Effect of ZnO and CuO Nanoparticles on Physiology, Steviol Glycosides and Antioxidant Activities in Invitro Grown Shoots of. Stevia rebaudiana Bertoni.
Effect of ZnO and CuO Nanoparticles on Physiology, Steviol Glycosides and Antioxidant Activities in Invitro Grown Shoots of Stevia rebaudiana Bertoni 1,2,4
Rabia Javed
2
3
5
, Muhammad Zia , Muhammad Usman , Buhara Yucesan , Ekrem Gurel
1
2
4 3
Department of Multidisciplinary Studies, National University, of Medical Sciences, Rawalpindi 46000, Pakistan Department of Biotechnology, Quaid-i-Azam University, Islamabad 45320, Pakistan Department of Physics, Quaid-i-Azam University, Islamabad 45320 , 4 5 Pakistan Department of Biology, Abant Izzet Baysel University , Bolu 14030, Turkey Department of Seed Science and Technology, Abant Izzet Baysel University, Bolu 14030, Turkey
Introduction
Materials & Methods
This study describes the effect of various concentrations (0, 0.1, 1.0, 10, 100 or 1000 mg/L) of ZnO (34 nm in size) and CuO (47 nm in size) nanoparticles on the physiological parameters, steviol glycosides (rebaudioside A and stevioside) content, and antioxidant
ZnO and CuO nanoparticles were synthesized by co-precipitation meth-
activities in tissue culture grown shoots of zero calorie highly valuable medicinal plant, Stevia rebaudiana Bertoni. In recent years,
od and characterized by:
the studies encompassing field of nanotechnology for determination of effects of environmental stress on plant physiology and bio-
chemistry have been finding a fast pace. Metallic oxide nanoparticles, specifically nano-scale ZnO and CuO have gained paramount
importance in this regard. These nanoparticles possess dynamic properties because of which these are employed to nodal explants to
examine their potential effect on growth and secondary metabolites production of Stevia rebaudiana.
X-ray diffraction (XRD) Fourier transform infra-red spectroscopy (FTIR) Scanning electron microscopy (SEM) Electron dispersive x-ray (EDX)
Shoot nodal explants of Stevia rebaudiana undergo direct shoot organogenesis in MS medium containing ZnO and CuO nanoparticles that cre-
Results
ate oxidative pressure. The physiological parameters are measured, and ZnLa
15
9
Error
C
6.1
1.26
O
24.54
0.43
Zn
64.91
0.93
Pd
1.35
0.15
Au
3.1
0.24
Total
100
the quantity of steviol glycosides (rebaudioside A and stevioside) is de-
ZnO
Microplate reader
0
1
ZnKa
2
ZnKb
0
Pd
Au
3
termined by high performance liquid chromatography (HPLC). Nonenzymatic antioxidant activities, i.e., total phenolic content (TPC), total
6 C OKa ZnL1
CPS/eV
12
Element Wt%
3
4
5
6
7
8
9
flavonoid content (TFC), total antioxidant capacity (TAC), total reducing
SEM
10
power (TRP) and DPPH-free radical scavenging activity are estimated
Energy (keV )
using microplate reader.
HPLC XRD and FTIR of ZnO nanoparticles
XRD and FTIR of CuO nanoparticles SEM of ZnO nanoparticles
SEM of CuO nanoparticles
EDX of ZnO and CuO nanoparticles
Conclusion Summing up, ZnO and CuO nanoparticles can be applied in invitro batch cultures of Stevia rebaudiana containing up to 1 mg/L and 10 mg/L of concentration, respectively. In this way, large quantity of secondary metabolites are obtained in bioreactors. Phytotoxic effects of ZnO and CuO nanoparticles appear once this threshold is achieved, and significantly highest level of phtotoxicity is exhibited at 1000 mg/L concentration. The phytotoxic effect is due to the excessive accumulation of reactive oxygen species (ROS). This research opens new avenues for studying the fate of nanoparticles in the medicinal plants in context of their uptake, translocation and alteration of metabolic pathways in a concentration-dependent manner.
HPLC results of CuO nanoparticles
HPLC results of ZnO nanoparticles
References
Physiology results of ZnO nanoparticles Conc. of ZnO TPC TFC nanoparticles (µg QE/mg) (µg GAE/mg) (ppm) Control 3.81±0.07d 4.52±0.03b 0.1 5.42±0.02b 4.54±0.02b
Physiology results of CuO nanoparticles
TAC(µg AAE/mg)
TRP(µg AAE/mg)
% DPPH inhibition
9.84±0.01c 10.8±0.01b
8.28±0.02c 8.63±0.02b
60.6 66.7
Conc. of CuO nanoparticles (ppm) Control 0.1
TPC (µg GAE/ mg)
TFC (µg QE/ mg)
TAC (µg TRP(µg % DPPH AAE/mg) AAE/mg) Inhibition
4.5±0.0c 4.6±0.0c
3.8±0.0d 6.3±0.0b
10.4±0.0b 10.9±0.0b
10.5±0.0c 10.7±0.0b
60.6 66.7
1.0 10
a
6.22±0.03 3.94±0.01c
a
4.67±0.03 4.24±0.05c
a
11.7±0.02 10.9±0.03b
a
10.3±0.01 10.3±0.01a
68.6 74.8
1.0 10
5.6±0.0b 6.2±0.0a
6.3±0.0b 7.5±0.0a
11.5±0.0a 11.9±0.0a
10.8±0.0b 11.5±0.0a
68.6 74.8
100
2.56±0.01e
3.01±0.01d
7.36±0.01d
9.99±0.04a
64.2
f
e
e
d
100
4.1±0.0d
5.7±0.0c
9.52±0.0c 10.6±0.0bc
64.2
59.5
1000
3.9±0.0e 2.1±0.0e 9.16±0.0d 10.3±0.0d Phytochemical results of CuO nanoparticles
58.5
1000
1.36±0.01
2.33±0.04
4.62±0.05
6.33±0.03
Phytochemical results of ZnO nanoparticles
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Acknowledgement Authors are highly indebted to TUBITAK fellowship (program # 2216) for providing the funds, and Quaid-i-Azam University, Islamabad, Pakistan and Abant Izzet Baysel University, Bolu, Turkey for providing all the research facilities to perform this work.
