“Curcumin: A natural product of biological importance,” Gomal Univ.
J. Res., vol. 27, no. 1, pp. 7–14, 2011.
[30] S. Gaikwad et al., “Antiviral activity of mycosynthesized silver nanopar-
ticles against herpes simplex virus and human parainfluenza virus type
3,” Int. J. Nanomed., vol. 8, pp. 47–53, Nov. 2013.
[31] N. Jain, A. Bhargava, J. C. Tarafdar, S. K. Singh, and J. Panwar,
“A biomimetic approach towards synthesis of zinc oxide nanopar-
ticles,” Appl. Microbiol. Biotechnol., vol. 97, no. 2, pp. 859–869,
Jan. 2013.
Authorized licensed use limited to: DELHI TECHNICAL UNIV. Downloaded on December 18,2020 at 08:53:44 UTC from IEEE Xplore. Restrictions apply.
639
[32] K. Xu, “Silicon MOS optoelectronic micro-nano structure based on
reverse-biased PN junction,” Phys. Status Solidi, vol. 216, no. 7, 2019,
Art. no. 1800868.
[33] J. S. Aprioku and I. Igbe, “Effects of aqueous Bryophyllum pinnatum
leaf extract on hematological, renal and sperm indices in Wistar rats,”
Indian J. Pharmaceutical Sci., vol. 79, no. 4, pp. 521–526, 2017.
[34] B. Derjaguin and L. Landau, “Theory of the stability of strongly
charged lyophobic sols and of the adhesion of strongly charged particles
in solutions of electrolytes,” Acta Physico-Chimica Sinica., vol. 43,
nos. 1–4, pp. 633–662, May/Aug. 1993.
[35] E. J. W. Verwey and J. T. G. Overbeek, Theory of the Stability of
Lyophobic Colloids. Amsterdam, The Netherlands: Elsevier, 1948.
[36] J. Farkas et al., “Effects of silver and gold nanoparticles on rainbow
trout (Oncorhynchus mykiss) hepatocytes,” Aquatic Toxicology, vol. 96,
no. 1, pp. 44–52, Jan. 2010.
[37] J. Farkas et al., “Uptake and effects of manufactured silver nanoparticles
in rainbow trout (Oncorhynchus mykiss) gill cells,” Aquatic Toxicology,
vol. 101, no. 1, pp. 117–125, Jan. 2011.
[38] A. K. Srivastav et al., “A comprehensive toxicity study of zinc oxide
nanoparticles versus their bulk in wistar rats: Toxicity study of zinc
oxide nanoparticles,” Human Experim. Toxicology, vol. 35, no. 12,
pp. 1286–1304, Dec. 2016.
[39] J. W. Ko et al., “Evaluation of 2-week repeated oral dose toxicity of 100
nm zinc oxide nanoparticles in rats,” Lab. Animal Res., vol. 31, no. 3,
pp. 139–147, 2015.
[40] J. Hau and S. J. Schapiro, Handbook of Laboratory Animal Science,
vol. 2, 3rd ed. Boca Raton, FL, USA: CRC Press, 2011.